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.

No abstract

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.

The luteinizing hormone receptor (LHR) is essential for elevated levels of progesterone to maintain pregnancy during the first trimester; the maintenance of the expression of LHR is a key factor controlling the duration of luteal function. Therefore, as the expression of LHR is most likely to be regulated by the stability of the receptor mRNA at the luteal phase of the human menstrual cycle, we focused on studies examining the stability of mRNA rather than the production of mRNA. In addition, LHR (exon 9), one of the splice variants of human LHR (hLHR), was cloned in the corpus luteum of a patient with a regular menstrual cycle. The results of Western blots using Percoll gradient fractionation indicated that hLHR formed complexes with hLHR (exon 9), which are transferred to the lysosome, where they are eventually degraded, instead of being translocated from the endoplasmic reticulum to the transducing organelle. These results showed that hLHR (exon 9) caused a reduction in the expression of functional receptor number and affected the signaling condition of wild-type hLHR. As the luteal phase progressed hLHR (exon 9) increased relative to hLHR, demonstrating that hLHR (exon 9) was expressed more than hLHR in the late luteal phase. This work reveals the essential function of the regulatory and structural elements involved in human LH receptor splicing, and that hLHR (exon 9) can negatively control the function of wild-type receptors. Moreover, this finding presented a novel mechanism of regulation of LHR in the human corpus luteum. (Reprod Med Biol 2008; 7: 11-16)

Abstract. Although the fallopian tube provides a sufficient environment for fertilization and early embryo development, the mechanism by which it does this is unclear. It is known that the transforming growth factorβ (TGFβ) superfamily plays important roles in various reproductive functions. Betaglycan, originally characterized as a TGFβ type III receptor lacking a clearly identifiable signaling motif, has been shown to be important for the high-affinity binding of TGFβs to the type II receptor. To our knowledge, there has been no study showing expression of betaglycan in the rat oviduct. Therefore, in this study, we examined the distribution of betaglycan in various rat tissues and its expression patterns in the oviduct during the estrous cycle. Northern blot analysis of various rat tissues showed that the adrenal gland, ovary and oviduct contained abundant amounts of 6.4-kb betaglycan mRNA. Furthermore, the mRNA level of betaglycan was highest after the LH surge that induced ovulation. The betaglycan protein, detected using immunohistochemistry, was especially abundant in the epithelium of the oviduct. Furthermore, in pregnant mare serum gonadotropin (PMSG) primed-rats, the expression of betaglycan was increased significantly by stimulation with human chorionic gonadotropin (hCG). RT-PCR analysis showed co-localization of other TGFβ family receptors (TGFβ types I and II, activin receptor types Ia and Ib and activin receptor types IIa and IIb) with betaglycan in the oviduct. Since betaglycan along with other TGFβ family receptors is abundantly expressed in the epithelium of the oviduct and its expression changes during estrous, it may also play an important role in the oviduct. Key words: Betaglycan, Estrous cycle, Oviduct, Rat, Transforming growth factor (TGF)-β (J. Reprod. Dev. 55: [200][201][202][203][204][205] 2009) etaglycan is a member-anchored proteoglycan originally characterized as a type III receptor for transforming growth factor-β1 (TGFβ), the prototype of a superfamily of growth factors involved in regulation of cell proliferation, differentiation and development. Betaglycan binds TGFβ isoforms with high affinity and increases the functional interaction between TGFβ and its type I and type II signaling receptors [1,2]. Rat betaglycan encodes a 91.6-kDa protein core that is further modified by N-linked glycosyl residues and by heparin and chondroitin sulfate side chains. The betaglycan protein consists of a large extracellular domain, a single transmembrane domain, and a short intracellular domain [3,4]. Although the short 43-amino acid cytoplasmic domain lacks an obvious signaling motif, it is enriched by serines and threonines [3,4]. Recently it has been demonstrated that betaglycan can bind inhibin A with high affinity and interfere with activin A to bind to its type II receptor [5]. It has been reported that betaglycan is expressed in the adrenal gland, ovary, uterus and pituitary, which have essential reproductive functions [6][7][8][9], whereas TGFβ are expressed in the ovary, testis, pre-a...

A 29-year-old man with von Recklinghausen's disease suddenly developed severe epigastric pain and was admitted to hospital. Physical examination revealed elevated blood pressure (200/130 mmHg) and tachycardia (162 bpm). Initially, he was suspected to have appendicitis, and appendectomy was performed immediately; however, appendicitis was not demonstrable pathologically. Retroperitoneal hematoma was found incidentally during the operation. Further clinical and laboratory examination demonstrated a markedincrease in the urinary excretion ofcatecholamines. Therewasno evidence of pheochromocytoma on computedtomographyor magneticresonance imaging; however, these imaging studies simply showed a hematoma at the right adrenal gland. Transient hypertension and tachycardia, resembling pheochromocytoma,was caused by adrenal hemorrhage. (Internal Medicine 36: 289-292, 1997)