Functional analysis of IGFBP-2 overexpression in mouse liver myofibroblasts: Therapeutic implication for liver fibrogenesis

Abstract

Liver fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM) proteins which are secreted from the cells of the fibroblast lineage during chronic liver injury. Different liver cell populations are involved in this process: activated hepatic stellate cells (HSCs) as well as portal and perivascular liver myofibroblasts (LMFs) which represent morphologically and functionally different fibroblast populations. 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 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. Significantly increased levels of IGFBP-2 in sera and hepatic tissue from patience suffering from liver cirrhosis of different etiology (Holt et al., 1996; Holt et al., 1997; Kratzsch et al., 1995; Ross et al., 1996; Wolf et al., 2000; Moller et al., 1995; Scharf et al., 1996). In general, IGFBP-2 has been shown either to inhibit or to enhance the effects of IGF-I in certain cell types studied. However, the precise role of IGFBP-2 overexpression in liver fibrogenesis is unknown. The aim of the present work is to examine the relationship between IGFBP-2 overexpression and cellular functions of LMFs. For this purpose, LMFs were isolated from the livers of wild type (wt) and CMV-IGFBP-2 transgenic (IGFBP-2 (+/-)) mice. IGFBP-2 (+/-) mLMFs showed an approximately four to five-fold increased expression of IGFBP-2 mRNA as compared with wt mLMFs during different time points of culture (days 2 to 5) that was confirmed at protein level by [125I]-IGF-I ligand. In wt mLMFs, the expression of IGFBP-3 mRNA was low at day 2 of culture but high at day 5 of culture whereas IGFBP-3 mRNA expression was reversibly decreased from high levels at day 2 of culture to low levels at day 5 of culture in IGFBP-2 (+/-) mLMFs. The expression of IGF-I, IGF-IR and IGF-II/M6-PR mRNA was increased in IGFBP-2 (+/-) mLMFs compared with wt mLMFs. In wt mLMFs, addition of IGF-I dose-dependently reduced IGFBP-2 mRNA and protein levels whereas in IGFBP-2 (+/-) mLMFs IGF-I showed a stimulatory effect on IGFBP-2 mRNA and protein levels. The IGF-I-dependent stimulation of IGFBP-3 mRNA and protein levels in IGFBP-2 (+/-) mLMFs were less pronounced than in wt LMFs. In contrast, the IGF-I-dependent decrease of IGF-IR mRNA and protein levels were not significantly different in wt and IGFBP-2 (+/-) mLMFs. Functionally, IGF-I dose-dependently stimulated DNA synthesis in wt mLMFs whereas in IGFBP-2 (+/-) mLMFs IGF-I-induced DNA synthesis was abrogated compared to untreated controls. Similarly, in wt LMFs, IGF-I stimulated mRNA expression of fibulin-2 and fibronectin 1, two of the ECM proteins deposited during liver fibrosis whereas IGF-I-induced mRNA expression of fibulin-2 and fibronectin 1 was inhibited compared to untreated controls in IGFBP-2 (+/-) mLMFs. Together, the data of present study demonstrate that overexpression of IGFBP-2 in LMFs is associated with alterations of DNA synthesis and of biosynthesis of ECM components in these cells. Our data point to a regulatory role of IGFBP-2 overexpression during liver fibrogenesis and indicate IGFBP-2 as a potential target in antifibrotic therapy.