Involvement Of C/EBP-α Gene In Activation Of Rat Hepatic Stellate Cells

Hepatic stellate cells (HSC) play key roles in hepatic fibrosis and liver cirrhosis, since activated HSC are the major sources of extracellular matrix (ECM), including collagen type I and type III, fibronectin, in fibrotic livers. Quiescent HSC are rich in cytoplasmic lipid storage, which containing retinoids, so they are also named by lipocytes. While HSC are activated by stimuli such as damaged hepatocytes, Kupffer cells, soluble cytokines or growth factors e. g. transforming growth factor type beta 1 (TGF-β1), they turn into highly proliferative, ECM- and cytokines-producing myofibroblast-like cells (MFb) charactered by expression of alpha type smooth muscle act in (a-SMA). Numerous studies have been focused on activation of HSC and some mechanisms have been elucidated, for instance, TGF-β1 is the most important cytokine which contributes to the activation of HSCs and even hepatic fibrosis or liver cirrhosis. However, since loss of cytoplasmic lipid droplets containing retinoids is one of the most important phenomenons during HSC activation, researchers are interested in the association of retinoids with HSC activat ion. Indeed, retinoids are shown to play important roles in HSC activation. Firstly, in vitro or in vivo activated HSC lose cytoplasmic retinoids, including all-trans-retinoic acid (atRA), 9-cis-retinoic acid (9cRA), as well as their nuclear receptors retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Secondly, extrogenous retinoids such as vitamin A or its acid metabolite retinoic acid (RA) at least partially reverse MFb into lipocytes. Finally, our previous studies have shown that transfection of RAR-p or RXR-α in activated HSC partially reversibly altered HSC phenotype, e.g. the cytoplasmic expression of α-SMA and collagens (type I and type III) diminished. But, as reported in our previous studies, RA could only partially change HSCphenotype, and alternatively some researches imply that retinoids may exacerbate HSC activation and even liver fibrosis.CCAAT/enhancer-binding proteins (C/EBPs) play key roles in differentiation of adipocytes, in which C/EBP-a controls preadipocyte maturation and cell growth. On one hand, C/EBP-a alone is sufficient to induce preadipocyte maturation and regulates adipogenic genes expression, such as lipoprotein lipase. On the other hand, C/EBP-a negatively regulates cell cycle of adipocytes and other cell types through its interaction with retinoblastoma protein (pRB), p21Cipl/WAFl, or even direct with E2F or cyclin-dependent kinase type 2 or type 4 (CDK2 or CDK4).Since quiescent HSC contain lipid droplets, just like adipocytes, it is conceivable to get into the hypothesis that C/EBPs may be involved in HSC activation. In order to test this hypothesis, C/EBP-a and -p mRNA or proteins were examined both in quiescent and in vitro naturally activated rat HSC. Furthermore, activated rat HSC were transfected with C/EBP-a gene, to see if over-expression of C/EBP-a could alter the activation phenotype of HSC.Part I Gene Expressions of some Members of C/EBPs Family during HSCActivationObjective To clarify gene expressions of some members of C/EBPs family duringHSC activation and to determine one of those genes which may play key rolesin rat HSC activation.Methods HSC were isolated from normal rats and primary cultures wereperformed. C/EBP-a and C/EBP-p mRNA levels in HSC at day 2 (quiescent HSC) orday 7(activated HSC) were determined by real-time RT-PCR assay and theirprotein levels by immunoflurorescence staining and fluroresconcequantitation.Results At day 7, HSC were auto-activated, which was confirmed by aSMAprotein, and the mRNA levels of C/EBP-a or C/EBP-p in activated HSC declinedby 92. 52% (p < 0. 001) and 21. 12% (p < 0. 05) , respectively. At the same time,the protein levels of C/EBP-a or C/EBP-p gene decreased by 57. 81% (p < 0. 001)and 10.83% (p < 0.05) , respectively.Conclusion The mRNA and protein levels of C/EBP-a gene and C/EBP-pgene decline in activated HSC, and our results indicate that C/EBP-a gene may involve more potently in the activation of HSCs.Part II Effects of C/EBP-a Gene Overexpression on Activation Status ofActivated HSCObjective To elucidate the effects of overexpression of C/EBP-a gene in HSC activation.Methods Activated HSC were transfected with C/EBP-a gene. Cell proliferation was determined by BrdU incorporation assay, a-SMA and collagen type I proteins were determined by immunoflurorescence staining and flurorescence quantitation by using laser scanning confocal microscope and free public domain NIH image software ImageJ, and oil red 0 staining was performed to determined cytopalsmic lipid droplets.Results In contrast with HSC transfected with pIRES2-EGFP vehicle vector, C/EBP-a gene transfected HSC had less BrdU incorporation (22.45% of control group, p < 0.05) and less collagen type I protein (21.16% of control group, p < 0. 001). No a-SMA protein was detected in C/EBP-a gene transfected HSC and cytoplasmic lipid droplets re-appeared in HSC transfected with C/EBP-a gene. Conclusion Overexpression of C/EBP-a gene can inhibit cell proliferation, gene expressions of a~SMA and collagen type I, induce rebound of cytoplasmic lipid droplets. C/EBP-a gene palys key roles in HSC activation.Part III Mechanisms of C/EBP-a Gene Inhibiting HSC ActivationObjective To study the molecular mechanisms of C/EBP-a gene in inhibitingHSC activation.Methods Activated HSC were transfected with pIRES2-EGFP-C/EBP-a gene andimmunoflurorescence staining and flurorescence quantitation by using laserscanning confocal microscope and free public domain NIH image software ImageJwas performed to determined some gene expressions.Results pl6INK4a, p21Cip1/WAF1 or p27Kipl protein levels was not affectedby C/EBP-a gene transfection (p> 0. 05). C/EBP-a gene suppressed the expressionof nuclear C/EBP-p and C/EBP-5 proteins by 13.94% (p < 0.05) and by 61.27%(p < 0.001), respectively, but nuclear C/EBP-p Pser"'\ active form of C/EBP-p...