Expression and matrix deposition of latent transforming growth factor β binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture
Expression and matrix deposition of latent transforming growth factor β binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture
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DOI：
10.1053/jhep.2001.21996
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发表时间：
2001-02-01
期刊：
影响因子：
13.5
通讯作者：
Gressner, AM
中科院分区：
文献类型：
--
作者：
Breitkopf, K;Lahme, B;Gressner, AM
潜伏性转化生长因子β结合蛋白（LTBP）是一种大的潜伏性TGF-β复合物的高分子量糖蛋白，被认为是细胞外基质中潜伏性TGF-β的锚，也是微纤维结构的组成部分。LTBP的蛋白水解裂解被认为是释放和产生生物活性（成熟）TGF-β的先决条件。我们研究了LTBP亚型在正常和纤维化大鼠肝脏和培养的大鼠肝星状细胞（HSC）转分化为肌成纤维细胞（MFB）中的表达。我们进一步确定了它们与基质的相互作用及其一些基本功能。正常和纤维化肝脏的免疫染色显示LTBP-1和-2的强信号，优选在实质中，但也在非实质细胞和纤维化细胞外基质中。然而，原位杂交点的限制，从纤维化肝脏的非实质细胞的转录，而肝细胞总是缺乏LTBP转录。结果证实了实时定量逆转录聚合酶链反应（RT-PCR），这表明在培养的星状细胞transdifferentiating MFB和北方印迹分析显示的LTBP-1 mRNA的情况下，在新鲜分离的肝细胞LTBP-1转录的异构体特异性增加。使用细胞酶联免疫吸附试验（ELISA），差分增加部分脱氧胆酸盐（DOC）耐药性，基质结合LTBP-1和-2在培养的星状细胞中进行测量。用纤溶酶处理产生可溶性LTBP-1和生物活性TGF-β，其能够以自分泌方式诱导Smad 7表达。我们的数据建议（转分化）星状细胞，分别MFB，作为LTBP在正常和纤维化肝脏，这在这里可能履行结构和TGF-β调节功能的非肝组织的主要来源。
Latent transforming growth factor beta binding protein (LTBP), a high-molecular-weight glycoprotein of the large latent TGF-beta complex is suggested to serve as an anchor for latent TGF-beta in the extracellular matrix and as a component of microfibrillar structures. Proteolytic cleavage of LTBP is supposed to be a prerequisite for the release and generation of bioactive (mature) TGF-beta. We investigated the expression of LTBP isoforms in normal and fibrotic rat liver and in cultured rat hepatic stellate cells (HSC) transdifferentiating to myofibroblasts (MFB). We further determined their interaction with the matrix and some of their basic functions. Immunostainings of normal and fibrotic livers demonstrate intense signals for LTBP-1 and -2, preferably in parenchymal, but also nonparenchymal, cells and in fibrotic extracellular matrix. However, in situ hybridization points to a restriction of transcripts to nonparenchymal cells from fibrotic livers, whereas hepatocytes were always devoid of LTBP transcripts. The findings were confirmed by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), which showed isoform-specific increases of LTBP transcripts in cultured stellate cells transdifferentiating to MFB and by Northern blot analyses showing the absence of LTBP-1 mRNA in freshly isolated hepatocytes. Using a cell enzyme-linked immunosorbent assay (ELISA), a differential increase of partly deoxycholate (DOC)-resistant, matrix-bound LTBP-1 and -2 was measured in cultured stellate cells. Treatment with plasmin generated soluble LTBP-1 and bioactive TGF-beta, which was able to induce Smad7 expression in an autocrine fashion. Our data propose (transdifferentiating) stellate cells, respectively MFB, as the major source of LTBP in normal and fibrotic liver, which here probably fulfills structural and TGF-beta -regulating functions as suggested for nonhepatic tissues.