Evidence for the Involvement of Protein Kinase C in the Inhibition of Prolactin Gene Expression by Transforming Growth Factor-β2
- 1Institute of Biochemistry (C.-C.C., S.-K.T., L.-K.T., J.-P.H., F.-F.W.) and 2Faculty of Medical Technology (S.-K.T.), National Yang-Ming University, Shih-Pai, Taipei, Taiwan 11221
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.
Footnotes
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Send reprint requests to: Dr. Fung-Fang Wang, Institute of Biochemistry, National Yang-Ming University, Shih-Pai, Taipei, Taiwan 11221. E-mail: ffwang{at}ym.edu.tw
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This work was supported by NSC Grants 83–0420-B010–006 M11 and 85–2331-B010–076 M10 from the National Science Council, Taiwan, Republic of China.
- Abbreviations:
- TGF
- transforming growth factor
- PMA
- phorbol-12-myristate-13-acetate
- PKC
- protein kinase C
- TRH
- thyrotropin releasing hormone
- MAP
- mitogen-activated protein kinase
- MBP
- myelin basic protein
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- GH
- growth hormone
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- SDS
- sodium dodecyl sulfate
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- Received August 25, 1997.
- Accepted March 2, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
