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Transcriptional Regulation of the Tissue Factor Gene by Progestins in Human Endometrial Stromal Cells¹

Departments of Obstetrics and Gynecology (G.K., S.G., C.J.L.) and Biochemistry (S.G.), New York University Medical Center, New York, New York 10016; and Departments of Immunology and Vascular Biology, The Scripps Research Institute (N.M.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Dr. Graciela Krikun, New York University Medical Center, Tisch Hospital, Room 533, 550 First Avenue, New York, New York 10016.

Decidualization of estradiol (E₂)-primed human endometrial cells (HESCs) by progesterone is associated with elevated levels of tissue factor (TF), the primary initiator of hemostasis. Similarly, in cultured human HESCs, the synthetic progestin, medroxyprogesterone acetate (MPA), enhances TF protein and messenger ribonucleic acid (mRNA) levels. Although ineffective alone, E₂ potentiates this progestin enhancement of TF expression by HESCs. The current study examines mechanisms underlying MPA enhancement of TF mRNA expression in HESCs. In the presence of the transcription-blocking agent dichlororibofuranosylbenzimidazole, no significant differences were noted in the half-lives of TF mRNA isolated from HESCs treated with E₂ alone or with E₂ plus MPA. This indicates that MPA-enhanced TF mRNA levels do not reflect changes in the stability of the TF message. To test the effect of progestin on TF promoter activity and to ascertain the mechanism of promoter regulation, primary or first passaged HESCs were transfected with TF promoter constructs spanning the regions -2106 to +121 (TFp_-2106), -278 to +121 (TFp_-278), and -111 to +14 (TFp_-111) bp upstream of the transcription start site. MPA was found to enhance TF transcription by 20-fold in HESCs transfected with TFp_-2106 after correcting for transfection efficiencies with a ß-galactosidase reporter plasmid. Interestingly, levels of E₂- plus MPA-stimulated transcription were significantly increased using TFp_-278 compared to TFp_-2106, suggesting that the region between -2106 and -278 bp may contain an inhibitory element. In addition, rates of MPA-stimulated transcription using TFp_-111 were significantly reduced compared to values obtained using TFp_-2106 and were even further reduced compared to values obtained using TFp_-278. This suggests that regulatory elements in the -111 bp region of the TF promoter are necessary for progestin-mediated regulation of the TF gene in HESCs, but are not sufficient to account for maximal rates of TF gene transcription.

Our results also demonstrated that induction of steady state TF mRNA by MPA was abolished by treating cells with E₂ plus MPA in conjunction with the protein synthesis inhibitor cycloheximide. In light of the absence of a complete progesterone or estrogen response element in the published 5'-sequence of the TF promoter, our results suggest that progestin-enhanced transcription of TF mRNA in stromal cells may be mediated by an uncharacterized protein intermediate(s).

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