Gong W, Pecci A, Roth S, Lahme B, Beato M, Gressner A M
Institute of Clinical Chemistry, Philipps-University, Marburg, Germany.
Hepatology. 1998 Aug;28(2):492-502. doi: 10.1002/hep.510280229.
Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.
细胞因子驱动的肝星状细胞（HSC）在组织损伤和炎症中的激活是肝纤维化发生的关键致病事件，导致产生基质的肌成纤维细胞（MFB）池扩大，而MFB是HSC的转化对应物。我们推测，MFB池的扩大也可能通过调节细胞凋亡来实现，细胞凋亡在细胞内环境稳定中与有丝分裂起着相反且互补的作用。我们对原代培养的HSC和传代培养的MFB对可溶性Fas配体（sFasL）诱导的细胞凋亡的易感性进行了表征，并将这些效应与Fas（APO-1/CD95）以及一些主要的促凋亡和抗凋亡原癌基因的表达水平相关联。MFB在暴露于sFasL后呈现出剂量依赖性的凋亡反应，这通过核小体DNA片段的显著增加、细胞DNA的丢失、阳性TUNEL反应和膜联蛋白染色得以证明。仅当蛋白质合成（放线菌酮）或RNA合成（放线菌素D）被阻断时才发现这种效应。在原代培养中维持不同时间的HSC与放线菌酮联合时对sFasL完全耐药，但在原代培养后期（第7天及以后）对sFasL介导的细胞凋亡的易感性增加。通过半定量逆转录聚合酶链反应（RT-PCR）分析和碱性磷酸酶-抗碱性磷酸酶染色，在HSC和MFB中均鉴定出Fas受体，其表达水平相当。通过核糖核酸酶保护试验发现，抗凋亡原癌基因bcl-2和bcl-xl在早期HSC中的表达比在晚期HSC和MFB中强得多。bcl-2的表达还通过半定量RT-PCR和免疫印迹得到了进一步证实。促凋亡基因bax在HSC和MFB的RNA水平上含量相当，但在蛋白质水平上MFB显示出bax表达增加。结论是，HSC向MFB的转化伴随着对sFasL介导的细胞凋亡的敏感性增加，这可能与bcl-2和bcl-xl表达的强烈降低有关，导致MFB中促凋亡基因表达占优势。调节转化中的HSC的凋亡易感性可能是纤维化发病机制中的一个重要补充途径。
