Stimulation of aromatase P450 promoter (II) activity in endometriosis and its inhibition in endometrium are regulated by competitive binding of steroidogenic factor-1 and chicken ovalbumin upstream promoter transcription factor to the same cis-acting element

In stromal cells of endometriosis, marked levels of aromatase P450 (P450aro m) mRNA and activity are present and can be vigorously stimulated by (Bu)(2 )cAMP or PGE(2) to give rise to physiologically significant estrogen biosyn thesis. Since eutopic endometrial tissue or stromal cells lack P450arom exp ression, we studied the molecular basis for differential P450arom expressio n in endometriosis and eutopic endometrium. First, we demonstrated by rapid amplification of cDNA 5'-ends that P450arom expression in pelvic endometri otic lesions is regulated almost exclusively via the alternative promoter I I. Then, luciferase reporter plasmids containing deletion mutations of the 5'-flanking region of promoter II were transfected into endometriotic strom al cells. We identified two critical regulatory regions for cAMP induction of promoter II activity: 1) a -214/-100 bp proximal region responsible for a 3.7-fold induction, and 2) a -517/-214 distal region responsible for pote ntiation of cAMP response up to 13-fold. In the -214/-100 region, we studie d eutopic endometrial and endometriotic nuclear protein binding to a nuclea r receptor half-site (NRHS, AGGTCA) and an imperfect cAMP response element (TGCACGTCA). Using electrophoretic mobility shift assay, cAMP response elem ent-binding activity in nuclear proteins from both endometriotic and eutopi c endometrial cells gave rise to formation of identical DNA-protein complex es. The NRHS probe, on the other hand, formed a distinct complex with nucle ar proteins from endometriotic cells, which migrated at a much faster rate compared with the complex formed with nuclear proteins from eutopic endomet rial cells. Employing recombinant proteins and antibodies against steroidog enic factor-1 (SF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF), we demonstrated that COUP-TF but not SF-1 bound to NRHS i n eutopic endometrial cells, whereas SF-1 was the primary NRHS-binding prot ein in endometriotic cells. In fact, COUP-TF transcripts were present in bo th eutopic endometrial (n = 12) and endometriotic tissues (n = 8), whereas SF-1 transcripts were detected in all endometriotic tissues (n = 12), but i n only 3 of 15 eutopic endometrial tissues. Moreover, we demonstrated a dos e-dependent direct competition between SF-1 and COUP-TF for occupancy of th e NRHS, to which SF-1 bound with a higher affinity. Finally, overexpression of SF-1 in eutopic endometrial and endometriotic cells strikingly potentia ted baseline and cAMP-induced activities of -517 promoter II construct, whe reas overexpression of COUP-TF almost completely abolished these activities . In conclusion, COUP-TF might be one of the factors responsible for the in hibition of P450arom expression in eutopic endometrial stromal cells, which lack SF-1 expression in the majority (80%) of the samples; in contrast, ab errant SF-1 expression in endometriotic stromal cells can override this inh ibition by competing for the same DNA-binding site, which is likely to acco unt for high levels of baseline and cAMP-induced aromatase activity.