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Cell-specific involvement of HNF-1 beta in alpha(1)-antitrypsin gene expression in human respiratory epithelial cells
Abstract
The synergistic action of hepatocyte nuclear factor (HNF)-1alpha and HNF-4 plays an important role in expression of the alpha(1)-antitrypsin (alpha(1)-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the alpha(1)-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that alpha(1)-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1alpha and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that alpha(1)-AT gene expression in H441 cells is predominantly driven by HNF-1beta, even though HNF-1beta has no effect on alpha(1)-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of alpha(1)-AT and HNF-1beta was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on alpha(1)-AT gene expression in H441 cells, even when it is cotransfected with HNF-1beta or HNF-1alpha. HNF-3 by itself has little effect on alpha(1)-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1beta in alpha(1)-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.
... Human bronchial epithelial 16HBE14o-cells express a 1 -AT It has been reported that a variety of human epithelial cells express a 1 -AT, including Calu-3, A549 and H441 [23][24][25]. We determined whether the 16HBE14o-human bronchial epithelial cell line belongs to this category. ...
... AT is produced locally in the lung by bronchial epithelial cells, amongst others[5,8,[23][24][25]. In non-a 1 -AT deficient ...
alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.
... 11,12 Gene therapy has also been directed to hepatocytes or skeletal muscles using an AAV vector system resulting in high levels of serum A1AT, 8,17,22 but the A1AT levels in the lung tissue were not determined. This may be important as A1AT can be secreted by alveolar macrophages 30 and alveolar epithelial cells, 31,32 and it has been speculated that high intra-alveolar level of A1AT may potentially dampen an acute inflammatory response in the lungs. 31 It is unclear how plasma A1AT diffuses across the alveolar capillary endothelial barrier into the interstitium and across the epithelial barrier into the alveolar spaces. ...
... This may be important as A1AT can be secreted by alveolar macrophages 30 and alveolar epithelial cells, 31,32 and it has been speculated that high intra-alveolar level of A1AT may potentially dampen an acute inflammatory response in the lungs. 31 It is unclear how plasma A1AT diffuses across the alveolar capillary endothelial barrier into the interstitium and across the epithelial barrier into the alveolar spaces. In humans, A1AT levels are 20-53 mM in the serum, 10-40 mM in the interstitial fluid and 2-5 mM in the alveolar spaces. ...
Inadequate antiprotease activity in the lungs due to alpha-1-antitrypsin (A1AT) deficiency is a factor of early-onset emphysema. We propose a new approach to gene therapy that involves the intratracheal delivery of macrophages expressing human A1AT (hA1AT). Recombinant adeno-associated virus (rAAV) plasmids encoding the hA1AT gene were packaged into virions using 293 cells, and transgenic progeny virus was purified from the cells. The murine macrophage cell line J774A.1 was infected in vitro with the recombinant hA1AT rAAV virus. The hA1AT-producing macrophages were delivered intratracheally into mechanically ventilated C57BL/6J mice, a strain with low endogenous levels of A1AT. Transcription of hA1AT mRNA was detected in the transfected cells by RT-PCR, and protein expression was verified by immunohistochemistry. Levels of hA1AT in the cell culture medium and in the bronchoalveolar lavage (BAL) were assayed by ELISA. The concentration of hA1AT in J774A.1 cell-conditioned medium increased from undetectable levels prior to transfection, to 60 mg/l at 24 h post-transfection. At 1, 3 and 7 days after intratracheal delivery of transfected macrophages, hA1AT protein in BAL from C57BL/6J mice increased from undetectable levels to 2.5+/-0.9, 2.6+/-1.1 and 2.2+/-0.8 mg/l, respectively. These results suggest that airway delivery of macrophages overexpressing hA1AT may be an effective approach to enhance alveolar protection in A1AT deficiency.
... Among these genes may be SERPINA1 which encodes a secretory protein alpha1-antitrypsin (AAT). Being predominantly liver-derived, it is produced by non-hepatic cells as well [6][7][8][9][10]. AAT plays multiple roles with the main in supporting protease-antiprotease balance in the organism [11], while others include anti-inflammatory and immunemodulatory [12][13][14][15], anti-apoptotic [16,17], anti-viral [18][19][20], and even chaperone-like activities [21,22]. ...
SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5'-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. CONCLUSION: Alternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression.
... It is also produced in small quantities by macrophages, monocytes, circulating neutrophils, and alveolar macrophages. Recently, it has been documented that it can also be synthesized by respiratory cells such as bronchial epithelial cells [3][4][5]6 & ]. This protein is codified by the highly polymorphic SERine Proteinase INhibitor 1 gene, located on frequent normal allelic variant associated with normal serum level is the protease inhibitor (PI)ÃM. ...
Purpose of review:
The aim of the article is to highlight the association between α1-antitrypsin deficiency (AATD) and asthma.
Recent findings:
AATD is one of the most common and underrecognized autosomal disorders associated with an increased risk of developing liver and lung diseases. An association between α1-antitrypsin and asthma has been suggested, especially with severe forms of this disease. Many studies have shown an increased prevalence of asthma in the α1-antitrypsin-deficient population overtime (4-38%). The biological mechanism underlying these two conditions and able to bind them has not yet been well investigated. As α1-antitrypsin is the main inhibitor of the serine proteinase and it is an important anti-inflammatory protein with pronounced immunomodulatory activities, it can be hypothesized that the link between AATD and asthma might be represented by the elastase/antielastase imbalance and the proinflammatory effect that occurs because of the reduction of this protein.
Summary:
There is a strong need for further researches to better understand the molecular mechanisms binding AATD and asthma. It is also recommendable to screen for AATD, late-onset asthma patients, and/or those with not fully reversible airways obstruction.
... These observations are consistent with early observations that COPD associated with AATD had a much higher degree of neutrophil infiltration and accelerated inflammatory destruction than COPD of other etiologies [107]. There is also evidence for synthesis of AAT in lung epithelial cells [108]. If accumulation of Z AAT has the same "cytostatic" consequences in respiratory epithelial cells as it has in liver cells, it could theoretically cause respiratory epithelial dysfunction that contributes to the progression of COPD. ...
Alpha-1-antitrypsin (AAT) is an acute phase secretory glycoprotein that inhibits neutrophil proteases like elastase and is considered as the archetype of a family of structurally related serine protease inhibitors termed serpins. Serum AAT predominantly originates from liver and increases three to five fold during host response to tissue injury and inflammation. The AAT deficiency is unique among the protein misfolding diseases in that it causes target organ injury by both loss-of-function and gain-of-toxic function mechanisms. Lack of its antiprotease activity is associated with premature development of pulmonary emphysema and loss of function due to accumulation of resultant aggregates in chronic obstructive pulmonary disease (COPD). This in turn markedly reduces the amount of AAT that is available to protect lungs against proteolytic attack by the enzyme neutrophil elastase. The coalescence of AAT deficiency, its reduced efficacy, and cigarette smoking or poor ventilation conditions have devastating effect on lung function. On the other hand, the accumulation of retained mutant proteins in endoplasmic reticulum of liver cells in a polymerized form rather than secreted into the blood in its monomeric form is associated with chronic liver disease and predisposition to hepatocellular carcinoma (HCC) by gain of toxic function. Liver injury resulting from this gain-of-toxic function mechanism in which mutant AAT retained in the ER initiates a series of pathologic events, eventually culminating at liver cirrhosis and HCC. Here in this review, we underline the structural, genetic, polymorphic, biochemical and pathological advances made in the field of AAT deficiency and further comprehensively emphasis on the therapeutic interventions available for the patient.
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The MaturityIOnset Diabetes of the Young type 5 (MODY5), also called Renal Cysts And Diabetes syndrome (RCAD), is a dominantly inherited diabetes mellitus and nephropathy. The phenotype, variable within a same family, also includes hepatic, pancreatic, urogenital, neurological, and neoplastic pathologies. The RCAD is associated with mutations or complete deletion (identical in proportions) of the hepatocyte nuclear
factor 1 homeobox B (HNF1B) gene, 50% of which appears de novo. HNF1B encodes a transcription factor involved in the embryogenesis and the homeostasis of the affected organs. The aim of this dissertation is to establish the role of the c.226G>T (G226T) substitution of HNF1B in the emergence of a RCAD. Using literature, we created a medical questionnaire and calculated a bioIclinical score in order to precisely describe the phenotype of 6 patients (5 families) carriers of this substitution. The latter has been analysed with bioinformatic tools able to distinguish a mutation from a polymorphism (impact on the protein stability, physicochemical properties and phylogenetic conservation of the residue, statistical learning using databases and occurrence among large cohorts). According to bioinformatic study the substitution is deleterious. It was found in 7 out of 8 169 patients (0.09%) in large genome research (ClinSeq and Exome Variant Server). In our cohort, we relate: (1) exclusive northern african origins of the patients, (2) associated pathologies never described in the RCAD, (3) no parental pathology except for one family in which there is (4) an absence of coIsegregation between the mutation and the phenotype. We therefore think that the G226T substitution is a variant with northern african origins which is not able to cause alone a RCAD. Systematic parental sequencing and further in vitro studies are needed to confirm this hypothesis.
... The regulation of basal and induced AT expression is cell-type dependent 18,[22][23][24][25][26] and in hepatocytes is regulated by three cis-acting elements 5' of the gene and is predominantly driven by the synergistic effects of the hepatocyte nuclear factors HNF1A and HNF4 and modulated by IL-6 during the acute phase reaction 17, 23, 27-31 . ...
In the classical form of α1-antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function, “proteotoxic” mechanism. Whereas some ATD patients develop severe liver disease that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPSCs) from ATD individuals with or without severe liver disease could model these personalized variations in hepatic disease phenotypes. Patient-specific iPSCs were generated from ATD patients and a control, and differentiated into hepatocyte-like cells (iHeps) having many characteristics of hepatocytes. Pulse-chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule, compared to the wild-type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from severe liver disease patients, compared to those from no liver disease patients. Transmission electron microscopy showed dilated rough endoplasmic reticulum in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with severe liver disease. These results provide definitive validation that iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that “proteostasis” mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPSCs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies.
... In A1AT deficiency, low level of plasma A1AT are believed to allow neutrophil elastase unfettered access to lung connective tissue inducing pulmonary emphysema. But A1AT is also actively transcribed and secreted in relatively smaller amounts by cells including neutrophils, mononuclear phagocytes, enterocytes [4,5], and human respiratory epithelial cells [6]. Although the primary role of A1AT is to inactivate neutrophil elastase [2], it may have other pathobiologically relevant functions. ...
BACKGROUND:
Alpha 1-antitrypsin (A1AT) is a 52 kDa serine protease inhibitor produced largely by hepatocytes but also by mononuclear phagocytes. A1AT chiefly inhibits neutrophil elastase and proteinase-3 but has also been reported to have immune modulatory functions including the ability to inhibit caspases. Its clinical availability for infusion suggests that A1AT therapy might modulate caspase related inflammation. Here we tested the ability of A1AT to modulate caspase-1 function in human mononuclear phagocytes.
METHODS:
Purified plasma derived A1AT was added to active caspase-1 in a cell-free system (THP-1 lysates) as well as added exogenously to cell-culture models and human whole blood models of caspase-1 activation. Functional caspase-1 activity was quantified by the cleavage of the caspase-1 specific fluorogenic tetrapeptide substrate (WEHD-afc) and the release of processed IL-18 and IL-1β.
RESULTS:
THP-1 cell lysates generated spontaneous activation of caspase-1 both by WEHD-afc cleavage and the generation of p20 caspase-1. A1AT added to this cell free system was unable to inhibit caspase-1 activity. Release of processed IL-18 by THP-1 cells was also unaffected by the addition of exogenous A1AT prior to stimulation with LPS/ATP, a standard caspase-1 activating signal. Importantly, the A1AT exhibited potent neutrophil elastase inhibitory capacity. Furthermore, A1AT complexed to NE (and hence conformationally modified) also did not affect THP-1 cell caspase-1 activation. Finally, exogenous A1AT did not inhibit the ability of human whole blood samples to process and release IL-1β.
CONCLUSIONS:
A1AT does not inhibit human monocyte caspase-1.
... Although it may also be produced in small amounts in a variety of extra-hepatic tissues (e.g. neutrophils, lymphocytes, mononuclear phagocytes, enterocytes, pulmonary alveolar cells) [22][23][24], renal tubular cell AAT transcription under basal or stress conditions has not been previously described. AAT's primary biologic activity is as a protease inhibitor [19,25]. ...
Alpha-1-antitrypsin (AAT) is a hepatic stress protein with protease inhibitor activity. Recent evidence indicates that ischemic or toxic injury can evoke selective changes within kidney that resemble a hepatic phenotype. Hence, we tested the following: i) Does acute kidney injury (AKI) up-regulate the normally renal silent AAT gene? ii) Does rapid urinary AAT excretion result? And iii) Can AAT's anti-protease/anti-neutrophil elastase (NE) activity protect injured proximal tubule cells? CD-1 mice were subjected to ischemic or nephrotoxic (glycerol, maleate, cisplatin) AKI. Renal functional and biochemical assessments were made 4-72 hrs later. Rapidly following injury, 5-10 fold renal cortical and isolated proximal tubule AAT mRNA and protein increases occurred. These were paralleled by rapid (>100 fold) increases in urinary AAT excretion. AKI also induced marked increases in renal cortical/isolated proximal tubule NE mRNA. However, sharp NE protein levels declines resulted, which strikingly correlated (r, -0.94) with rising AAT protein levels (reflecting NE complexing by AAT/destruction). NE addition to HK-2 cells evoked ∼95% cell death. AAT completely blocked this NE toxicity, as well as Fe induced oxidant HK-2 cell attack. Translational relevance of experimental AAT gene induction was indicated by ∼100-1000 fold urinary AAT increases in 22 AKI patients (matching urine NGAL increases). We conclude: i) AKI rapidly up-regulates the renal cortical/proximal tubule AAT gene; ii) NE gene induction also results; iii) AAT can confer cytoprotection, potentially by blocking/reducing cytotoxic NE accumulation; and iv) marked increases in urinary AAT excretion in AKI patients implies clinical relevance of the AKI- AAT induction pathway.
... TCF2, also known as HNF1β, is a downstream transcription activator of the Wnt signaling pathway that is widely expressed in a variety of tissues, and it is crucial in embryonic development [15,16] . It also regulates the expression of many other tissue-specific genes by binding the proximal region of the promoter sequences of genes such as alpha 1-antitrypsin [17,18] , which, when overexpressed, is associated with lung cancer development [19] . Interestingly, the SNP of TCF2 is associated with renal cancer and prostate cancer [20,21] , but the association with lung cancer remains unknown. ...
The Wnt signaling pathway is crucial for pulmonary development and differentiation; dysregulation of the Wnt signaling pathway may impair lung function. Indeed, single nucleotide polymorphisms (SNPs) of Wnt pathway-related genes have been suggested as risk factors for certain types of cancers. In this study, we aimed to evaluate the influence of SNPs in Wnt-related genes (, ) on susceptibility to lung cancer.
Polymorphisms of rs4430796, rs2250889, and 9 rs17576 were studied in Han Chinese subjects, including 135 patients with lung cancer and 176 controls, using the Sequenom MassARRAY platform. The association of genotypes with susceptibility to lung cancer was analyzed using odds ratio (OR), with 95% confidence interval (95% CI) and .
The three SNPs (rs4430796, rs2250889, and rs17576) were found to be significantly associated with an increased risk of lung cancer. The AA genotype and AG+AA genotype of rs4430796 showed a significantly increased susceptibility to lung cancer compared with the GG genotype (adjusted OR=6.03, 95% CI: 1.30-28.09, =0.022; 5.55, 95% CI: 1.20-25.58, =0.028). Compared with the rs17576 GG genotype, the AG and AG+AA genotypes were also associated with a significant risk (adjusted OR=2.65, 95% CI: 1.60-4.37, ≤0.001; 2.57, 95% CI: 1.59-4.19, ≤0.001) whereas the rs2250889 CG and CG+GG genotypes had 2.97-fold (95% CI: 1.81-4.85; ≤0.001) and 2.80-fold increased associations with lung cancer (95% CI: 1.73-4.54; ≤0.001), respectively, compared with the rs2250889 CC genotype. Furthermore, the association of rs4430796 with lung cancer became insignificant (>0.05) after adjusting for gender and rs2250889.
The three SNPs may play a role in the predisposition of members of the Han Chinese population to lung cancer.
... AAT is a secretory glycoprotein produced by the liver and is the most abundant serum antiprotease in circulation (Kueppers 1971). While the majority of AAT in the body is hepatocyte-derived, it is actively transcribed and secreted by other cell types including monocytes (), macrophages (Mornex et al. 1986), neutrophils (Bergin et al. 2010), intestinal epithelial cells (Perlmutter et al. 1989), and various epithelial cells in the lung (Hu and Perlmutter 2002; Venembre et al. 1994; Cichy, Potempa, and Travis 1997), albeit in smaller quantities. In keeping with its role as an acute phase reactant, the hepatocyte expresses approximately 200 times more AAT mRNA than other cells (Rogers et al. 1983) and serum levels can rapidly increase by between two-and five-fold during infection, trauma, surgery and burns (Kossmann et al. 1995; Voulgari et al. 1982; Sandford et al. 1999; Jeschke, Barrow, and Herndon 2004). ...
... For example, ATZ polymers deposited in the lung from the extracellular fluid may attract neutrophil and neutrophilic inflammatory effectors that probably increase the severity of lung damage (Mahadeva et al. 2005). Gain-of-toxic function could also theoretically result from the effects of mutant ATZ expression in bronchoalveolar macrophages and respiratory epithelial cells in the same way it happens in hepatocytes (Hu and Perlmutter 2002 ). These effects could explain the fact that replacement therapy has had relatively limited clinical efficacy to date. ...
In α1-antitrypsin (AT) deficiency, a point mutation renders a hepatic secretory glycoprotein prone to misfolding and polymerization. The mutant protein accumulates in the endoplasmic reticulum of liver cells and causes hepatic fibrosis and hepatocellular carcinoma by a gain-of-function mechanism. Genetic and/or environmental modifiers determine whether an affected homozygote is susceptible to hepatic fibrosis/carcinoma. Two types of proteostasis mechanisms for such modifiers have been postulated: variation in the function of intracellular degradative mechanisms and/or variation in the signal transduction pathways that are activated to protect the cell from protein mislocalization and/or aggregation. In recent studies we found that carbamazepine, a drug that has been used safely as an anticonvulsant and mood stabilizer, reduces the hepatic load of mutant AT and hepatic fibrosis in a mouse model by enhancing autophagic disposal of this mutant protein. These results provide evidence that pharmacological manipulation of endogenous proteostasis mechanisms is an appealing strategy for chemoprophylaxis in disorders involving gain-of-function mechanisms.
... A1AT is an acute phase 52 kDa 418 amino acid glycoprotein that is primarily synthesized and secreted by hepatocytes (Rogers et al., 1983), although it is also actively transcribed and secreted in smaller amounts by cells including neutrophils , mononuclear phagocytes and enterocytes (Molmenti et al., 1993). A1AT is also produced locally in the lung by bronchial epithelial cells (Mason et al., 1991; Venembre et al., 1994; Cichy et al., 1997; Hu and Perlmutter, 2002; Mulgrew et al., 2004). It is present in all tissues of the body and its primary role is to inhibit NE (Travis et al., 1985). ...
Chronic inflammatory lung diseases such as cystic fibrosis and emphysema are characterized by higher‐than‐normal levels of pulmonary proteases. While these enzymes play important roles such as bacterial killing, their dysregulated expression or activity can adversely impact on the inflammatory process. The existence of efficient endogenous control mechanisms that can dampen or halt this overexuberant protease activity in vivo is essential for the effective resolution of inflammatory lung disease. The function of pulmonary antiproteases is to fulfil this role. Interestingly, in addition to their antiprotease activity, protease inhibitors in the lung also often possess other intrinsic properties that contribute to microbial killing or termination of the inflammatory process. This review will outline important features of chronic inflammation that are regulated by pulmonary proteases and will describe the various mechanisms by which antiproteases attempt to counterbalance exaggerated protease‐mediated inflammatory events. These proteases, antiproteases and their modifiers represent interesting targets for therapeutic intervention.
This article is part of a themed issue on Mediators and Receptors in the Resolution of Inflammation. To view this issue visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009
... Alpha-1 antitrypsin (A1AT) is an acute phase 52kDa 418 amino acid glycoprotein that is primarily synthesized and secreted by the hepatocytes in the liver1 though it is also actively transcribed and secreted in smaller amounts by cells including neutrophils, mononuclear phagocytes, enterocytes,2 and human respiratory epithelial cells.3 ...
Alpha-1 antitrypsin (A1AT) is a 52 kDa serine protease inhibitor that is synthesized in and secreted from the liver. Although it is present in all tissues in the body the present consensus is that its main role is to inhibit neutrophil elastase in the lung. A1AT deficiency occurs due to mutations of the A1AT gene that reduce serum A1AT levels to <35% of normal. The most clinically significant form of A1AT deficiency is caused by the Z mutation (Glu342Lys). ZA1AT polymerizes in the endoplasmic reticulum of liver cells and the resulting accumulation of the mutant protein can lead to liver disease, while the reduction in circulating A1AT can result in lung disease including early onset emphysema. There is currently no available treatment for the liver disease other than transplantation and therapies for the lung manifestations of the disease remain limited. Gene therapy is an evolving field which may be of use as a treatment for A1AT deficiency. As the liver disease associated with A1AT deficiency may represent a gain of function possible gene therapies for this condition include the use of ribozymes, peptide nucleic acids (PNAs) and RNA interference (RNAi), which by decreasing the amount of aberrant protein in cells may impact on the pathogenesis of the condition.
... These transcription factors have diverse functions on lung development, morphogenesis, and immune functions. [27][28][29][30] Whether these SNPs with transcription factors binding sites affect arginase expression in the airways or leukocytes remains to be explored. ...
Arginases (encoded by ARG1 and ARG2 genes) might play an important role in asthma pathogenesis through effects on nitrosative stress. Arginase expression is upregulated in asthma and varies with T(H)2 cytokine levels and oxidative stress.
We aimed to examine whether variants in these genes are associated with asthma and whether atopy and exposures to smoking and air pollution influence the associations.
Among non-Hispanic and Hispanic white participants of the Children's Health Study (n = 2946), we characterized variation in each locus (including promoter region) with 6 tag single nucleotide polymorphisms for ARG1 and 10 for ARG2. Asthma was defined by parental report of physician-diagnosed asthma at study entry.
Both ARG1 and ARG2 genetic loci were significantly associated with asthma (global locus level P = .02 and .04, respectively). Compared with the most common haplotype within each locus, 1 ARG1 haplotype was associated with reduced risk (odds ratio [OR] per haplotype copy, 0.55; 95% CI, 0.36-0.84), and 1 ARG2 haplotype was associated with increased risk (OR per haplotype copy, 1.35; 95% CI, 1.04-1.76) of asthma. The effect of the ARG1 haplotype that was significantly associated with asthma varied by the child's history of atopy and ambient ozone (P(interaction) = .04 and .02, respectively). Among atopic children living in high-ozone communities, those carrying the ARG1 haplotype had reduced asthma risk (OR per haplotype copy, 0.12; 95% CI, 0.04-0.43; P(heterogeneity) across atopy/ozone categories = .008).
ARG1 and ARG2 loci are associated with childhood asthma. The association between ARG1 variation and asthma might depend on atopy and ambient ozone levels.
... Activation of NF-B has potentially important implications for target organ injury in AT deficiency. First, through NF-B, accumulation of ATZ in liver cells and respiratory epithelial cells (40) could mediate inflammation in the liver and the lung, particularly neutrophil infiltration in response to the NF-B target interleukin-8. Second, activation of NF-B has been shown to play a key role in inflammationassociated carcinogenesis (41)(42)(43) and therein could be involved in the pathogenesis of hepatocellular carcinoma in AT deficiency. ...
Alpha-1-antitrypsin (AT) deficiency is the most common genetic cause of liver disease in children. In addition to chronic liver inflammation and injury, it has a predilection to cause hepatocellular carcinoma later in life. The deficiency is caused by a mutant protein, ATZ, which is retained in the endoplasmic reticulum (ER) in a polymerized form rather than secreted into the blood in its monomeric form. The histologic hallmark of the disease is ATZ-containing globules in some, but not all, hepatocytes. Liver injury results from a gain-of-toxic function mechanism in which mutant ATZ retained in the ER initiates a series of pathologic events, but little is known about the mechanism by which this leads to carcinogenesis. Several recent observations from my laboratory have led to a novel hypothetical paradigm for carcinogenesis in AT deficiency in which globule-containing hepatocytes are "sick," relatively growth suppressed, but also elaborating trans-acting regenerative signals. These signals are received and transduced by globule-devoid hepatocytes, which, because they are younger and have a lesser load of accumulated ATZ, have a selective proliferative advantage. Chronic regeneration in the presence of tissue injury leads to adenomas and ultimately carcinomas. Aspects of this hypothetical paradigm may also explain the proclivity for hepatocarcinogenesis in other chronic liver diseases, including other genetic diseases, viral hepatitis, and nonalcoholic steatohepatitis.
In the classical form of α1-antitrypsin deficiency (ATD) a point mutation leads to protein misfolding such that the mutant protein accumulates in liver cells with a marked decrease in levels of this protein in the blood and body fluids. Because the major function of α1-antitrypsin is inhibition of neutrophil elastase and several other neutrophil proteases, individuals with ATD are susceptible to destructive lung disease/emphysema, now called chronic obstructive pulmonary disease (COPD), by a loss-of-function mechanism in which uninhibited neutrophil proteases destroy the connective tissue matrix of the lung. Homozygous individuals are also susceptible to liver disease by a gain-of-toxic function mechanism triggered by the intracellular accumulation of the misfolded protein. The only therapeutic strategies currently available are protein replacement therapy and lung transplantation for COPD and liver transplantation for the subgroup with progressive liver involvement. New therapeutic strategies that mitigate the intracellular accumulation/proteotoxicity have advanced to clinical trials and corrective genetic strategies are in earlier pre-clinical phases of development.
Alpha-1 antitrypsin (AAT), alternately referred to as alpha-1 proteinase inhibitor or alpha-1 protease inhibitor, is a member of the serine protease inhibitor (serpin) superfamily comprised of alpha-1 antichymotrypsin, C1 inhibitor, antithrombin, neuroserpin, and others (Stoller and Aboussouan, Am J Respir Crit Care Med 185:246–259, 2012; Ekeowa et al., Clin Sci (Lond) 116:837–850, 2009). AAT is considered the major anti-elastase of the lower respiratory tract based on its unsurpassed ability to inhibit the serine protease, neutrophil elastase (Perlmutter and Pierce, Am J Physiol 257:L147–62, 1989; Gadek et al., J Clin Investig 68:889–898, 1981). In addition to its antiprotease activity, AAT has likewise been shown to have other biological effects, including the ability to modulate both inflammation and apoptosis (Petrache et al., Am J Pathol 169:1155–1166, 2006; Janciauskiene et al., Respir Med 105:1129–1139, 2011). Mutant forms of AAT play a well-documented role in disease pathogenesis: misfolding of AAT protein, accumulation of misfolded protein polymers, and an associated decrease in secreted, functional monomers are known to cause clinical dysfunction and disease through both gain-of-function and loss-of-function mechanisms and are collectively known as “AAT deficiency.” In this section, we review the protein’s history, structural composition, regulation, and functional characteristics.
The function of α1 -antitrypsin depends upon the protein folding to a native structure that is only partially stabilised. This renders disease mutants such as the common Z (Glu342Lys) variant vulnerable to misfolding and polymerisation that underlie the disease mechanisms of α1 -antitrypsin deficiency. Combinations of gain- and loss-of-function effects explain the observed disease behaviour. Detailed studies of these processes lead to hypotheses as to why disease variants may arise and persist. They continue to generate and inform novel therapeutic strategies for the treatment of α1 -antitrypsin deficiency, whilst providing insights into other diseases resulting from protein misfolding and abnormal conformational behaviour.
Homozygous PIZZ α1-antitrypsin (α1-AT) deficiency is a relatively common genetic disorder, affecting 1 in 1600 to 1 in 2000 live births [1, 2]. It is an autosomal codominant disorder associated with 85–90% reduction in serum concentrations of α1-AT. A single amino acid substitution results in an abnormally folded protein that is unable to traverse the secretory pathway. The mutant α1-ATZ protein is retained in the endoplasmic reticulum (ER) rather than secreted into the blood and body fluids. α1-Antitrypsin is an approximately 55-kDa secretory glycoprotein that inhibits destructive neutrophil proteases, elastase, cathepsin G, and proteinase 3. Plasma α1-AT is derived predominantly from the liver and increases three- to fivefold during the host response to tissue injury or inflammation. It is the archetype of a family of structurally related circulating serine protease inhibitors called serpins. Nationwide prospective screening studies done by Sveger [1, 3] in Sweden have shown that only 8–10% of the PIZZ population develops clinically significant liver disease over the first 20 years of life. Nevertheless, this deficiency is the most frequent genetic cause of liver disease in children and the most frequent genetic disease for which children undergo orthotropic liver transplantation. It also has been associated with chronic hepatitis, cirrhosis, and hepatocellular carcinoma in adults [4]. Although the condition does not affect children, many α1-AT-deficient individuals develop destructive lung disease and emphysema.
The classic form of α1-antitrypsin (α1-AT) deficiency is the most common genetic cause of liver disease in childhood. It also causes chronic liver disease and hepatocellular carcinoma in adults. The protein that is altered in this deficiency is a secretory glycoprotein predominantly derived from the liver and predominantly functioning as an inhibitor of neutrophil elastase. In the deficiency, a point mutation in the α1-AT Z molecule leads to altered folding and polymerization/aggregation such that soluble and insoluble α1-AT Z accumulate in the endoplasmic reticulum of liver cells. Liver inflammation and carcinogenesis are caused by a gain-of-toxic function mechanism involving the toxic effects of polymerized/aggregated mutant α1-AT retained within liver cells. Nevertheless, only 8% of the homozygous population develops clinically significant liver disease in the first 30 years of life, indicating that genetic and/or environmental modifiers determine the incidence and severity of liver disease in α1-AT deficiency. Altered migration of the abnormal α1-AT molecule in isoelectric focusing gels is the basis of the diagnosis of this deficiency. Although several new concepts for pharmacologic treatment are currently being investigated management of this liver disease is mostly supportive. Liver replacement therapy has been used successfully for severe liver disease.
Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal
muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the
lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies,
surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic
for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous
pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model
resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional
restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional
activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent
model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous
proteostasis mechanism and an attractive target for therapy.
The classic form of α1-antitrypsin (α1-AT) deficiency is the most common genetic cause of liver disease in childhood. It also causes chronic liver disease and hepatocellular carcinoma in adults. The protein that is altered in this deficiency is a secretory glycoprotein predominantly derived from the liver and predominantly functioning as an inhibitor of neutrophil elastase. In the deficiency, a point mutation in the α1-AT Z molecule leads to altered folding and polymerization/aggregation such that soluble and insoluble α1-AT Z accumulate in the endoplasmic reticulum of liver cells. Liver inflammation and carcinogenesis are caused by a gain-of-toxic function mechanism involving the toxic effects of polymerized/aggregated mutant α1-AT retained within liver cells. Nevertheless, only 8% of the homozygous population develops clinically significant liver disease in the first 30 years of life, indicating that genetic and/or environmental modifiers determine the incidence and severity of liver disease in α1-AT deficiency. Altered migration of the abnormal α1-AT molecule in isoelectric focusing gels is the basis of the diagnosis of this deficiency. Although several new concepts for pharmacologic treatment are currently being investigated management of this liver disease is mostly supportive. Liver replacement therapy has been used successfully for severe liver disease.
Nicotinamide N-methyltransferase (NNMT) catalyzes N-methylation of nicotinamide and other structural analogues. NNMT gene expression is enhanced in many papillary thyroid cancer cells and activated by hepatocyte nuclear factor (HNF)-1 beta. In this work, we studied the effects of depsipeptide, a histone deacetylase inhibitor, on NNMT gene expression in BHP 18-21 papillary thyroid cancer cells. Depsipeptide reduced NNMT mRNA level in a dose-dependent and time-dependent manner as determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In contrast, expression of the sodium iodide symporter (NIS), a gene with differentiated function, was enhanced in the treated cells. NNMT protein level determined by Western blot analysis and NNMT catalytic activity was also reduced significantly in the depsipeptide-treated cells. To study the mechanism of NNMT gene repression by depsipeptide, effects of depsipeptide on NNMT promoter activity were determined by luciferase reporter gene assay. NNMT promoter activity was significantly reduced in the HNF-1 beta-positive BHP 18-21 cells but not in the HNF-1 beta-negative BHP 14-9 papillary cancer cells. A mutant reporter construct with mutations in a HNF-1 site in the NNMT basal promoter region did not respond to depsipeptide in both HNF-1 beta-positive and -negative cells. Depsipeptide reduced steady-state HNF-1 beta mRNA level, depleted nuclear HNF-1,6 protein levels, and abolished activity of DNA binding to the HNF-1 site in the NNMT promoter region. Protein synthesis inhibitor cycloheximide and proteasome inhibitor MG-132 enhanced HNF-1 beta stability in the depsipeptide-treated cells. In summary, depsipeptide represses NNMT and HNF-1 beta gene expression in some papillary thyroid cancer cells. The repression of NNMT by depsipeptide is at the transcription level through downregulation of transcription activator HNF-1 beta.
The classical form of α1-antitrypsin (AT) deficiency, homozygous for the Z allele, is the most common genetic cause of liver disease in children and
the most frequent genetic disease for which people undergo liver transplantation. As a cause of chronic hepatitis, cryptogenic
cirrhosis, and hepatocellular carcinoma with new onset in the adult, this diagnosis has been under-appreciated. Among the
liver diseases it has a unique and fascinating pathobiology, related to the hepatotoxic consequences of an aggregation-prone
mutant protein.
The serine proteinase inhibitor α-1 anti-trypsin (AAT) provides an antiprotease protective screen throughout the body. Mutations in the AAT gene (SERPINA1) that lead to deficiency in AAT are associated with chronic obstructive pulmonary diseases. The Z mutation encodes a misfolded variant of AAT that is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum of hepatocytes and other AAT-producing cells. Until recently, it was thought that loss of antiprotease function was the major cause of ZAAT-related lung disease. However, the contribution of gain-of-function effects is now being recognized. Here we describe how both loss- and gain-of-function effects can contribute to ZAAT-related lung disease. In addition, we explore how SERPINA1 heterozygosity could contribute to smoking-induced chronic obstructive pulmonary diseases and consider the consequences.
Alpha-1-antitrypsin (AT) deficiency is the most common genetic cause of liver disease in children. The primary pathological issue is a point mutation that renders an abundant hepatic secretory glycoprotein prone to altered folding and a tendency to polymerize and aggregate. However, the expression of serious liver damage among homozygotes is dependent on genetic and/or environmental modifiers. Several studies have validated the concept that endogenous hepatic pathways for disposal of aggregation-prone proteins, including the proteasomal and autophagic degradative pathways, could play a key role in the variation in hepatic damage and be the target of the modifiers. Exciting recent results have shown that a drug that enhances autophagy can reduce the hepatic load of aggregated protein and reverse fibrosis in a mouse model of this disease.
The hereditary disorder alpha-1 antitrypsin (AAT) deficiency results from mutations in the SERPINA1 gene and presents with emphysema in young adults and liver disease in childhood. The most common form of AAT deficiency occurs because of the Z mutation, causing the protein to fold aberrantly and accumulate in the endoplasmic reticulum (ER). This leads to ER stress and contributes significantly to the liver disease associated with the condition. In addition to hepatocytes, AAT is also synthesized by monocytes, neutrophils, and epithelial cells. In this study we show for the first time that the unfolded protein response (UPR) is activated in quiescent monocytes from ZZ individuals. Activating transcription factor 4, X-box binding protein 1, and a subset of genes involved in the UPR are increased in monocytes from ZZ compared with MM individuals. This contributes to an inflammatory phenotype with ZZ monocytes exhibiting enhanced cytokine production and activation of the NF-kappaB pathway when compared with MM monocytes. In addition, we demonstrate intracellular accumulation of AAT within the ER of ZZ monocytes. These are the first data showing that Z AAT protein accumulation induces UPR activation in peripheral blood monocytes. These findings change the current paradigm regarding lung inflammation in AAT deficiency, which up until now was derived from the protease-anti-protease hypothesis, but which now must include the exaggerated inflammatory response generated by accumulated aberrantly folded AAT in circulating blood cells.
Secretory IgA and IgM, which protect the mucosal surfaces, are generated by selective transport of locally produced polymeric (p)Igs through the epithelial barrier by the pIgR. The expression of this receptor, and hence the generation of secretory Igs, is modulated by numerous extracellular factors. We have previously identified a STAT6 site in intron 1 of the human pIgR gene that is required for the slow and de novo protein synthesis-dependent IL-4-mediated transcriptional activation of the gene. In this study, we show that this intronic IL-4-responsive enhancer is confined to a 250-bp region that is highly conserved in the murine pIgR gene. The enhancer was dependent on the cooperation between the STAT6 site and at least four additional DNA elements. EMSA experiments demonstrated binding by hepatocyte NF-1 to one of these DNA elements. Extensive overlap in the tissue distribution of hepatocyte NF-1 and pIgR suggests that this transcription factor contributes to tissue-specific pIgR expression. Changing the helical phase between the STAT6 site and downstream DNA elements greatly reduced the strength of the IL-4 response, suggesting that the precise organization of this enhancer is important for its proper function. Thus, several transcription factors cooperate in this enhanceosome to mediate IL-4 responsiveness in HT-29 epithelial cells.
For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors.
HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines.
Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes.
Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.
We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT protein levels. In summary, HNF-1beta functions as a transcription activator for NNMT gene expression in some papillary thyroid cancer cells.
Liver disease in alpha-1-antitrypsin (alpha1AT) deficiency is caused by a gain-of-toxic function mechanism engendered by the accumulation of a mutant glycoprotein in the endoplasmic reticulum (ER). The extraordinary degree of variation in phenotypical expression of this liver disease is believed to be determined by genetic modifiers and/or environmental factors that influence the intracellular disposal of the mutant glycoprotein or the signal transduction pathways that are activated. Recent investigations suggest that a specific repertoire of signaling pathways are involved, including the autophagic response, mitochondrial- and ER-caspase activation, and nuclear factor kappaB (NFkappaB) activation. Whether activation of these signaling pathways, presumably to protect the cell, inadvertently contributes to liver injury or perhaps protects the cell from one injury and, in so doing, predisposes it to another type of injury, such as hepatocarcinogenesis, is not yet known. Recent studies also suggest that hepatocytes with marked accumulation of alpha1ATZ, globule-containing hepatocytes, engender a cancer-prone state by surviving with intrinsic damage and by chronically stimulating in 'trans' adjacent relatively undamaged hepatocytes that have a selective proliferative advantage. Further, this paradigm may apply to other genetic and infectious liver diseases that are predisposed to hepatocellular carcinoma.
In alpha(1)-antitrypsin (alpha1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein alpha1AT, alpha1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant alpha1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant alpha1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant alpha1AT. The results show that ER retention of polymerogenic mutant alpha1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of alpha1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic alpha1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant alpha1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFkappaB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic alpha1AT mutants.
An imbalance between neutrophil protease and surrounding antiprotease levels has been shown to be important in the pathogenesis of chronic obstructive pulmonary disease (COPD). Adenoviral E1A DNA and protein are frequently detected in the lungs of COPD patients. As secretory leukoprotease inhibitor (SLPI) and elafin/skin-derived antileukoproteinase (SKALP) are locally produced in the lung and inhibit neutrophil elastase activity, we hypothesized that adenoviral E1A might affect the production of these antiproteases.
To examine the effect of E1A on SLPI and elafin/SKALP secretion in A549 (alveolar epithelial) cells and primary human bronchial epithelial (HBE) cells.
SLPI and elafin/SKALP were quantitated from cell culture supernatants using an ELISA. SLPI mRNA expression was examined by Northern blotting, and SLPI promoter activity was measured using a reporter gene assay.
E1A significantly suppressed SLPI and elafin/SKALP secretion by A549 cells upon interleukin (IL)-1beta stimulation. E1A also suppressed SLPI and elafin/SKALP secretion by HBE cells. SLPI mRNA expression in A549 cells was suppressed by E1A regardless of IL-1beta stimulation. IL-1beta-induced SLPI promoter activity was suppressed by E1A gene transfection into A549 cells.
Our findings of adenoviral E1A-mediated suppression of SLPI and elafin/SKALP secretion suggest that E1A may be involved in the enhancement of alveolar damage and play a role in the COPD process.
Hepatocyte nuclear factor (HNF)-1alpha and HNF-1beta are related transcription factors that are mainly expressed in liver cells. Our previous study showed that HNF-1beta was highly expressed in papillary thyroid cancer cell lines and tumors. HNF-1alpha mRNA, however, was not detected in differentiated thyroid cancer cell lines. The objective of this study was to determine whether HNF-1alpha is expressed in dedifferentiated anaplastic thyroid cancer cells.
Total RNA isolated from six anaplastic thyroid cancer cell lines and 38 surgical samples was analyzed for HNF-1alpha mRNA by conventional reverse-transcription polymerase chain reaction (RT-PCR) or real-time RT-PCR. HNF-1alpha DNA binding activity was measured by gel retardation assay and HNF-1alpha protein was identified by Western blotting.
HNF-1alpha mRNA was expressed in four of the six anaplastic cell lines. The presence of HNF-1alpha protein and DNA binding activity was detected in three lines with higher HNF-1alpha mRNA level. Three cell lines also expressed HNF-1beta. HNF-1alpha transcripts were also detected in five out of six anaplastic tumors, but not in the papillary tumors except one with weak PCR signal.
HNF-1alpha mRNA was detected in high frequency in anaplastic thyroid cancer cell lines and tumors. HNF-1alpha might play a role in the pathogenesis of anaplastic thyroid cancer.
Maintenance of the intestinal epithelium is based on well-balanced molecular mechanisms that confer the stable and continuous supply of specialized epithelial cell lineages from multipotent progenitors. Lineage commitment decisions in the intestinal epithelium system involve multiple regulatory systems that interplay with each other to establish the cellular identities. Here, we demonstrate that the microRNA system could be involved in intestinal epithelial cell differentiation, and that microRNA-194 (miR-194) is highly induced during this process. To investigate this inducible expression mechanism, we identified the genomic structure of the miR-194-2, -192 gene, one of the inducible class of miR-194 parental genes. Furthermore, we identified its transcriptional regulatory region that contains a consensus-binding motif for hepatocyte nuclear factor-1alpha (HNF-1alpha), which is well known as a transcription factor to regulate gene expression in intestinal epithelial cells. By chromatin immunoprecipitation assay and luciferase reporter analysis, we revealed that pri-miR-194-2 expression is controlled by HNF-1alpha, and its consensus binding region is required for the transcription of pri-miR-194-2 in vivo in an intestinal epithelial cell line, Caco-2. Our observations indicate that microRNA genes could be targets of lineage-specific transcription factors and that microRNAs are regulated by a tissue-specific manner in the intestinal epithelium. Therefore, our work suggests that induced expression of these microRNAs have important roles in intestinal epithelium maturation.
Apolipoprotein AI (apoAI) gene expression in liver depends on synergistic interactions between transcription factors bound to three distinct sites (A, B, and C) within a hepatocyte-specific enhancer in the 5′-flanking region of the gene. In this study, we showed that a segment spanning sites A and B retains substantial levels of enhancer activity in hepatoblastoma HepG2 cells and that sites A and B are occupied by the liver-enriched hepatocyte nuclear factors (HNFs) 4 and 3, respectively, in these cells. In non-hepatic CV-1 cells, HNF-4 and HNF-3β activated this minimal enhancer synergistically. This synergy was dependent upon simultaneous binding of these factors to their cognate sites, but it was not due to cooperativity in DNA binding. Separation of these sites by varying helical turns of DNA did not affect simultaneous binding of HNF-3β and HNF-4 nor did it influence their functional synergy. The synergy was, however, dependent upon the cell type used for functional analysis. In addition, this synergy was further potentiated by estrogen treatment of cells cotransfected with the estrogen receptor. These data indicate that a cell type-restricted intermediary factor jointly recruited by HNF-4 and HNF-3 participates in activation of the apoAI enhancer in liver cells and suggest that the activity of this factor is regulated by estrogen.
The transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1beta gene have been identified that lead to early onset of diabetes and severe primary renal defects. The degree and type of renal defects seem to depend on the specific mutation. We show that the frameshift mutant P328L329fsdelCCTCT associated with nephron agenesis retains its DNA-binding properties and acts as a gain-of-function mutation with increased transactivation potential in transfection experiments. Expression of this mutated factor in the Xenopus embryo leads to defective development and agenesis of the pronephros, the first kidney form of amphibians. Very similar defects are generated by overexpressing in Xenopus the wild-type HNF1beta, which is consistent with the gain-of-function property of the mutant. In contrast, introduction of the human HNF1beta mutant R137-K161del, which is associated with a reduced number of nephrons with hypertrophy of the remaining ones and which has an impaired DNA binding, shows only a minor effect on pronephros development in Xenopus. Thus, the overexpression of both human mutants has a different effect on renal development in Xenopus, reflecting the variation in renal phenotype seen with these mutations. We conclude that mutations in human HNF1beta can be functionally characterized in Xenopus. Our findings imply that HNF1beta not only is an early marker of kidney development but also is functionally involved in morphogenetic events, and these processes can be investigated in lower vertebrates.
It has been reported that respiratory epithelium-specific transcription is mediated by thyroid transcription factor 1 and members of the HNF3/forkhead family of transcription factors. Here, we show that the uteroglobin/Clara cell 10-kDa promoters from rabbit and man are regulated by HNF3alpha and HNF3beta but not by HFH-4 and TTF-1. We have identified two HNF3-responsive elements in the rabbit uteroglobin/CC10 promoter located around 95 and 130 base pairs upstream of the transcriptional start site. Both elements contribute to promoter activity in H441 cells expressing uteroglobin/CC10 and HNF3alpha. Gene transfer experiments into Drosophila Schneider cells that lack many mammalian transcription factor homologs revealed that HNF3alpha and HNF3beta on their own cannot activate the uteroglobin/CC10 promoter. However, HNF3alpha and HNF3beta strongly enhanced Sp1-mediated promoter activation. Synergistic activation by HNF3alpha and Sp1 was absolutely dependent on the integrity of two Sp1 sites located at around -65 and -230. We show further that multiple activation domains of Sp1 are required for cooperativity with HNF3alpha. These studies demonstrate that transcription from the rabbit uteroglobin/CC10 promoter in lung epithelium is controlled by the combinatorial action of the cell-specific factor HNF3alpha and the ubiquitous factor Sp1.
In situ hybridization is a powerful means of identifying sites of gene expression. We used this technique to examine the spatial and developmental control of transcription of the human alpha 1-antitrypsin (alpha 1 AT) gene in transgenic mice carrying this gene and extensive 5'- and 3'-flanking sequences. In addition to expression in yolk sac and liver, human alpha 1AT RNA was detected in gut, stomach, pancreas, nasal epithelium, pharynx, bronchi, spinal ganglia, and ossifying cartilage of transgenic fetuses at 14.5 days post coitum (dpc). In transgenic adults, expression was no longer found in the pancreas but was found in the kidney and salivary gland. In each tissue, expression was confined to a specific cell population. This pattern of alpha 1AT expression was found to correlate with that seen in several fetal and adult human tissues. These results suggest a wider role of alpha 1AT in human physiology and development than previously suspected, and they demonstrated the potential value of this approach in delineating the physiological role of human proteins. Expression of the endogenous alpha 1AT gene in mice was confined to a limited, but overlapping, set of tissues, suggesting that the cis-acting DNA sequences that regulate the expression of the human and mouse genes interact differently with transcription factors present in mouse cells.
alpha 1-Antitrypsin (alpha 1AT) is an abundant serum protein whose major site of synthesis is in the hepatocyte. alpha 1AT transcripts are also present, albeit at a lower level, in a variety of other human tissues. This pattern of expression is partly related to initiation of transcription at sites with distinct tissue specificities. The mouse alpha 1AT gene, in contrast, is more strictly liver specific in its expression. To explore the regulation of the alpha 1AT gene we have microinjected a cosmid insert carrying the human gene into fertilized mouse eggs. In three lines obtained from transgenic mice, inheritance of copies of the human gene is accompanied by a high serum concentration of the human protein. Human alpha 1AT RNA accumulates to the highest level in liver of transgenic animals. The presence of transcripts in other tissues indicates that the human pattern of expression is maintained, whereas the temporal activity of the introduced gene parallels that of the endogenous one during mouse embryogenesis.
Expression of the alpha 1-proteinase inhibitor (alpha 1PI) gene was studied in human mononuclear cells. Using RNA blot and dot hybridization, alpha 1PI mRNA was detected in human peripheral blood monocytes, bronchoalveolar and breast milk macrophages, but not in B or T lymphocytes. Using incorporation of a radiolabeled amino acid precursor, synthesis and secretion of alpha 1PI were demonstrated in human monocytes and macrophages, but not in lymphocytes. In addition, alpha 1PI was secreted in functionally active form as shown by complexing with serine proteases. Biosynthesis of alpha 1PI by mononuclear phagocytes was greatest during the first 24 hr in culture and progressively decreased over the next 10 days. The reduction in alpha 1PI biosynthesis in vitro involved a mechanism acting at the pretranslational level as alpha 1PI mRNA content also progressively declined over 10 days in culture. The ease of sampling human monocytes and macrophages now permits examination of the biochemical defect in homozygous PiZ and PiS alpha 1PI deficiencies and study of the functional significance of locally produced alpha 1PI in normal tissues and sites of injury or inflammation.
We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription
initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using
the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus
promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA
and Ad 2 VA).
Cis-acting elements determining lung epithelial cell-selective transcription of the murine surfactant protein A (SP-A) gene were identified between nucleotide positions -255 and -57. This region of the murine SP-A gene contained nucleotide sequences consistent with thyroid transcription factor-1 (TTF-1) binding motifs. An SP-A-CAT plasmid containing the TTF-1 binding sites was transcriptionally active in mouse lung epithelial (MLE-15) cells but not in HeLa, 3T3, or H441 cells. However, transcription of the SP-A-CAT construct was activated after cotransfection of HeLa cells with a vector expressing recombinant TTF-1, pCMV-TTF-1. Recombinant TTF-1 homeodomain protein bound to four distinct binding sites located between nucleotides -166 to -117 [corrected]. Proteins in nuclear extracts of MLE-15 cells bound TTF-1 binding sites and were supershifted by TTF-1 antibody. Mutations of three of the TTF-1 binding sites in this region reduced expression of the SP-A-CAT construct in transfected MLE-15 cells and reduced transactivation in HeLa cells. TTF-1 interacts with complex protein/DNA binding sites located in the 5'-flanking region of the murine SP-A gene enhancing lung epithelial cell-specific expression in vitro.
The intestinal fatty acid binding protein gene (Fabpi) provides a good model system for studying how gene transcription is regulated in enterocytes as a function of their differentiation program and location along the duodenal-to-colonic axis. We have compared and contrasted the transcriptional activity of four fusion genes composed of elements from the 5'-nontranscribed domain of rat Fabpi linked to the human growth hormone gene (I-FABP/hGH) in transgenic mice and in five primate epithelial cell lines derived from intestine, liver, kidney, and cervix. Nucleotides -103 to +28 of rat Fabpi are able to direct appropriate lineage-specific and geographic patterns of hGH expression in transgenic mice. I-FABP-103 to +28/hGH is preferentially expressed in Caco-2 cells, which emulate some of the features of differentiated small intestinal enterocytes after they reach confluence. However, other I-FABP/hGH fusion genes that exhibit differentiation-dependent changes in their expression along the crypt-to-villus axis do not manifest the same pattern of differentiation-dependent change in activity in this cell line. Correlation of their patterns of expression in vivo and ex vivo suggest that nonproliferating Caco-2 cells mimic some of the features of the transcriptional regulatory environment of enterocytes located in the upper crypt. Nucleotides -103 to +28 of rat Fabpi contain one copy of a repeated 14-base pair element that is conserved in the orthologous mouse and human genes and represented in several other homologous and nonhomologous genes, which are expressed in villus-associated enterocytes. This element binds to two members of the steroid hormone receptor superfamily of transcription factors produced in enterocytes and Caco-2 cells: hepatic nuclear factor-4 (HNF-4) and apolipoprotein regulatory protein-1 (ARP-1). Co-transfection studies performed in Caco-2 cells and in a monkey kidney cell line (CV-1) that lacks endogenous pools of ARP-1 and HNF-4 suggest that ARP-1 and HNF-4 can function to activate I-FABP-103 to +28/hGH+3 through their interactions with the 14-base pair element. This activation appears to be affected by elements located between nucleotides -277 and -104 and other transcription factors.
This report defines the elements between bp -800 and -166 that regulate the quantitative level of mouse CC10 (mCC10) transcription in the lungs. The elements in this promoter domain are the response elements for the NKx2.1 homeobox protein, thyroid transcription factor 1 (TTF1). DNase I footprint analysis identified five binding sites for TTF1 between bp -800 and - 166. These sites are located at bp -344 to -335, - 282 to -273, -268 to -263, -258 to -249, and - 199 to - 190. In addition to these enhancer elements, two TTF1 binding sites were identified in the proximal promoter region (bp - 166 to + 1), at bp -74 to -69 and -49 to -39. An identical footprint of the mCC10 promoter region was also observed with another member of the NKx family, NKx 2.5, the cardiac muscle-specific homeobox protein (CSX). Deletion and linker-scanner mutational analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the major regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249. The importance of the NKx binding site at bp -282 to -273 was verified in vivo. Transgenic mice generated with the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 showed a reduction in transgene expression equal to that of the mice generated with only 166 bp of 5'-flanking DNA. This report emphasizes the importance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes.
Liver-specific gene expression in adult hepatocytes relies on four families of evolutionary conserved transcription factors that are liver-enriched but not restricted to this tissue. These factors function in unique combinations, often synergistically, to stimulate cell-specific transcription. Each family is composed of several members displaying similar, if not identical, DNA recognition properties and sharing structural homology in their DNA binding domains. The homo- and heterodimerization between members of a particular transcription factor family adds an additional level of complexity in gene regulation. The consequences of inactivating different family members in the mouse by homologous recombination, together with recent studies of their regulation, suggest a model for liver differentiation involving a regulatory network rather than a completely hierarchical genetic circuitry. These studies also indicate that individual regulators appear to serve multiple developmental functions. Their possible role in the progression through different stages of hepatic cell commitment and differentiation is discussed.
Immediately prior to gastrulation the murine embryo consists of an outer layer of visceral endoderm (VE) and an inner layer of ectoderm. Differentiation and migration of the ectoderm then occurs to produce the three germ layers (ectoderm, embryonic endoderm and mesoderm) from which the fetus is derived. An indication that the VE might have a critical role in this process emerged from studies of Hnf-4(-/-) mouse embryos which fail to undergo normal gastrulation. Since expression of the transcription factor HNF-4 is restricted to the VE during this phase of development, we proposed that HNF-4-regulated gene expression in the VE creates an environment capable of supporting gastrulation. To address this directly we have exploited the versatility of embryonic stem (ES) cells which are amenable to genetic manipulation and can be induced to form VE in vitro. Moreover, embryos derived solely from ES cells can be generated by aggregation with tetraploid morulae. Using Hnf-4(-/-) ES cells we demonstrate that HNF-4 is a key regulator of tissue-specific gene expression in the VE, required for normal expression of secreted factors including alphafetoprotein, apolipoproteins, transthyretin, retinol binding protein, and transferrin. Furthermore, specific complementation of Hnf-4(-/-) embryos with tetraploid-derived Hnf-4(+/+) VE rescues their early developmental arrest, showing conclusively that a functional VE is mandatory for gastrulation.
Destruction of components of the extracellular matrix of the lung by neutrophil elastase is believed to be a critical event
in the development of obstructive lung disease. The local synthesis of α1-proteinase inhibitor, the controlling inhibitor of this enzyme, may provide a partial mechanism for neutrophil elastase regulation,
especially during inflammation, when proteolytic enzymes are released from phagocytes. In this study, we show that lung-derived
epithelial cells not only have the capacity to synthesize functional α1-PI but also to increase the rate of its production when stimulated by specific inflammatory mediators, including oncostatin
M, interleukin-1, and the glucocorticoid analogue, dexamethasone.
Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.
vHNF1/HNF1beta, a member of the divergent HNF1/vHNF1 homeoprotein family, is expressed in polarized epithelia of several adult organs and may participate in controlling the transcription of specific genes. In addition to this late requirement, vHNF1 may play earlier roles during development, as it is first expressed in the visceral endoderm at the onset of gastrulation. In order to shed light on its function during embryogenesis, we have inactivated the murine gene by homologous recombination. The homozygous mutation results in embryonic lethality by day 7.5 of development and vHNF1(-)(/)(-) embryos display a disorganized visceral endoderm and a significantly reduced size. Studies of ES cell differentiation and aggregation with tetraploid morulae establish that vHNF1 expression is essential for visceral endoderm differentiation, both in vitro and in vivo. Analysis of differentiation markers confirms that vHNF1 is part of a genetic network that directs the expression of HNF4 and downstream endodermal genes. Furthermore, the complementation of the mutant embryos with wild-type visceral endoderm rescues the day 7.5 lethality and reveals an additional phenotype linked to vHNF1 later expression. The examination of chimeric embryos suggests that vHNF1 expression might be cell-autonomously required in the gut for the proper morphogenesis of the embryo.
There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha(1)-antitrypsin (alpha(1)-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha(1)-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-lot (HNF-1 alpha), HNF-1 beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha(1)-AT gene. Cotransfection studies showed that HNF-1 alpha and HNF-4 had a synergistic effect on alpha(1)-AT gene expression. RNA blot analysis showed that HNF-1 alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1 alpha binding activity increase coordinately with alpha(1)-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1 alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha(1)-AT gene expression. Together, these results provide evidence that HNF-1 alpha and HNF-4 play a role in the mechanism by which the alpha(1)-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.
There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized α1- antitrypsin (α1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans- acting DNA-binding proteins responsible for expression of the α1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1α (HNF-1α), HNF-1β, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the α1-AT gene. Cotransfection studies showed that HNF-1α and HNF-4 had a synergistic effect on α1-AT gene expression. RNA blot analysis showed that HNF-1α and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1α binding activity increase coordinately with α1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1α in Caco-2 cells resulted in a selective decrease in endogenous α1-AT gene expression. Together, these results provide evidence that HNF-1α and HNF-4 play a role in the mechanism by which the α1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.
We have investigated the ability of alveolar epithelial cells (human A549 cell line and rat type-II pneumocytes) to produce al-antitrypsin (AAT). Northern blot analysis demonstrated the presence of an AAT-specific mRNA transcript in A549 cells. Unstimulated A549 cells secreted immunoreactive AAT at a rate of 0.51 +/- 0.04 ng/10(6) cells/h, with a modified glycosylation compared to serum AAT. AAT formed a complex with neutrophil elastase. Rat type-II pneumocytes secreted immunoreactive AAT. Our results suggest that alveolar epithelial cells could participate in antiprotease defense within the lung through local AAT production.
Hepatocyte nuclear factors (HNFs) are a heterogeneous class of evolutionarily conserved transcription factors that are required
for cellular differentiation and metabolism. Mutations in HNF-1αand HNF-4α genes impair insulin secretion and cause type 2 diabetes. Regulation of HNF-4/HNF-1 expression byHNF-3α and HNF-3β was studied in embryoid bodies in which one or both HNF-3α or HNF-3βalleles were inactivated. HNF-3β positively regulated the expression of HNF-4α/HNF-1α and their downstream targets, implicating a role in diabetes. HNF-3β was also necessary for expression of HNF-3α. In contrast, HNF-3α acts as a negative regulator of HNF-4α/HNF-1α demonstrating thatHNF-3α and HNF-3β have antagonistic transcriptional regulatory functions in vivo. HNF-3α does not appear to act as a classic biochemical repressor but rather exerts its negative effect by competing for HNF-3 binding
sites with the more efficient activatorHNF-3β. In addition, the HNF-3α/HNF-3β ratio is modulated by the presence of insulin, providing evidence that the HNF network may have important roles in mediating
the action of insulin.
Serum protein types were determined in eight recipients and donors in cases of hepatic homotransplantation. A change from recipient type to donor type was observed for factor B, C8, orosomucoid, haptoglobin, transferrin, α1-antitrypsin, C3 and C6, but not for Gm and Inv immunoglobulin markers. The results indicate that all the proteins studied (except immunoglobulins) are produced primarily by the liver in vivo.
To evaluate the factors controlling migration of leukocytes into pulmonary airway epithelium, we determined the biochemical mechanisms responsible for the regulation of intercellular adhesion molecule-1 (ICAM-1) expression on cultured monolayers of human tracheal epithelial cells (HTECs) or SV40 virus-transformed human bronchial epithelial cells (BEAS-2B). Validation experiments with human umbilical vein endothelial cells (HUVECs) demonstrated little detectable ICAM-1 expression on unstimulated cells or on cells incubated with interferon-gamma (IFN-gamma), but HUVEC monolayers responded to interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) with significant increases in ICAM-1 and ICAM-1-dependent adherence of polymorphonuclear leukocytes (PMNs). HTEC monolayers also exhibited no significant basal ICAM-1 expression but, in contrast to HUVEC monolayers, had marked increases in ICAM-1 expression and ICAM-1-dependent PMN adherence only after incubation with IFN-gamma (and not after IL-1 beta or TNF-alpha) treatment. BEAS-2B cells also exhibited relatively selective IFN-gamma stimulation of ICAM-1 expression and ICAM-1-dependent PMN adherence but (like late passage HTEC) showed significant basal ICAM-1 expression. Differences in IFN-gamma effect on ICAM-1 levels between HUVEC and HTEC monolayers were not due to differences in number or responsiveness of IFN-gamma receptors, because both cell types exhibited a similar number of receptors and other IFN-gamma-dependent responses of HUVECs remained active. In all analyses, ICAM-1 mRNA levels correlated closely with detection of ICAM-1 on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
α
1-Antitrypsin (α
1AT) deficiency, one of the most common lethal hereditary disorders among Caucasians, is associated with emphysema in adults, while in children it is associated with liver disease. Produced in the liver and released into the plasma, α
1AT serves as the body’s major inhibitor of neutrophil elastase, a powerful proteolytic enzyme capable of degrading extracellular structural proteins. The pathogenesis of the liver disease associated with α
1AT deficiency is not as well understood, but is clearly linked to specific mutations in coding exons of the α
1AT gene, and the resulting accumulation of α
1AT within hepatocytes. At present, therapy for the liver disease associated with α
1AT deficiency is symptomatic, with liver transplantation as a last resort. New strategies are being developed to suppress the accumulation of α
1AT by transferring the normal gene into the liver.
Article synthese sur l'α1-antitrypsine: caracteristiques structurales et fonctionnelles (capacite de liaison a des modulateurs); utilisation de l'α1-antitrypsine en tant que modele pour toutes les serpines
Surfactant proteolipid SP-B is a hydrophobic protein of Mr = 8000 identified in organic solvent extracts of pulmonary surfactant. Analysis of the human SP-B RNA predicts that the active surfactant peptide is derived by proteolysis of an Mr = 40,000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenocarcinoma cell line by immunoprecipitation of radiolabelled precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of Mr = 39,000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A)+ RNA detected an Mr = 40,000 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of Mr = 41,000-43,000, pI = 5.1-5.4 were the first isoforms detected within the cells and were processed to endoglycosidase-H-resistant isoforms and secreted. Neuraminidase and endoglycosidase-F-sensitive forms of proSP-B were first detected in the media at 60 min as Mr = 42-46,000 isoforms with pI = 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal Mr = 16,000 peptide resulting in Mr = 27,000-33,000 isoforms (pH = 5.6-6.8). The Mr = 27,000-33,000 isoforms were sensitive to neuraminidase, resulting in isoforms with pH = 6.0-6.8. Digestion of the Mr = 27,000-33,000 peptide with endoglycosidase-F resulted in isoforms of Mr = 23,000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of Mr = 16,000, pI = 4.2-4.4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further proteolytic processing of the Mr = 27,000-33,000 isoforms to the Mr = 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with morphologic features of Clara cells), SP-B is synthesized as a preproprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of Mr = 27,000-33,000 and Mr = 16,000, representing carboxy and amino-terminal domains, accumulate in the media.
Normal liver and differentiated hepatoma cell lines contain a nuclear factor, HNF-1, which binds functional sequences within the promoters of the alpha and beta chains of fibrinogen and alpha 1-antitrypsin. In UV cross-linking studies we find that HNF-1 has an apparent mol. wt of 92 kd in differentiated hepatocytes. Nuclear extracts from a dedifferentiated hepatoma cell line, Fao flC2 (C2), selected on the basis of morphological and biochemical dedifferentiation from Fao contains a protein, vHNF, which binds to the same DNA sequence motif as HNF-1 but has an apparent mol. wt of 72 rather than 92 kd. Mixing experiments indicate that this variant nuclear factor does not arise from HNF-1 by proteolysis. Reversion to the differentiated phenotype in C2-Rev7 (Rev7), selected by growth in glucose-free media, results in the re-expression of many liver-specific functions including the fibrinogen genes. In Rev7, HNF-1 is indistinguishable from that in the original differentiated cell line Fao. Transfection studies and nuclear run-on experiments indicate that reduced expression of fibrinogen RNA in C2 relative to Fao is related to reduced transcription. vHNF but not HNF-1 is present in somatic hybrids between fibroblasts and liver cells which show extinction of liver specific traits and it can also be detected in normal tissue, predominantly in lung nuclear extracts. Since vHNF and HNF-1 are not co-expressed yet correlate with the non-hepatic and hepatic phenotype, respectively, we suggest that the expression of these variant forms reflects determination events in establishing the hepatic phenotype.
This report will examine the place of orthotopic liver transplantation in the treatment of seven white patients who had end-stage liver disease due to alpha-1-antitrypsin deficiency. After transplantation, the phenotypes of the recipients became those of the donors, and alpha-1-antitrypsin levels were restored to normal. Thus, the metabolic basis of the disease was corrected for as long as the patients survived the transplantation procedure. Three of the patients are still alive after 13, 21, and 36 months. The others died between 12 days and 28 months after transplantation.
Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.
The 5' flanking region of the Clara cell secretory protein (CCSP) gene contains two cis-acting elements which bind hepatocyte nuclear factor (HNF)-3 alpha and HNF-3 beta in vitro. To determine the role of these proteins in mediating CCSP gene expression in the bronchiolar epithelium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5' flanking region were co-transfected into H-441 cells with HNF-3 alpha or HNF-3 beta expression plasmids. These studies indicate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is sufficient to mediate this effect. Gel-mobility-shift assays with oligonucleotides corresponding to CCSP region I, nuclear extract from bronchiolar epithelial cells and HNF-3-specific antibodies indicate that HNF-3 alpha and HNF-3 beta are the only proteins in bronchiolar epithelial cells which directly interact with this region. Consistent with these observations, HNF-3 alpha and HNF-3 beta transcripts were found to be enriched in this cell population and in situ hybridization of adult lung revealed HNF-3 gene expression in non-ciliated bronchiolar epithelial cells expressing the CCSP gene. Finally, experiments with CCSP region I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interacting via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bronchiolar epithelium.
Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.
alpha 1-Antitrypsin (alpha 1-AT) is an acute phase plasma protein predominantly derived from the liver which inhibits neutrophil elastase. Previous studies have suggested that alpha 1-AT is also expressed in human enterocytes because alpha 1-AT mRNA could be detected in human jejunum by RNA blot analysis, and alpha 1-AT synthesis could be detected in a human intestinal adenocarcinoma cell line Caco2, which spontaneously differentiates into villous-like enterocytes in tissue culture. To definitively determine that the alpha 1-AT gene is expressed in human enterocytes in vivo, we examined tissue slices of human jejunum and ileum by in situ hybridization. The results demonstrate specific hybridization to enterocytes from the bases to the tips of the villi. Although there was no hybridization to enterocytes in most of the crypt epithelium, there was intense specific hybridization in one region of the crypt. Double-label immunohistochemical studies showed that alpha 1-AT and lysozyme co-localized to this region, indicating that it represented Paneth cells. Finally, there was a marked increase in hybridization to alpha 1-AT mRNA in villous enterocytes and Paneth cells in Crohn's disease. The results of this study provide definitive evidence that alpha 1-AT is expressed in human jejunal and ileal enterocytes in vivo, and show that alpha 1-AT is also a product of Paneth cells. Together with the results of other studies, these data raise the possibility that alpha 1-AT detected in fecal alpha 1-AT clearance assays for diagnosing protein-losing enteropathies is predominantly derived from sloughed enterocytes.
Apolipoprotein AI (apoAI) gene expression in liver depends on synergistic interactions between transcription factors bound to three distinct sites (A, B, and C) within a hepatocyte-specific enhancer in the 5'-flanking region of the gene. In this study, we showed that a segment spanning sites A and B retains substantial levels of enhancer activity in hepatoblastoma HepG2 cells and that sites A and B are occupied by the liver-enriched hepatocyte nuclear factors (HNFs) 4 and 3, respectively, in these cells. In non-hepatic CV-1 cells, HNF-4 and HNF-3beta activated this minimal enhancer synergistically. This synergy was dependent upon simultaneous binding of these factors to their cognate sites, but it was not due to cooperativity in DNA binding. Separation of these sites by varying helical turns of DNA did not affect simultaneous binding of HNF-3beta and HNF-4 nor did it influence their functional synergy. The synergy was, however, dependent upon the cell type used for functional analysis. In addition, this synergy was further potentiated by estrogen treatment of cells cotransfected with the estrogen receptor. These data indicate that a cell type-restricted intermediary factor jointly recruited by HNF-4 and HNF-3 participates in activation of the apoAI enhancer in liver cells and suggest that the activity of this factor is regulated by estrogen.
The transcription factors of the hepatocyte nuclear factor 3 (HNF3) family, which are active in the liver, are expressed early during endoderm differentiation. To study their involvement in early murine development, we examined their role in embryonic stem (ES) cells. HNF3alpha or HNF3beta mRNA transcripts were not detected in ES cells before differentiation, and only low levels of HNF3beta mRNA were detected at a late stage of differentiation of ES cells to embryoid bodies (EB) (20 days after induction of differentiation). To examine the consequences of overexpressing HNF3alpha or -beta in ES cells, we transfected the two genes into these cells and determined the levels of expression of tissue-specific genes during EB differentiation. Specifically, we examined expression of albumin, cystic fibrosis transmembrane conductance regulator (CFTR), phosphoenolpyruvate carboxykinase (PEPCK), alpha1-antitrypsin, transthyretin, zeta-globin, and neurofilament 68kd as markers for different cell lineages. Overexpression of HNF3beta (and to a lesser extent of HNF3alpha) induced the expression of genes associated with endodermal lineage, namely, the genes for CFTR and albumin, but did not induce the expression of genes involved in late endoderm differentiation, such as the genes for PEPCK and alpha1-antitrypsin. Moreover, expression of HNF1beta was highly induced in HNF3-overexpressing cells, while expression of HNF1alpha and HNF4 was only mildly induced in these cells. Therefore, HNF3alpha and -beta seem to be involved in early endoderm differentiation of ES cells and together with other developmental factors are apparently needed for the induction of the endodermal lineage in vivo.
Hepatocyte nuclear factors 1 and 4 (HNF-1 and HNF-4) are liver-enriched transcription factors that function in the regulation of several liver-specific genes. HNF-1 activates genes containing promoters with HNF-1 binding sites. However, this factor negatively regulates its own expression and that of other HNF-4-dependent genes that lack HNF-1 binding sites in their promoter region. This repression is exerted by a direct interaction of HNF-1 with AF2, the main activation domain of HNF-4. The dual functions of gene activation and repression suggest that HNF-1 is a global regulator of the transcriptional network involved in the maintenance of hepatocyte-specific phenotype.
The development of our knowledge of the serpins illustrates the advantages of considering a protein superfamily as a whole. The serpins have all retained a common tertiary structure despite the individual evolution of diverse functions; for example, the homology of the plasma protease inhibitor α1-antitrypsin is closer to that of corticosteroid binding globulin than is the homology of the two heparin-binding plasma inhibitors — antithrombin and heparin cofactor II — one to another. This retention of a well conserved structure necessarily requires the retention of strong homologies in primary and secondary structures in all the members of the family, across functions as well as species. For this reason, from the beginning, the study of the serpins has been a collective process with our understanding of the function of each member being greatly strengthened by parallel studies of other serpins. This has been particularly true of the lessons learnt from the human dysfunctional variants; one by one they have provided clues as to the normal function in individual members but when considered together with structural studies, in terms of the family as a whole, they have opened our understanding to a degree that far surpasses the contribution of more conventional approaches.
This review concerns the reasons why only an estimated 10-15% of patients with alpha-1-antitrypsin (A1AT) deficiency develop the destructive lung disease known as emphysema. The arguments presented revolve around the proteinase-antiproteinase balance in the 'microenvironment' of the epithelial space of the lung. Attention is focused on the balance between destructive enzymes such as neutrophil elastase and protective proteins such as A1AT, secretory leucocyte proteinase inhibitor (SLPI), human elastase inhibitor (HEI) and elafin. When neutrophil elastase is already attached to the elastin fibres the smaller molecules SLPI and elafin appear to be better inhibitors of this enzyme than larger inhibitors such as A1AT and HEI. Furthermore, SLPI and elafin may provide the first line of defence against proteinase attack from neutrophil elastase. In trying to explain the variability in the clinical expression of A1AT-deficiency and the development of emphysema, the importance of changes to A1AT, SLPI and elafin molecules induced by smoking and/or oxygen free radicals has been considered. It is possible that emphysema only develops in patients who have SLPI/elafin deficiency as well as A1AT deficiency.
There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha1-antitrypsin (alpha1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1alpha (HNF-1alpha), HNF-1beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha1-AT gene. Cotransfection studies showed that HNF-1alpha and HNF-4 had a synergistic effect on alpha1-AT gene expression. RNA blot analysis showed that HNF-1alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1alpha binding activity increase coordinately with alpha1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha1-AT gene expression. Together, these results provide evidence that HNF-1alpha and HNF-4 play a role in the mechanism by which the alpha1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.
Expression of the ␣1-proteinase inhibitor gene in human monocytes and macrophages
- D H Perlmutter
- F S Cole
- P Kilbridge
- T H Rossing
- Colten Hr
Perlmutter DH, Cole FS, Kilbridge P, Rossing TH, and
Colten HR. Expression of the ␣1-proteinase inhibitor gene in
human monocytes and macrophages. Proc Natl Acad Sci USA
82: 795-799, 1985.