MOLECULAR DISSECTION OF THE MITOGENIC EFFECT OF HEPATOCYTES ON CULTURED HEPATIC STELLATE CELLS

The activation of proliferation of rat liver hepatic stellate cells (H SC) in cooperation with hepatocytes (PC) was studied using a coculture system and cell conditioned media, respectively. The proliferation of HSC was followed by incorporation of [H-3] thymidine and BrdU into DN A and by DNA content per culture. Strong stimulation of HSC proliferat ion was noticed under reduced fetal calf serum (FCS) conditions (0.2%) during a 48-hour coculture with PC, rat hepatoma, human hepatoma, and transforming growth factor (TGF)-alpha-transgenic mouse PC, respectiv ely. The extent of stimulation was frequently higher than that observe d by the addition of 10% FCS. Transformed HSC (myofibroblasts) could a lso be stimulated by cocultured PC, but the magnitude of activation wa s lower than that of (untransformed) HSC. Using radioreceptor assays, we could demonstrate significant concentrations of insulinlike growth factor (IGF)-1 (300 ng/10(6) cells x 48 hours) and quite lower concent rations of bFGF and TGF-alpha in the hepatocyte-conditioned media (PCc M), whereas IGF-2 was not detectable. With anti-IGF-1 neutralizing ant ibody, the stimulatory activity of PCcM could be reduced by approximat ely 50%. PCcM, which mimics the effects of cocultures and supports str ongly the action of exogenous IGF-1 on HSC proliferation, leaving that of other cytokines (TGF-alpha, IL-1 alpha, bFGE, aFGF, TNF-alpha), ad ded either separately or in various combinations, uninfluenced. The la tter cytokines were without significant effects on HSC proliferation. The mitogenic activity of cytokine combinations containing IGF-1 could be enhanced severalfold by limiting amounts of PCcM. Maximum stimulat ion of cell proliferation of 40-fold above control cultures was reache d by IGF-1 in combination with TGF-alpha and bFGF in presence of dilut ed PCcM, which is approximately 6-fold higher than in the absence of P CcM. [I-125] IGF-1 added to PCcM was bound by more than 90% to carrier proteins. The results confirm. in cocultures strong mitogenic activat ion of HSC by PC. It is suggested that IGF-1 and respective IGF-bindin g proteins are of great importance in the mitogenic signal transfer be tween hepatocytes and hepatic stellate cells.