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Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells
Abstract
Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.
... 78 To determine the biological mechanism underlying the influence of HHIP on COPD susceptibility, a knockdown cell model was used. 79 Silencing of the gene resulted in differential expression of nearly 300 genes in the bronchial epithelial cell line Beas-2B. 79 Interestingly, there was enrichment for variants associated with COPD and seven of these genes were found to be differentially expressed in human COPD and control lung tissues. ...
... 79 Silencing of the gene resulted in differential expression of nearly 300 genes in the bronchial epithelial cell line Beas-2B. 79 Interestingly, there was enrichment for variants associated with COPD and seven of these genes were found to be differentially expressed in human COPD and control lung tissues. 79 Additional analyses found enrichment for extracellular matrix genes and genes involved in cell growth. ...
... 79 Interestingly, there was enrichment for variants associated with COPD and seven of these genes were found to be differentially expressed in human COPD and control lung tissues. 79 Additional analyses found enrichment for extracellular matrix genes and genes involved in cell growth. 79 The functionality of the specific SNP in the long range enhancer was also demonstrated using promoter-reporter approaches. ...
Chronic respiratory diseases are a major cause of morbidity and mortality. Asthma and chronic obstructive pulmonary disease (COPD) combined affect over 500 million people worldwide. While environmental factors are important in disease progression, asthma and COPD have long been known to be heritable with genetic components playing an important role in the risk of developing disease. Identification of genetic variation contributing to disease progression is important for a number of reasons including identification of risk alleles, understanding underlying disease mechanisms and development of novel therapies. Genome‐wide association studies (GWAS) have been successful in identifying many loci associated with lung function, COPD and asthma. In recent years, meta‐analyses and improved imputation have facilitated the growth of GWAS in terms of numbers of subjects and the number of single nucleotide polymorphisms (SNP) that can be interrogated. As a consequence, there has been a significant increase in the number of signals associated with asthma, COPD and lung function. SNP that have shown association with lung function reassuringly show a significant overlap with SNP associated with COPD giving a glimpse at pathways that may be involved in COPD mechanisms including genes in, for example, developmental pathways. In asthma, association signals are often in or near genes involved in both adaptive and innate immune response pathways, epithelial cell homeostasis and airway structural changes. The challenges now are translating these genetic signals into a new understanding of lung biology, understanding how variants impact health and disease and how they may provide opportunities for therapeutic intervention.
... The SNPs map to an intergenic region upstream of the HHIP gene and regulates HHIP gene expression in carriers. HHIP is expressed in epithelial cells and considered to modulate epithelial barrier structure as reported by Zhou et al. [15]. Zhou and workers [15] reported that modulation of HHIP expression results in differential expression of 296 epithelial genes, most of which associated with ECM matrix interactions, junctional complexes or cell growth. ...
... HHIP is expressed in epithelial cells and considered to modulate epithelial barrier structure as reported by Zhou et al. [15]. Zhou and workers [15] reported that modulation of HHIP expression results in differential expression of 296 epithelial genes, most of which associated with ECM matrix interactions, junctional complexes or cell growth. Analysis of epithelial cells derived from human lung tissue showed significantly decreased expression of these genes in cells from COPD patients as compared to cells from healthy controls. ...
... Analysis of epithelial cells derived from human lung tissue showed significantly decreased expression of these genes in cells from COPD patients as compared to cells from healthy controls. These observations indicate loss of HHIP function in human bronchial epithelial cells may contribute to susceptibility to smoke-induced COPD by regulating genes important for epithelial barrier integrity and function [15]. ...
... Real-time PCR with gene-specific TaqMan primers/ probes was performed as described previously [18]. Relative gene expression was calculated based on the standard 2 -ΔΔCT method, using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a reference gene. ...
... Application of network models in mice exposed chronically to cigarette smoke To interrogate potential regulatory mechanisms that differed in Hhip +/vs. Hhip +/+ mice and infer molecular driving factors that may contribute to the murine phenotypes described above, we constructed network models from microarray gene expression data using the PANDA (Passing Attributes between Networks for Data Assimilation) method [18]. PANDA integrates multiple sources of information, including protein-protein interaction, gene expression, and sequence motif data, to construct genome-wide, condition-specific regulatory networks. ...
... As a larger number of TFs are included in the existing motif database, our future network modeling studies using the PANDA method may reveal additional insights into the activities of Hhip in regulating CS-induced lung effects in mice. Third, we also attempted to compare gene expression changes caused by Hhip haploinsufficiency in murine lungs (Hhip +/--air vs. Hhip +/+ -air) with our previous microarray analysis in an airway epithelial cell line (Beas-2B cells), treated with either non-targeting shRNA controls or HHIP shRNAs [18]. No overlapping genes were identified (data not shown). ...
The HHIP gene, encoding Hedgehog interacting protein, has been implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS), and our subsequent studies identified a functional upstream genetic variant that decreased HHIP transcription. However, little is known about how HHIP contributes to COPD pathogenesis.
We exposed Hhip haploinsufficient mice (Hhip (+/-) ) to cigarette smoke (CS) for 6 months to model the biological consequences caused by CS in human COPD risk-allele carriers at the HHIP locus. Gene expression profiling in murine lungs was performed followed by an integrative network inference analysis, PANDA (Passing Attributes between Networks for Data Assimilation) analysis.
We detected more severe airspace enlargement in Hhip (+/-) mice vs. wild-type littermates (Hhip (+/+) ) exposed to CS. Gene expression profiling in murine lungs suggested enhanced lymphocyte activation pathways in CS-exposed Hhip (+/-) vs. Hhip (+/+) mice, which was supported by increased numbers of lymphoid aggregates and enhanced activation of CD8+ T cells after CS-exposure in the lungs of Hhip (+/-) mice compared to Hhip (+/+) mice. Mechanistically, results from PANDA network analysis suggested a rewired and dampened Klf4 signaling network in Hhip (+/-) mice after CS exposure.
In summary, HHIP haploinsufficiency exaggerated CS-induced airspace enlargement, which models CS-induced emphysema in human smokers carrying COPD risk alleles at the HHIP locus. Network modeling suggested rewired lymphocyte activation signaling circuits in the HHIP haploinsufficiency state.
... The single nucleotide polymorphisms map to an intergenic region upstream of the HHIP gene and result in reduced HHIP gene expression in carriers. HHIP is mainly expressed in epithelial cells and considered to modulate epithelial barrier structure and function [20,21]. Zhou et al. [21] reported that modulation of HHIP expression results in differential expression of 296 epithelial genes, most of which associated with extracellular matrix interactions, junctional complexes or cell growth. ...
... HHIP is mainly expressed in epithelial cells and considered to modulate epithelial barrier structure and function [20,21]. Zhou et al. [21] reported that modulation of HHIP expression results in differential expression of 296 epithelial genes, most of which associated with extracellular matrix interactions, junctional complexes or cell growth. Analysis of epithelial cells derived from human lung tissue demonstrated significantly decreased expression of these genes in cells from COPD patients as compared with cells from healthy controls. ...
... Analysis of epithelial cells derived from human lung tissue demonstrated significantly decreased expression of these genes in cells from COPD patients as compared with cells from healthy controls. These observations indicate loss of HHIP function in human bronchial epithelial cells may contribute to susceptibility to smoke-induced COPD by regulating genes important for epithelial barrier integrity and function [21]. These association studies point to the importance of effective immune surveillance and an intact and functional epithelial barrier in COPD. ...
Chronic obstructive pulmonary disease (COPD) is characterized by sustained inflammation of the airways, leading to destruction of lung tissue and declining pulmonary function. Although smoking is the most obvious risk factor for COPD, only about 20% of smokers develop COPD and smoking cessation does not reverse progression of COPD, indicating that while smoking is an important cause or initiating factor, it is not the only driver of ongoing chronic inflammation and disease progression in COPD patients. We hypothesize that smoking-induced changes in lung microbiota, epithelial integrity and epigenetic control of gene expression result in autoantigen induction and perturbed immune regulation in genetically vunerable individuals. In our view, COPD patients may be stratified according to their immunological and inflammatory status related to specific changes in the lung microbiota (innate and adaptive immunity), presence of autoantigens (adaptive immunity: Th1-B-cell axis) and epigenetic modifications (inflammation and structural changes).
... RNA samples were extracted using RNAeasy Kit from each condition and gene expression profiling was performed using one Illumina HumanHT-12_V4_0_R2_15002873_B chip. Regular data QC check was performed as previously described 44 . Gene expression levels for the two subjects were then batch-corrected using ComBat in the sva Bioconductor package 45 . ...
... In order to validation our initial ChIP-Seq and microarray analysis, we performed qPCR validation on selected H3K27Ac ChIP-Seq peaks or differentially expressed genes after intermittent smoke exposure as described previously 44,49 . ...
Cigarette smoke (CS) is one of the major risk factors for many pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. The first line of defense for CS exposure is the bronchial epithelial cells. Elucidation of the epigenetic changes during CS exposure is key to gaining a mechanistic understanding into how mature and differentiated bronchial epithelial cells respond to CS. Therefore, we performed epigenomic profiling in conjunction with transcriptional profiling in well-differentiated human bronchial epithelial (HBE) cells cultured in air-liquid interface (ALI) exposed to the vapor phase of CS. The genome-wide enrichment of histone 3 lysine 27 acetylation was detected by chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) in HBE cells and suggested the plausible binding of specific transcription factors related to CS exposure. Additionally, interrogation of ChIP-Seq data with gene expression profiling of HBE cells after CS exposure for different durations (3 hours, 2 days, 4 days) suggested that earlier epigenetic changes (3 hours after CS exposure) may be associated with later gene expression changes induced by CS exposure (4 days). The integration of epigenetics and gene expression data revealed signaling pathways related to CS-induced epigenetic changes in HBE cells that may identify novel regulatory pathways related to CS-induced COPD.
... Using chromosomal conformation capture studies, our group demonstrated that this GWAS region interacts with the hedgehog interacting protein (HHIP) promoter through a functional genetic variant within a distal enhancer which alters binding to the SP3 transcription factor 3 . Subsequent studies of human lung epithelial cells exposed to cigarette smoke in vitro suggested that, in addition to its established roles in morphogenesis and embryonic development through the hedgehog pathway 4 , HHIP may alter extracellular matrix and cell growth pathways 5 . Recently, we also demonstrated a role for HHIP in the development of spontaneous, age-related emphysema in murine models of Hhip haploinsufficiency 6 . ...
... Hedgehog interacting protein (HHIP) is a highly conserved, vertebrate-specific protein which is both induced by and serves as a negative regulator of hedgehog signaling 11 . HHIP has an established role in branching morphogenesis of the lung during embryonic development 4 ; based on subsequent RNA interference studies in a human airway epithelial cell line, HHIP was also implicated in lung extracellular matrix and cell growth pathways 5 . In addition to associations with adult height 12 and several malignancies 13 , variants annotated to the HHIP locus have been robustly associated with lung function and the development of COPD 2, 14 . ...
Genetic variants annotated to the hedgehog interacting protein (HHIP) are robustly associated with chronic obstructive pulmonary disease (COPD). Hhip haploinsufficiency in mice leads to increased susceptibility towards the development of emphysema following exposure to chronic cigarette smoke (CS). To explore the molecular pathways which contribute to increased susceptibility, we performed metabolomic profiling using high performance liquid chromatography tandem mass spectroscopy (LC/MS-MS) on plasma, urine, and lung tissue of Hhip+/− heterozygotes and wild type (Hhip+/+) C57/BL6 mice exposed to either room-air or CS for six months. Univariate comparisons between groups were made with a combined fold change ≥2 and Student’s t-test p-value < 0.05 to denote significance; associations with mean alveolar chord length (MACL), a quantitative measure of emphysema, and gene-by-environment interactions were examined using empiric Bayes-mediated linear models. Decreased urinary excretion of cotinine despite comparable plasma levels was observed in Hhip+/− heterozygotes; a strong gene-by-smoking association was also observed. Correlations between MACL and markers of oxidative stress such as urinary methionine sulfoxide were observed in Hhip+/− but not in Hhip+/+ mice. Metabolite set enrichment analyses suggest reduced antioxidant capacity and alterations in macronutrient metabolism contribute to increased susceptibility to chronic CS-induced oxidative stress in Hhip haploinsufficiency states.
... All samples were assayed in duplicates by quantitative real-time PCR. Expression levels of target genes were calculated based on the 2 ÀDDCt method as we performed previously [30]. ...
Background:
Family with Sequence Similarity 13, Member A (FAM13A) gene has been consistently associated with COPD by Genome-wide association studies (GWAS). Our previous study demonstrated that FAM13A was mainly expressed in the lung epithelial progenitors including Club cells and alveolar type II epithelial (ATII) cells. Fam13a-/- mice were resistant to cigarette smoke (CS)-induced emphysema through promoting β-catenin/Wnt activation. Given the important roles of β-catenin/Wnt activation in alveolar regeneration during injury, it is unclear when and where FAM13A regulates the Wnt pathway, the requisite pathway for alveolar epithelial repair, in vivo during CS exposure in lung epithelial progenitors.
Methods:
Fam13a+/+ or Fam13a-/- mice were crossed with TCF/Lef:H2B-GFP Wnt-signaling reporter mouse line to indicate β-catenin/Wnt-activated cells labeled with GFP followed by acute (1 month) or chronic (7 months) CS exposure. Fluorescence-activated flow cytometry analysis, immunofluorescence and organoid culture system were performed to identify the β-catenin/Wnt-activated cells in Fam13a+/+ or Fam13a-/- mice exposed to CS. Fam13a;SftpcCreERT2;Rosa26RmTmG mouse line, where GFP labels ATII cells, was generated for alveolar organoid culture followed by analyses of organoid number, immunofluorescence and gene expression. Single cell RNA-seq data from COPD ever smokers and nonsmoker control lungs were further analyzed.
Findings:
We found that FAM13A-deficiency significantly increased Wnt activation mainly in lung epithelial cells. Consistently, after long-term CS exposure in vivo, FAM13A deficiency bestows alveolar epithelial progenitor cells with enhanced proliferation and differentiation in the ex vivo organoid model. Importantly, expression of FAM13A is significantly increased in human COPD-derived ATII cells compared to healthy ATII cells as suggested by single cell RNA-sequencing data.
Interpretation:
Our findings suggest that FAM13A-deficiency promotes the Wnt pathway-mediated ATII cell repair/regeneration, and thereby possibly mitigating CS-induced alveolar destruction. FUND: This project is funded by the National Institutes of Health of United States of America (NIH) grants R01HL127200, R01HL137927, R01HL148667 and R01HL147148 (XZ).
... Specific amino acid residues on collagen and elastin can be oxidized by LOX in the extracellular matrix, forming covalent bonds between collagen and elastin, with the participation of Cu2+ (58)(59)(60)(61)(62). Single-nucleotide polymorphisms (SNP), such as rs1828591 and rs13118928 on the gene of Hedgehog Interacting Protein (HHIP), are intensively related to COPD (63). HHIP negatively regulates cell proliferation and matrix repair under normal conditions (64). By comparing mesenchymal cells in the lung tissue of emphysema and normal subjects through singlecell sequencing, it was found that the Hh signaling pathways were atopically activated in the alveolar matrix, impairing alveolar stem cells. ...
The chronic obstructive pulmonary disease (COPD) emphysematous phenotype is characterized by destruction of lung tissue structure. Patients with this phenotype usually present with typical emphysema-like changes on chest computed Tomography CT, experience higher mortality and poorer prognosis, and are insensitive to routine pharmacological COPD therapy. However, the pathogenesis for the COPD emphysematous phenotype remains unclear, resulting in diagnostic and therapeutic challenges. The imbalance between injury and defense mechanisms is essential in the progression of many pulmonary diseases. Thus, in this review, we focus on the pathogenesis of the COPD emphysematous phenotype and discuss the pathophysiological processes involved in disease progression, from the perspective of injury and defense imbalance.
... HHIP, mapping to chromosome 4q31.21-31.3, negatively regulates activation of the hedgehog signaling pathway and functions as an anti-oncogene in many kinds of tumors, including gastric, colon, lung, and breast cancers (17)(18)(19)(20)(21). HHIP is critical to many biological functions linked to tumor development, such as epithelial-mesenchymal transition, metastasis, apoptosis and proliferation (18)(19)(20)(21)(22). ...
Background:
MicroRNAs are found to be aberrantly expressed in multiple cancers, including glioblastoma (GBM), and microRNA-221 (miR-221) has been verified as an oncogene in various human cancers. Nevertheless, the role of miR-221 in GBM is unclear. This study aimed to investigate the miR-221 expression level in GBM and to evaluate its function and underlying mechanisms.
Methods:
Western blotting and qPCR were used to determine the expression of human hedgehog-interacting protein (HHIP) and miR-221 levels. MiR-221-inhibited cell models were constructed, and siRNA was used for HHIP silencing. Cell proliferation was analyzed by MTT and colony formation assays and a subcutaneous xenograft model. Cell migration and invasion was analyzed by wound healing and Transwell invasion assays. A dual luciferase reporter assay system was used to clarify the relationship between miR-221 and HHIP.
Results:
The results of this study revealed that miR-221 expression was upregulated in GBM tissues and A172, U251, as well as T98G cells, as detected by real-time PCR analysis. MTT, Transwell, and colony formation assays revealed that miR-221 knockdown could suppress GBM cells from proliferating, migrating, and invading in vitro. Moreover, animal experiments showed that tumor growth in vivo was inhibited when miR-221 expression decreased. Furthermore, HHIP was predicted and verified to be a target of miR-221 by bioinformatics analysis, and luciferase and western blot assays. In addition, HHIP silencing rescued the suppressive effect of a miR-221 inhibitor on the proliferation, migration, and invasion of GBM cells.
Conclusions:
Our results indicated that miR-221 is upregulated in GBM and enhances tumor progression by targeting HHIP, which suggests this may be a potential therapeutic target for GBM.
... Only one locus near HHIP was identified in the three GWAS. The HHIP gene, which encodes a member of the hedgehog-interacting protein family, is a well-known COPD susceptibility locus that has been identified in GWAS and gene expression studies 17,31,32 and has also been associated with COPD exacerbations 2 . Two additional loci near CHRNA3 and CHRNA4 were associated with both ICD-coded and GOLD-based COPD. ...
Biobanks have facilitated the conduct of large-scale genomics studies, but they are challenged by the difficulty of validating some phenotypes, particularly for complex traits that represent heterogeneous groups ofpatients. The guideline definition of COPD, based on objective spirometry measures, has been preferred in genome-wide association studies (GWAS) conducted with epidemiological cohorts, but spirometry measures are seldom available for biobank participants. Defining COPD based on International Classification of Disease (ICD) codes or self-reported measures is highly feasible in biobanks, but it remains unclear whether the misclassification inherent in these definitions prevent the discovery of genetic variants that contribute to COPD. We found that while there was poor agreement in classification of UK Biobank participants as having COPD based on ICD diagnosis codes, self-reported doctor diagnosis or spirometry measures, contrasting GWAS results for these definitions provided insights into what patient characteristics each trait may capture.
... HHIP competes with Ptch1 (which is the membrane receptor for IHH) for the binding of IHH and DHH. Ptch1 binding to IHH and DHH triggers the hedgehog signaling pathway, therefore the binding of HHIP with IHH negatively regulates the hedgehog pathway which is known to have a crucial role in lung development 38,40 . ...
The molecular and clinical features of a complex disease can be influenced by other diseases affecting the same individual. Understanding disease-disease interactions is therefore crucial for revealing shared molecular mechanisms among diseases and designing effective treatments. Here we introduce Flow Centrality (FC), a network-based approach to identify the genes mediating the interaction between two diseases in a protein-protein interaction network. We focus on asthma and COPD, two chronic respiratory diseases that have been long hypothesized to share common genetic determinants and mechanisms. We show that FC highlights potential mediator genes between the two diseases, and observe similar outcomes when applying FC to 66 additional pairs of related diseases. Further, we perform in vitro perturbation experiments on a widely replicated asthma gene, GSDMB, showing that FC identifies candidate mediators of the interactions between GSDMB and COPD-associated genes. Our results indicate that FC predicts promising gene candidates for further study of disease-disease interactions. Complex diseases often share genetic determinants and symptoms, but the mechanistic basis of disease interactions remains elusive. Here, the authors propose a network topological measure to identify proteins linking complex diseases in the interactome, and identify mediators between COPD and asthma.
... First, the top sentinel variant for both COPD/RHR and COPD/HBP was rs7655625 near HHIP, known to be associated with COPD susceptibility by influencing crucial lung development signaling pathway [51]. HHIP is also downregulated during angiogenesis and under oxidative stress [52], and its knockdown in late endothelial progenitor cells improves endothelial angiogenesis, promoting vascular repair [53]. ...
Background
A growing number of studies clearly demonstrate a substantial association between chronic obstructive pulmonary disease (COPD) and cardiovascular diseases (CVD), although little is known about the shared genetics that contribute to this association.
Methods
We conducted a large-scale cross-trait genome-wide association study to investigate genetic overlap between COPD (Ncase = 12,550, Ncontrol = 46,368) from the International COPD Genetics Consortium and four primary cardiac traits: resting heart rate (RHR) (N = 458,969), high blood pressure (HBP) (Ncase = 144,793, Ncontrol = 313,761), coronary artery disease (CAD)(Ncase = 60,801, Ncontrol = 123,504), and stroke (Ncase = 40,585, Ncontrol = 406,111) from UK Biobank, CARDIoGRAMplusC4D Consortium, and International Stroke Genetics Consortium data.
Results
RHR and HBP had modest genetic correlation, and CAD had borderline evidence with COPD at a genome-wide level. We found evidence of local genetic correlation with particular regions of the genome. Cross-trait meta-analysis of COPD identified 21 loci jointly associated with RHR, 22 loci with HBP, and 3 loci with CAD. Functional analysis revealed that shared genes were enriched in smoking-related pathways and in cardiovascular, nervous, and immune system tissues. An examination of smoking-related genetic variants identified SNPs located in 15q25.1 region associated with cigarettes per day, with effects on RHR and CAD. A Mendelian randomization analysis showed a significant positive causal effect of COPD on RHR (causal estimate = 0.1374, P = 0.008).
Conclusion
In a set of large-scale GWAS, we identify evidence of shared genetics between COPD and cardiac traits.
... TULP3 is a known target of the Hedgehog pathway. Notably, GWASs and follow-up functional studies have identified an important role for HHIP in the development of COPD (7); TULP3 has been shown to change expression after HHIP silencing (37). Also, WDR35 and IFT140 were associated with respiratory system abnormalities in mouse models (WDR35 leads to lung hypoplasia and mutations in IFT140 produces severely misshapen lungs). ...
Chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide, is substantially influenced by genetic factors. Alpha-1 antitrypsin deficiency demonstrates that rare coding variants of large effect can influence COPD susceptibility. To identify additional rare coding variants in patients with severe COPD, we conducted whole exome sequencing analysis in 2,543 subjects from two family-based studies (Boston Early-Onset COPD Study and International COPD Genetics Network) and one case-control study (COPDGene). Applying a gene-based segregation test in the family-based data, we identified significant segregation of rare loss of function variants in TBC1D10A and RFPL1 (P < 2x10-6), but were unable to find similar variants in the case-control study. In single variant, gene-based, and pathway association analyses, we were unable to find significant findings that replicated or were significant in meta-analysis. However, we found that the top results in the two datasets were in proximity to each other in the protein-protein interaction network (p = 0.014), suggesting enrichment of these results for similar biological processes. A network of these association results and their neighbors was significantly enriched in the transforming growth factor beta-receptor binding and cilia-related pathways. Finally, in a more detailed examination of candidate genes, we identified individuals with putative high-risk variants, including patients harboring homozygous mutations in genes associated with cutis laxa and Niemann-Pick Disease Type C. Our results likely reflect heterogeneity of genetic risk for COPD along with limitations of statistical power and functional annotation, and highlight the potential of network analysis to gain insight into genetic association studies.
... Kif13b promotes Shh signalling (Schou et al., 2017) Tgfb1 TGF-β-increases Shh signalling to induced fibrosis. (Chung and Fu, 2013) Itgav Itgav is a target of Hedgehog signalling and can modulate Hedgehog signalling (Kosinski et al., 2010;Zhou et al., 2013) Aliahmad, P., Kadavallore, A., de la Torre, B., Kappes, D., Kaye, J., 2011. TOX is required for development of the CD4 T cell lineage gene program. ...
... Kif13b promotes Shh signalling (Schou et al., 2017) Tgfb1 TGF-β-increases Shh signalling to induced fibrosis. (Chung and Fu, 2013) Itgav Itgav is a target of Hedgehog signalling and can modulate Hedgehog signalling (Kosinski et al., 2010;Zhou et al., 2013) Aliahmad, P., Kadavallore, A., de la Torre, B., Kappes, D., Kaye, J., 2011. TOX is required for development of the CD4 T cell lineage gene program. ...
... It also triggers GLI transcription factors, including activators GLI1 and GLI2, and an inhibitor GLI3 that regulate transcription of target genes, such as HOX and WNT in the nucleus [17,18]. Modifiers of SHh signaling pathway include, among other, SUFU, HHIP, GPC3 and RAB23 proteins [19]. ...
Aim of the study
Examination of copy number changes in a group of intracranial germ cell tumors (GCTs) with particular focus on putative aberrations of the main genes coding SHh pathway proteins.
Material and methods
The study was performed on DNA isolated from fresh-frozen tumor tissue samples from eight GCTs, including six intracranial GCTs. The intracranial group consisted of three germinomas, two mature teratomas and one mixed germ cell tumor. Comparative genomic profiling analysis was carried out using microarray-CGH method (Cytosure ISCA UPD 4×180k, OGT). The results were analyzed with Feature Extraction (Agilent Technologies) and Nexus Copy Number (BioDiscovery) softwares.
Results and conclusions
Chromosomal aberrations were found in two intracranial germinomas. These tumors were characterized by complex genomic profiles encompassing chromosomes 7, 8, 9, 10, 11, 12, 16, 17 and 19. Common findings were gain at 12p13.33p11.1 of 35 Mbp and gain at 17q11.1q25.3 of 55 Mbp. In one tumor, also SHh (7q36.3), SMO (7q32.1) and GLI3 (7p14.1) copy gains occurred together with 9q21.11q34.3 loss, including PTCH1, all being elements of SHh signaling pathway. Moreover, both tumors showed various copy gain of genes being ligands, regulators, receptors or target genes of SHh (MTSS1; PRKACA and FKBP8) as well as gain of genes of SHh coopting WNT pathway (WNT3, WNT5B, WNT9B in both tumors; WNT16, WNT2 in pineal lesion). Further studies on larger group are needed to characterize SHh-related gene alterations in intracranial GCTs and for searching genotype-phenotype relations.
... Kif13b promotes Shh signalling (Schou et al., 2017) Tgfb1 TGF-β-increases Shh signalling to induced fibrosis. (Chung and Fu, 2013) Itgav Itgav is a target of Hedgehog signalling and can modulate Hedgehog signalling (Kosinski et al., 2010;Zhou et al., 2013) Aliahmad, P., Kadavallore, A., de la Torre, B., Kappes, D., Kaye, J., 2011. TOX is required for development of the CD4 T cell lineage gene program. ...
Gli3 is a Hedgehog (Hh) responsive transcription factor that can function as a transcriptional repressor or activator. We show that Gli3 activity in thymic epithelial cells (TEC) promotes positive selection and differentiation from CD4+CD8+ to CD4+CD8- single positive (SP4) cell in the fetal thymus and that Gli3 represses Shh Constitutive deletion of Gli3, and conditional deletion of Gli3 from TEC, reduced differentiation to SP4, whereas conditional deletion of Gli3 from thymocytes did not. Conditional deletion of Shh from TEC increased differentiation to SP4, and expression of Shh was upregulated in the Gli3-deficient thymus. Use of a transgenic Hh-reporter showed that the Hh pathway was active in thymocytes, and increased in the Gli3-deficient fetal thymus. Neutralisation of endogenous Hh proteins in the Gli3-/- thymus restored SP4 differentiation, indicating that Gli3 in TEC promotes SP4 differentiation by repression of Shh Transcriptome analysis showed that Hh-mediated transcription was increased but TCR-mediated transcription decreased in Gli3-/- thymocytes compared to WT.
... [10][11][12] Additionally, the expression of Shh pathway members, including Shh, Ptch1, and Gli2, is significantly decreased in lung tissues from COPD patients compared to that in control smokers with normal lung function. 13 This suggests that the Shh pathway is involved in COPD pathogenesis and may be related to lung stem cells. ...
Background
COPD is a leading cause of mortality worldwide, and cigarette smoke is a pivotal risk factor. Adenovirus is a common cause of acute exacerbations of COPD and expedites COPD progression. Lung stem/progenitor cells play an important role in the development of COPD, while the relevant mechanism remains elusive. Here, we investigated the number of lung CD31?CD45?Sca-1? cells and sonic hedgehog (Shh) signaling pathway expression levels in cigarette smoke extract (CSE)-induced emphysema mice, as well as the relevant effects of acute adenovirus infection (AAI).
Materials and methods
BALB/c mice were treated with CSE by intraperitoneal injection and/or adenovirus endotracheal instillation at different time points for 28 days. Lung function, lung histomorphology, CD31?CD45?Sca-1? cell count, and expression levels of major components in the Shh signaling pathway in the lungs were measured.
Results
CSE intraperitoneal injection and adenovirus endotracheal instillation successfully induced emphysema and AAI in mice, respectively. In the lungs of emphysema mice, both the number of CD31?CD45?Sca-1? cells and expression levels of Shh signaling pathway molecules were reduced. However, AAI increased the number of inhibited CD31?CD45?Sca-1? cells and activated the suppression of the Shh signaling pathway.
Conclusion
Both CD31?CD45?Sca-1? cell numbers and Shh signaling pathway expression levels were downregulated in the lungs of emphysema mice induced by CSE intraperitoneal injection, which likely contributes to the pathogenesis of emphysema. Additionally, these inhibited lung CD31?CD45?Sca-1? cells and Shh signaling pathway molecules were upregulated during AAI, indicating that they play a protective role in the epithelial repair process after AAI injury.
... External Datasets. As previously reported, microarray gene expression profiling was performed in human bronchial epithelial cells (Beas-2B) stably infected with four different lentivirus-based shRNAs against HHIP and one non-targeting shRNA (control), using the Illumina HT12 gene expression platform 31 . To identify differentially expressed probes after HHIP silencing, two linear regression models were used. ...
In comparison to genome-wide association studies (GWAS), there has been poor replication of gene expression studies in chronic obstructive pulmonary disease (COPD). We performed microarray gene expression profiling on a large sample of resected lung tissues from subjects with severe COPD. Comparing 111 COPD cases and 40 control smokers, 204 genes were differentially expressed; none were at significant GWAS loci. The top differentially expressed gene was HMGB1, which interacts with AGER, a known COPD GWAS gene. Differentially expressed genes showed enrichment for putative interactors of the first three identified COPD GWAS genes IREB2, HHIP, and FAM13A, based on gene sets derived from protein and RNA binding studies, RNA-interference, a murine smoking model, and expression quantitative trait locus analyses. The gene module most highly associated for COPD in Weighted Gene Co-Expression Network Analysis (WGCNA) was enriched for B cell pathways, and shared seventeen genes with a mouse smoking model and twenty genes with previous emphysema studies. As in other common diseases, genes at COPD GWAS loci were not differentially expressed; however, using a combination of network methods, experimental studies and careful phenotype definition, we found differential expression of putative interactors of these genes, and we replicated previous human and mouse microarray results.
... Unexpectedly, the authors did not observe a significant enrichment of canonical HH pathway members other than HHIP among genes differentially expressed by microarray analysis after HHIP knockdown. Similar to our data, these results appear to support the idea of a crosstalk between HHIP and genes from distinct pathways, probably determining reciprocal influences among each other [38]. On the other hand, the expression of WNT1, an antagonist of the Hh pathway in epithelial cells [13], was markedly increased in IBD samples. ...
Hedgehog (Hh) signaling is essential for intestinal homeostasis and has been associated with inflammation and tissue repair. We hypothesized that Hh signaling could affect the inflammatory process in inflammatory bowel disease (IBD). For this purpose, colon specimens from the inflamed and non-inflamed mucosa of 15 patients with Crohn?s disease (CD), 15 with ulcerative colitis, and 15 controls were analyzed by immunohistochemistry and real-time PCR. The production and modulation of cytokines were measured by ELISA from culture explants. Apoptosis was assessed by TUNEL and caspase-3 activity assays. Chemotaxis was evaluated using a transwell system. Primary human intestinal and skin fibroblasts were used for analyzing migration and BrdU incorporation. Hh proteins were generally expressed at the superficial epithelium, and a marked reduction was observed in CD. In the lamina propria, Gli-1 predominantly co-localized with vimentin- and alpha-smooth muscle actin-positive cells, with lower levels observed in CD. In colon explants, Hh stimulation resulted in reduction, while blockade increased, TNF ?, IL-17, and TGF ? levels. Apoptotic rates were higher in inflamed samples, and they increased after Hh blockade. Levels of Gli-1 mRNA were negatively correlated with caspase-3 activity. Hh blockade increased chemoattraction of monocytes. Primary fibroblasts incorporated more BrdU, but migrated less after Hh blockade. These results suggest that Hh signaling provides a negative feedback to the lamina propria, down-regulating inflammatory cytokines, and inhibiting leukocyte migration and fibroblast proliferation, while favoring fibroblast migration. Therefore, Hh signaling is strongly implicated in the pathogenesis of intestinal inflammation, and it may represent a novel therapeutic target for IBD.
... 32 Silencing of HHIP leads to differential expression of about 300 genes enriched for cell growth, lung extracellular matrix, and genes associated with COPD. 33 Together, these findings point toward a plausible role for HHIP as an airway obstruction susceptibility gene, regardless of smoking. It was suggested that FAM13A might play a role in Rho GTPase signaling based on the presence of a Rho-GAP (Ras homologous GTPase-activation protein) domain in the protein sequence. ...
Background:
Although a striking proportion (25-45%) of subjects with chronic obstructive pulmonary disease (COPD) are never-smokers, most genetic susceptibility studies have not focused on this group exclusively.
Objective:
The aim of this study was to identify common genetic variants associated with the level of forced expiratory volume in one second (FEV1) and its ratio to forced vital capacity (FEV1/FVC) in never-smokers.
Methods:
Genome-wide association studies (GWAS) were performed in 5,070 never-smokers of the identification cohort LifeLines, and results (p-value <10(-5)) were verified by a meta-analysis of the Vlagtwedde-Vlaardingen study and the Rotterdam Study I-III (total n=1,966). Furthermore, we aimed to assess the effects of the replicated variants in more detail by performing genetic risk score (GRS), expression quantitative trait loci (eQTL), and variant*ever-smoking interaction analyses.
Results:
We identified associations between FEV1/FVC and five common genetic variants in the identification cohort and two of these associations were replicated. The two variants annotated to the genes HHIP and FAM13A showed to have an additive effect on FEV1/FVC level in the GRS analysis, were associated with gene expression of HHIP and FAM13A in lung tissue, respectively, and were genome-wide significant in a meta-analysis including both identification and four verification cohorts (p-value <2.19x10(-7)). Finally, we did not identify significant interactions between the variants and ever-smoking. Results of the FEV1 identification analysis were not replicated.
Conclusion:
The genes HHIP and FAM13A confer a risk for airway obstruction in general that is not driven exclusively by cigarette smoking, the main risk factor for COPD.
... HHIP silencing in an airway epithelial cell line leads to a change in gene expression, and these differentially expressed genes are enriched in pathways related to the extracellular matrix and cell growth, which are processes relevant to COPD pathogenesis. 38 Recently, Lao et al 39 found that Hhip-haploinsufficient mice have increased airspace size after cigarette smoke exposure, increased lung compliance, and increased numbers of lymphoid aggregates. The functions of the genes with altered expression in Hhip +/mice exposed to cigarette smoke were enriched in the pathway of lymphocyte activation. ...
COPD is a common complex disease characterized by progressive airflow limitation. Several genome-wide association studies (GWASs) have discovered genes that are associated with COPD. Recently, candidate genes for COPD identified by GWASs include CHRNA3/5 (cholinergic nicotine receptor alpha 3/5), IREB2 (iron regulatory binding protein 2), HHIP (hedgehog-interacting protein), FAM13A (family with sequence similarity 13, member A), and AGER (advanced glycosylation end product–specific receptor). Their association with COPD susceptibility has been replicated in multiple populations. Since these candidate genes have not been considered in COPD, their pathological roles are still largely unknown. Herein, we review some evidences that they can be effective drug targets or serve as biomarkers for diagnosis or subtyping. However, more study is required to understand the functional roles of these candidate genes. Future research is needed to characterize the effect of genetic variants, validate gene function in humans and model systems, and elucidate the genes’ transcriptional and posttranscriptional regulatory mechanisms.
... 6 Other studies of HHIP, which encode an inhibitory protein for sonic hedgehog, revealed that single nucleotide polymorphisms (SNPs) upstream of HHIP both modulate the expression of HHIP and functionally link reduced HHIP expression to COPD pathogenesis, 8 and are crucial for the development of the lungs and other organs. 9 Additional studies identified three SNPs on HHIP (rs13118928, rs13141641, and rs1828591) that were associated with COPD and lung function in non-Asian populations. 7,10,11 Recently, the association of HHIP polymorphisms with COPD and COPD-related phenotypes has been found in Chinese Han population. ...
It is known that the contribution of risk alleles to chronic obstructive pulmonary disease (COPD) may vary between populations. Further, previous studies involving various ethnic groups have revealed associations between COPD and genetic polymorphisms in families with sequence similarity 13, member A (FAM13A), micro-RNA 2054 (MIR2054), SET domain containing protein 7 (SETD7), ring finger protein 150 (RNF150), hedgehog interacting protein (HHIP), and vascular endothelial growth factor A (VEGFA). Our objective was to explore the association between these gene polymorphism and COPD in members of Chinese Li minority population.
The Chinese Li population case-control study was conducted to assess genetic associations with COPD risk. Seven single nucleotide polymorphisms (SNPs) located on chromosome 4, including FAM13A, MIR2054, SETD7, RNF150, and HHIP, and nine SNPs in the VEGFA gene were genotyped among 234 cases and 240 controls using Sequenom Mass-ARRAY(®) platform. Linkage disequilibrium (LD) analysis was performed using Haploview software and the associations of the SNP frequencies with COPD were analyzed using chi-square (χ (2)) tests, genetic models analysis, and haplotype analysis.
By χ (2) we found the minor allele "G" of rs17050782 was with increased COPD risk in allele model. In genetic models, we found the minor allele of rs7671167 (P=0.028 by dominant model) and rs17050782 (P=0.008 by recessive model) was associated with the increased risk of COPD disease. Likewise, an increased risk of developing COPD was associated with the "GGCGC" haplotype of VEGFA (odds ratio =1.48, 95% confidence interval =1.02-2.12, P=0.037).
Our results were the first time to reveal that SNPs from FAM13A (rs7671167), SETD7 (rs17050782), and a haplotype of VEGFA ("GGCGC") are potential susceptibility loci associated with increased COPD risk in Chinese Li minority population.
... To determine the regulation of GAB1 on the NFkb pathway, we performed knockdown of GAB1 in Beas-2B cells, a human bronchial epithelial cell line using two individual siRNAs as we have done previously (59). Briefly, 40% confluent Beas-2B cells were transfected with 100 pmol/well of GAB1-specific siRNAs or a non-targeting siRNA control (Life Technologies) using lipofectamine 2000 (Life Technologies). ...
Recent advances in genetics have spurred rapid progress towards the systematic identification of genes involved in complex diseases. Still, the detailed understanding of the molecular and physiological mechanisms through which these genes impact disease phenotypes remains a major challenge. Here, we identify the asthma disease module, i.e. the local neighborhood of the interactome whose perturbation is associated with asthma, and validate it for functional and pathophysiological relevance, using both computational and experimental approaches. We find that the asthma disease module is enriched with modest GWAS p-values against the background of random variation, and with differentially expressed genes from normal and asthmatic fibroblast cells treated with an asthma specific drug. The asthma module also contains immune-response mechanisms that are shared with other immune-related disease modules. Further, using diverse omics (genomics, gene-expression, drug response) data, we identify the GAB1 signaling pathway as an important novel modulator in asthma. The wiring diagram of the uncovered asthma module suggests a relatively close link between GAB1 and glucocorticoids, that we experimentally validate, observing an increase in the level of GAB1 after glucocorticoid treatment in BEAS-2B bronchial epithelial cells. The siRNA knockdown of GAB1 in the BEAS-2B cell line resulted in a decrease in NFkB level, suggesting a novel regulatory path of the pro-inflammatory factor NFkB by GAB1 in asthma.
Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
... Genetic studies may also help us to understand disease heterogeneity and to define disease subtypes (e.g., six genomic regions so far have been associated with COPD [46]). Finally, the use of network analysis allows the creation of molecular (11,47) and phenotypic (34,48,49) networks that could provide new insights into the pathobiology of the disease. Development of new drugs. ...
This Pulmonary Perspective summarizes the content and main conclusions of an international workshop on personalized respiratory medicine co-organized by the Barcelona Respiratory Network (www.brn.cat) and the AJRCCM in June 2014. It discusses: (1) its definition, historical, social, legal and ethical aspects; (2) the view from different disciplines, including basic science, epidemiology, bioinformatics and network/systems medicine.; (3) the bottlenecks and opportunities identified by some currently ongoing projects; and, (4) the implications for the individual, the health-care system and the pharmaceutical industry. Authors hope that, albeit not a systematic review on the subject, this document can be a useful reference for researchers, clinicians, health-care managers, policy-makers, and industry parties interested in personalized respiratory medicine.
... Even though GWASs of GOLD 1 relative to smoking controls revealed no genome-wide significant associations in both NHWs and AAs, the top SNP in AA subjects, rs114095670, was nearly genome-wide significant and is located within RECK, a negative regulator of MMP9 [43,44], which was previously reported by our group to be regulated by HHIP in human bronchial epithelial cells [45]. In both NHWs and AAs, the airway-predominant GOLD 1 cluster did not show any significant associations with previously identified COPD risk alleles. ...
Background
Chronic obstructive pulmonary disease (COPD) is characterized by marked phenotypic heterogeneity. Most previous studies have focused on COPD subjects with FEV1 < 80% predicted. We investigated the clinical and genetic heterogeneity in subjects with mild airflow limitation in spirometry grade 1 defined by the Global Initiative for chronic Obstructive Lung Disease (GOLD 1).
Methods
Data from current and former smokers participating in the COPDGene Study (NCT00608764) were analyzed. K-means clustering was performed to explore subtypes within 794 GOLD 1 subjects. For all subjects with GOLD 1 and with each cluster, a genome-wide association study and candidate gene testing were performed using smokers with normal lung function as a control group. Combinations of COPD genome-wide significant single nucleotide polymorphisms (SNPs) were tested for association with FEV1 (% predicted) in GOLD 1 and in a combined group of GOLD1 and smoking control subjects.
Results
K-means clustering of GOLD 1 subjects identified putative “near-normal”, “airway-predominant”, “emphysema-predominant” and “lowest FEV1 % predicted” subtypes. In non-Hispanic whites, the only SNP nominally associated with GOLD 1 status relative to smoking controls was rs7671167 (FAM13A) in logistic regression models with adjustment for age, sex, pack-years of smoking, and genetic ancestry. The emphysema-predominant GOLD 1 cluster was nominally associated with rs7671167 (FAM13A) and rs161976 (BICD1). The lowest FEV1 % predicted cluster was nominally associated with rs1980057 (HHIP) and rs1051730 (CHRNA3). Combinations of COPD genome-wide significant SNPs were associated with FEV1 (% predicted) in a combined group of GOLD 1 and smoking control subjects.
Conclusions
Our results indicate that GOLD 1 subjects show substantial clinical heterogeneity, which is at least partially related to genetic heterogeneity.
... In a follow-up paper, the authors performed microarray expression analysis following in vitro silencing of HHIP using short hairpin RNAs. The differentially expressed genes were functionally enriched for extracellular matrix and cell proliferation pathways [26]. ...
... Additional analyses using the meta-analysis results included gene-based testing using VEGAS 42 and the literature mining using GRAIL 43 . Gene expression levels of TGFB2 and RIN3 were measured in lung tissue samples from 15 COPD patients -8 with moderate (FEV 1 < 80% predicted), and 7 with severe (FEV 1 < 50% predicted) disease -and 15 control subjects with normal lung function, obtained from the NHLBI Lung Tissue Research Consortium (LTRC), as described previously 44 . ...
Background
The genetic risk factors for susceptibility to chronic obstructive pulmonary disease (COPD) are still largely unknown. Additional genetic variants are likely to be identified by genome-wide association studies in larger cohorts or specific subgroups. We sought to identify risk loci for moderate to severe and severe COPD with data from several cohort studies.
Methods
We combined genome-wide association analysis data from participants in the COPDGene study (non-Hispanic white and African-American ethnic origin) and the ECLIPSE, NETT/NAS, and Norway GenKOLS studies (self-described white ethnic origin). We did analyses comparing control individuals with individuals with moderate to severe COPD and with a subset of individuals with severe COPD. Single nucleotide polymorphisms yielding a p value of less than 5 × 10−7 in the meta-analysis at loci not previously described were genotyped in individuals from the family-based ICGN study. We combined results in a joint meta-analysis (threshold for significance p<5 × 10−8).
Findings
Analysis of 6633 individuals with moderate to severe COPD and 5704 control individuals confirmed association at three known loci: CHRNA3 (p=6·38 × 10−14), FAM13A (p=1·12 × 10−14), and HHIP (p=1·57 × 10−12). We also showed significant evidence of association at a novel locus near RIN3 (p=5·25 × 10−9). In the overall meta-analysis (ie, including data from 2859 ICGN participants), the association with RIN3 remained significant (p=5·4 × 10−9). 3497 individuals were included in our analysis of severe COPD. The effect estimates for the loci near HHIP and CHRNA3 were significantly stronger in severe disease than in moderate to severe disease (p<0·01). We also identified associations at two additional loci: MMP12 (overall joint meta-analysis p=2·6 × 10−9) and TGFB2 (overall joint meta-analysis p=8·3 × 10−9).
Interpretation
We have confirmed associations with COPD at three known loci and identified three new genome-wide significant associations. Genetic variants other than in α-1 antitrypsin increase the risk of COPD.
Funding
US National Heart, Lung, and Blood Institute; the Alpha-1 Foundation; the COPD Foundation through contributions from AstraZeneca, Boehringer Ingelheim, Novartis, and Sepracor; GlaxoSmithKline; Centers for Medicare and Medicaid Services; Agency for Healthcare Research and Quality; and US Department of Veterans Affairs.
... HHIP encodes a membrane glycoprotein that is an inhibitor of hedgehog signalling, which is involved in development processes. Gene expression studies in BEAS-2B bronchial epithelial cell lines implicate HHIP in extracellular matrix and cell proliferation (77). At 4q24, FAM13A (family with sequence similarity 13, member A) has been detected in two GWAS (73,74), and also in a genetic association in which SNPs were related to both COPD and lung cancer, indicating a possible shared genetic pathway (78). ...
Lung cancer is a leading cause of cancer death and disease burden in many countries. Understanding of the biological pathways involved in lung cancer aetiology is required to identify key biomolecules that could be of significant clinical value, either as predictive, prognostic or diagnostic markers, or as targets for the development of novel therapies to treat this disease, in addition to smoking avoidance strategies. Genome-wide association studies (GWAS) have enabled significant progress in the past 5 years in investigating genetic susceptibility to lung cancer. Large scale, multi-cohort GWAS of mainly Caucasian, smoking, populations have identified strong associations for lung cancer mapped to chromosomal regions 15q [nicotinic acetylcholine receptor (nAChR) subunits: CHRNA3, CHRNA5], 5p (TERT-CLPTM1L locus) and 6p (BAT3-MSH5). Some studies in Asian populations of smokers have found similar risk loci, whereas GWAS in never smoking Asian females have identified associations in other chromosomal regions, e.g., 3q (TP63), that are distinct from smoking-related lung cancer risk loci. GWAS of smoking behaviour have identified risk loci for smoking quantity at 15q (similar genes to lung cancer susceptibility: CHRNA3, CHRNA5) and 19q (CYP2A6). Other genes have been mapped for smoking initiation and smoking cessation. In chronic obstructive pulmonary disease (COPD), which is a known risk factor for lung cancer, GWAS in large cohorts have also found CHRNA3 and CHRNA5 single nucleotide polymorphisms (SNPs) mapping at 15q as risk loci, as well as other regions at 4q31 (HHIP), 4q24 (FAM13A) and 5q (HTR4). The overlap in risk loci between lung cancer, smoking behaviour and COPD may be due to the effects of nicotine addiction; however, more work needs to be undertaken to explore the potential direct effects of nicotine and its metabolites in gene-environment interaction in these phenotypes. Goals of future genetic susceptibility studies of lung cancer should focus on refining the strongest risk loci in a wide range of populations with lung cancer, and integrating other clinical and biomarker information, in order to achieve the aim of personalised therapy for lung cancer.
Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. The primary causes of COPD are environmental, including cigarette smoking; however, genetic susceptibility also contributes to COPD risk. Genome-Wide Association Studies (GWASes) have revealed more than 80 genetic loci associated with COPD, leading to the identification of multiple COPD GWAS genes. However, the biological relationships between the identified COPD susceptibility genes are largely unknown. Genes associated with a complex disease are often in close network proximity, i.e. their protein products often interact directly with each other and/or similar proteins. In this study, we use affinity purification mass spectrometry (AP-MS) to identify protein interactions with HHIP , a well-established COPD GWAS gene which is part of the sonic hedgehog pathway, in two disease-relevant lung cell lines (IMR90 and 16HBE). To better understand the network neighborhood of HHIP , its proximity to the protein products of other COPD GWAS genes, and its functional role in COPD pathogenesis, we create HUBRIS, a protein-protein interaction network compiled from 8 publicly available databases. We identified both common and cell type-specific protein-protein interactors of HHIP. We find that our newly identified interactions shorten the network distance between HHIP and the protein products of several COPD GWAS genes, including DSP, MFAP2, TET2 , and FBLN5 . These new shorter paths include proteins that are encoded by genes involved in extracellular matrix and tissue organization. We found and validated interactions to proteins that provide new insights into COPD pathobiology, including CAVIN1 (IMR90) and TP53 (16HBE). The newly discovered HHIP interactions with CAVIN1 and TP53 implicate HHIP in response to oxidative stress.
Rationale
Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of IFN-stimulated genes (ISGs). How asthma genetic susceptible genes modulate cellular response upon viral infection through fine-tuning ISGs induction and subsequent airway inflammation in genetically susceptible asthmatics remains largely unknown.
Objectives
To decipher the functions of GSDMB in respiratory virus-induced lung inflammation.
Methods
In two independent cohorts, we analyzed expression correlation between GSDMB and ISGs . In human bronchial epithelial cell line or primary cells, we generated GSDMB-overexpressing and -deficient cells. A series of qPCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISGs induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and applied respiratory syncytial virus (RSV) infection to determine the role of GSDMB on RSV-induced lung inflammation in vivo .
Measurements and Main Results
Gasdermin B encoded by GSDMB, one of the most significant asthma-susceptible genes at 17q21, acts as a novel RNA sensor, promoting MAVS-TBK1 signaling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB gene in mouse airway epithelium leads to enhanced ISGs induction, increased airway inflammation with mucus hyper-secretion upon RSV infection.
Conclusions
GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.
Single-nucleotide polymorphisms (SNPs) within FAM13A gene are significantly associated with chronic obstructive pulmonary disease (COPD) and lung function in genome-wide association studies (GWAS). However, how FAM13A protein is regulated under physiological and pathological condition remains largely elusive. Herein, we report that FAM13A is phosphorylated at the Serine 312 residue by AKT kinase after cigarette smoke extract treatment and thereby recognized by the CULLIN4A/DCAF1 E3 ligase complex, rendering the ubiquitination-mediated degradation of FAM13A. More broadly, downregulation of FAM13A protein upon AKT activation, as a general cellular response to acute stress, was also detected in influenza- or naphthalene-injured lungs in mice. Functionally, reduced protein levels of FAM13A leads to accelerated epithelial cell proliferation in murine lungs during recovery phase after injury. In summary, we characterized a novel molecular mechanism that regulates the stability of FAM13A protein, which enables the fine-tuning of lung epithelial repair post injuries. These significant findings will expand our molecular understanding on the regulation of protein stability that may modulate lung epithelial repair implicated in the development of COPD and other lung diseases.
Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the Hhip gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.
COPD affects millions of people and is now ranked as the third leading cause of death worldwide. This largely untreatable chronic airway disease results in irreversible destruction of lung architecture. The small lung hypothesis is now supported by epidemiological, physiological and clinical studies. Accordingly, the early and severe COPD phenotype carries the most dreadful prognosis and finds its roots during lung growth. Pathophysiological mechanisms remain poorly understood and implicate individual susceptibility (genetics), a large part of environmental factors (viral infections, tobacco consumption, air pollution) and the combined effects of those triggers on gene expression. Genetic susceptibility is most likely involved as the disease is severe and starts early in life. The latter observation led to the identification of Mendelian inheritance via disease-causing variants of SERPINA1 – known as the basis for alpha-1 anti-trypsin deficiency, and TERT. In the last two decades multiple genome wide association studies (GWAS) identified many single nucleotide polymorphisms (SNPs) associated with COPD. High significance SNPs are located in 4q31 near HHIP which encodes an evolutionarily highly conserved physiological inhibitor of the Hedgehog signaling pathway (HH). HHIP is critical to several in utero developmental lung processes. It is also implicated in homeostasis, injury response, epithelial-mesenchymal transition and tumor resistance to apoptosis. A few studies have reported decreased HHIP RNA and protein levels in human adult COPD lungs. HHIP+/- murine models led to emphysema. HH pathway inhibitors, such as vismodegib and sonidegib, are already validated in oncology, whereas other drugs have evidenced in vitro effects. Targeting the Hedgehog pathway could lead to a new therapeutic avenue in COPD. In this review, we focused on the early and severe COPD phenotype and the small lung hypothesis by exploring genetic susceptibility traits that are potentially treatable, thus summarizing promising therapeutics for the future.
Background
Genome wide association study identified hedgehog interacting protein gene (HHIP) variants with chronic obstructive pulmonary disease and asthma. Loss of HHIP, a key regulator of the hedgehog signaling pathway, leads to impaired lung morphogenesis and lethality in animal models, through unimpeded sonic hedgehog expression blocking mesenchymal-expressed fibroblast growth factor 10 (FGF10). Since bronchopulmonary dysplasia (BPD) is also associated with altered lung development and worsens with stimuli including mechanical ventilation, reactive oxygen species, and inflammation, HHIP and FGF10 may be candidate genes.Methods
This was an observational, cohort study including extremely low birth weight infants that who developed BPD and those who did not. DNA was isolated from buccal swabs and subjected to allelic discrimination, using specific HHIP and FGF10 probes. Protein levels were measured in tracheal aspirates. Student's t test, Chi-square, Z test and logistic regression were used.ResultsDemographic characteristics did not differ except that birth weight (715 ± 153 vs. 835 ± 132 g) and gestational age (25 vs. 26 weeks) were less in babies with BPD. HHIP variant rs13147758 (GG genotype) was found to be independently protective for BPD (odds ratio 0.35, 95% confidence interval 0.15–0.82, P = − 0.02). Early airway HHIP protein levels were increased in infants with BPD compared to those without [median (interquartile range) 130.6 (55.6–297.0) and 41.2 (22.1–145.6) pg/mL, respectively; P = 0.05]. The FGF10 single nucleotide polymorphisms were not associated with BPD.ConclusionHHIP, as a regulator of lung bud formation, affects BPD susceptibility, and may be valuable in understanding the specific mechanisms for this disease as well as for identifying therapeutic targets in the era of personalized medicine.
Background
Genetic association studies have identified single nucleotide polymorphisms (SNPs) related to chronic obstructive pulmonary disease (COPD) susceptibility. The aim of this study was to identify HHIP genetic variants associated with COPD, pulmonary function, and serum and sputum HHIP protein levels in Mexican mestizo smokers.
Materials and Methods
Association analysis was performed by carrying out a case–control study in Mexican mestizo smokers comprised of two groups: tobacco-smoking subjects with COPD (COPD-TS, n = 222) and smokers without COPD (SWOC, n = 333). We evaluated three SNPs (rs13147758, rs1828591, and rs13118928) in the HHIP gene. Allele discrimination was accomplished by qPCR using TaqMan probes, and determination of protein levels in the serum and sputum supernatants (SS) was performed using ELISA.
Results
Statistically significant differences were observed in the rs13147758 GG genotype (adjusted p = 0.014, OR = 1.95) and the rs13147758-rs1828591 GA haplotype (p = 6.6E-06, OR = 2.65) in the case–control comparison. HHIP protein levels were elevated in SS samples from the COPD-TS group compared to those from the SWOC group (p = 0.03). Based on genotype analysis, HHIP protein levels were lower in the serum samples of rs13147758 GG genotype carriers in the COPD-TS group than in the serum samples of rs13147758 GG genotype carriers from the SWOC group (p < 0.05), but there were no differences in the sputum samples.
Conclusion
The rs13147758 GG genotype and the rs13147758-rs1828591 GA haplotype are associated with susceptibility to COPD. Furthermore, an association in protein levels was observed between the HHIP rs13147758 genotype and COPD in Mexican mestizo smokers.
Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are generally progressive and irreversible chronic-degenerative disorders. COPD is associated with chronic inflammation, and changes in both the alveolar and airway compartments, resulting in small airways disease and emphysema. IPF is driven by an abnormal activation of the alveolar lung epithelium resulting in progressive scarring of the lung parenchyma. Both diseases are heterogeneous with several clinical phenotypes, and usually occur in aged individuals, mostly smokers. Although drastically different but similarly devastating, COPD and IPF share a number of accelerating lung aging processes, smoking injury and even some convergent molecular pathways. A key question remains why an older smoking individual develops IPF instead of COPD? Moreover, why do some individuals develop both diseases? Here we examine the common and divergent pathogenic mechanisms, highlighting the distinct relevant self-determining molecular mechanisms that made the pathological pathways and outcome radically different.
Although environmental factors, including cigarette smoking and biomass smoke exposure, are major risk factors of COPD, genetic risk factors are also important [1]. In addition, an interaction between genetics and environment is believed to drive the development of COPD.
Lung function is a heritable trait. Heritability estimates posit that approximately30%–50% of the phenotypic variation in FEV1 is explained by genetics,1–3 and genome-wide association studies (GWAS) of lung function4–11 have discovered multiple genetic variants that are associated with cross-sectional measurements in adults of FEV1, FVC and FEV1/FVC ratio at genome-wide significance levels. Many of these same loci have also been implicated in GWAS of COPD. Additional work is required to link these loci to pathophysiology. Functional studies on GWAS loci involving HHIP ,12 ,13 FAM13A ,14 HTR4 ,15 AGER 16 and IREB2 17 have already provided important insights into the biological mechanisms for genetic determinants of COPD and/or lung function.
Longitudinal change in lung function (ΔFEV1, ΔFEV1/FVC) is also heritable,18 albeit perhaps to a lesser degree than cross-sectional lung function levels. Although studies of longitudinal lung function have discovered a few loci associated with ΔFEV1 at genome-wide significance levels, none were robust to replication in the available populations. While at first glance the genetic determinants of FEV1 level and ΔFEV1 might be expected to show significant overlap, a recent study of COPD by Lange et al 19 and a complementary study in asthma by McGeachie et al 20 have suggested that low maximum attained lung function and accelerated lung function decline are distinct processes that can exert independent effects on the risk for chronic airflow obstruction. Therefore, it would not be surprising if different genetic determinants influenced cross-sectional and longitudinal lung function change. Longitudinal measurements of lung function are highly informative for identifying these separate processes. In contrast, one …
Chronic obstructive pulmonary disease (COPD) is a complex disease with both genetic and environmental determinants, and case–control association studies on candidate genes and also genomic approaches such as genome-wide association studies (GWASs) have been used to discover genes involved in COPD pathogenesis. Though the 15q25 locus which encodes a family of nicotinic cholinergic receptors including CHRNA3 and CHRNA5 and also the other novel loci were reported to be associated with COPD susceptibility, it is uncertain through which molecular pathways the genetic variants of these genes affect the pathogenesis in a concrete manner and whether the genetic effects are on susceptibility to smoking behavior and/or to lung destruction as emphysematous change of the lungs induced by smoking. Recent studies showed the functional genetic variations related to COPD pathogenesis by using two different types of omics data, such as GWAS and gene expression profiling in the lungs. A recent study with more than 50,000 individuals of European ancestry in the United Kingdom investigated the genes associated with COPD and reported the genes related to nicotine addiction, impaired lung development, and accelerated lung function decline, respectively. The genetic variations associated with COPD exacerbations and the ethnic difference of COPD pathogenesis also should be elucidated.
Chronic obstructive pulmonary disease (COPD) is a complex clinical entity. In contrast to previously limited diagnostic definitions, it is now apparent that COPD is a clinically and biologically heterogeneous disease process, overlapping with other airways diseases like chronic asthma. As such, symptomatic response to current standard treatment practices is variable. New clinical guidelines have been altered to reflect this, with the inclusion of symptoms and risk factors in diagnostic and management algorithms. However, as our understanding of COPD pathophysiology deepens, many novel physiological, cellular, proteomic, and genetic markers have been identified. Several have been observed to be independently predictive of distinct clinical disease patterns, which at present are not illustrated by conventional measurements of lung impairment. The potential use of these predictive biomarkers to stratify this diverse patient population could transform the care we offer. We should aim for precision medicine to optimize diagnosis and treatment choices and to monitor and improve clinical outcomes in this disease.
© 2015 New York Academy of Sciences.
Chronic obstructive pulmonary disease (COPD) affects over 10 million Americans.(1) This complex disorder demonstrates many different presentations in a wide variety of patients, and results from a combination of environmental exposures and genetic risk factors. Smoking alone does not result in COPD: not all smokers develop COPD and lung function decline among smokers is highly variable. There is growing evidence for genetic risk factors for COPD: early familial aggregation and linkage analysis studies strongly suggested genetic contributions to COPD, and recent genome-wide association studies have identified several genomic regions that are clearly related to COPD susceptibility. However, despite recent advances in COPD genetics, much of the heritability of COPD remains unexplained, and functional studies are only beginning to elucidate a role for the genetic associations that have been identified. Despite this, the future is bright for understanding the genetics of COPD. Improvements in COPD phenotyping, collaborations among COPD study cohorts, and novel integrative approaches to identifying genetic markers all promise to unravel much of this missing heritability and ultimately lead to improvements in our understanding of COPD susceptibility and treatment.
ETS family proteins play a role in immune responses. A unique member of this family, Elk-3, is a transcriptional repressor that regulates the expression of HO-1. Elk-3 is very sensitive to the effects of inflammatory mediators and is down-regulated by bacterial endotoxin (LPS). In the present study, exposure of mouse macrophages to Escherichia coli LPS resulted in decreased, full-length, and splice-variant isoforms of Elk-3. We isolated the Elk-3 promoter and demonstrated that LPS also decreased promoter activity. The Elk-3 promoter contains GC-rich regions that are putative binding sites for zinc-finger transcription factors, such as Sp1 and KLFs. Mutation of the GC-rich region from bp -613 to -603 blunted LPS-induced down-regulation of the Elk-3 promoter. Similar to the LPS response, coexpression of KLF4 led to repression of Elk-3 promoter activity, whereas coexpression of Sp1 increased activity. ChIP assays revealed that KLF4 binding to the Elk-3 promoter was increased by LPS exposure, and Sp1 binding was decreased. Thus, down-regulation of Elk-3 by bacterial LPS is regulated, in part, by the transcriptional repressor KLF4. Overexpression of Elk-3, in the presence of E. coli bacteria, resulted in decreased macrophage phagocytosis. To determine whether limited expression of HO-1 may contribute to this response, we exposed HO-1-deficient bone marrow-derived macrophages to E. coli and found a comparable reduction in bacterial phagocytosis. These data suggest that down-regulation of Elk-3 and the subsequent induction of HO-1 are important for macrophage function during the inflammatory response to infection.
The response to drug treatment in asthma is a complex trait and is markedly variable even in patients with apparently similar clinical features. Pharmacogenomics, which is the study of variations of human genome characteristics as related to drug response, can play a role in asthma therapy. Both a traditional candidate-gene approach to conducting genetic association studies and genome-wide association studies have provided an increasing list of genes and variants associated with the three major classes of asthma medications: β2-agonists, inhaled corticosteroids, and leukotriene modifiers. Moreover, a recent integrative, systems-level approach has offered a promising opportunity to identify important pharmacogenomics loci in asthma treatment. However, we are still a long way away from making this discipline directly relevant to patients. The combination of network modeling, functional validation, and integrative omics technologies will likely be needed to move asthma pharmacogenomics closer to clinical relevance. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 55 is January 06, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Despite public health campaigns discouraging smoking, 1,000 American children every day become smokers, ensuring that tobacco-related health complications will be with us for decades to come. Smoking is the greatest risk factor for both chronic obstructive lung disease and interstitial lung disease. The facts that not every smoker develops chronic lung disease and that lung pathology differs markedly among smokers indicate that individual susceptibility must be a central determinant of lung injury responses to cigarette smoke. Comparative examination of pathogenic mechanisms of smoke-induced lung disease can shed light on the homeostatic pathways critical to maintaining lung health. In this review, we explore common and divergent biological forces tilting the lung homeostatic balance away from health and toward emphysema or pulmonary fibrosis. We emphasize recent insights that highlight the greatest contrasts or similarities in the pathogenesis of these two chronic lung disease phenotypes. Expected final online publication date for the Annual Review of Physiology Volume 76 is February 10, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
The Hedgehog signaling pathway plays an important role in lung morphogenesis and cellular responses to lung injury. Genome-wide association studies (GWAS) and integrative genomics approaches have demonstrated the associations between HHIP polymorphisms and chronic obstructive pulmonary disease (COPD) and in non-Asian populations. Here we investigated whether HHIP polymorphisms would also be associated with COPD susceptibility and COPD-related phenotypes in a Chinese Han population. In the present case-control study a total of 680 COPD patients and 687 healthy control subjects were recruited. Six single nucleotide polymorphisms (SNPs) (rs1828591, rs13118928, rs6817273, rs10519717, rs12504628, rs13147758) were selected for genotyping. Allele frequencies and genotype distributions were compared between patients and controls. To estimate the strength of association, odds ratios (OR) (with 95% CI) were calculated and potential confounding variables were tested by using logistic regression analysis. Association between haplotypes and COPD outcome was also assessed. We identified that SNP rs12504628 was associated with FEV1/FVC ratio among cases (P=0.0460). Moreover, the HHIP SNP rs10519717 was associated with the severity of disease (adjusted P-value=0.0300). The six SNPs showed strong linkage disequilibrium (r(2)≥0.9). Three major haplotypes were observed but showed no significant difference between case and control groups (P=0.4532, 0.0875, and 0.3484, respectively). In conclusion, our study suggests that the HHIP gene may be involved in COPD susceptibility in Chinese Han population.
Polysialic acid (polySia) is a unique polysaccharide that modifies neural cell adhesion molecule (NCAM) spatiotemporally. Recently, we demonstrated that polySia functions as a reservoir for several neurotrophic factors and neurotransmitters. Here, we showed the direct interaction between polySia and fibroblast growth factor-2 (FGF2) by native-PAGE, gel filtration, and surface plasmon resonance. The minimum chain length of polySia required for the interaction with FGF2 was 17. Compared with heparan sulfate, a well known glycosaminoglycan capable of forming a complex with FGF2, polySia formed a larger complex with distinct properties in facilitating oligomerization of FGF2, as well as in binding to FGF receptors. In polySia-NCAM-expressing NIH-3T3 cells, which were established by transfecting cells with either of the plasmids for the expression of the polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST that can polysialylate NCAM, FGF2-stimulated cell growth, but not cell survival, was inhibited. Taken together, these results suggest that polySia-NCAM might be involved in the regulation of FGF2-FGF receptor signaling through the direct binding of FGF2 in a manner distinct from heparan sulfate.
This report presents 2008 data on U.S. births according to a wide variety of characteristics. Data are presented for maternal demographic characteristics including age, live-birth order, race and Hispanic origin, marital status, attendant at birth, method of delivery, and infant characteristics (period of gestation, birthweight, and multiple births). Birth and fertility rates by age, live-birth order, race and Hispanic origin, and marital status also are presented. Selected data by mother's state of residence are shown, as well as data on age of father. Trends in fertility patterns and maternal and infant characteristics are described and interpreted.
Descriptive tabulations of data reported on the birth certificates of the 4.25 million births that occurred in 2008 are presented. Denominators for population-based rates are postcensal estimates derived from the U.S. 2000 census.
A total of 4,247,694 births were registered in the United States in 2008, 2 percent less than in 2007. The general fertility rate declined 1 percent to 68.6 per 1,000. The teenage birth rate declined 2 percent to 41.5 per 1,000. Birth rates for women aged 20 to 39 years were down 1-3 percent, whereas the birth rate for women aged 40-44 rose to the highest level reported in more than 40 years. The total fertility rate declined 2 percent to 2,084.5 per 1,000 women. All measures of unmarried childbearing reached record levels-40.6 percent of births were to unmarried women in 2008. The cesarean delivery rate rose again to 32.3 percent. The preterm birth rate declined for the second consecutive year to 12.3 percent; the low birthweight rate was down very slightly. The twin birth rate increased 1 percent to 32.6 per 1,000; the triplet and higher-order multiple birth rate was stable.
Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive
pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study
(GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb
upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with
statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated
allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus,
increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal
one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.
The vertebrate Hedgehog (Hh) pathway has essential functions during development and tissue homeostasis in normal physiology, and its dysregulation is a common theme in cancer. The Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) bind to the receptors Patched1 and Patched2, resulting in inhibition of their repressive effect on Smoothened (Smo). Smo is a seven-transmembrane protein, which was only recently shown to function as a G protein-coupled receptor (GPCR) with specificity toward the heterotrimeric guanine nucleotide-binding protein G(i). In addition to activating G(i), Smo signals through its C-terminal tail to inhibit Suppressor of Fused, resulting in stabilization and activation of the Gli family of transcription factors, which execute a transcriptional response to so-called "canonical Hh signaling." In this Presentation, we illustrate two outcomes of Hh signaling that are independent of Gli transcriptional activity and, thus, are defined as "noncanonical." One outcome is dependent on Smo coupling to G(i) proteins and exerts changes to the actin cytoskeleton through stimulation of the small guanosine triphosphatases (GTPases) RhoA and Rac1. These cytoskeletal changes promote migration in fibroblasts and tubulogenesis in endothelial cells. Signaling through the other noncanonical Hh pathway is independent of Smo and inhibits Patched1-induced cell death.
Genomics provided us with an unprecedented quantity of data on the genes that are activated or repressed in a wide range of
phenotypes. We have increasingly come to recognize that defining the networks and pathways underlying these phenotypes requires
both the integration of multiple data types and the development of advanced computational methods to infer relationships between
the genes and to estimate the predictive power of the networks through which they interact. To address these issues we have
developed Predictive Networks (PN), a flexible, open-source, web-based application and data services framework that enables the integration, navigation,
visualization and analysis of gene interaction networks. The primary goal of PN is to allow biomedical researchers to evaluate
experimentally derived gene lists in the context of large-scale gene interaction networks. The PN analytical pipeline involves
two key steps. The first is the collection of a comprehensive set of known gene interactions derived from a variety of publicly
available sources. The second is to use these ‘known’ interactions together with gene expression data to infer robust gene
networks. The PN web application is accessible from http://predictivenetworks.org. The PN code base is freely available at https://sourceforge.net/projects/predictivenets/.
The genetic risk factors for chronic obstructive pulmonary disease (COPD) are still largely unknown. To date, genome-wide
association studies (GWASs) of limited size have identified several novel risk loci for COPD at CHRNA3/CHRNA5/IREB2, HHIP and FAM13A; additional loci may be identified through larger studies. We performed a GWAS using a total of 3499 cases and 1922 control
subjects from four cohorts: the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE); the
Normative Aging Study (NAS) and National Emphysema Treatment Trial (NETT); Bergen, Norway (GenKOLS); and the COPDGene study.
Genotyping was performed on Illumina platforms with additional markers imputed using 1000 Genomes data; results were summarized
using fixed-effect meta-analysis. We identified a new genome-wide significant locus on chromosome 19q13 (rs7937, OR = 0.74,
P = 2.9 × 10−9). Genotyping this single nucleotide polymorphism (SNP) and another nearby SNP in linkage disequilibrium (rs2604894) in 2859
subjects from the family-based International COPD Genetics Network study (ICGN) demonstrated supportive evidence for association
for COPD (P = 0.28 and 0.11 for rs7937 and rs2604894), pre-bronchodilator FEV1 (P = 0.08 and 0.04) and severe (GOLD 3&4) COPD (P = 0.09 and 0.017). This region includes RAB4B, EGLN2, MIA and CYP2A6, and has previously been identified in association with cigarette smoking behavior.
Kaposi's sarcoma (KS) is a vascular tumor frequently occurring in Human Immunodeficiency Virus- (HIV-) 1-infected individuals.
Our previous work indicated that the angiogenic fibroblast growth factor (FGF)-2 and the Tat protein of HIV-1, both expressed in KS lesions of HIV-infected patients, synergize at inducing angioproliferative, KS-like lesions in mice.
Here we show that the development of angioproliferative lesions promoted in mice by combined Tat and FGF-2 associates with an increase in the levels of expression of the antiapoptotic Bcl-2 protein. Upregulation of Bcl-2 expression by combined FGF-2 and Tat occurs also in vitro, and this protects human primary endothelial cells from programmed cell death.
As Bcl-2 is expressed in human KS lesions in a fashion paralleling the progression of the disease, these findings suggest a molecular mechanism by which Tat and FGF-2 cooperate in KS maintenance and progression in HIV-infected individuals.
A common difficulty in large-scale microarray studies is the presence of confounding factors, which may significantly skew estimates of statistical significance, cause unreliable feature selection and high false negative rates. To deal with these difficulties, an algorithmic framework known as Surrogate Variable Analysis (SVA) was recently proposed.
Based on the notion that data can be viewed as an interference pattern, reflecting the superposition of independent effects and random noise, we present a modified SVA, called Independent Surrogate Variable Analysis (ISVA), to identify features correlating with a phenotype of interest in the presence of potential confounding factors. Using simulated data, we show that ISVA performs well in identifying confounders as well as outperforming methods which do not adjust for confounding. Using four large-scale Illumina Infinium DNA methylation datasets subject to low signal to noise ratios and substantial confounding by beadchip effects and variable bisulfite conversion efficiency, we show that ISVA improves the identifiability of confounders and that this enables a framework for feature selection that is more robust to model misspecification and heterogeneous phenotypes. Finally, we demonstrate similar improvements of ISVA across four mRNA expression datasets. Thus, ISVA should be useful as a feature selection tool in studies that are subject to confounding.
An R-package isva is available from www.cran.r-project.org.
ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.
Survivin is a member of the inhibitors-of-apoptosis protein (IAPs) family. It promotes cell survival through interference with multiple cell cycle-related proteins such as INCENP and Aurora B kinase. Survivin also inhibits cell death through interference with both caspase-dependent and -independent cell apoptosis. Interestingly, recent evidence suggests that survivin may also play a role in the regulation of cancer cell autophagy. At the clinical level, studies on clinical specimens have shown that survivin expression is up-regulated in various human cancers and its up-regulation is associated with tumour resistance to both chemotherapy and radiation therapy. On the basis of these findings, survivin has been proposed as an attractive target for new anti-cancer interventions. However, despite the role that survivin plays in cancer cell survival and anti-drug response, the development of survivin inhibitors is relatively slow as compared to other therapeutic inhibitors for cancer treatment. In this review, the relationships between survivin expression and the causation of drug resistance in cancers are re-addressed. This review also summarizes the recent development of survivin inhibitors for clinical usage.
Chronic obstructive pulmonary disease (COPD) is the single greatest risk factor for lung cancer in smokers and is found in 50-90% of lung cancer cases. The link between COPD and lung cancer may stem in part from the matrix remodelling and repair processes underlying COPD, and the development of epithelial-mesenchymal transition (EMT) that underlies lung carcinogenesis. The Hedgehog-interacting protein (HHIP), which mediates the epithelial response (EMT) to smoking, has been implicated in COPD and lung cancer. Recent genome-wide and candidate gene studies of COPD implicate genetic variants on the chromosomal 4q31 (HHIP/glycophorin A (GYPA)) locus. In a case-control study of smokers with normal lung function, COPD and lung cancer (subphenotyped for COPD), we show the GG genotype of the rs 1489759 HHIP single-nucleotide polymorphism (SNP) and the CC genotype of the rs 2202507 GYPA SNP confers a "protective" effect on COPD (OR 0.59, p = 0.006 for HHIP and OR = 0.65, p = 0.006 for GYPA) and lung cancer (OR = 0.70 (p = 0.05) for HHIP and OR 0.70 (p = 0.02) for GYPA). This study suggests that, in smokers, genetic variants of the 4q31 locus conferring a protective effect for COPD are also protective in lung cancer. We conclude that genetic susceptibility to lung cancer includes COPD-related gene variants.
Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.
Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS-mitogen-activated protein kinase or phosphoinositide-3-kinase signaling cascade leads to induction of the transcription factor cAMP response element-binding protein-3-like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma.
Spirometric measures of lung function are heritable traits that reflect respiratory health and predict morbidity and mortality. We meta-analyzed genome-wide association studies for two clinically important lung-function measures: forced expiratory volume in the first second (FEV(1)) and its ratio to forced vital capacity (FEV(1)/FVC), an indicator of airflow obstruction. This meta-analysis included 20,890 participants of European ancestry from four CHARGE Consortium studies: Atherosclerosis Risk in Communities, Cardiovascular Health Study, Framingham Heart Study and Rotterdam Study. We identified eight loci associated with FEV(1)/FVC (HHIP, GPR126, ADAM19, AGER-PPT2, FAM13A, PTCH1, PID1 and HTR4) and one locus associated with FEV(1) (INTS12-GSTCD-NPNT) at or near genome-wide significance (P < 5 x 10(-8)) in the CHARGE Consortium dataset. Our findings may offer insights into pulmonary function and pathogenesis of chronic lung disease.
Pulmonary function measures are heritable traits that predict morbidity and mortality and define chronic obstructive pulmonary disease (COPD). We tested genome-wide association with forced expiratory volume in 1 s (FEV(1)) and the ratio of FEV(1) to forced vital capacity (FVC) in the SpiroMeta consortium (n = 20,288 individuals of European ancestry). We conducted a meta-analysis of top signals with data from direct genotyping (n < or = 32,184 additional individuals) and in silico summary association data from the CHARGE Consortium (n = 21,209) and the Health 2000 survey (n < or = 883). We confirmed the reported locus at 4q31 and identified associations with FEV(1) or FEV(1)/FVC and common variants at five additional loci: 2q35 in TNS1 (P = 1.11 x 10(-12)), 4q24 in GSTCD (2.18 x 10(-23)), 5q33 in HTR4 (P = 4.29 x 10(-9)), 6p21 in AGER (P = 3.07 x 10(-15)) and 15q23 in THSD4 (P = 7.24 x 10(-15)). mRNA analyses showed expression of TNS1, GSTCD, AGER, HTR4 and THSD4 in human lung tissue. These associations offer mechanistic insight into pulmonary function regulation and indicate potential targets for interventions to alleviate respiratory disease.
Heparan sulfate binds to and regulates many inflammatory mediators in vitro, suggesting that it serves an important role in directing the progression and outcome of inflammatory responses in vivo. Here, we evaluated the role of syndecan-1, a major heparan sulfate proteoglycan, in modulating multiorgan host injury responses in murine endotoxemia. The extent of systemic inflammation was similar between endotoxemic syndecan-1-null and wild-type mice. However, high levels of CXC chemokines (KC and MIP-2), particularly at later times after LPS, were specifically sustained in multiple organs in syndecan-1-null mice and associated with exaggerated neutrophilic inflammation, organ damage, and lethality. Syndecan-1 shedding was activated in several organs of endotoxemic wild-type mice, and this associated closely with the removal of tissue-bound CXC chemokines and resolution of accumulated neutrophils. Moreover, administration of a shedding inhibitor exacerbated disease by impeding the removal of CXC chemokines and neutrophils, whereas administration of heparan sulfate inhibited the accumulation of CXC chemokines and neutrophils in tissues and attenuated multiorgan injury and lethality. These data show that syndecan-1 shedding is a critical endogenous mechanism that facilitates the resolution of neutrophilic inflammation by aiding the clearance of proinflammatory chemokines in a heparan sulfate-dependent manner.
Hedgehog (Hh) signaling is crucial for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hedgehog-interacting protein (Hhip) is a surface receptor antagonist that is equipotent against all three mammalian Hh homologs. The crystal structures of human HHIP alone and bound to Sonic hedgehog (SHH) now reveal that HHIP is comprised of two EGF domains and a six-bladed beta-propeller domain. In the complex structure, a critical loop from HHIP binds the pseudo active site groove of SHH and directly coordinates its Zn2+ cation. Notably, sequence comparisons of this SHH binding loop with the Hh receptor Patched (Ptc1) ectodomains and HHIP- and PTC1-peptide binding studies suggest a 'patch for Patched' at the Shh pseudo active site; thus, we propose a role for Hhip as a structural decoy receptor for vertebrate Hh.
There is considerable variability in the susceptibility of smokers to develop chronic obstructive pulmonary disease (COPD). The only known genetic risk factor is severe deficiency of alpha(1)-antitrypsin, which is present in 1-2% of individuals with COPD. We conducted a genome-wide association study (GWAS) in a homogenous case-control cohort from Bergen, Norway (823 COPD cases and 810 smoking controls) and evaluated the top 100 single nucleotide polymorphisms (SNPs) in the family-based International COPD Genetics Network (ICGN; 1891 Caucasian individuals from 606 pedigrees) study. The polymorphisms that showed replication were further evaluated in 389 subjects from the US National Emphysema Treatment Trial (NETT) and 472 controls from the Normative Aging Study (NAS) and then in a fourth cohort of 949 individuals from 127 extended pedigrees from the Boston Early-Onset COPD population. Logistic regression models with adjustments of covariates were used to analyze the case-control populations. Family-based association analyses were conducted for a diagnosis of COPD and lung function in the family populations. Two SNPs at the alpha-nicotinic acetylcholine receptor (CHRNA 3/5) locus were identified in the genome-wide association study. They showed unambiguous replication in the ICGN family-based analysis and in the NETT case-control analysis with combined p-values of 1.48 x 10(-10), (rs8034191) and 5.74 x 10(-10) (rs1051730). Furthermore, these SNPs were significantly associated with lung function in both the ICGN and Boston Early-Onset COPD populations. The C allele of the rs8034191 SNP was estimated to have a population attributable risk for COPD of 12.2%. The association of hedgehog interacting protein (HHIP) locus on chromosome 4 was also consistently replicated, but did not reach genome-wide significance levels. Genome-wide significant association of the HHIP locus with lung function was identified in the Framingham Heart study (Wilk et al., companion article in this issue of PLoS Genetics; doi:10.1371/journal.pgen.1000429). The CHRNA 3/5 and the HHIP loci make a significant contribution to the risk of COPD. CHRNA3/5 is the same locus that has been implicated in the risk of lung cancer.
Recent genome-wide association (GWA) studies have identified dozens of common variants associated with adult height. However, it is unknown how these variants influence height growth during childhood. We derived peak height velocity in infancy (PHV1) and puberty (PHV2) and timing of pubertal height growth spurt from parametric growth curves fitted to longitudinal height growth data to test their association with known height variants. The study consisted of N = 3,538 singletons from the prospective Northern Finland Birth Cohort 1966 with genotype data and frequent height measurements (on average 20 measurements per person) from 0-20 years. Twenty-six of the 48 variants tested associated with adult height (p<0.05, adjusted for sex and principal components) in this sample, all in the same direction as in previous GWA scans. Seven SNPs in or near the genes HHIP, DLEU7, UQCC, SF3B4/SV2A, LCORL, and HIST1H1D associated with PHV1 and five SNPs in or near SOCS2, SF3B4/SV2A, C17orf67, CABLES1, and DOT1L with PHV2 (p<0.05). We formally tested variants for interaction with age (infancy versus puberty) and found biologically meaningful evidence for an age-dependent effect for the SNP in SOCS2 (p = 0.0030) and for the SNP in HHIP (p = 0.045). We did not have similar prior evidence for the association between height variants and timing of pubertal height growth spurt as we had for PHVs, and none of the associations were statistically significant after correction for multiple testing. The fact that in this sample, less than half of the variants associated with adult height had a measurable effect on PHV1 or PHV2 is likely to reflect limited power to detect these associations in this dataset. Our study is the first genetic association analysis on longitudinal height growth in a prospective cohort from birth to adulthood and gives grounding for future research on the genetic regulation of human height during different periods of growth.
The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data.
RECK, a glycosylphosphatidylinositol-anchored glycoprotein, inhibits the enzymatic activities of some matrix metalloproteinases (MMP), thereby suppressing tumor cell metastasis; however, the detailed mechanism is still obscure. In this study, we compared the gene expression profiles between mock- and RECK-transfected HT1080 cells and showed that RECK decreases MMP-9 mRNA levels but not other MMP mRNA levels. Moreover, treatment with RECK-specific siRNA increased MMP-9 mRNA in RECK-expressing cells. The promoter assay showed that MMP-9 promoter activity was suppressed by RECK and that RECK-mediated suppression of MMP-9 promoter activity requires 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) and kappaB sites. Moreover, the binding ability of Fra-1 and c-Jun to TRE within the MMP-9 promoter region was suppressed by RECK. Thus, these results show that RECK is a negative regulator of MMP-9 transcription.
Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics
scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput
strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately
68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely
categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections,
unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding
the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the
survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details
of particular tool categories/tools, enabling them to make the best choices for their particular research interests.
Human papillomavirus (HPV) oncoproteins subvert cellular signaling pathways, including kinase pathways, during the carcinogenic process. To identify kinases targeted by the HPV16 E7 oncoprotein, shRNA kinase screens were performed in RKO colorectal carcinoma cell lines that differ only in their expression of HPV16 E7. Our screens identified kinases that were essential for the survival of RKO cells, but not essential for RKO cells expressing HPV16 E7. These kinases include CDK6, ERBB3, FYN, AAK1, and TSSK2. We show that, as predicted, CDK6 knockdown inhibits pRb phosphorylation and induces S-phase depletion, thereby inhibiting cell viability. Knockdown of ERBB3, FYN, AAK1, and TSSK2 induces a similar loss of cell viability through an unknown mechanism. Expression of the HPV16 E7 oncoprotein, known to bind and degrade pRb, relieves the requirement of these kinases. These studies demonstate that expression of a single oncoprotein can dramatically alter kinase sensitivity in human cells. The shRNA screens used here perform analogously to genetic interaction screens commonly used in genetically tractable organisms such as yeast, and thus represent an exciting method for unbiased identification of cellular signaling pathways targeted by cancer mutations.
• essential kinases
• shRNA screening
• E7 oncoprotein
• retinoblastoma tumor suppressor
Histone deacetylase (HDAC) inhibitors are known to exert antimetastatic and antiangiogenic activity in vitro and in vivo. RECK is a membrane-anchored glycoprotein that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor metastasis and angiogenesis. In this study, we test the possibility that HDAC inhibitor may increase RECK expression to inhibit MMP activation and cancer cell invasion. Our results showed that trichostatin A (TSA) up-regulated RECK via transcriptional activation in CL-1 human lung cancer cells. Flow cytometric analysis demonstrated that RECK protein on cell surface was increased after treatment of TSA. Moreover, up-regulation of RECK expression by TSA attenuated MMP-2 activity. To explore whether HDAC inhibitor-induced inhibition of MMP-2 activation is indeed mediated via RECK, we used small interference RNA (siRNA) to block RECK expression and found that inhibition of RECK by siRNA abolished the inhibitory effect of TSA on MMP-2 activation. In addition, TSA suppressed the invasive ability of CL-1 cells. Taken together, this study reveals a novel mechanism by which HDAC inhibitors suppress tumor invasion and provides a new strategy for cancer therapy.
Integrins are heterodimeric cell-surface proteins that regulate cell growth, migration and survival. We have shown previously that the epithelial-restricted integrin alpha(v)beta6 has another critical function; that is, it binds and activates latent transforming growth factor-beta (TGF-beta). Through a global analysis of pulmonary gene expression in the lungs of mice lacking this integrin (Itgb6 null mice) we have identified a marked induction of macrophage metalloelastase (Mmp12)--a metalloproteinase that preferentially degrades elastin and has been implicated in the chronic lung disease emphysema. Here we report that Itgb6-null mice develop age-related emphysema that is completely abrogated either by transgenic expression of versions of the beta6 integrin subunit that support TGF-beta activation, or by the loss of Mmp12. Furthermore, we show that the effects of Itgb6 deletion are overcome by simultaneous transgenic expression of active TGF-beta1. We have uncovered a pathway in which the loss of integrin-mediated activation of latent TGF-beta causes age-dependent pulmonary emphysema through alterations of macrophage Mmp12 expression. Furthermore, we show that a functional alteration in the TGF-beta activation pathway affects susceptibility to this disease.
Pulmonary function measures are heritable traits that predict morbidity and mortality and define chronic obstructive pulmonary disease (COPD). We tested genome-wide association with forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) in the SpiroMeta consortium (n = 20,288 individuals of European ancestry). We conducted a meta-analysis of top signals with data from direct genotyping (n ? 32,184 additional individuals) and in silico summary association data from the CHARGE Consortium (n = 21,209) and the Health 2000 survey (n ? 883). We confirmed the reported locus at 4q31 and identified associations with FEV1 or FEV1/FVC and common variants at five additional loci: 2q35 in TNS1 (P = 1.11 × 10?12), 4q24 in GSTCD (2.18 × 10?23), 5q33 in HTR4 (P = 4.29 × 10?9), 6p21 in AGER (P = 3.07 × 10?15) and 15q23 in THSD4 (P = 7.24 × 10?15). mRNA analyses showed expression of TNS1, GSTCD, AGER, HTR4 and THSD4 in human lung tissue. These associations offer mechanistic insight into pulmonary function regulation and indicate potential targets for interventions to alleviate respiratory disease
It has unambiguously been shown that genetic, environmental, demographic, and technical factors may have substantial effects on gene expression levels. In addition to the measured variable(s) of interest, there will tend to be sources of signal due to factors that are unknown, unmeasured, or too complicated to capture through simple models. We show that failing to incorporate these sources of heterogeneity into an analysis can have widespread and detrimental effects on the study. Not only can this reduce power or induce unwanted dependence across genes, but it can also introduce sources of spurious signal to many genes. This phenomenon is true even for well-designed, randomized studies. We introduce “surrogate variable analysis” (SVA) to overcome the problems caused by heterogeneity in expression studies. SVA can be applied in conjunction with standard analysis techniques to accurately capture the relationship between expression and any modeled variables of interest. We apply SVA to disease class, time course, and genetics of gene expression studies. We show that SVA increases the biological accuracy and reproducibility of analyses in genome-wide expression studies.
Melanoma is the deadliest form of skin cancer. Melanoma develops in response to genetic and environmental pressures which lead to oncogenic transformation of normal human melanocytes, the pigment-producing cells in the body. The majority of melanoma-associated deaths are due to metastases, highlighting the importance of understanding the molecular mechanisms driving melanoma development and progression. This review focuses on survivin, and its involvement in the melanoma biology. Since its identification in the late 1990s, a vast body of work has been generated, demonstrating the role of survivin in various malignancies. This review discusses the established mitotic and cytoprotective properties of survivin, and its potential role in melanoma development and progression. A newly recognized functional property of survivin is also discussed, namely enhancement of cellular motility, which may underlie its role in promoting melanoma metastasis. Finally, various therapeutic strategies targeting survivin in melanoma are reviewed.
Cyclin D1, and to a lesser extent the other D-type cyclins, is frequently deregulated in cancer and is a biomarker of cancer phenotype and disease progression. The ability of these cyclins to activate the cyclin-dependent kinases (CDKs) CDK4 and CDK6 is the most extensively documented mechanism for their oncogenic actions and provides an attractive therapeutic target. Is this an effective means of targeting the cyclin D oncogenes, and how might the patient subgroups that are most likely to benefit be identified?
Hedgehog proteins constitute one of a small number of families of secreted signals that have a central role in the development of metazoans. Genetic analyses in flies, fish and mice have uncovered the major components of the pathway that transduces Hedgehog signals, and recent genome sequence projects have provided clues about its evolutionary origins. In this Review we provide an updated overview of the mechanisms and functions of this signalling pathway, highlighting the conserved and divergent features of the pathway, as well as some of the common themes in its deployment that have emerged from recent studies.
Even in large-scale genome-wide association studies (GWASs), only a fraction of the true associations are detected at the genome-wide significance level. When few or no associations reach the significance threshold, one strategy is to follow up on the most promising candidates, i.e. the single nucleotide polymorphisms (SNPs) with the smallest association-test P-values, by genotyping them in additional studies. In this communication, we propose an overall test for GWASs that analyzes the SNPs with the most promising P-values simultaneously and therefore allows an early assessment of whether the follow-up of the selected SNPs is likely promising. We theoretically derive the properties of the proposed overall test under the null hypothesis and assess its power based on simulation studies. An application to a GWAS for chronic obstructive pulmonary disease suggests that there are true association signals among the top SNPs and that an additional follow-up study is promising.
The hedgehog (Hh) signaling pathway is activated in various types of cancer including pancreatic ductal adenocarcinoma. It has been shown that extremely low oxygen tension (below 1% O2) is found in tumor tissue including pancreatic ductal adenocarcinoma cells (PDAC) and increases the invasiveness of PDAC. To investigate the contribution of the Hh pathway to hypoxia-induced invasiveness, we examined how hypoxia affects Hh pathway activation and the invasiveness of PDAC. In the present study, three human PDAC lines were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. Hypoxia upregulated the transcription of Sonic hedgehog (Shh), Smoothened (Smo), Gli1 and matrix metalloproteinase9 (MMP9) and increased the invasiveness of PDAC. Significantly, neither the addition of recombinant Shh (rhShh) nor the silencing of Shh affected the transcription of these genes and the invasiveness of PDAC. On the other hand, silencing of Smo decreased the transcription of Gli1 and MMP9 and PDAC invasiveness. Silencing of Gli1 or MMP9 decreased PDAC invasiveness. These results suggest that hypoxia activates the Hh pathway of PDAC by increasing the transcription of Smo in a ligand-independent manner and increases PDAC invasiveness.
Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) involves diverse processes characterized by epithelial disruption, smooth muscle hypertrophy/hyperplasia, airway wall fibrosis, and alveolar destruction. According to the accepted current theory of COPD pathogenesis, tissue remodeling in COPD is predominantly a consequence of an imbalance between proteolytic and antiproteolytic activities. However, most of the studies carried out during the last few years have focused on mechanisms related to degradation of extracellular matrix (ECM) structural proteins, neglecting those involved in ECM protein deposition. This review revisits some of the latest findings related to fibrotic changes that occur in the airway wall of COPD patients, as well as the main cellular phenotypes relevant to these processes.
An increasing number of functions for syndecan cell surface heparan sulfate proteoglycans have been proposed over the last decade. Moreover, aberrant syndecan regulation has been found to play a critical role in multiple pathologies, including cancers, as well as wound healing and inflammation. As receptors, they have much in common with other molecules on the cell surface. Syndecans are type I transmembrane molecules with cytoplasmic domains that link to the actin cytoskeleton and can interact with a number of regulators. However, they are also highly complex by virtue of their external glycosaminoglycan chains, especially heparan sulfate. This heterodisperse polysaccharide has the potential to interact with many ligands from diverse protein families. Here, we relate the structural features of syndecans to some of their known functions.
Key findings:
Data from the National Vital Statistics System, Mortality In 2007, the age-adjusted death rate for the United States reached a record low of 760.3 per 100,000 population. Life expectancy at birth reached a record high of 77.9 years. States in the southeast region have higher death rates than those in other regions of the country. In 2007, the five leading causes of death were heart disease, cancer, stroke, chronic lower respiratory diseases, and accidents. These accounted for over 64 percent of all deaths in the United States. White females have the longest life expectancy (80.7 years), followed by black females (77.0 years). The gap in life expectancy between white persons and black persons declined by 35 percent between 1989 and 2007. The race differential was 4.6 years in 2007.
The Hedgehog signalling pathway plays an important role in lung morphogenesis and cellular responses to lung injury. A genome- wide association study has demonstrated that two single nucleotide polymorphisms (SNPs) near the Hedgehog- interacting protein (Hip) gene, SNP identifiers rs1828591 and rs13118928, are associated with risk of chronic obstructive pulmonary disease (COPD). The aim of the present study was to validate the observed association between genetic variation near the Hip gene and COPD, and to investigate whether risk estimates were modified by smoking behaviour.
The association between the Hip gene SNPs and COPD was investigated in the Rotterdam Study by logistic regression analyses, adjusted for several covariates. In addition, an association meta- analysis was performed that included data from the genome-wide association study on COPD.
Both SNPs were significantly associated with risk of COPD (OR 0.80; 95% CI 0.72-0.91). Homozygosity for the minor G allele resulted in a decreased risk of COPD of similar to 40% (95% CI 0.47-.78). There was a significant interaction with the number of pack-years of smoking (p=0.004). The meta- analysis yielded an odds ratio for COPD of 0.80 per additional G allele (p=3.4x10(-9)).
Genetic variation near the Hip gene was significantly associated with risk of COPD, depending on the number of pack-years of smoking.
The sonic hedgehog (Shh) signaling pathway is crucial for normal lung development. In the lung, epithelial-produced Shh signals via mesenchymal Gli1-3 transcription factors. Gli-null lung phenotypes suggest that Gli2 is the primary Gli transcription factor transducing Shh-regulated lung growth, although the mechanism has yet to be elucidated. To clarify the role of Gli2 during lung development, we overexpressed gli2 in the lung mesenchyme of mice, to investigate for changes in Shh signaling, and cellular proliferation. The ectopic overexpression of gli2 resulted in increased Shh pathway activation as evident by increased expression of shh, ptc1, ptc2, smo, hhip, and gli1. Interestingly, we also observed increased expression of gli3 transcripts. Using two different mouse models, gli3-null and gli3Delta699 (Gli3 constitutive repressor), it was found that Gli3 activity does not affect the levels of gli2 in the developing lung. Real-time PCR and immunoblotting revealed that there is increased expression of cyclins D1, D2, and E1 associated with increased gli2 levels. Furthermore, the increase and decrease of cyclins (associated with changes in gli2 levels) positively correlated with cellular proliferation, as assessed by phospho-histone H3 immunohistochemistry. To determine if Gli3 has an effect on cyclin expression in the developing lung, we measured the levels of cyclin D1, D2, and E1, in gli3-null and gli3Delta699 mice and compared them to their wild-type counterparts. However, no change in the levels of cyclins D1, D2, or E1 due to altered Gli3 was observed. These findings suggest that Gli2 and not Gli3 is the primary mediator of Shh signaling influencing fetal lung growth through cyclin regulation.
Chronic obstructive pulmonary disease (COPD) is a highly prevalent airway disease characterized by an abnormal inflammatory response of the lungs to noxious particles and gases. Cigarette smoking remains a major risk factor for COPD development; however, little is known about its effect on human airway smooth muscle cells (HASMCs). The aim of this study is to examine whether apoptosis is involved in cigarette smoke extract (CSE)-induced HASMC death and the molecular mechanisms underlying it. Our studies have shown that CSE increased the level of reactive oxygen species (ROS) and cell apoptosis of HASMCs in a dose- and time-dependent manner, and the ROS scavenger N-acetyl-cysteine abrogated the effect of ROS level and apoptosis on HASMCs. Further, the expression of Bax, Bad, and Fas was increased but Bcl-2 and nuclear factor-kappaB (NF-kappaB) was decreased in a dose- and time-dependent fashion in CSE-induced apoptosis in HASMCs. Taken together, CSE could inhibit the cell growth and induce apoptosis of HASMCs through both the mitochondrial pathway and death receptor pathway. Oxidative stress and inhibition of NF-kappaB expression caused by CSE may play important roles in apoptosis and inhibition of cell growth in HASMCs.
Severe alpha-1-antitrypsin deficiency is the only proven genetic risk factor for chronic obstructive pulmonary disease (COPD). We have assembled a cohort of 44 probands with severe, early-onset COPD, who do not have severe alpha-1-antitrypsin deficiency. A surprisingly high prevalence of females (79.6%) was found. Assessment of the risk to relatives of these early-onset COPD probands for airflow obstruction and chronic bronchitis was performed to determine whether significant familial aggregation for COPD, independent of alpha-1-antitrypsin deficiency, could be demonstrated. First- degree relatives of early-onset COPD probands had significantly lower FEV1 and FEV1/FVC values than control subjects (p < 0.01), despite similar pack-years of smoking. Reduced spirometric values in first-degree relatives of early-onset COPD probands were found only in current or ex-cigarette smokers. The mean FEV1 in current or ex-smoking first-degree relatives was 76.1 +/- 20.9% predicted compared to 89.2 +/- 14.4% predicted in current or ex-smoking control subjects (p < 0.01); in lifelong nonsmokers, the mean FEV1 was 93.4% predicted for both control subjects and first-degree relatives of early-onset COPD probands. Generalized estimating equations, adjusting for age and pack-years of smoking, demonstrated increased odds of reduced FEV1 and chronic bronchitis in current or ex-smoking first-degree relatives of early-onset COPD probands. Using a new method to estimate relative risk from relative odds, we estimate that the relative risks for FEV1 below 60%, FEV1 below 80%, and chronic bronchitis are each approximately three in current or ex-smoking first-degree relatives of early-onset COPD probands. The increased risk to relatives of early-onset COPD probands for reduced FEV1 and chronic bronchitis, limited to current or ex-smokers, suggests genetic risk factor(s) for COPD that are expressed in response to cigarette smoking.
The Hedgehog signalling pathway is essential for the development of diverse tissues during embryogenesis. Signalling is activated by binding of Hedgehog protein to the multipass membrane protein Patched (Ptc). We have now identified a novel component in the vertebrate signalling pathway, which we name Hip (for Hedgehog-interacting protein) because of its ability to bind Hedgehog proteins. Hip encodes a membrane glycoprotein that binds to all three mammalian Hedgehog proteins with an affinity comparable to that of Ptc-1. Hip-expressing cells are located next to cells that express each Hedgehog gene. Hip expression is induced by ectopic Hedgehog signalling and is lost in Hedgehog mutants. Thus, Hip, like Ptc-1, is a general transcriptional target of Hedgehog signalling. Overexpression of Hip in cartilage, where Indian hedgehog (Ihh) controls growth, leads to a shortened skeleton that resembles that seen when Ihh function is lost (B. St-Jacques, M. Hammerschmidt & A.P.M., in preparation). Our findings support a model in which Hip attenuates Hedgehog signalling as a result of binding to Hedgehog proteins: a negative regulatory feedback loop established in this way could thus modulate the responses to any Hedgehog signal.
Although familial clustering has been described, few studies have quantified the risk of airflow obstruction in siblings of patients with chronic obstructive pulmonary disease (COPD). One hundred fifty-two subjects with airflow obstruction and a low gas transfer factor (but without PiZ alpha(1)-antitrypsin deficiency) were identified and 150 were enrolled in the study. Complete data were obtained from 173 of 221 siblings of these subjects. Forty-four of 126 current or ex-smoking siblings had airflow obstruction (FEV(1)/FVC < 0.7) and 36 also had a FEV(1) < 80% predicted, in keeping with COPD. One hundred eleven current or ex-smoking siblings were matched for age, sex, and smoking history with 419 subjects, without a known family history of COPD, from the European Prospective Investigation of Cancer (EPIC)-Norfolk cohort. The prevalence of COPD was much lower in the EPIC group (9.3%) when compared with the siblings (31.5%; odds ratio, 4.70; 95% confidence interval, 2.63 to 8.41). The odds ratio for COPD in siblings with less than a 30 pack-year smoking history was 5.39 (95% confidence interval, 2.49 to 11.67) when compared with matched control subjects. Taken together these results demonstrate a significant familial risk of airflow obstruction in smoking siblings of patients with severe COPD.
Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.
Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 in the context of Hh signaling, we generated Hip1 mutants in the mouse. Loss of Hip1 function results in specific defects in two Hh target issues, the lung, a target of Sonic hedgehog (Shh) signaling, and the endochondral skeleton, a target of Indian hedgehog (Ihh) signaling. Hh signaling was up-regulated in Hip1 mutants, substantiating Hip1's general role in negatively regulating Hh signaling. Our studies focused on Hip1 in the lung. Here, a dynamic interaction between Hh and fibroblast growth factor (Fgf) signaling, modulated at least in part by Hip1, controls early lung branching.
Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.
Pulmonary emphysema is characterized by alveolar wall destruction and airspace enlargement. Recent evidence indicates that epithelial or endothelial apoptosis may be involved in the pathogenesis of emphysema. Here, we describe the induction of emphysematous changes, including airspace enlargement, alveolar wall destruction, and enhanced lung distensibility, in mice receiving a single intratracheal injection of active caspase-3 and Chariot, a newly developed protein transfection reagent. Epithelial apoptosis and enhanced elastolytic activity (optimal at pH 5.5) in bronchoalveolar lavage were noted. Emphysematous changes were also generated in mice receiving an intratracheal injection of nodularin, a proapoptotic serine/threonine kinase inhibitor. This murine model provides direct evidence that confirms that alveolar wall apoptosis causes emphysematous changes. Furthermore, this simple technique for protein transfection of lung tissue can be used in a variety of future applications.
Morphological development of the small intestinal mucosa involves the stepwise remodeling of a smooth-surfaced endodermal tube to form finger-like luminal projections (villi) and flask-shaped invaginations (crypts). These remodeling processes are orchestrated by instructive signals that pass bidirectionally between the epithelium and underlying mesenchyme. Sonic (Shh) and Indian (Ihh) hedgehog are expressed in the epithelium throughout these morphogenic events, and mice lacking either factor exhibit intestinal abnormalities. To examine the combined role of Shh and Ihh in intestinal morphogenesis, we generated transgenic mice expressing the pan-hedgehog inhibitor, Hhip (hedgehog interacting protein) in the epithelium. We demonstrate that hedgehog (Hh) signaling in the neonatal intestine is paracrine, from epithelium to Ptch1-expressing subepithelial myofibroblasts (ISEMFs) and smooth muscle cells (SMCs). Strong inhibition of this signal compromises epithelial remodeling and villus formation. Surprisingly, modest attenuation of Hh also perturbs villus patterning. Desmin-positive smooth muscle progenitors are expanded, and ISEMFs are mislocalized. This mesenchymal change secondarily affects the epithelium: Tcf4/beta-catenin target gene activity is enhanced, proliferation is increased, and ectopic precrypt structures form on villus tips. Thus, through a combined Hh signal to underlying ISEMFs, the epithelium patterns the crypt-villus axis, ensuring the proper size and location of the emerging precrypt compartment.
Lung disease is the leading and second-leading cause of death in women and men in Taiwan, respectively. Epidemiological studies conducted in Taiwan have shown that cigarette smoking is the principal risk factor of lung disease, but little is known about the association between apoptosis and cigarette smoke (CS)-induced lung pathogenesis. We designed an animal exposure system to study signal proteins involved in the process of apoptosis induced by smoking in rat terminal bronchiole. Rats were exposed to CS in doses of 5, 10, and 15 cigarettes, respectively, and the exposure lasted for 30 min, twice a day, 6 days a week for 1 month. Following which the rats were sacrificed and the lung tissues were analyzed by histopathological methods. The terminal bronchioles revealed mild to severe inflammation according to the doses of CS and marked lipid peroxidation, lymphocyte infiltration, congestion, and epithelial emphysema of alveolar spaces were also noted. Using an in situ cell death detection kit (TA300), the association of CS with apoptosis was determined in a concentration-dependent manner. Immunohistochemical evaluation showed that CS treatment produced an increase in the cellular levels of Bax, t-Bid, cleaved caspase-3, phospho-p53, phospho-JNK, and FasL but a decline in Bcl-2 and Mcl-1 (p<0.001 for all) in rat terminal bronchioles. The results provided evidences suggesting that exposure to CS not only induced apoptosis, but also involved p53/Bax and JNK/FasL cascade pathway.
Hedgehog signaling is known to regulate tissue morphogenesis and cell differentiation in a dose-dependent manner. Loss of Indian hedgehog (Ihh) results in reduction in pancreas size, indicating a requirement for hedgehog signaling during pancreas development. By contrast, ectopic expression of sonic hedgehog (Shh) inhibits pancreatic marker expression and results in transformation of pancreatic mesenchyme into duodenal mesoderm. These observations suggest that hedgehog signaling activity has to be regulated tightly to ensure proper pancreas development. We have analyzed the function of two hedgehog inhibitors, Hhip and patched 1 (Ptch), during pancreas formation. Our results indicated that loss of Hhip results in increased hedgehog signaling within the pancreas anlage. Pancreas morphogenesis, islet formation and endocrine cell proliferation is impaired in Hhip mutant embryos. Additional loss of one Ptch allele in Hhip-/-Ptch+/- embryos further impairs pancreatic growth and endodermal cell differentiation. These results demonstrate combined requirements for Hhip and Ptch during pancreas development and point to a dose-dependent response to hedgehog signaling within pancreatic tissue. Reduction of Fgf10 expression in Hhip homozygous mutants suggests that at least some of the observed phenotypes result from hedgehog-mediated inhibition of Fgf signaling at early stages.