Acyl-CoA: retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activation during the lipocyte phenotype induction in hepatic stellate cells

We have examined retinol esterification in the established GRX cell line, representative of hepatic stellate cells, and in primary cultures of ex vivo purified murine hepatic stellate cells. The metabolism of [3H]retinol was compared in cells expressing the myofibroblast or the lipocyte phenotype, under the physiological retinol concentrations. Retinyl esters were the major metabolites, whose production was dependent upon both acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). Lipocytes had a significantly higher esterification capacity than myofibroblasts. In order to distinguish the intrinsic enzyme activity from modulation of retinol uptake and CRBP-retinol content of the cytosol in the studied cells, we monitored enzyme kinetics in the purified microsomal fraction. We found that both LRAT and ARAT activities were induced during the conversion of myofibroblasts to lipocytes. LRAT induction was dependent upon retinoic acid, while that of ARAT was dependent upon the overall induction of the fat storing phenotype. The fatty acid composition of retinyl-esters suggested a preferential inclusion of exogenous fatty acids into retinyl esters. We conclude that both LRAT and ARAT participate in retinol esterification in hepatic stellate cells: LRAT's activity correlates with the vitamin A status, while ARAT depends upon the availability of fatty acyl-CoA and the overall lipid metabolism in hepatic stellate cells.     

Replacement of retinyl esters by polyunsaturated triacylglycerol species in lipid droplets of hepatic stellate cells during activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation.     

Opposite regulation of hepatic lipase and lecithin: cholesterol acyltransferase by glucocorticoids in rats.

Rats were treated with hydrocortisone, dexamethasone or triamcinolone for 4 days. The effect of treatment on hepatic lipase and lecithin:cholesterol acyltransferase (LCAT) mRNA levels and catalytic activities was determined. Hepatic lipase mRNA was not affected by hydrocortisone, but was decreased after dexamethasone (-28%) and triamcinolone (-54%). Hepatic lipase activity followed the same pattern, it was not affected by hydrocortisone and lowered by dexamethasone (-38%) and triamcinolone (-70%). The LCAT mRNA level in the liver was also not affected by hydrocortisone, but increased upon treatment with dexamethasone (+22%) and triamcinolone (+72%). Plasma LCAT, determined with an excess exogenous substrate (designated LCAT-II), tended to decrease after hydrocortisone treatment (-11%) and was higher after dexamethasone (+21%) and triamcinolone (+22%). The plasma cholesterol esterification rate (designated LCAT-I), determined by incubation of the plasma at 37 degrees C, followed the same pattern. The activity ratio of hepatic lipase/LCAT-II decreased from 1 in the controls to 0.51 after dexamethasone and 0.25 in the triamcinolone-treated animals. The plasma HDL cholesterol concentration in the different groups changed oppositely to the hepatic lipase/LCAT activity ratio. It is concluded that HDL cholesterol is raised by synthetic glucocorticoids due, among other factors, to a lowered hepatic lipase and an increased plasma LCAT activity. The influence of glucocorticoids on these enzymes is, at least partly, explained by the effects on the hepatic mRNA contents.     

Retinol uptake and metabolism,and cellular retinol binding protein expression in an in vitro model of hepatic stellate cells

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 M, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.     

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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β (TGF-β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-β stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF-I and TGF-β differentially regulate IGFBP-3, IGFBP-4, and IGFBP-5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF-I. J. Cell. Physiol. 174:240–250, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of acid-sensing ion channel 1a in hepatic stellate cells attenuates PDGF-induced activation of HSCs through MAPK pathway

Acid-sensing ion channels (ASICs), a group of Na⁺-selective and Ca²⁺-permeant ligand-gated cation channels, can be transiently activated by extracellular acid. Among seven subunits of ASICs, acid-sensing ion channel 1a (ASIC1a), which is responsible for Ca²⁺ transportation, is elevated in response to inflammation, tumor, and ischemic injury in central nervous system and non-neuronal tissues. In this study, we demonstrated for the first time the presence of ASIC1a in rat liver and hepatic stellate cells (HSCs). Furthermore, the expression of ASIC1a was increased in primary HSCs and liver tissues of CCl₄-treated rats, suggesting that ASIC1a may play certain role in liver fibrosis. Interestingly, we identified that the level of ASIC1a was significantly elevated in response to platelet-derived growth factor (PDGF) induction in a time- and dose-dependent manner. It was also established that Ca²⁺-transporting ASIC1a was involved in acid-induced injury of different cell types. Moreover, inhibition or silencing of ASIC1a was able to inhibit PDGF-induced pro-fibrogenic effects of activated rat HSCs, including cell activation, de novo synthesis of extracellular matrix components through mitogen-activated protein kinase signaling pathway. Collectively, our studies identified that ASIC1a was expressed in rat liver and HSCs and provided a strong evidence for the involvement of the ASIC1a in the progression of hepatic fibrosis.     

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT^−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT^−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT^−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT^−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.     

Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells

Hepatic stellate cells (HSCs) play a central role in the development of hepatic fibrosis. Recent evidence has revealed that HSCs also play a role in its resolution, where HSC apoptosis was determined. Moreover, induction of HSC apoptosis caused a reduction of experimental hepatic fibrosis in rats. Thus knowing the mechanism of HSC apoptosis might be important to clarify the pathophysiology and establish the therapeutic strategy for hepatic fibrosis. In HSCs, Rho and Rho kinase are known to regulate contraction, migration, and proliferation with modulation of cell morphology. Controversy exists as to the participation of Rho and Rho kinase on cell survival, and little is known regarding this matter in HSCs. In this study, we directed our focus on the role of the Rho pathway in the regulation of HSC survival. C3, an inhibitor of Rho, increased histone-associated DNA fragmentation and caspase 3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs. Moreover, Y-27632, an inhibitor of Rho kinase, had the same effects, suggesting that inhibition of the Rho/Rho kinase pathway causes HSC apoptosis. On the other hand, lysophosphatidic acid, which stimulates the Rho/Rho kinase pathway, decreased histone-associated DNA fragmentation in HSCs. Inhibition of the Rho/Rho kinase pathway did not affect p53, Bcl-2, or Bax levels in HSCs. Thus we concluded that the Rho/Rho kinase pathway may play a role in the regulation of HSC survival.     

Hypoxia induces the activation of hepatic stellate cells through the PVT1-miR-152-ATG14 signaling pathway

Molecular and Cellular Biochemistry - Shrm4 is a protein that is exclusively expressed in polarized tissues. The physiological function of Shrm4 in the brain was required to be elucidated. Thus, we...     

GATA binding protein 3 is correlated with leptin regulation of PPARγ1 in hepatic stellate cells

Accumulating evidence reveals that hormone leptin, mainly produced by adipocyte, plays a unique role in promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a key step in liver fibrosis and peroxisome‐proliferator activated receptor γ (PPARγ) exerts a crucial role in inhibition of HSC activation. Our previous researches demonstrated that leptin reduced PPARγ1 (a major subtype of PPARγ in HSCs) expression through GATA binding protein 2 (GATA2) binding to a site around ?2323 in PPARγ1 promoter. The present researches aimed to examine the effect of GATA3 on leptin‐induced inhibition of PPARγ1 and elucidate the relationship between GATA3 and GATA2. Gene expressions were analysed by real‐time PCR, western blot, luciferase assay and immunostaining. C57BL/6J ob/ob mouse model of thioacetamide‐induced liver injury was used in vivo. Results demonstrate that leptin significantly induces GATA3 expression in HSCs by multiple signalling pathways including NADPH oxidase pathway. There exist crosstalks between NADPH oxidase pathway and the other pathways. GATA3 can bind to GATA2‐binding site in PPARγ1 promoter and interacts with GATA2, contributing to leptin inhibition of PPARγ1 expression in HSCs. These data demonstrated novel molecular events for leptin inhibition of PPARγ1 expression in HSCs and thus might have potential implications for clarifying the detailed mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as non‐alcoholic steatohepatitis in obese patients.     

Transforming growth factor-beta1 downregulation of Smad1 gene expression in rat hepatic stellate cells

Smads are intracellular signaling molecules of the transforming growth factor-beta (TGF-beta) superfamily that play an important role in the activation of hepatic stellate cells (HSCs) and hepatic fibrosis. Excepting the regulation of Smad7, receptor-regulated Smad gene expression is still unclear. We employed rat HSCs to investigate the expression and regulation of the Smad1 gene, which is a bone morphogenetic protein (BMP) receptor-regulated Smad. We found that the expression and phosphorylation of Smad1 are increased during the activation of HSCs. Moreover, TGF-beta significantly inhibits Smad1 gene expression in HSCs in a time- and dose-dependent manner. Furthermore, although both TGF-beta1 and BMP2 stimulate the activation of HSCs, they have different effects on HSC proliferation. In conclusion, Smad1 expression and phosphorylation are increased during the activation of HSCs and TGF-beta1 significantly inhibits the expression of the Smad1 gene.