Abstract

We investigated the mechanisms by which transforming growth factor (TGF)-β₂ inhibited prolactin mRNA expression in GH₃ rat pituitary tumor cells. Maximal inhibition was observed with cells exposed to 5 ng/ml TGF-β₂ for 24 hr. Continuous presence of the hormone during the entire period was not necessary because exposure of cells to TGF-β₂ 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-β₂ 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-β₂ 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-β₂ 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-β₂. Inhibition of PKC did not require the sustained presence of TGF-β₂. In vitro kinase assays of the immunoprecipitated PKC demonstrated that the activities of α, ε, μ, and ζ isozymes were significantly decreased in the TGF-β₂-treated cells, whereas that of PKCλ was not affected. Western blotting did not reveal any change in PKCε steady state protein levels, suggesting TGF-β₂ inhibits PKC activity through a post-translational mechanism. Our results support that inhibition of PKC activity is an early event mediating TGF-β₂-inhibited prolactin mRNA expression in GH₃ cells.