Vol. 53, Issue 6, 1054-1061, June 1998

Evidence for the Involvement of Protein Kinase C in the Inhibition of Prolactin Gene Expression by Transforming Growth Factor-₂

Chuan-Chang Chuang, Sai-Koong Tan, Lung-Kuo Tai, Jin-Ping Hsin, and Fung-Fang Wang

Institute of Biochemistry (C.-C.C., S.-K.T., L.-K.T., J.-P.H., F.-F.W.) and Faculty of Medical Technology (S.-K.T.), National Yang-Ming University, Shih-Pai, Taipei, Taiwan 11221

We investigated the mechanisms by which transforming growth factor (TGF)-₂ inhibited prolactin mRNA expression in GH₃ rat pituitary tumor cells. Maximal inhibition was observed with cells exposed to 5 ng/ml TGF-₂ for 24 hr. Continuous presence of the hormone during the entire period was not necessary because exposure of cells to TGF-₂ for 20 min was sufficient to trigger the same extent of prolactin mRNA inhibition at 24 hr as with its persistent presence. The action of TGF-₂ could be abolished by cycloheximide or EGTA, suggesting the requirement of a newly synthesized protein and extracellular Ca²⁺. The response of prolactin mRNA to TGF-₂ was inhibited by preincubation of cells with phorbol-12-myristate-13-acetate, which down-regulated protein kinase C (PKC). The activities of both the cytosolic and membrane PKC were significantly reduced at 20 min after TGF-₂ addition, and inhibition continued to 24 hr, the last time point analyzed. However, the ratio of cytosolic to membrane PKC was not altered by TGF-₂. Inhibition of PKC did not require the sustained presence of TGF-₂. In vitro kinase assays of the immunoprecipitated PKC demonstrated that the activities of , , µ, and  isozymes were significantly decreased in the TGF-₂-treated cells, whereas that of PKC was not affected. Western blotting did not reveal any change in PKC steady state protein levels, suggesting TGF-₂ inhibits PKC activity through a post-translational mechanism. Our results support that inhibition of PKC activity is an early event mediating TGF-₂-inhibited prolactin mRNA expression in GH₃ cells.