Evidence for the involvement of protein kinase C in the inhibition of prolactin gene expression by transforming growth factor-beta2.
We investigated the mechanisms by which transforming growth factor (TGF)-beta2 inhibited prolactin mRNA expression in GH3 rat pituitary tumor cells. Maximal inhibition was observed with cells exposed to 5 ng/ml TGF-beta2 for 24 hr. Continuous presence of the hormone during the entire period was not necessary because exposure of cells to TGF-beta2 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-beta2 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-beta2 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-beta2 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-beta2. Inhibition of PKC did not require the sustained presence of TGF-beta2. In vitro kinase assays of the immunoprecipitated PKC demonstrated that the activities of alpha, epsilon, mu, and zeta isozymes were significantly decreased in the TGF-beta2-treated cells, whereas that of PKClambda was not affected. Western blotting did not reveal any change in PKCepsilon steady state protein levels, suggesting TGF-beta2 inhibits PKC activity through a post-translational mechanism. Our results support that inhibition of PKC activity is an early event mediating TGF-beta2-inhibited prolactin mRNA expression in GH3 cells.
Chuang
C C
CC
Institute of Biochemistry, National Yang-Ming University, Shih-Pai, Taipei, Taiwan 11221.
Tan
S K
SK
Tai
L K
LK
Hsin
J P
JP
Wang
F F
FF
D000818:Animals; D002118:Calcium / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D007527:Isoenzymes / Q000502:physiology; D011388:Prolactin / Q000235:genetics*; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D011388:Prolactin; D011493:Protein Kinase C; D002118:Calcium