Separate and synergistic effect of progesterone and estradiol on induction of annexin 2 and its interaction protein p11 in pregnant sheep myometrium

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Abstract
To search for myometrial candidate genes regulated by progesterone, we isolated annexin 2 cDNA by subtractive hybridization and cloning. We also examined the effect of estradiol and/or progesterone, individually or combined, on expressions of annexin 2 and its ligand protein, p11 in pregnant sheep intrauterine tissues. Annexin 2 is a Ca2+-dependent phospholipid-binding protein which interacts with p11 to form a bivalent heterotetramer. The heterotetramer was indicated in the production of prostaglandins through the regulation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Thus, annexin 2 and p11 could be the important players in Ca2+ signaling and prostaglandin production in uterine smooth muscles. Twenty-two ewes were treated with vehicle (n = 6), or 5 mg estradiol administered intramuscularly twice a day for 2 days (n = 6), or 100 mg progesterone administered intramuscularly twice a day for 14 days (n = 5), or estradiol plus progesterone with 100 mg progesterone administered intramuscularly twice a day for 10 days (n = 5) and then with vehicle for 2 days followed by estradiol for 2 days (5 mg administered intramuscularly twice a day). At 121 days of gestation age (dGA), endometrium, myometrium, placenta, and cervix were obtained under halothane anesthesia. Subtractive hybridization was used to isolate differentially expressed mRNAs from myometrial tissues treated with progesterone, which was confirmed by western blot at protein level. Annexin 2 and p11 interaction was validated by immunoprecipitation and their expressions in intrauterine tissues were determined by western blot analysis. Data were analyzed by ANOVA. A cDNA clone from myometrial-subtracted library representing differentially expressed mRNA in the pregnant sheep myometrium with progesterone treatment was identified as annexin 2 by sequence analysis and BLASTN search. Annexin 2 was present in both cytosolic and membrane-associated fractions of intrauterine tissues. In contrast, p11 was detectable only in myometrial cytosolic fraction. Both annexin 2 and p11 significantly increased in myometrial cytosolic fraction after progesterone or progesterone plus estradiol treatment. Furthermore, estradiol and progesterone combined had a more profound effect on induction of annexin 2 than progesterone alone. Annexin 2 was immunoprecipitated specifically by p11 antibody from the myometrium. This study indicates that progesterone may play an important role in the control of myometrial contractility by modifying protein expression associated with Ca2+ and prostaglandin signaling systems during pregnancy. Enhanced expression of myometrial annexin 2 in progesterone plus estradiol group supports our hypothesis that estrogen’s stimulation is optimized by progesterone’s priming in the pregnant sheep uterus.

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