title: Progesterone receptor activates its promoter activity in human endometrial stromal cells.
abstract: Previous studies have shown that progestin increases the content of progesterone receptor (hPRA and hPRB) and the hPR mRNA during decidualization of human endometrial stromal cells suggesting that endogenous hPR enhances the transcription of the hPR gene. In the present study, we provide evidence that hPR regulates the promoter activity mediated through an active Sp1 site. In stromal cells treated with medroxyprogesterone acetate, the promoter activity was significantly increased when cells were co-transfected with hPR expression vector. Progressive deletion analysis showed that the highest activity was derived from the promoter region between -55 and +31 bp. Transactivation by hPR was dose dependent. The capacity of hPRA was stronger than that of hPRB. The ligand binding domain, but not DNA binding domain of the hPR was required for the transactivation. The proximal promoter region lacks a canonical progesterone response element. Instead, an active Sp1 site (-49 to -43 bp) has been confirmed. Mutation of the Sp1 site eliminated the effect of hPR activation. The promoter activity was increased by over expression of Sp1, whereas Sp3 had no effect. Electrophoretic mobility shift assay showed that the promoter region between -55 and +31 bp bound to Sp1 family proteins, Sp1 (C2 complex) and Sp3 (C1 and C3 complexes) identified by antibodies to Sp1 and Sp3. Sp1 complex formed by extracts of stromal cells was less intense than that formed by progestin-decidualized stromal cells. Sp1/DNA binding was enhanced when stromal cell extracts were incubated with calf intestine alkaline phosphatase (CIP) suggesting that dephosphorylation of Sp1 enhances the DNA binding. Addition of protein kinase inhibitor, H-89 or H-7, enhanced the hPR stimulated promoter activity. Western blot analysis showed that endometrial stromal/decidual cell extracts contained a wide band of Sp1 spanning from approximately 105 to 96 kDa and was resolved into one band at 96 kDa by CIP. Decidual cell extracts are abundant with the 96 kDa Sp1. In addition, the 96 kDa Sp1 was co-precipitated with ligand-activated hPRA or hPRB in the decidual cell nuclear extracts. These data suggest that dephosphorylated Sp1, abundant in decidual cells, enhances the binding to both DNA and hPR resulting in a robust increase of the hPR promoter activity.
authors.affiliation: Department of Obstetrics/Gynecology and Reproductive Medicine, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.
authors.forename: Meiyi
authors.initials: M
authors.lastname: Tang
authors.forename: James
authors.initials: J
authors.lastname: Mazella
authors.forename: Jiaguo
authors.initials: J
authors.lastname: Gao
authors.forename: Linda
authors.initials: L
authors.lastname: Tseng
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003656:Decidua / Q000166:cytology / Q000378:metabolism; D004717:Endometrium / Q000378:metabolism*; D005260:Female; D006801:Humans; D008875:Middle Aged; D010766:Phosphorylation / Q000187:drug effects; D011374:Progesterone / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011980:Receptors, Progesterone / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051705:Sp3 Transcription Factor; D017154:Stromal Cells / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D011374:Progesterone; D000469:Alkaline Phosphatase