Abstract
The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell proliferation was investigated by using the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin. The hepatoma cells (wild type) and stable regucalcin/pCXN2 transfectants were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayters. The proliferation of cells was significantly suppressed in transfectants cultured for 24–72 h. The proliferation of wild‐type cells was significantly inhibited when the cells were cultured for 72 h in a medium containing an inhibitor of transcriptional activity or protein synthesis. Such an effect was not seen in transfectants. The presence of various inhibitors of protein kinase including PD 98059 (10^−7^ or 10^−6^ M), dibucaine (10^−6^ M), wortmannin (10^−8^ or 10^−6^ M), or genistein (10^−5^ M) caused a significant inhibition of the proliferation of wild‐type cells. These inhibitory effects were not seen in transfectants. Staurosporine (10^−8^ − 10^−7^ M) significantly inhibited the proliferation of wild‐type cells and transfectants. Also, the effect of vanadate (10^−5^ M), an inhibitor of protein tyrosine phosphatase, or Bay K 8644 (10^−6^ M), an agonist of calcium entry into cells, in inhibiting the proliferation of wild‐type cells was not observed in transfectants. Moreover, the proliferation of wild‐type cells was significantly inhibited in the presence of roscovitine (10^−7^ or 10^−6^ M) or sulforaphane (10^−7^ M), which induces cell‐cycle arrest. Such effect was not seen in transfectants. The inhibitory effect of sodium butyrate (8.3 × 10^−4^ M) on proliferation of wild‐type cells was also induced in transfectants. Gene expression in hepatoma cells cultured for 72 h with 10% FBS was determined by using reverse transcription‐polymerase chain reaction (RT‐PCR). The expression of p21 mRNA was significantly enhanced in transfectants, while cdc2a and chk2 mRNA expression were not significantly changed. Insulin‐like growth factor‐I (IGF‐I) mRNA expression was significantly suppressed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell proliferation that is partly mediated through various intracellular signaling‐related factors, and that the effect may be partly involved in the change in p21 or IGF‐I mRNA expression. The finding further supports that regucalcin plays an important role as a suppressor in the enhancement of cell proliferation. © 2005 Wiley‐Liss, Inc.