Cathepsin L gene expression and promoter activation in rodent granulosa cells.
The cysteine protease cathepsin L exhibits hormone-regulated expression during ovulation. In situ hybridization analyses of immature and pregnant mare serum gonadotropin-treated mouse and rat ovaries showed that cathepsin L expression in granulosa cells of small, growing follicles increased in periovulatory follicles after human chorionic gonadotropin stimulation. In the rat ovary, cathepsin L was also expressed in follicles with signs of atresia. To determine the molecular mechanisms that mediate the diverse regulation of this gene in granulosa cells, rat cathepsin L promoter-reporter constructs were analyzed by transient transfection assays in rat granulosa cells and EMSAs. A construct containing the transcriptional start site and -244 bp of upstream promoter sequence (-244/+33 bp) exhibited inducibility by forskolin, the phorbol ester phorbol myristate acetate, and an additive effect of both. Within this region, three functional specificity protein 1 (Sp1) sites, an overlapping early growth response protein-1 site, and a cAMP regulatory element-binding protein site were identified. Single or double mutants of the above-mentioned sites did not alter forskolin/phorbol myristate acetate inducibility of the promoter. Mutation of all three Sp1/specificity protein 3 (Sp3) sites, which also mutated the early growth response protein-1 site, reduced the promoter activation. Mutation of the cAMP regulatory element-binding protein site in the triple Sp1 mutant construct completely blocked the inducibility of the promoter. When these same constructs were transfected into MCF-7 human breast cancer cells or were cotransfected with an Sp1 expression vector in Drosophila SL2 cells, similar results were obtained. Collectively, the data document that three Sp1/specificity protein 3 binding GC-rich regions and a functional cAMP regulatory element constitute an important transcriptional regulatory complex for expression of the cathepsin L gene in rat granulosa cells.
Sriraman
Venkataraman
V
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Place, Houston, TX 77030, USA.
Richards
JoAnne S
JS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms; D050882:CREB-Binding Protein; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics*; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila; D005260:Female; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects; D006064:Gonadotropins, Equine / Q000494:pharmacology; D006107:Granulosa Cells / Q000201:enzymology*; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D004268:DNA-Binding Proteins; D006064:Gonadotropins, Equine; D009687:Nuclear Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; C497123:Sp3 protein, rat; D015534:Trans-Activators; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D005576:Colforsin; D004247:DNA; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; C494365:Crebbp protein, rat; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse; C534280:Ctsl protein, rat; D013755:Tetradecanoylphorbol Acetate