Characterization of hepatocyte-derived mitogenic activity on hepatic stellate cells

Background/Aims: Hepatic stellate cells (HSC) are located in close proximit y to hepatocytes in Disse's space. Hepatocyte derived factors have earlier been implicated in the paracrine regulation of HSC proliferation. The aim o f the present study was to further characterize this mitogenic activity of the parenchymal cell conditioned medium (PCcM). Methods: Primary rat HSC we re cultured for 4 days. DNA synthesis was measured by H-3-thymidine incorpo ration. TGF beta(1) immunoreactivity was quantified by ELISA. PCcM was obta ined from hepatocytes cultured in medium without serum or hormones for two days. Results: Incubation of 4-day-old HSC on plastic surface with PCcM for 2 days increased DNA synthesis, while no effect was seen in HSC cultured o n Matrigel. Heat-, acid-, and protease-treatment of PCcM abolished its stim ulatory effect. Size fractionations with spin columns indicated that the st imulatory effect was contained in the fractions of a molecular size between 30 and 100 kD. The addition of LY 294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, dose-dependently inhibited the PCcM induced increase in DNA synthesis to about 9% of the control values. The specific MAP kinase (M APK) inhibitor, PD 98059 only suppressed the PCcM induced DNA synthesis to 35% of control cultures at the highest dose (10 mu M). DNA content in the c ultures was not affected by either blocker. HSC seemed to produce immunorea ctive TGF beta(1). However, addition of latency-associated peptide (LAP), a potent TGF beta(1) blocker, stimulated DNA synthesis to a much less extent than PCcM. Conclusions: The factor(s) that stimulate DNA synthesis in HSC from hepatocytes are most likely protein(s) with a molecular size between 3 0-100 kD. These factor(s) rely more on PI3-K than on MAPK for their mitogen ic effect and are probably not acting via TGF beta(1) inhibition.