CHARACTERIZATION OF THE IGF AXIS COMPONENTS IN ISOLATED RAT HEPATIC STELLATE CELLS

The insulin-like growth factors I and II (IGF-I, -II) are circulating peptides known to participate in the regulation of metabolism, growth, and cellular differentiation. In the present study, ''early cultured' ' (days 2-3 of culture) and ''culture-activated'' (days 6-7 of culture ) rat hepatic stellate cells (HSCs) were analyzed for expression of in dividual components of the IGF axis. Northern blot analysis of IGF-I m essenger RNA (mRNA) revealed transcripts of 7.5, 4, 2, and 1.0 to 1.5 kb in culture-activated HSCs, while early cultured HSCs did not expres s IGF-I mRNA. In culture-activated HSCs, an IGF-I secretion of 8.3 +/- 2.5 ng/10(6) cells per 24 hours was determined radioimmunologically. In media from early cultured HSCs, IGF-I was not detectable. The IGF-I receptor (IGF-I-R) mRNA expression was threefold higher in early cult ured HSCs than in culture-activated HSCs. By immunohistochemistry, a d ecrease of IGF-I-R expression of HSCs in vivo following CCl4-induced l iver damage was noted as well. IGF binding proteins (IGFBPs) were dete cted in conditioned media from HSCs by I-125- IGF-I ligand blotting at apparent molecular masses of 24 and 41 to 45 kd that were immunologic ally identified as IGFBP-4 and -3, respectively Synthesis of these IGF BPs increased with time of culture. At neutral pH, no IGFBP proteolysi s was observed in conditioned media of early cultured and culture-acti vated HSCs, whereas at acidic pH, protease activities against IGFBP-3 and -4 were detectable. IGFBP protease activities were completely abol ished by inhibitors of aspartyl and cysteine proteases. Addition of 10 0 nmol/L IGF-I stimulated cell proliferation of early cultured HSCs 5. 6 +/- 1.1- and 4.6 +/- 0.2-fold as measured by [H-3]thymidine and 5-br omo-2'-deoxyuridine incorporation, respectively In culture-activated H SCs, proliferation was increased 1.2 +/- 0.1-fold in the presence of 1 00 nmol/L IGF-I in both proliferation assays. It can be concluded that due to a higher expression of the IGF-I-R and lower levels of IGFBPs, early cultured HSCs are more susceptible to the mitogenic actions of IGFs than the culture-activated HSCs. The present data suggest a role for the IGF axis components in the initiation rather than the perpetua tion of HSC proliferation during hepatic fibrogenesis.