Evidence for the Involvement of Protein Kinase C in the Inhibition of Prolactin Gene Expression by Transforming Growth Factor-b2

We investigated the mechanisms by which transforming growth factor (TGF)-b2 inhibited prolactin mRNA expression in GH3 rat pituitary tumor cells. Maximal inhibition was observed with cells exposed to 5 ng/ml TGF-b2 for 24 hr. Continuous presence of the hormone during the entire period was not necessary because exposure of cells to TGF-b2 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-b2 could be abolished by cycloheximide or EGTA, suggesting the requirement of a newly synthesized protein and extracellular Ca. The response of prolactin mRNA to TGF-b2 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-b2 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-b2. Inhibition of PKC did not require the sustained presence of TGF-b2. In vitro kinase assays of the immunoprecipitated PKC demonstrated that the activities of a, e, m, and z isozymes were significantly decreased in the TGF-b2-treated cells, whereas that of PKCl was not affected. Western blotting did not reveal any change in PKCe steady state protein levels, suggesting TGF-b2 inhibits PKC activity through a post-translational mechanism. Our results support that inhibition of PKC activity is an early event mediating TGF-b2-inhibited prolactin mRNA expression in GH3 cells. Prolactin is a lactotropic polypeptide hormone originally found to be secreted by the pituitary. Recent studies indicate that thymocytes (Montgomery et al., 1992) and lymphocytes (Pellegrini et al., 1992) also express and release prolactin. The prolactin receptor belongs to the cytokine receptor superfamily and is present in B and T lymphocytes and macrophages (Gagnerault et al., 1993). These findings have led to the suggestion of a novel role for prolactin as a cytokine capable of stimulating immune responses. The secretion and synthesis of prolactin in the pituitary are regulated by neurotransmitters and neuropeptides. For example, estrogen (Maurer, 1981) and TRH (Albert and Tashjian, 1984) have been shown to stimulate prolactin secretion or gene expression, whereas dopamine (Ben-Jonathan, 1985) and somatostatin (Lamberts et al., 1989) negatively regulate prolactin release. These factors act through a variety of signaling pathways to modulate the concentrations of this physiologically