Expression and regulation of transient receptor potential cation channel, subfamily M, member 2 (TRPM2) in human endometrium.

To identify estrogen-responsive genes, we previously isolated estrogen receptor (ER)-binding DNA fragments from human genomic DNA using a recombinant ER protein. Six DNA fragments, each including a perfect palindromic estrogen response element (ERE), were obtained. The nucleotide sequence of one of the six fragments (E1 fragment) showed that the ERE of the E1 fragment is located in the 3'-untranslated region (UTR) of transient receptor potential cation channel, subfamily M, member 2 (TRPM2). Here, we confirmed the estrogen-dependent enhancer activity of the ERE of the E1 fragment by chloramphenicol acetyltransferase assay. TRPM2 mRNA expression was investigated in human endometrium, cultured human endometrial stromal cells (ESCs), and cultured human endometrial epithelial cells (EECs) using RT-PCR. Quantitative RT-PCR revealed that TRPM2 mRNA expression in ESCs increased after 17ß-estradiol (E2) treatment. This study demonstrated for the first time that TRPM2 is an estrogen-responsive gene expressed in human endometrial cells.

The progesterone-responsive gene 14-3-3τ enhances the transcriptional activity of progesterone receptor in uterine cells.

Members of the 14-3-3 family are intracellular dimeric phosphoserine-binding proteins that can associate with and modulate the activities of many proteins. In our efforts to isolate the genes regulated by progesterone (P(4)) using suppressive subtractive hybridization, we previously found that 14-3-3τ is one of the genes upregulated by P(4). In this study, we demonstrated by quantitative RT-PCR (qRT-PCR), western blot analyses, and immunohistochemistry that 14-3-3τ mRNA and protein levels were increased in the rat uterus after P(4) treatment. Furthermore, qRT-PCR indicated that P(4) increased 14-3-3τ mRNA levels in human endometrial epithelial cells and endometrial stromal cells (ESCs). Western blot and qRT-PCR analyses revealed that in vitro decidualization using cAMP and medroxyprogesterone 17-acetate increased levels of 14-3-3τ mRNA and protein in ESCs. We have shown by qRT-PCR and western blot analyses that P(4) increased the mRNA and protein levels of 14-3-3τ in Ishikawa cells that stably express P(4) receptor-B (PR-B). Immunocytochemistry revealed that 14-3-3τ colocalizes with PR and translocates from the cytoplasm to the nucleus in response to P(4). Moreover, by luciferase reporter assay, we demonstrated that 14-3-3τ enhances the transcriptional activity of PR-B. Taken together, we propose that 14-3-3τ is a P(4)-responsive gene in uterine cells that modulates P(4) signaling.

Expression of retinoic acid-related orphan receptor alpha and its responsive genes in human endometrium regulated by cholesterol sulfate.

Cholesterol sulfate (CS) is a major sterol sulfate in human plasma that is detected in the uterine endometrium. CS plays a role in steroidogenesis, cellular membrane stabilization, and regulation of the skin barrier. We previously reported that CS increased in rabbit endometrium during the implantation period. Recently, CS has been reported to be a ligand of retinoic acid receptor-related orphan receptor alpha (RORA). NR1D1 is one of the genes regulated by RORA. In the present study, we investigated the regulation of RORA and NR1D1 by CS in human endometrium. We determined the association-dissociation curves for the interaction of CS with RORA and the kinetic rates by surface plasmon resonance. Immunohistochemical staining and in situ hybridization revealed that RORA and NR1D1 were expressed in human endometrial stromal and epithelial cells. CS treatment significantly induced the mRNA expression of RORA and NR1D1 mRNA in ESCs. The results of a luciferase assay showed that RORA significantly activated the human NR1D1 promoter regardless of CS. Our results suggest that CS regulates the expression of RORA responsive genes in human endometrial cells but not as a ligand for RORA.