The insulin-like growth factor (IGF) autocrine/paracrine system is believed to play a role in endometrial differentiation and trophoblast growth. The IGFs bind with high affinity to a family of binding proteins (IGFBPs) that regulate the actions of the IGFs at their target cells. IGFBP-1, one of three well-characterized IGFBPs in humans, has been shown by numerous investigators to be present in secretory but not proliferative endometrium; however, no information is available with regard to two other human IGFBPs, IGFBP-2 and IGFBP-3, in normal human endometrium. We have examined expression of the messenger RNAs (mRNAs) encoding IGFBP-2 and IGFBP-3 in human proliferative endometrium [under the influence of estradiol (E2)] compared to secretory endometrium (under the influence of E2 and progesterone). Using a complementary DNA probe specific for IGFBP-2, by Northern analysis, expression of a 1.4 kilobase mRNA was found to be differentially expressed in secretory compared to proliferative phase endometrium. Using a complementary DNA probe encoding IGFBP-3, a 2.4 kilobase mRNA was found in endometrium and was also found to be differentially expressed in secretory compared to proliferative phase endometrium. Endometrial stromal cells were established in culture and revealed constitutive synthesis and secretion of IGFBP-2 and IGFBP-3 into the conditioned medium (CM), as detected by Western ligand blot analysis of the CM. Identification of the IGFBPs in the CM was made using IGFBP-2 and IGFBP-3 specific antiserum. In the presence of E2 and progesterone, there was an enhancement in the amount of IGFBP-3 and a marked enhancement of IGFBP-2 in the conditioned media, suggesting sex steroid-dependence of IGFBP-2 and, to a lesser extent, IGFBP-3 protein synthesis. Western ligand blot analysis of IGFBPs in serum throughout the menstrual cycle revealed no hormonal dependence in the serum IGFBPs, suggesting local action of the IGFBPs in endometrium. These data thus show that mRNAs encoding IGFBP-2 and IGFBP-3 are expressed in human endometrium, that their expression is differentially regulated in secretory compared to proliferative phase endometrium (different steroidogenic environments), and that the synthesis of both IGFBP-2 and IGFBP-3 proteins is regulated by steroid hormones.
The insulin-like growth factors (IGF-I and -II) are believed to be important in endometrial differentiation and blastocyst nidation, and proteins that regulate IGF action (IGF-binding proteins, IGFBPs) are hormonally regulated in endometrium during the menstrual cycle. To characterize further steroid-dependence of the IGFBPs, we established endometrial stromal cells in culture in the absence and presence of oestradiol (E2) and progesterone (P) and examined the conditioned medium for IGFBPs by Western ligand blot analysis. Stromal cells constitutively synthesized IGFBP-3, IGFBP-2, a 27 kd, and a 24 kd IGFBP. In the presence of E2 and P, a 10- to 15-fold increase in IGFBP-2 was detected in the conditioned medium beginning after about 7 days in culture, when cells decidualized and steroid-mediated prolactin secretion began. Withdrawal of steroids resulted in a marked decrease in IGFBP-2, comparable to control levels, and cells increased their IGFBP-2 production when rechallenged with E2 and P. Total RNA was isolated from stromal cells, and Northern blot analysis using a cDNA probe specific for IGFBP-2 revealed differential expression of a 1.4 kb mRNA transcript in steroid-treated compared to control cells. The effects of progestational agents alone on IGFBP synthesis was also examined. Progesterone, medroxyprogesterone acetate and norethindrone all stimulated IGFBP-2 synthesis 12- to 15-fold compared to controls, and a progesterone receptor antagonist, RU 486, blocked the stimulatory effect of progesterone. IGFBP-2 synthesis was increased two-fold above controls by 17-alpha-hydroxyprogesterone, and RU 486 alone and hydrocortisone were without effect. Identification of IGFBP-2 in conditioned medium was made using IGFBP-specific antiserum. These data show that (a) endometrial stromal cells synthesize and secrete IGFBP-2, (b) IGFBP-2 protein synthesis is controlled by steroid hormones, (c) P, by interacting with its receptor, modulates IGFBP-2 synthesis and (d) expression of IGFBP-2 mRNA is controlled by sex steroids.
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