[Show abstract][Hide abstract] ABSTRACT: CXXC5 is a member of the CXXC-type zinc-finger domain containing protein family, which is suggested to function in gene transcription, cell adhesion and cytoskeleton organization. Previous studies have revealed that CXXC5 is expressed in skeletal muscle, but whether it regulates skeletal myogenesis is yet unknown. Here, we screened for the possible signaling pathways in which CXXC5 might participate using luciferase gene reporters. The results indicated that CXXC5 significantly increased the activities of the promoters of genes involved in skeletal muscle differentiation. We therefore studied the role of CXXC5 during skeletal myogenesis in C2C12 myoblasts. Our findings suggest that overexpression of CXXC5 in C2C12 myoblasts facilitated myocyte differentiation, while RNAi interference of CXXC5 significantly inhibited the differentiation of C2C12 myoblasts. This study suggests that CXXC5 plays a significant role in regulating skeletal myogenesis.
Journal of Muscle Research and Cell Motility 11/2014; 35(5-6). DOI:10.1007/s10974-014-9400-2 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Leucine-rich repeat (LRR) containing proteins play an essential role in signal transduction, cell adhesion, cell development, DNA repair and RNA processing. Here we cloned a novel gene, Spata34, encoding a LRR containing protein of 415 aa. Spata34 gene consisted of 9 exons and 8 introns and mapped to chromosome 3qA3. Spata34 is conserved across species in evolution. The Spata34 gene was expressed at various levels, faintly before first weeks postpartum and strongly from 2 weeks postpartum in adult testes. Western blot analysis showed that Spata34 protein was specially expressed in mouse testis. Immunohistochemical analysis revealed that Spata34 protein was most abundant in the cytoplasm of round spermatids and elongating spermatids within seminiferous tubules of the adult testis. Overexpression of Spata34 in COS7 cells inhibited the transcriptional activity of AP-1, p53 and p21 which suggested that Spata34 protein may act as a transcriptional repressor in p53 and p21 pathway.
[Show abstract][Hide abstract] ABSTRACT: -Heart function declines with age, but the genetic factors underlying such deterioration are largely unknown. Wnt signaling is known to play a role in heart development but it has not been shown to be important in adult heart function. We have investigated the nuclear adapter protein encoded by pygopus (pygo), which mediates canonical Wnt signaling, for roles in aging-related cardiac dysfunction.
-Using the Drosophila heart model, we show that cardiac-specific pygo knockdown (KD) in adult flies causes a significant (4-5 fold) increase in cardiac arrhythmias (p<0.001) that worsened with age and caused a significant decrease in contractility (-54%, p<0.001) with systolic dysfunction. Immunohistochemistry revealed structural abnormalities that worsened with age and both functional and morphological alterations were ameliorated by pygo overexpression. Unexpectedly, KD of two other Wnt signaling components, β-cat/armadillo or TCF/pangolin, had relatively milder effects on cardiac function. Double-heterozygous combinations of mutants for pygo and canonical Wnt-signaling components had no additional effect on heart function over pygo heterozygotes alone. However, double KD of pygo and Ca(2+)/calmodulin-dependent protein kinase II caused additional arrhythmia compared to pygo KD alone suggesting that some of the effects of pygo are mediated by Ca(2+) signaling. In the isoproterenol-induced hypertrophic mouse model we show that Pygo1 protein levels are increased.
-Our data indicate that Pygo plays a critical role in adult heart function that is Wnt signaling-independent and is likely conserved in mammals.
[Show abstract][Hide abstract] ABSTRACT: Apoptosis is a widespread phenomenon and its dysregulation may result in a variety of human pathologies, such as cancer, autoimmune diseases and neurodegenerative disorders. CXXC-type zinc finger protein 5 (CXXC5) is commonly considered as a tumor suppressor undergoing deregulation or deletion in hematonosis. But it has implied involvement in apoptosis indirectly and the molecular mechanism remains unknown. In this study, we investigated CXXC5-induced apoptosis as well as its underlying mechanism. A fluorescence resonance energy transfer (FRET) assay suggested that CXXC5 induced cell death and caspase-3 activity in primary rat cortical neurons. Further colorimetric TUNEL assay, Hoechst staining and flow cytometric assay indicated a time-dependent apoptosis in which the activities of caspase-8 and caspase-3 were both regulated via CXXC5 according to enzymatic activity assay, Hoechst staining and Western blotting. Transcription reporter assay and Western blotting showed that CXXC5 resulted in activation of tumor necrosis factor-α (TNF-α), initiated the extrinsic apoptosis pathway and cross-linked with the intrinsic mitochondrial pathway. Being a bone morphogenetic protein 4 (BMP-4) downstream regulator, and also a transcription factor, cellular co-localization and co-immunoprecipitation results indicate that CXXC5 co-localized and interacted with Smads. Western blotting and nuclear fraction extraction implied that CXXC5 facilitated Smad3 phosphorylation and Smad4 nuclear translocation. Transcription reporter assay showed that Smad3 can activate TNF-α but CXXC5 was dominant when they were co-expressed. Smad4 can facilitate TNF-α activity induced by CXXC5. In sum, CXXC5 regulated the TNF-α apoptosis pathway associated with Smads.
Current Molecular Medicine 07/2013; 13(8). DOI:10.2174/15665240113139990069 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Published data on the association between tumor necrosis factor-α (TNF-α) G-308A gene polymorphism and dilated cardiomyopathy (DCM) risk are inconclusive. To clarify the association of TNF-α G-308A gene polymorphism and DCM, a meta-analysis of case-control studies was performed. Some databases, such as PubMed and Embase, were searched to indentify related studies. Search terms included dilated cardiomyopathy, tumor necrosis factor-alpha or TNF-α or TNF alpha or tumor necrosis factor alpha, and polymorphism or mutation. Eight case-control studies involving 1487 DCM cases and 1734 normal controls were included in the meta-analysis to assess the purported association between the TNF-α G-308A gene polymorphism and the risk of DCM. A dominant genetic model was used and the comparison of GA/AA genotype versus GG genotype was performed in the present meta-analysis. The odds ratio was 1.42 (95% confidence interval: 1.05, 1.93, P=0.02), manifesting frequency of the TNF-α-308 GA/AA genotype was higher in DCM patients than the control group. TNF-α G-308A nucleotide transition might be associated with the risk of DCM.
DNA and cell biology 03/2013; 32(3):130-7. DOI:10.1089/dna.2012.1911 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Four-and-a-half LIM proteins FHL1-3 play important roles in cardiovascular pathophysiology. However, their roles in heart development remain unclear. Here, we report that fhlA, the zebrafish homolog of human FHL1, was found to be expressed around the 22-somite stage. After 24 hpf, expression was restricted to the heart. fhlA knockdown caused an enlarged cardiac chamber phenotype with up-regulated expression of the cardiac markers, but fhlA overexpression reduced the sizes of the cardiac chambers and down-regulated expression of the markers. The morphology associated with the cmlc2, amhc, and vmhc expression patterns at the 22 somite and 24 hpf stages included a broadened domain in embryos lacking fhlA and a smaller domain in embryos overexpressing fhlA. The changes in the sizes of the chambers were attributed to the changes in the number of ventricular and atrial cells. Loss of fhlA caused a longer heart period and pause between heartbeats in M-modes than in controls, but fhlA overexpression caused shorter systolic and diastolic intervals. Abnormal cardiac chambers and physiological function were found to be largely rescued. We also showed the expression of fhlA in the heart to be increased by retinoic acid (RA) and decreased by the RA synthase inhibitor DEAB. Both fhlA and RA signaling caused a phenotype characterized by the morphological alterations in the chamber sizes, suggesting that the role of fhlA in heart development is probably regulated by RA signaling. Taken together, these results showed that fhlA regulates the size of the heart chamber by reducing the number of cardiac cells.
Current Molecular Medicine 02/2013; 13(6). DOI:10.2174/1566524011313060010 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza A virus (IAV) is a widespread human pathogen which plays an important role in the development and exacerbation of myocarditis and causes a significant impairment of the cardiac function and even mortality. Curcumin is an important component of traditional Chinese and Indian medicine with well-documented anti-inflammatory effects to prevent the cardiovascular disease. However, no study to date has addressed the effects and possible mechanism of curcumin on IAV-induced myocarditis in mice. In this study, a mouse model was firstly established for the study of IAV-induced myocarditis. H1N1-infected mice were apparently ill with lethargy, poor coat condition, anorexia, irritability, back arching and even survival. After treatment with curcumin (100 mg/kg/day), the weight loss and survival rate were ameliorated throughout the study. On day 5 after infection, both the heart weight to body weight ratio and the left ventricular weight to body weight ratio were significantly decreased in mice treated with curcumin. The area of myocardial necrosis was significantly smaller in the hearts of mice treated with curcumin compared to IVA-infected mice. Gene expression of NS1, TNF-α, IL-6 and IL-1β, I collagen, and MMP-2 mRNA in the heart tissue was markedly increased in IAV-infected mice, while curcumin significantly attenuated the expression of these genes. RT-PCR analysis revealed that curcumin inhibited the mRNA expression of Wnt3, Tcf4 and β-catenin. Curcumin also suppressed mRNA levels of Wnt target genes, c-Myc and cyclin D1. Our results provided the first evidence for the effect of curcumin to treat IVA-induced myocardial damage in particular via inhibiting Wnt/β-catenin signaling.
[Show abstract][Hide abstract] ABSTRACT: HMGB1 is associated with human cancers and is an activator of autophagy which mediates chemotherapy resistance. We here show that the mRNA levels of HMGB1 are high in leukemia cells and it is involved in the progression of childhood chronic myeloid leukemia (CML). HMGB1 decreases the sensitivity of human myeloid leukemia cells K562 to anti-cancer drug induced death through up-regulating the autophagy pathway, which is confirmed by the observation with an increase in fusion of autophagosomes and autophagolysosomes. When overexpressing HMGB1, both mRNA levels of Beclin-1, VSP34 and UVRAG which are key genes involved in mammalian autophagy and protein levels of p-Bcl-2 and LC3-II are increased. Luciferase assays document that over-expression of HMGB1 increases the transcriptional activity of JNK and ERK, which may be silenced by siRNA. The results suggest that HMGB1 regulates JNK and ERK required for autophagy, which provides a potential drug target for therapeutic interventions in childhood CML.
[Show abstract][Hide abstract] ABSTRACT: Leucine-rich repeat containing protein 10 (LRRC10) is characterized as a cardiac-specific gene, suggesting a role in heart development and disease. A severe cardiac morphogenic defect in zebrafish morphants was recently reported but a contradictory result was found in mice, suggesting a more complicated molecular mechanism exists during mouse embryonic development. To elucidate how LRRC10 is regulated, we analyzed the 5'enhancer region approximately 3 kilo bases (kb) upstream of the Lrrc10 start site using luciferase reporter gene assays. Our characterization of the Lrrc10 promoter indicates it possesses complicated cis-and trans-acting elements. We show that GATA4 and MEF2C could both increase transcriptional activity of Lrrc10 promoter individually but that they do not act synergistically, suggesting that there exists a more complex regulation pattern. Surprisingly, knockout of Gata4 and Mef2c binding sites in the 5'enhancer region (-2,894/-2,889) didn't change the transcriptional activity of the Lrrc10 promoter and the likely GATA4 binding site identified was located in a region only 100 base pair (bp) upstream of the promoter. Our data provides insight into the molecular regulation of Lrrc10 expression, which probably also contributes to its tissue-specific expression.
[Show abstract][Hide abstract] ABSTRACT: Zinc finger (ZNF) proteins play a critical role in cell growth, proliferation, apoptosis, and intracellular signal transduction. In this paper, we cloned and characterized a novel human KRAB-related zinc finger gene, ZNF425, which encodes a protein of 752 amino acids. ZNF425 is strongly expressed in the three month old human embryos and then is almost undetectable in six month old embryos and in adult tissues. An EGFP-ZNF425 fusion protein can be found in both the nucleus and the cytoplasm. ZNF425 appears to act as a transcription repressor. Over-expression of ZNF425 inhibits the transcriptional activities of SRE, AP-1, and SRF. Deletion analysis indicates that the C2H2 domain is the main region responsible for the repression. Our results suggest that the ZNF425 gene is a new transcriptional inhibitor that functions in the MAPK signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: Human YPEL4 is a member of YPEL family. It contains a Yippee domain, which is a putative zinc-finger-like, metal-binding domain. The human YPEL4 gene maps to chromosome 11q12.1, is ubiquitously expressed in adult tissues, and encodes a nuclear protein of 127 amino acids, the function of which remains unknown. To gain insights into the cellular function of this protein, we searched for YPEL4-interacting proteins using a yeast two-hybrid screen. The major vault protein (MVP), a lung resistance associated protein, was identified as a binding partner of YPEL4. The interaction between YPEL4 and MVP in mammalian cells was further demonstrated by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay, and immunocytochemistry. Using a reporter system, we found that MVP can inhibit YPEL4's ability to activate Elk-1 in the MAPK signaling pathway. This study provides new clues for understanding the molecular mechanism of YPEL4 in cell division and signal transduction pathways and should be helpful for understanding molecular functions of the YPEL family.
[Show abstract][Hide abstract] ABSTRACT: LBH is a transcription factor as a candidate gene for CHD associated with partial trisomy 2p syndrome. To identify potential LBH-interacting partners, a yeast two-hybrid screen using LBH as a bait was performed with a human heart cDNA library. One of the clones identified encodes alphaB-crystallin. Co-immunoprecipitation and GST pull-down assays showed that LBH interacts with alphaB-crystallin, which is further confirmed by mammalian two-hybrid assays. Co-localization analysis showed that in COS-7 cells, alphaB-crystallin that is cytoplasmic alone, accumulates partialy in the nucleus when co-transfected with LBH. Transient transfection assays indicated that overexpression of LBH or alphaB-crystallin reduced the transcriptional activities of p53 and p21, respectively, Overexpression of both alphaB-crystallin and LBH together resulted in a stronger repression of the transcriptional activities of p21 and p53. These results showed that the interaction of LBH and alphaB-crystallin may inhibit synergistically the transcriptional regulation of p53 and p21.
[Show abstract][Hide abstract] ABSTRACT: The sterile alpha motif (SAM) is a putative protein interaction domain involved in a wide variety of biological processes. Here we report the identification and characterization of a novel gene, SAMD4B, which encodes a putative protein of 694 amino acids with a SAM domain. Northern blot and RT-PCR analysis showed that SAMD4B is widely expressed in human embryonic and adult tissues. Transcriptional activity assays show SAMD4B suppresses transcriptional activity of L8G5-luciferase. Over-expression of SAMD4B in mammalian cells inhibited the transcriptional activities of activator protein-1 (AP-1), p53 and p21, and the inhibitory effects can be relieved by siRNA. Deletion analysis indicates that the SAM domain is the main region for transcriptional suppression. The results suggest that SAMD4B is a widely expressed gene involved in AP-1-, p53-and p21-mediated transcriptional signaling activity.
[Show abstract][Hide abstract] ABSTRACT: In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.
[Show abstract][Hide abstract] ABSTRACT: Zinc finger-containing transcription factors are the largest single family of transcriptional regulators in mammals, which play an essential role in cell differentiation, cell proliferation, apoptosis, and neoplastic transformation. Here we have cloned a novel KRAB-related zinc finger gene, ZNF424, encoding a protein of 555aa. ZNF424 gene consisted of 4 exons and 3 introns, and mapped to chromosome 19p13.3. ZNF424 gene was ubiquitously expressed in human embryo tissues by Northern blot analysis. ZNF424 is conserved across species in evolution. Using a GFP-labeled ZNF424 protein, we demonstrate that ZNF424 localizes mostly in the nucleus. Transcriptional activity assays shows ZNF424 suppresses transcriptional activity of L8G5-luciferase. Overexpression of ZNF424 in HEK- 293 cells inhibited the transcriptional activity of NFAT and p21, which may be silenced by siRNA. The results suggest that ZNF424 protein may act as a transcriptional repressor that suppresses NFAT and p21 pathway to mediate cellular functions.
[Show abstract][Hide abstract] ABSTRACT: In this study, we report the identification and characterization of a novel C2H2 zinc finger protein, ZNF552, from a human embryonic heart cDNA library. ZNF552 is composed of three exons and two introns and maps to chromosome 19q13.43. The cDNA of ZNF552 is 2.3 kb, encoding 407 amino acids with an amino-terminal KRAB domain and seven carboxyl-terminal C2H2 zinc finger motifs in the nucleus and cytoplasm. Northern blotting analysis indicated that a 2.3 kb transcript specific for ZNF552 was expressed in liver, lung, spleen, testis and kidney, especially with a higher level in the lung and testis in human adult tissues. Reporter gene assays showed that ZNF552 was a transcriptional repressor, and overexpression of ZNF552 in the COS-7 cells inhibited the transcriptional activities of AP-1 and SRE, which could be relieved through RNAi analysis. Deletion studies showed that the KRAB domain of ZNF552 may be involved in this inhibition.
[Show abstract][Hide abstract] ABSTRACT: In this study, a novel member of BTB-kelch proteins, named KBTBD7, was cloned from a human embryonic heart cDNA library. The cDNA of KBTBD7 is 3,008 bp long and encodes a protein product of 684 amino acids (77.2 kD). This protein is highly conserved in evolution across different species. Western blot analysis indicates that a 77 kD protein specific for KBTBD7 is wildly expressed in all embryonic tissues examined. In COS-7 cells, KBTBD7 proteins are localized to the cytoplasm. KBTBD7 is a transcription activator when fused to GAL4 DNA-binding domain. Deletion analysis indicates that the BTB domain and kelch repeat motif are main regions for transcriptional activation. Overexpression of KBTBD7 in MCF-7 cells activates the transcriptional activities of activator protein-1 (AP-1) and serum response element (SRE), which can be relieved by siRNA. These results suggest that KBTBD7 proteins may act as a new transcriptional activator in mitogen-activated protein kinase (MAPK) signaling. [BMB reports 2010; 43(1): 17-22].
[Show abstract][Hide abstract] ABSTRACT: The Bric-a-brac, Tramtrack, Broad-complex (BTB) domain is a protein-protein interaction domain that is found in many zinc finger transcription factors. BTB containing proteins play important roles in a variety of cellular functions including regulation of transcription, regulation of the cytoskeleton, protein ubiquitination, angiogenesis, and apoptosis. Here, we report the cloning and characterization of a novel human gene, KLHL31, from a human embryonic heart cDNA library. The cDNA of KLHL31 is 5743 bp long, encoding a protein product of 634 amino acids containing a BTB domain. The protein is highly conserved across different species. Western blot analysis indicates that the KLHL31 protein is abundantly expressed in both embryonic skeletal and heart tissue. In COS-7 cells, KLHL31 proteins are localized to both the nucleus and the cytoplasm. In primary cultures of nascent mouse cardiomyocytes, the majority of endogenous KLHL31 proteins are localized to the cytoplasm. KLHL31 acts as a transcription repressor when fused to GAL4 DNA-binding domain and deletion analysis indicates that the BTB domain is the main region responsible for this repression. Overexpression of KLHL31 in COS-7 cells inhibits the transcriptional activities of both the TPA-response element (TRE) and serum response element (SRE). KLHL31 also significantly reduces JNK activation leading to decreased phosphorylation and protein levels of the JNK target c-Jun in both COS-7 and Hela cells. These results suggest that KLHL31 protein may act as a new transcriptional repressor in MAPK/JNK signaling pathway to regulate cellular functions.
Molecules and Cells 09/2008; 26(5):443-53. · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HNULP1, a new member of the basic helixloop-helix transcription factors, contains a DUF654 domain in its C-terminus and is
highly conserved from Drosophilae, yeast, zebrafish to mouse. The function of this motif, however, is currently unknown. In this research, we fused five deletion
fragments of the DUF654 domain to the GAL4 DNA-binding domain and then co-transfected with plasmids L8G5-Luc and VP-16. The
analysis of the GAL4 luciferase reporter gene indicated that fragments from 228 to 407 amino acids in the DUF654 domain had
a strong transcription repression activity. Therefore, this study lays a solid foundation for research on the mechanism of
hnulp1 transcriptional regulation and the function of the DUF654 domain.
Frontiers of Biology in China 06/2008; 3(2):137-142. DOI:10.1007/s11515-008-0038-6