Cirrhotic Human Liver Research Articles

Aromatase in Nontumoral and Malignant Human Liver Tissues and Cells

We have investigated activity and expression of key steroids enzymes, including aromatase, in nontumoral, cirrhotic, and malignant human liver tissues and cells. Following 24 and 72 h incubation of malignant human liver cell lines HepG2, HuH7, and HA22T cells with testosterone (T) used as androgen precursor, we observed an increasingly high proportion of T oxidation to androstenedione, with aromatase being the prevalent enzyme activity in HepG2 and HuH7 cells at 72 h, while no aromatase could be detected in HA22T cells. On the other hand, balance of 5alpha- and 5beta-pathways was largely in favor of the 5alpha-pathway in HA22T cells and in favor of the 5beta-pathways in HuH7 cells. In in vivo studies conducted in nontumoral, cirrhotic, and malignant human liver tissue samples, aromatase activity was, respectively, undetectable, moderate, and elevated. RT-PCR analysis revealed high, intermediate, and very low levels of aromatase expression in HepG2, HuH7, and HA22T cells, respectively. Interestingly, both amphiregulin, a member of the EGF family of EGFR ligands, and an estrogen receptor (ER)-alpha, the hERalpha46, exhibited a corresponding figure of expression, being very high in HepG2 cells and markedly lower in HA22T and HuH7 cells. We propose here that locally elevated estrogen formation, brought about by high aromatase expression and activity, may result in the promotion of liver tumor cell growth through the induction of AREG via an ER-mediated mechanism.

Androgen metabolism and biotransformation in nontumoral and malignant human liver tissues and cells

There is indirect multiple evidence that hints at a potential role of sex steroids in development and progression of human hepatocellular carcinoma (HCC). In the present study, we have investigated androgen metabolism in a panel of human liver cancer cell lines (HA22T, Huh7, HepG2) and in normal, cirrhotic and malignant human liver tissues aiming to dissect the potential impact of individual enzyme activities and their products in normal and diseased human liver, both in vivo and in vitro. Using our intact cell analysis we were able to assess rates and pathways of androgen metabolism in living conditions. Overall, incubation of cultured cells or tissue minces with either testosterone (T) or androstenedione (Ad) used as precursor resulted in a large extent of 17βoxidation of T to Ad (cells: 28–77%; tissues: 35–50%). In malignant liver cell lines, both HA22T and Huh7 cells showed consistent amounts of the 5α-reductase enzyme products (18% and 15%, respectively), while 5β-reductase activity was more pronounced in Huh7 cells (18%) than in HA22T cells (1.8%). Interestingly, a significant extent of estrogen formation could be observed in Huh7 cells (5.4–11.5%), while no aromatase activity could be detected in HA22T cells. In HepG2 cells, along with a relatively high proportion of Ad, estrogens represented the most prominent (50–55%) end product of androgen metabolism, regardless of the precursor used. In liver tissues, equivalent results could be obtained, with a consistent proportion of 17βoxidation of T to Ad (35–50%) being observed in the majority of samples. However, while normal liver tissue samples exhibited a minor proportion of bioactive androgens (3.4%) with no aromatase products, HCC tissues showed a significant extent of aromatase activity (nearly 20%) with estrogen representing the most prominent metabolic product after 24 h incubation with either T or Ad. HCV and alcoholic cirrhotic tissues displayed different patterns of androgen metabolism. The former produced limited amounts of bioactive androgens (5.3%) and considerable levels of the intermediate aromatase product 19OH-Ad (up to 28%), the latter exhibited a prevalence of androgen degradation through the 5β-reductase pathway (9.8%) and a significant extent of aromatase activity (16% as a whole). In conclusion, three major metabolic states could be depicted, depending on prevalent pathways of androgen metabolism and steroid receptor status: estrogenic, androgenic, and mixed. This model supports the idea that local estrogen biosynthesis may be implicated in human HCC and provides a basis for the exploitation of aromatase inhibitors and/or ER antagonists or selective estrogen receptor modulators (SERMs) as a new therapeutic strategy in HCC patients.

Altered expression of iron regulatory genes in cirrhotic human livers: clues to the cause of hemosiderosis?

Hepatic iron deposition unrelated to hereditary hemochromatosis occurs commonly in cirrhosis but the pathogenesis of this condition is unknown. The aim of this study was to compare the expression of genes involved in the regulation of iron metabolism in cirrhotic (n=22) and control human livers (n=5). Transcripts were quantitated by real-time RT-PCR and protein levels were assessed by western blot. Hepatic iron concentrations (HICs) were measured by a spectrophotometric method. Levels of hepcidin mRNA did not differ between controls and cirrhotic livers; there was a highly significant correlation between hepcidin transcript levels and HIC in the latter group. Ferroportin, divalent metal transporter-1 (DMT1), and ferritin heavy chain mRNA levels were significantly higher in cirrhotic human livers than in controls (P=0.007, 0.039, and 0.025, respectively). By western blot, ferroportin and DMT1 levels were generally diminished in the cirrhotic livers compared to controls; neither correlated with HIC. In contrast, the abundance of ferritin increased with increasing HIC in the cirrhotic livers, whereas transferrin receptor decreased, indicating physiologically appropriate regulation. In conclusion, hepcidin expression appears to be appropriately responsive to iron status in cirrhosis. However, there are complex alterations in DMT1 and ferroportin expression in cirrhotic liver, including decreases in ferroportin and DMT1 at the protein level that may play a role in aberrant regulation of iron metabolism in cirrhosis.

Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury

Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl4. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1+ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1+ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl4-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of "activated" sinusoidal HSC, but it is a marker of "activated" (myo)fibroblasts found in portal areas and in scar tissue.

Expression of Matrilin-2 in Liver Cirrhosis and Hepatocellular Carcinoma

The recently described matrilin protein family is part of the extracellular matrix, their pathophysiological role as well as distribution in liver diseases, however, have not yet been studied. Considering that matrilins have been found to play role in cell growth and tissue remodeling, their possible involvement in carcinogenesis has been raised. The main objective of this study was to investigate the changes in matrilin-2 expression which is one of the main components of basement membranes. Thirty-five cases of surgically resected hepatocellular carcinomas, 35 corresponding surrounding liver tissues and 10 normal liver samples were used for the study. In 15 of 35 cases the tumor developed on the basis of cirrhosis. Matrilin-2 protein expression was detected in normal liver around bile ducts, portal blood vessels, while sinusoids were negative by immunohistochemistry. Cirrhotic surrounding tissue showed intensive matrilin-2 staining along the sinusoids. Tumorous neovasculature was found strongly positive by immunohistochemistry. No differences, however, were detected by morphometry regarding the amount of protein expression based on the grade of hepatocellular carcinomas. Real-time RT-PCR did not show significant differences in matrilin-2 mRNA expression between normal, cirrhotic and tumor samples. This suggests posttranslational modification of matrilin-2 manifesting in altered distribution in liver fibrosis. Our data indicate that matrilin-2 is a novel basement membrane component in the liver, which is synthetised during sinusoidal "capillarization" in cirrhosis and in hepatocellular carcinoma. This is the first report to describe the expression and distribution of matrilin-2 in human normal and cirrhotic liver as well as in hepatocellular carcinoma.

Effects of phosphodiesterase 5 inhibitors on hepatic hemodynamics in the rat

Aims: Clinical data published by our group suggest that phosphodiesterase 5 (PDE5) inhibitors lower portal pressure and increase portal venous flow in healthy and cirrhotic human liver. Animal data reported by others have been conflicting.

Major HGF-mediated regenerative pathways are similarly affected in human and canine cirrhosis

BackgroundThe availability of non-rodent animal models for human cirrhosis is limited. We investigated whether privately-owned dogs (Canis familiaris) are potential model animals for liver disease focusing on regenerative pathways. Several forms of canine hepatitis were examined: Acute Hepatitis (AH), Chronic Hepatitis (CH), Lobular Dissecting Hepatitis (LDH, a specific form of micronodulair cirrhosis), and Cirrhosis (CIRR). Canine cirrhotic samples were compared to human liver samples from cirrhotic stages of alcoholic liver disease (hALC) and chronic hepatitis C infection (hHC).ResultsCanine specific mRNA expression of the regenerative hepatocyte growth factor (HGF) signaling pathway and relevant down-stream pathways were measured by semi-quantitative PCR and Western blot (STAT3, PKB, ERK1/2, and p38-MAPK). In all canine groups, levels of c-MET mRNA (proto-oncogenic receptor for HGF) were significantly decreased (p < 0.05). Surprisingly, ERK1/2 and p38-MAPK were increased in CH and LDH. In the human liver samples Western blotting indicated a high homology of down-stream pathways between different etiologies (hALC and hHC). Similarly activated pathways were found in CIRR, hALC, and hHC.ConclusionIn canine hepatitis and cirrhosis the major regenerative downstream pathways were activated. Signaling pathways are similarly activated in human cirrhotic liver samples, irrespective of the differences in etiology in the human samples (alcohol abuse and HCV-infection). Therefore, canine hepatitis and cirrhosis could be an important clinical model to evaluate novel interventions prior to human clinical trials.

Dielectric properties of human normal, malignant and cirrhotic liver tissue: in vivo and ex vivo measurements from 0.5 to 20 GHz using a precision open-ended coaxial probe

Hepatic malignancies have historically been treated with surgical resection. Due to the shortcomings of this technique, there is interest in other, less invasive, treatment modalities, such as microwave hepatic ablation. Crucial to the development of this technique is the accurate knowledge of the dielectric properties of human liver tissue at microwave frequencies. To this end, we characterized the dielectric properties of in vivo and ex vivo normal, malignant and cirrhotic human liver tissues from 0.5 to 20 GHz. Analysis of our data at 915 MHz and 2.45 GHz indicates that the dielectric properties of ex vivo malignant liver tissue are 19 to 30% higher than normal tissue. The differences in the dielectric properties of in vivo malignant and normal liver tissue are not statistically significant (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 16% higher than normal). Also, the dielectric properties of in vivo normal liver tissue at 915 MHz and 2.45 GHz are 16 to 43% higher than ex vivo. No statistically significant differences were found between the dielectric properties of in vivo and ex vivo malignant tissue (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 28% higher than normal). We report the one-pole Cole–Cole parameters for ex vivo normal, malignant and cirrhotic liver tissue in this frequency range. We observe that wideband dielectric properties of in vivo liver tissue are different from the wideband dielectric properties of ex vivo liver tissue, and that the in vivo data cannot be represented in terms of a Cole–Cole model. Further work is needed to uncover the mechanisms responsible for the observed wideband trends in the in vivo liver data.

E-cadherin as a reliable cell surface marker for the identification of liver specific stem cells

Oval cells are liver-specific bipotent stem cells which accumulate in injured liver when proliferation of mature hepatocytes and/or cholangiocytes is impaired. They represent an intermediary cell type with phenotypical characteristics of both, hepatocytes and cholangiocytes. Oval cells express specific cell surface proteins allowing their identification in situ. Most of these cell surface proteins, however, are recognized by antibodies in mouse liver tissue that are not commercially available or work only on frozen sections. We show herein the unequivocal identification of oval cells in paraffin-embedded mouse liver samples based on strong E-cadherin expression different from that of hepatocytes and bile duct cells. By comparing the pattern of E-cadherin expression with that of both, A6-antigen and CD44, we suggest a tight control of E-cadherin expression depending on the differentiation stage of the progenitor cells. In human cirrhotic liver samples E-cadherin expression was found as a common feature of both, typical and atypical reactions, and, thus, can also serve as an indication of the progenitor cell compartment activation.

Bone Morphogenetic Protein 7 is Elevated in Patients with Chronic Liver Disease and Exerts Fibrogenic Effects on Human Hepatic Stellate Cells

Hepatic stellate cells (HSCs) are the main extracellular matrix (ECM)-producing cells in liver fibrogenesis. The excessive synthesis of ECM proteins deteriorates hepatic architecture and results in liver fibrosis and cirrhosis. This study investigated the role of bone morphogenetic protein 7 (BMP7) as a member of the transforming growth factor (TGF)-beta superfamily in chronic liver disease. Plasma levels of BMP7 were significantly elevated in patients with chronic liver disease compared with healthy controls. Immunohistochemistry of cirrhotic human liver demonstrated upregulated BMP7 protein expression in hepatocytes as compared with normal human liver. Because gene expression for all putative BMP7 receptors was induced during the culture activation process of primary human HSCs, we studied the effects of BMP7 on hTERT immortalized human HSCs in vitro. BMP7, as expressed and secreted after infection with adenoviruses encoding BMP7 (AdBMP7), increased proliferation of HSCs. The mRNA and protein expression of type I collagen and fibronectin was increased in BMP7-stimulated HSCs. Elevated systemic and hepatic levels of BMP7 in patients with chronic liver disease may contribute to progression of liver fibrogenesis in vivo.

Expression of MMPs and TIMPs in liver fibrosis – a systematic review with special emphasis on anti-fibrotic strategies

In liver tissue matrix metalloproteinases (MMPs) and their specific inhibitors (tissue inhibitors of metalloproteinases, TIMPs) play a pivotal role in both, fibrogenesis and fibrolysis. The current knowledge of the pathophysiology of liver fibrogenesis with special emphasis on MMPs and TIMPs is presented. A systematic literature search was conducted. All experimental models of liver fibrosis that evaluated a defined anti-fibrotic intervention in vivo or in vitro considering MMPs and TIMPs were selected. The methodological quality of all these publications has been critically appraised using an objective scoring system and the content has been summarized in a table.

Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver

Hepatocyte function and regeneration are severely compromised in severe liver disease, and a common sequela is cirrhosis. Structural changes caused by cirrhosis create a cellular environment conducive to the formation of ductular reactions (DRs). Ductular reactions are primarily composed of oval cells also known as "intermediate hepatobiliary cells". We have conducted single, double, and triple staining to study lineages of oval cells present in DRs. Staining with NCAM, CK19, and HepPar1 has revealed a distinctly bipolar structure to DRs that are embedded in cirrhotic tissue. Spatial analysis of cells that are singly HepPar1-positive, or CK19-positive, has revealed hepatocytic and biliary poles, respectively, in the DRs. Also, the location of singly NCAM-positive cells in DRs suggests that they may be bipotent liver stem/progenitor cells. The locations of other intermediate hepatobiliary cells, which have combinations of markers, suggest that CK19+/NCAM+ cells are transitional cells in the biliary lineage and that rare cells that are negative for all three markers are transitional cells in the hepatocytic lineage. A working cell lineage model for DRs is presented.

Targeting 15d-Prostaglandin J2 to Hepatic Stellate Cells: Two Options Evaluated

PurposeDelivery of apoptosis-inducing compounds to hepatic stellate cells (HSC) may be an effective strategy to reverse liver fibrosis. The aim of this study was therefore to examine the selective targeting of the apoptosis-inducing drug 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) with two different HSC-carriers: human serum albumin modified with the sugar mannose-6-phosphate (M6PHSA) or albumin modified with PDGF-receptor recognizing peptides (pPBHSA).Methods and ResultsAfter chemical conjugation of 15dPGJ2 to the carriers, the constructs displayed pharmacological activity and specific receptor-mediated binding to HSC in vitro. Unlike 15dPGJ2-pPBHSA, the cellular binding of 15dPGJ2-M6PHSA was reduced by a scavenger receptor antagonist. In vivo, both conjugates rapidly accumulated in fibrotic livers. Intrahepatic analysis revealed that 15dPGJ2-M6PHSA mainly accumulated in HSC, and to a lesser extent in Kupffer cells. 15dPGJ2-pPBHSA also predominantly accumulated in HSC with additional uptake in hepatocytes. Assessment of target receptors in human cirrhotic livers revealed that M6P/IGFII-receptor expression was present in fibrotic areas. PDGF-β receptor expression was abundantly expressed on human fibroblasts.ConclusionsThese studies show that 15dPGJ2 coupled to either M6PHSA or pPBHSA is specifically taken up by HSC and is highly effective within these cells. Both carriers differ with respect to receptor specificity, leading to differences in intrahepatic distribution. Nevertheless, both carriers can be used to deliver the apoptosis-inducing drug 15dPGJ2 to HSC in vivo.

Dispensability and dynamics of caveolin-1 during liver regeneration and in isolated hepatic cells

Caveolae participate in several cellular processes such as vesicular transport, cholesterol homeostasis, regulation of signal transduction, integrin signaling, and cell growth. The expression and functional role of caveolin (Cav), the most abundant protein of caveolae, has been reported in liver and in different hepatocyte cell lines, in human cirrhotic liver, and in hepatocellular carcinomas. The role of Cav-1 in liver regeneration after partial hepatectomy (PH) has been investigated as a model of liver proliferation in vivo. Our results show that Cav-1 increases in liver after PH with a redistribution of the protein from the caveola-enriched domain to the noncaveolar fraction. Moreover, the Cav-1 located in the noncaveolar fraction is phosphorylated in tyrosine 14, even though the Cav-1 gene is dispensable for liver regeneration after PH, as deduced from data obtained with commercially available animals lacking this gene. In addition to this, the proinflammatory stimulation of hepatocytes induces Cav-1 translocation to a noncaveolar fraction and tyrosine 14 phosphorylation mainly through the activation of tyrosine kinases such as Src. These results support a dynamic role for Cav-1 in liver proliferation both in vivo after PH and in vitro in cultured hepatic cell lines, but with minimal implications for the liver regeneration process.

Identification and Functional Characterization of the Hepatic Stellate Cell CD38 Cell Surface Molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.

Expression and Distribution of Prolactin Receptor in Normal, Fibrotic, and Cirrhotic Human Liver

Liver cirrhosis is often associated with elevated levels of prolactin (PRL). This is commonly attributed to impaired hepatic metabolism of estrogens. However, there is evidence suggesting that PRL may be an important factor in hepatic tissue regeneration. To investigate the role of PRL in the pathogenesis of liver cirrhosis, we used RT-PCR and immunhistochemical staining to analyze changes in the expression and the histological distribution of the prolactin receptor (PRLR) in normal, fibrotic and cirrhotic hepatic tissue. Liver tissue was obtained from 29 surgically explanted human livers. The histological examination demonstrated normal liver tissue (n=9) as well as different grades of fibrosis (n=10) and cirrhosis (n=10). In liver cirrhosis and fibrosis, PRLR-mRNA was expressed at a higher level compared to normal liver specimens. Immunohistochemical staining of normal liver tissue demonstrated homogeneous distribution of the PRLR in the hepatocytes and in the epithelial cells of the bile ducts. This pattern of distribution was lost in fibrosis, where an accumulation of the PRLR was observed in the damaged hepatocytes. As no PRL-mRNA was detectable in normal, fibrotic or cirrhotic tissue, PRL does not act through autocrine or paracrine mechanisms. These data confirm previous results, which we obtained using an animal model for experimental liver cirrhosis in rats suggesting a metabolic function of PRL in normal liver and a regenerative function in fibrotic and cirrhotic liver. In conclusion, PRL might be involved in the pathogenesis of liver cirrhosis.

De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane

Basement membrane (BM) is a highly specialized extracellular matrix (ECM), which is associated with epithelia and endothelia. BM provide epithelia with structural support and also regulate cell behavior. The liver contains a unique ECM within the space of Disse, which consists of basement membrane constituents as well as fibrillar ECM molecules. Changes in composition of this ECM are considered detrimental for viability of hepatocytes during progression of liver disease. Mouse tumor-derived BM preparations, such as Matrigel, which are commonly used as a model for BM in vitro, differ significantly in their composition from liver BM present in vivo. In order to gain further insights into the role of BM in the regulation of hepatocyte behavior in health and disease, we generated a liver-derived basement membrane matrix (LBLM). LBLM allowed investigation of BM-hepatocyte interactions in vitro. Here we report a novel approach of generating a liver-derived basement membrane matrix by separate isolation of type IV collagen, laminin, nidogen, and heparan sulfate proteoglycans, and subsequent reconstitution into a matrix-like gel. Adhesion of primary human hepatocytes to LBLM was increased and the rate of de-differentiation was decreased compared to hepatocyte cultivation on Matrigel or type I collagen matrix. Primary human hepatocytes maintained their differentiated epithelial phenotype on LBLM isolated from normal human livers for more than 21 days, whereas they de-differentiated rapidly on LBLM isolated from cirrhotic human livers. Normal human LBLM contains a unique isoform composition of type IV collagen, namely alpha1 (IV), alpha2(IV), alpha4(IV), and alpha6(IV) chains, whereas cirrhotic LBLM contains only alpha1(IV) and alpha2(IV) isoforms, albeit present in increased amounts. These findings suggest that the composition of liver basement membrane is important for the maintenance of hepatocyte viability and provide anti-de-differentiation clues.

Cholangiocyte Endothelin 1 and Transforming Growth Factor β1 Production in Rat Experimental Hepatopulmonary Syndrome

Cholangiocyte Endothelin 1 and Transforming Growth Factor β1 Production in Rat Experimental Hepatopulmonary Syndrome

Expression of oncostatin M and its receptors in normal and cirrhotic human liver

In the cirrhotic liver, gene expression of the multifunctional cytokine oncostatin M (OSM) is up-regulated, but its cellular origin is unknown. Therefore, we investigated the expression of OSM protein and its specific receptor subunits, OSMRbeta and LIFRbeta in normal and cirrhotic human liver using immunohistochemical and Western blot analysis. OSM protein was expressed in Kupffer cells, variably in normal liver but consistently in cirrhosis. OSMRbeta was expressed at low level in hepatocytes of all normal livers examined, but in no cirrhotic sample. In contrast, LIFRbeta receptor was expressed weakly in normal livers, but much more intensely in cirrhosis, in reactive ductules, bile duct epithelial cells and perisinusoidal areas. Double immunostaining showed co-localization of LIFRbeta with cytokeratin 7, proliferating cell nuclear antigen (PCNA) and leukemia inhibitory factor (LIF), in bile duct epithelial cells, but not with alpha-smooth muscle actin, a myofibroblast marker. In human liver, OSM protein is expressed in Kupffer cells, variably in normals but universally in cirrhosis. The differential expression pattern of OSM and its receptors could allow for differential OSM signaling by alternative utilization of receptors to promote hepatocyte proliferation in acute injury and, with its homologue LIF, for the bile ductular reaction in cirrhosis.

Zinc finger protein 267 is up-regulated during the activation process of human hepatic stellate cells and functions as a negative transcriptional regulator of MMP-10

Activation of hepatic stellate cells (HSCs) is the central event in the development of liver fibrosis and cirrhosis. The transdifferentiation process of quiescent into activated HSCs requires a complete reprogramming in gene expression, which is governed by modulation of transcriptional activators or repressors. Using microarray analysis to identify genes differentially expressed during the activation process of human HSCs, zinc finger protein 267 (ZNF267) mRNA was up-regulated in activated HSCs and in cirrhotic human liver. ZNF267 belongs to the family of Kruppel-like zinc fingers and contains a conserved KRAB (Kruppel associated box) A and B domain in the N-terminal part outside the C-terminal region of zinc fingers. ZNF267 constructs containing enhanced cyan fluorescence protein were constitutively localized in the nucleus. When fused to GAL4 DNA binding domain, full-length ZNF267 and all constructs encompassing KRAB A domain showed transcriptional repressor activity. Microarray analysis and RNase protection assays showed that ZNF267 represses MMP-10 gene expression, which was confirmed by reporter gene assays. Furthermore, ZNF267 binds to the MMP-10 promoter region as demonstrated by chromatin immunoprecipitation assays. In conclusion, our results suggest that ZNF267 as a negative transcriptional regulator of MMP-10 might promote liver fibrogenesis through alteration of matrix degradation in vivo.