Expression of cyclins D1, D2 and E correlates with proliferation of rat stellate cells in culture

Background/Aims: Regulation of cell cycle progression of cultured rat stellate cells was studied. Methods: DNA synthesis was determined by the uptake of [3H]thymidine or 5-bromo-2′-deoxyuridine. Cell cycle distribution was analyzed using FACScan of cellular DNA stained with propidium iodide. Expression of cyclins and cyclin-dependent kinase 4 was evaluated by Western and Northern blotting. Results: DNA synthesis of primary-cultured stellate cells was found to accelerate 48 h after plating. Cell cycle analysis revealed that more than 93% of the cells were in G0/G1 phase during the first 48 h after plating. The cell population in S phase abruptly increased to about 16% 72 h after culture and shifted to G2/M phase thereafter. The level of proteins and mRNAs for cyclins D1, D2 and E started to increase 48 h after culture with a concomitant expression of platelet-derived growth factor receptor β, while the level of cyclin-dependent kinase 4 and its mRNA remained unchanged. On the other hand, stellate cells remained in G1 phase when they were cultured in the presence of 3-isobutyl-1-methylxanthine or dibutyryl cAMP after plating. Attenuation of the expression of cyclins D1, D2 and E and platelet-derived growth factor receptor β, but not cyclin-dependent kinase 4 was found in stellate cells cultured with these agents. Further analysis revealed that LY294002, a selective inhibitor of phosphatidylinositol 3-kinase, suppressed DNA synthesis and cyclin D1 expression in a dosedependent manner without affecting platelet-derived growth factor receptor β expression. Conclusions: Induction of G1 cyclins may play crucial roles in cell cycle transition of cultured stellate cells from G1 to S. Expression of platelet-derived growth factor receptor β and activation of phosphatidylinositol 3-kinase may be involved in the process.