Endometrial nuclear receptor co-factors SRC-1 and N-CoR are increased in human endometrium during menstruation

Molecular Human Reproduction Vol.8, No.7 pp. 644–650, 2002 Endometrial nuclear receptor co-factors SRC-1 and N-CoR are increased in human endometrium during menstruation F.Wieser1, C.Schneeberger1, G.Hudelist2, C.Singer2, C.Kurz1, F.Nagele1, C.Gruber1, J.C.Huber1 and W.Tschugguel1,3 1Division of Gynaecological Endocrinology and Reproductive Medicine and 2Division of Special Gynaecology, Department of Obstetrics and Gynaecology, University of Vienna Medical School, Vienna, Austria 3To whom correspondence should be addressed. E-mail: Steroid hormone receptor co-factors are abundantly expressed in the uterus in order to modify steroid hormone receptor action, either leading to activation or repression of transcription in the endometrium. However, the role of co-factors in remodelling of the human endometrium has not been established. We therefore endeavoured to evaluate the presence of the co-activator SRC (steroid receptor co-activator)-1 and the co-repressors N-CoR (nuclear receptor co-repressor) and steroid co-repressor SMRT (silencing mediator of retinod and thyroid) receptors in the human endometrium during the different phases of the menstrual cycle. By using a real-time RT–PCR assay, we showed that SRC-1, N-CoR and SMRT mRNA are expressed in human endometrium during all phases of the menstrual cycle, as well as in inactive endometrium. Moreover, endometrial expression of SRC-1 and N-CoR mRNA increased during menstruation when compared with the other phases of the menstrual cycle (P < 0.001). Immunohistochemistry demonstrated that SRC-1 and N-CoR stain positive in the glandular epithelium and stroma in menstrual phase endometrium. The staining was weak in proliferative and secretory endometrium and absent in inactive endometrium. Our results suggest that differential expression of endometrial steroid receptor co-factors probably play a role in the regulation of human endometrium remodelling. Key words: endometrium/menstrual cycle/N-CoR/SMRT/SRC-1 Introduction In the human menstrual cycle, the endometrium undergoes a regular sequence of proliferation, differentiation and degeneration in response to the fluctuation of steroid hormone levels (Irwin et al., 1991). Steroid hormones are known to exert their effects on cell growth, development and differentiation in the human endometrium through intracellular steroid hormone receptors (SHRs), which are present in endometrial epithelium and stroma (Lessey et al., 1988; Tseng and Zhu, 1997; Matsuzaki et al., 1999). Several models have been proposed to explain the exact mechanisms by which ligand binding to SHRs activates gene transcription in hormone responsive tissues such as the endometrium and the breast (Thenot et al., 1999; Fu et al., 2000; Wang et al., 2000). Unliganded SHRs are attached to receptor-associated proteins termed heat shock proteins, which stabilize the receptor in an inactivated state. The binding of steroids to SHRs leads to dissociation of heat shock proteins, receptor dimerization, phosphorylation and binding of the activated SHR to hormone response elements (HREs) in the promoter region of target genes. The ligand–SHR complexes activate or repress gene transcription machinery through protein–protein interaction with other sequencespecific transcription factors or chromatin factors, or cross-talk with other signal transduction pathways (Beato and Klug, 2000; Nilsson et al., 2001). Katzenellenbogen and O’Malley proposed a tripartite model (ligand–receptor–co-factor)with liganded steroid receptors working through complexing proteins, i.e. hormone co-activators enhancing transcription (Katzenellenbogen et al., 1996). 644 A growing number of co-factors have been identified to mediate initiation (co-activators) or repression (co-repressors) of target gene transcription mediated by nuclear receptors in several organs (Horwitz et al., 1996; Torchia et al., 1998). In terms of transcriptional activation of estrogen receptors, the 160 kDa steroid receptor co-activator proteins which include SRC (steroid receptor co-activator)-1 and the p300/CBP (cAMP-response-element binding proteins) (Smith et al., 1996; Beato and Klug, 2000) are the most important. The mechanisms of co-factors imply several modes of action. SRC-1 enhances stabilization of the pre-initiation complex by direct or indirect interaction with general transcription factors (Liu et al., 1999). A second activating mechanism of SRC-1 is that it displays histone acetylase activity (Sterner and Berger, 2000). Acetylation of histones is thought to remodel the chromatin, stabilize the pre-initiation complex, and hence facilitate transcription. In contrast, the repression of transcription is yielded by association of agonist- or antagonist-occupied receptors with co-repressors, including N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid receptors) (Jackson et al., 1997; Lavinsky et al., 1998). The co-repressors SMRT and N-CoR establish an unfavourable chromatin structure for transcriptional activation as a result of their histone deacetylase activity (Yang et al., 1996), thereby finally leading to the repression of transcription of SHRs (Labrie et al., 1999). These co-repressors may also compete with the SRC proteins, demonstrating that the balance of co-activators and co-repressors may finally regulate transcription (Torchia et al., 1998; © European Society of Human Reproduction and Embryology Increased SRC-1 and N-CoR expression during menstruation Beato et al., 2000). SHRs can exist in different conformational states, each of which exhibits a different degree of transcriptional activity. The degree of these ligand-induced conformational changes in the SHRs regulates the interaction of the receptor with co-activators (Wagner et al., 1995). Co-activator mRNA for SRC-1, GRIP (glucocorticoid receptor interacting protein)-1, RAC (receptor-associated co-activator)-3 and transcriptional co-factor p300 and a co-repressor mRNA for SMRT have been shown to be expressed in human endometrial stromal cells and in the female reproductive system in rat uteri (Brosens et al., 1999; Nephew et al., 2000). However, whether or not mRNA expression levels of SHR co-factors in human endometrium might vary in response to the action of steroid hormones during the natural menstrual cycle remains to be clarified. Therefore, the objective of this study was to assess the presence of the steroid hormone receptor co-factors SRC-1, N-CoR and SMRT in human endometrium during the menstrual cycle at mRNA and protein levels using a real-time RT–PCR assay and immunohistochemistry respectively. Materials and methods Tissue collection Samples of proliferative and secretory endometrium were obtained by hysterectomy or curettage specimens from premenopausal women who underwent surgery for benign disease including uterine leiomyoma or ovarian lesions and who had no history of endometriosis. The samples of menstrual phase endometrium were taken fro (...truncated)