EVIDENCE FOR THE INVOLVEMENT OF PROTEIN-KINASE-C IN THE INHIBITION OFPROLACTIN GENE-EXPRESSION BY TRANSFORMING GROWTH FACTOR-BETA(2)

We investigated the mechanisms by which transforming growth factor (TG F)-beta(2) inhibited prolactin mRNA expression in GH(3) rat pituitary tumor cells. Maximal inhibition was observed with cells exposed to 5 n g/ml TGF-beta(2) for 24 hr. Continuous presence of the hormone during the entire period was not necessary because exposure of cells to TGF-b eta(2) for 20 min was sufficient to trigger the same extent of prolact in mRNA inhibition at 24 hr as with its persistent presence. The actio n of TGF-beta(2) could be abolished by cycloheximide or EGTA, suggesti ng the requirement of a newly synthesized protein and extracellular Ca 2+. The response oi prolactin mRNA to TGF-beta(2) was inhibited by pre incubation of cells with phorbol-12-myristate-13-acetate, which down-r egulated protein kinase C (PKC). The activities of both the cytosolic and membrane PKC were significantly reduced at 20 min after TGF-beta(2 ) addition, and inhibition continued to 24 hr, the last lime point ana lyzed. However, the ratio of cytosolic to membrane PKC was not altered by TGF-beta(2). Inhibition of PKC did not require the sustained prese nce of TGF-beta(2). In vitro kinase assays of the immunoprecipitated P KC demonstrated that the activities of alpha, epsilon, mu, and zeta is ozymes were significantly decreased in the TGF-beta(2)-treated cells, whereas that of PKC lambda was not affected. Western blotting did not reveal any change in PKC epsilon steady state protein levels, suggesti ng TGF-beta(2) inhibits PKC activity through a post-translational mech anism. Our results support that inhibition of PKC activity is an early event mediating TGF-beta(2)-inhibited prolactin mRNA expression in GH (3) cells.