Gonadotropin regulation of the rat proopiomelanocortin promoter: characterization by transfection of primary ovarian granulosa cells.
To characterize the transcriptional effects of human (h)FSH and hCG on the POMC gene, primary rat granulosa cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid under the control of the POMC promoter and 5' region. POMC-CAT contains a fragment of the rat POMC gene, extending from nucleotide -704 to nucleotide +63, fused to the CAT gene. Treatment of POMC-CAT-transfected cells with either hFSH (20 ng/ml) or hCG (10 ng/ml) significantly increased CAT enzyme activity; however, neither hCG nor hFSH increased CAT enzyme activity in cells transfected with pSV2-CAT, a reporter plasmid under the control of the SV40 virus promoter and 5' region. The phosphodiesterase inhibitor isobutylmethylxanthine or the nonhydrolyzable cAMP analog cAMP-chlorothiophenyl significantly increased CAT activity in POMC-CAT-transfected granulosa cells. Human FSH stimulated transcription 10, 20, and 40 h after treatment, but FSH stimulation at the two earlier time points was 2.5- to 5.5-fold greater than that at 40 h. Gonadotropin-stimulated steroidogenesis was equivalent in POMC-CAT-transfected granulosa cells, untransfected, and mock-transfected cells. This indicates that transfection left the physiological hormone response intact. These data demonstrate the following. 1) 767 basepairs of the rat POMC gene are enough to confer gonadotropin stimulation on the CAT marker gene in granulosa cells. 2) Although the POMC promotor lacks a well conserved cAMP response element, either of two different pharmacological manipulations of granulosa cells that raise intracellular cAMP can also stimulate POMC-driven CAT expression. 3) Transfected primary cultures of granulosa cells provide a nontransformed, physiologically relevant model with which to study hormone-regulated gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Young
S L
SL
Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton 97006.
Nielsen
C P
CP
Lundblad
J R
JR
Roberts
J L
JL
Melner
M H
MH
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006107:Granulosa Cells / Q000378:metabolism*; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D011374:Progesterone / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D051381:Rats; D013873:Thionucleotides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D006063:Chorionic Gonadotropin; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D011374:Progesterone; D011333:Pro-Opiomelanocortin; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D015056:1-Methyl-3-isobutylxanthine