Estrogen has been considered to enhance FSH actions in the ovary, including the induction of the LH receptor (LHR). In this study, we elucidated the mechanism underlying the effect of estrogen on the induction of LHR by FSH in rat granulosa cells. Estradiol clearly enhanced the FSH-induced LHR mRNA increase in a time- and dose-dependent manner, with a maximum increase of approximately 3.5-fold at 72 h, compared with the level of LHR mRNA solely induced by FSH. We then investigated whether the effect of estrogen on LHR mRNA was due to increased transcription and/or altered mRNA stability. A luciferase assay with the plasmid containing the LHR 5'-flanking region did not show that estradiol increased the promoter activity induced by FSH. In contrast, the decay curves for LHR mRNA showed a significant increase in half-life with FSH and estradiol, suggesting that the increased stability of LHR mRNA is at least responsible for the regulation of LHR mRNA by estrogen. Recently mevalonate kinase (Mvk) was identified as a trans-factor that binds to LHR mRNA and alters LHR mRNA stability in the ovary. We found that estradiol, with FSH, decreased Mvk mRNA levels in rat granulosa cell culture, resulting in up-regulation of LHR mRNA that was inversely correlated to Mvk mRNA expression. Furthermore, the augmentation of FSH-induced LHR expression in the presence of estrogen was erased with the overexpression of Mvk by transient transfection. Taken together, these data indicate that LHR mRNA is up-regulated due to increased stability when estrogen negatively controls Mvk.

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Glucose-regulated protein, 78-kilodalton (GRP78) is a molecular chaperone that exists in the endoplasmic reticulum and is involved in the assembly, transportation, and folding of proteins. Previously, GRP78 was reported to associate with gonadotropin receptors. However, little is known about how GRP78 is involved in the regulation of luteinizing hormone receptor (LHR). Thus, in this study, we investigated the significance of GRP78 for the induction of LHR in rat luteinizing granulosa cells. Western blot analysis of rat LHR expressed in HEK293 cells revealed that the protein levels of LHR were increased, depending on the increment of GRP78 protein. In both in vivo and in vitro experiments, the GRP78 mRNA level peaked while LHR mRNA was down-regulated by human chorionic gonadotropin (hCG). To examine the time-dependent localization of GRP78 in vivo, immunohistochemistry was performed. GRP78 was expressed mainly in granulosa cells, and the GRP78 protein peaked 18 h after the ovulatory dose of hCG injection in equine chorionic gonadotropin-primed immature rats. To ascertain the role of GRP78 in LHR after down-regulation, small interfering GRP78 was transfected to cultured rat granulosa cells, demonstrating that knockdown of the GRP78 protein level impaired the recovery of cell surface LHR from down-regulation that negatively affected progesterone synthesis. Moreover, luciferase assays showed that CRE mediated the hCG-induced promoter activity of GRP78 in rat luteinizing granulosa cells. These results reveal a novel mechanism of LHR by GRP78 in the early stage of corpus lustrum formation, which may be an important factor in the recovery of LHR after the down-regulation.

BackgroundThe epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, has been clinically applied for the treatment of a variety of tumors with EGFR overexpression. A phase II clinical study of erlotinib (NCIC IND-148) for recurrent or metastatic endometrial carcinoma (EC) resulted in an unfavorable result. However, in that study, the expression levels of EGFR were not accurately analyzed. Thus, the aim of this study was to re-examine the efficacy of erlotinib in EC cells by utilizing in vitro and in vivo models.MethodsTissue samples obtained from patients histologically diagnosed with EC of the uterine corpus were subjected to immunohistochemistry and RT-PCR to determine the protein and mRNA expression levels of EGFR. Western blot and WST-1 assays of EGFR siRNA-transfected HEC-1A, KLE, and Ishikawa cells were used to evaluate the efficacy of erlotinib in tumor cell lines expressing different EGFR levels. Furthermore, HEC-1A and Ishikawa cells were implanted into athymic mice treated with either erlotinib or trastuzumab.ResultsAt our institution, 20.9 % of endometrial cancer patients with low grade endometrioid histology have been diagnosed as stage III and IV. Immunohistochemical analysis and RT-PCR revealed the presence of significant EGFR and EGFR mRNA expression in low-grade endometrioid carcinoma in comparison with high-grade endometrioid carcinoma. In vitro study, WST-1 assay and Western blot analysis revealed that EGFR expression levels were correlated with tumor cell viability. Erlotinib reduced the proliferation of HEC-1A expressing high levels of EGFR, while trastuzumab showed similar effect in Ishikawa cells dominantly expressing human epidermal growth factor receptor type2 (HER2). In vivo erlotinib decreased tumor growth in mice xenografted with HEC-1A cells, whereas this tumor-growth inhibition was not observed in trastuzumab-treated mice xenografted with Ishikawa cell.ConclusionsEGF contributed to tumor proliferation in EC cell lines along with EGFR expression in vitro. Erlotinib also demonstrated anti-tumor effects in xenograft mice models. Our results suggest that erlotinib continues to have clinical usefulness in specific cases, after taking into consideration the EGFR expression levels.

The results of this study suggest that high UCHL1 expression is a strong marker of poor prognosis of endometrial cancer. Furthermore, we suggest that UCHL1 may be involved in the development of distant metastasis in endometrial cancer.