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 individual components of the IGF axis. Northern blot analysis of IGF-I messenger 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 express 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 three-fold higher in early cultured HSCs than in culture-activated HSCs. By immunohistochemistry, a decrease of IGF-I-R expression of HSCs in vivo following CCl4-induced liver damage was noted as well. IGF binding proteins (IGFBPs) were detected in conditioned media from HSCs by 125I-IGF-I ligand blotting at apparent molecular masses of 24 and 41 to 45 kd that were immunologically identified as IGFBP-4 and -3, respectively. Synthesis of these IGFBPs increased with time of culture. At neutral pH, no IGFBP proteolysis was observed in conditioned media of early cultured and culture-activated HSCs, whereas at acidic pH, protease activities against IGFBP-3 and -4 were detectable. IGFBP protease activities were completely abolished by inhibitors of aspartyl and cysteine proteases. Addition of 100 nmol/L IGF-I stimulated cell proliferation of early cultured HSCs 5.6+/-1.1- and 4.6+/-0.2-fold as measured by [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation, respectively. In culture-activated HSCs, proliferation was increased 1.2+/-0.1-fold in the presence of 100 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 perpetuation of HSC proliferation during hepatic fibrogenesis.
Scharf
J G
JG
Department of Medicine, Georg-August-Universität, Göttingen, Germany.
Knittel
T
T
Dombrowski
F
F
Müller
L
L
Saile
B
B
Braulke
T
T
Hartmann
H
H
Ramadori
G
G
D000818:Animals; D002252:Carbon Tetrachloride Poisoning / Q000378:metabolism; D002455:Cell Division; D002478:Cells, Cultured; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D018969:Insulin-Like Growth Factor Binding Proteins / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D007335:Insulin-Like Growth Factor II / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000502:physiology*; D017527:Receptor, IGF Type 2 / Q000502:physiology*; D013997:Time Factors
D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D017527:Receptor, IGF Type 2; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1