Expression and Regulation of the Insulin-like Growth Factor Axis Components in Rat Liver Myofibroblasts

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Hepatic stellate cells (HSCs) and liver myofibroblasts (LMFs) represent major cell populations involved in liver fibrogenesis. Several lines of evidence demonstrate that in contrast to LMFs, HSCs undergo spontaneous apoptosis both in vitro and in vivo, in parallel with their activation. Therefore, LMFs appear to be an essential cell type with the fibrogenic potential in the liver. The IGF system including the insulin-like growth factors I and II (IGF-I, -II), their receptors (IGF-I receptor, IGF-IR; IGF-II/mannose 6-phosphate receptor, IGF-II/M6-PR) and six high affinity IGF binding proteins (IGFBPs) participate in the regulation of growth and differentiation of cells of the fibroblast lineage, possibly contributing to the fibrogenic process. Therefore, the purpose of the current work was to study the expression and regulation of the IGF axis components in rat LMFs. Since IGF-I is known as a progression factor for the growth-promoting effects of platelet-derived growth factor (PDGF) in many cell types, the aim of this work was also to study the role of PDGF in proliferation of LMFs and to investigate a possible cross-talk between PDGFR and IGF-IR signalling systems in rat LMFs.LMFs from passages 1 to 7 constitutively expressed transcripts encoding IGF-I, IGF-IR and IGF-II/M6-PR. A soluble form of the IGF-II/M6-PR was abundantly produced by LMFs, and its release was stimulated by IGF-II and TGF-ß. In LMFs, biosynthesis of IGFBP-3 and -2 was observed that was stimulated by IGF-I, insulin and TGF-ß and inhibited by PDGF-BB. During cultivation of LMFs IGFBP-3 gene expression was down-regulated, whereas that of IGFBP-2 was up-regulated.IGF-I stimulated de novo synthesis of type I collagen and had mitogenic activity, whereas long-R3-IGF-I, an IGF-I analogue which binds to the IGF receptors but not to IGFBPs, had no effect on DNA synthesis in LMFs. Simultaneous addition of recombinant human IGFBP-2 or -3 with IGF-I diminished the mitogenic effects of IGF-I on LMFs, whereas preincubation of LMFs with IGFBP-2 or -3 potentiated DNA synthesis induced by IGF-I. Exogenous IGFBP-3 revealed also mitoinhibitory activity in LMFs that was independent from IGF-I. Moreover, a relatively high amount of endogenous IGFBP-3 in LMFs was accumulated in the nucleus that might be linked with the intrinsic antiproliferative activity of IGFBP-3.Recombinant PDGF-BB stimulated DNA synthesis in LMFs and this effect was similar to that of IGF-I. Blockade of the IGF-IR with a selective inhibitor completely abrogated IGF-I- and PDGF-induced mitogenesis in cultures of rat LMFs. In rat liver, alpha and beta subunits of the PDGF receptor (PDGFR) were exclusively expressed in HSCs and LMFs, and were substantially up-regulated during their in vitro cultivation. IGF-I and PDGF-BB differentially affected the IGF-IR and PDGFR signalling systems. High concentrations of IGF-I induced down-regulation of the IGF-IR and decreased amount of IRS-1, a principal adaptor protein of the IGF-IR. Expression and activation of the PDGFR-alpha was also inhibited by IGF-I. In contrast PDGF-BB increased the IGF-IR expression and effectively prevented its IGF-I-induced down-regulation. However, PDGF-BB inhibited the IGF-I-induced tyrosine phosphorylation of IRS-1 and substantially decreased the abundance of several IRS proteins in the cell, in particular IRS-1, IRS-2 and Gab-1. PDGF-BB did not affect expression of the PDGFR. Transphosphorylation of the PDGFR and the IGF-IR was not observed in LMFs. PDGF-BB effectively induced phosphorylation of all terminal MAP kinases (ERK1/2, JNK, p38 kinase) in LMFs in contrast to IGF-I, which had only a weak effect. Inhibition of MEK, p38 kinase and JNK effectively blocked IGF-I-induced DNA synthesis in LMFs. Inactivation of JNK and p38 kinase also resulted in abrogation of mitogenic effects induced by PDGF-BB. However, the rate of PDGF-induced DNA synthesis was unaffected when phosphorylation of ERK1/2 was blocked. Inhibition of phospholipase C (PLC) in LMFs was associated with a substantial reduction of both PDGF- and IGF-I-induced DNA synthesis, although in LMFs PLC-gamma-1 was activated only in response to PDGF-BB, but not to IGF-I. Blockade of the IGF-IR kinase considerably impaired the ability of PDGF-BB to stimulate PLC-gamma-1 activity in LMFs.In conclusion, the present study demonstrates that the IGF axis via complex interactions with the PDGFR signalling system may play an important role in the proliferation of LMFs in vitro that might be relevant in vivo for fibroproliferative response during acute and chronic liver injury.