Z Gastroenterol 2007; 45 - A1_24
DOI: 10.1055/s-2007-967778

Functional analysis of IGFBP–2 overexpression in mouse liver myofibroblats: implications for liver fibrogenesis

R Pannem 1, A Hoeflich 2, E Wolf 2, G Ramadori 3, JG Scharf 1
  • 1Abteilung für Gastroenterologie und Endokrinologie, Göttingen
  • 2Institut für Molekulare Tierzucht und Biotechnologie, Genzentrum der LMU München, München
  • 3Abteilung Gastroenterologie und Endokrinologie, Georg-August-Universität, Göttingen

Liver fibrosis results from excessive accumulation of extracellular matrix (ECM). Different liver cell populations are involved in this process: activated hepatic stellate cells as well as portal and perivascular liver myofibroblasts (LMFs) which represent morphologically and functionally different fibroblast populations. To examine the relationship between IGFBP–2 overexpression and cellular functions of LMFs, LMFs were isolated from wild type (wt) and IGFBP–2 transgenic (BP–2 (±)) mice.

Methods: LMFs were obtained by outgrowth from primary hepatocytes isolated from wt or CMV-IGFBP–2 transgenic mice. Expression of IGF-I, IGF-IR, IGF-II/M6PR, IGFBP–2 and –3 messenger RNA (mRNA) was investigated by Northern blot hybridization. IGF-IRβ protein expression was confirmed by Western immunoblot. IGFBP secretion was evaluated by [125I]-IGF-I ligand and immunoblotting. Determination of DNA synthesis in mLMF was assessed by means of BrdU incorporation assay. mRNA expression of collagen type 2 and fibulin–2 was assessed by real-time PCR.

Results: BP–2 (±) LMFs demonstrated a 5–fold increased mRNA expression of IGFBP–2 as compared with wt LMFs that was confirmed at protein level by [125I]-IGF-I ligand and immunoblotting. Expression of IGF-I, IGF-IR, IGF-II/M6PR and IGFBP–3 was not significantly different between wt and BP–2(±) LMFs. In wt LMFs, addition of IGF-I dose-dependently reduced IGFBP–2 mRNA and protein whereas in BP–2 (±) LMFs IGF-I showed a strong stimulatory effect on IGFBP–2 expression. The IGF-I-dependent stimulation of IGFBP–3 in BP–2 (±) LMFs was less pronounced than in wt LMFs. In contrast, the IGF-I-dependent decrease of IGF-IR was not significantly different in wt and BP–2 (±) LMFs. Functionally, IGF-I dose-dependently stimulated DNA synthesis in wt LMFs whereas in BP–2 (±) LMFs IGF-I-induced DNA synthesis was reduced to 50% of untreated controls. Similarly, IGF-I increased expression of collagen type 2 and fibulin–2, one of the ECM proteins deposited during liver fibrosis, in wt LMFs whereas IGF-I-dependent expression of collagen type 2 and fibulin–2 was diminished to approx. 50% of untreated controls.

Conclusion: Overexpression of IGFBP–2 in LMFs is associated with alterations of DNA replication and of biosynthesis of ECM components in these cells. Our data point to an important role of IGFBP–2 during liver fibrogenesis and indicate IGFBP–2 as a potential target in antifibrotic therapy.