Susumu Takekoshi

Expression and localization of leptin receptor in the normal rat pituitary gland

Abstract. Leptin receptor (leptin-R) is a polypeptide consisting of a single transmembrane-spanning component. Recent studies performed by reverse transcriptase polymerase chain reaction (RT-PCR) have shown the production of leptin-R in various tissues including the pituitary, hypothalamus and reproductive organs. The localization of leptin-R protein in the pituitary gland, however, has not been extensively studied. This study deals with the expression of leptin-R in the normal rat pituitary gland, which was disclosed primarily in the plasma membrane fraction by immunoblotting and immunohistochemical staining methods. Double immunohistochemical staining revealed that the colocalization of leptin-R and anterior pituitary hormone expression was seen mainly in growth hormone (GH)-secreting cells (97.4±1.3%; GH-positive cells/leptin-R-positive cells), but in less than 1% of prolactin (PRL)-, adrenocorticotropic hormone (ACTH)-, thyroid-stimulating hormone-β (TSHβ)- and follicle-stimulating hormone-β (FSHβ)/luteinizing hormone-β (LHβ)-positive cells. In contrast, leptin was localized most frequently in FSHβ/LHβ- and less frequently in TSHβ-positive cells. The above findings suggest that, in the rat anterior pituitary gland, there are paracrine relationships between leptin-producing cells and cells with leptin-R, which may regulate the function of GH cells.

Determination of nucleotide sequence of cDNA coding rat glutathione peroxidase and diminished expression of the mRNA in selenium deficient rat liver

The cDNA for rat glutathione peroxidase mRNA was isolated from liver cDNA library in lambda gt11 by cross-hybridization using the mouse cDNA, and its nucleotide sequence was determined. The selenocysteine which constitutes an active center of this enzyme was encoded by TGA, a nonsense codon in general, as was the cases with mouse and human glutathione peroxidase. Northern blot analysis elucidated that the mRNA for glutathione peroxidase was markedly diminished in selenium deficient rat liver as compared with that of normal rat livers. The result suggested that the de novo synthesis of the mRNA would be regulated by selenium.

Therapeutic strategy targeting the mTOR-HIF-1α-VEGF pathway in ovarian clear cell adenocarcinoma

Malignant tumors usually involve a relatively hypoxic state, which induces overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) to satisfactorily enable the tumor to survive. Thus, inhibition of the mammalian target of rapamycin (mTOR) pathway including HIF‐1α is expected to play a major role in suppression of tumor cell growth, having recently drawn much attention as an anti‐cancer therapeutic strategy for various malignant tumors. In the present study, which compared clear cell adenocarcinoma (CLA) of the ovary with serous adenocarcinoma (SEA), the immunohistochemical expression of mTOR, phosphorylated‐mTOR (p‐mTOR), HIF‐1α, and vascular endothelial growth factor (VEGF) was examined in surgically resected specimens of 29 SEA and 47 CLA. There were no significant differences in expression of mTOR, HIF‐1α and VEGF between SEA and CLA, but it was noted that p‐mTOR expression was more prominent in CLA than SEA. Then, using the cell lines of CLA (RMG‐1 and W3uF), an experimental study was designed to clarify whether tumor suppression due to downregulation of mTOR activity could represent a promising therapeutic strategy for CLA. After treatment of an analogue of rapamycin (everolimus), expression of mTOR, p‐mTOR, HIF‐1α and VEGF was examined on western blot. As a result, although mTOR expression remained unchangeable, expression of p‐mTOR, HIF‐1α and VEGF was shown to be sharply depressed. The same expression alterations were demonstrated in the xenograft model treated with everolimus. In conclusion, mTOR‐targeted therapy through usage of drugs such as everolimus may be more effective for CLA of the ovary because of its significant expression of p‐mTOR.

Pathology of the human pituitary adenomas

This article describes pertinent aspects of histochemical and molecular changes of the human pituitary adenomas. The article outlines individual tumor groups with general, specific and molecular findings. The discussion further extends to the unusual adenomas or carcinomas. The description in this article are pertinent not only for the practicing pathologists who are in the position of making proper diagnosis, but also for the pituitary research scientists who engage in solving basic problems in pituitary neoplasms by histochemistry and molecular biology.

Angiotensin II type 2 receptor signaling significantly attenuates growth of murine pancreatic carcinoma grafts in syngeneic mice

BackgroundPancreatic cancer is one of the most aggressive human malignancies, with a very poor prognosis. To evaluate the effect of angiotensin II (Ang II) type 2 receptor (AT2) expression in the hosts body on the growth of pancreatic carcinoma, we have investigated the growth of mouse pancreatic ductal carcinoma grafts in syngeneic wild type and AT2 receptor-deficient (AT2-KO) mice.MethodsThe role of AT2 receptor-signaling in stromal cells on the growth of murine pancreatic carcinoma cells (PAN02) was studied using various in vitro and in vivo assays. In vivo cell proliferation, apoptosis, and vasculature in tumors were monitored by Ki-67 immunostaining, TUNEL assay, and von Willebrand factor immunostaining, respectively. In the co-culture study, cell proliferation was measured by MTT cell viability assay. All the data were analyzed using t-test and data were treated as significant when p < 0.05.ResultsOur results show that the growth of subcutaneously transplanted syngeneic xenografts of PAN02 cells, mouse pancreatic ductal carcinoma cells derived from the C57/BL6 strain, was significantly faster in AT2-KO mice compared to control wild type mice. Immunohistochemical analysis of tumor tissue revealed significantly more Ki-67 positive cells in xenografts grown in AT2-KO mice than in wild type mice. The index of apoptosis is slightly higher in wild type mice than in AT2-KO mice as evaluated by TUNEL assay. Tumor vasculature number was significantly higher in AT2-KO mice than in wild type mice. In vitro co-culture studies revealed that the growth of PAN02 cells was significantly decreased when grown with AT2 receptor gene transfected wild type and AT2-KO mouse-derived fibroblasts. Faster tumor growth in AT2-KO mice may be associated with higher VEGF production in stromal cells.ConclusionsThese results suggest that Ang II regulates the growth of pancreatic carcinoma cells through modulating functions of host stromal cells; Moreover, Ang II AT2 receptor signaling is a negative regulator in the growth of pancreatic carcinoma cells. These findings indicate that the AT2 receptor in stromal fibroblasts is a potentially important target for chemotherapy for pancreatic cancer.

Expression of Neuro D1 in Human Normal Pituitaries and Pituitary Adenomas

Many transcription factors have important roles in the function and differentiation of the human pituitary adenomas. Forkhead box gene transcription factor L2, Foxl2, is expressed during mouse pituitary development and co-localizes with the expression of α-glycoprotein hormone subunit (αGSU). In addition, Foxl2 regulates expression of the αGSU gene (Cga) in cell culture. To elucidate the functional role of FOXL2 in the human pituitary, we examined the expression and localization of FOXL2 in normal human pituitaries and various types of pituitary adenomas. Human pituitary adenomas were obtained by trans-sphenoidal surgery from 67 patients. Three normal adult pituitaries were obtained from autopsies of non-endocrine cases. The localization of FOXL2 and pituitary hormones in these pituitary patients was examined by immunohistochemical staining and RT–PCR. Quantitative analysis of FOXL2 protein was performed by immunoblotting. FOXL2 was localized in the nuclei of ∼20% of normal pituitary cells that also co-expressed gonadotropins including follicule-stimulating hormone β (FSHβ), luteinizing hormone β (LHβ), and αGSU, whereas it was observed in minor proportion of thyroid-stimulating hormone (TSH)-producing cells, prolactin (PRL)-producing cells, and precursor of adrenocorticotropic hormone (ACTH)-producing cells. FOXL2 immunoreactivity was not detected in growth hormone (GH)-producing cells or S100-positive folliculo-stellate cells. In human pituitary adenomas, FOXL2 was expressed in the nuclei of the adenoma cells. FOXL2 was detected in 13 of 15 gonadotropin-subunit-producing adenoma (Gn-oma) cases and 8 of 11 null cell adenoma cases, but its incidence was reduced or not detected in the other types of adenomas. The results of this study suggest that FOXL2 contributes to the human-specific functional expression and the differentiation of gonadotroph cells and adenomas.

Prohormone convertases (PC1/3 and PC2) in rat and human pancreas and islet cell tumors: Subcellular immunohistochemical analysis

Prohormons convertase 1/3 (PC1/3; also termed PC1 or PC3) and PC2 are enzymes that activate prohormones by cleaving the pairs of basic amlno acids. This mechanlsm was inltlally Interred lrom the series of several endocrine and neuroendocrine precursor protoh, inciudlng proinsulin and prolusion. To determine the cellular and sub cellular distribution of PC1/3 and PC2 in the rat snd human pancreas, Immunohlstochemistry was performed using polyclonal antlers against mouse PC1/3 (ST‐28) and mouse PC2 (ST‐29). These studles showed light and dsctron mlcroacoplc co‐locailzation of Insulln, PC1/3 and PC2, and the coexistence of glucagons and PC2 In the pancreatic islets. This tendency of colocalizstion was also depicted In one case of human insulin and three cam of human glucagonomas, as well as In rat Insullnomas. in two cases of human Insullnomas, Incomplete processing of proinsulin was suggested by the absence of PC2. At the sub cellular level in the rat pancreatic lslet, the colocalizstion of PC1/3 and insulin, and that of PC2 and glucagons, were observed in the same secretor granules by immunoelectron, microscopy and Image analysis. These studles suggest that PC1/3 and PC2 can functlon with the specifictties In the processing of proinsulin and proglucagon Into their active forms, respectively, in the normal and neoplastic pancreatic islets.

Expression of Wnt4 in Human Pituitary Adenomas Regulates Activation of the β-Catenin-Independent Pathway

The Wnt signaling pathway has been implicated in the genesis of numerous human cancers. A member of the Wnt family of genes, Wnt4, has been known to regulate proliferation of anterior pituitary cell types in the mouse during embryonic development. In order to elucidate the roles of Wnt signaling in human pituitary adenomas, we examined the expression of Wnt4 and its putative receptor Frizzled6 (Fzd6) by immunohistochemistry in pituitary adenomas and normal pituitaries. Expression of Wnt4 was higher in growth hormone-producing adenomas (GHomas), prolactin-producing adenomas (PRLomas), and thyroid-stimulating hormone-producing adenomas (TSHomas) than in the normal pituitary. Fzd6 was widely expressed in GHomas, PRLomas, TSHomas, and gonadotropin subunit (GnSU)-positive adenomas. In normal pituitary glands, Wnt4 and Fzd6 were colocalized predominantly in follicle-stimulating hormone-, luteinizing hormone-, and α-subunits of glycoprotein hormone-positive cells. The canonical Wnt/β-catenin signaling pathway was analyzed by β-catenin immunohistochemistry. β-Catenin was localized at the cell membrane in all pituitary adenomas, but not in the nuclei. On the other hand, Erk1/2 was highly activated in GHomas and TSHomas. These results suggested that activation of Wnt4/Fzd6 signaling through a “β-catenin-independent” pathway played a role in proliferation and survival of the pituitary adenoma cells. Detailed involvement of transcription factors including Pit-1 remains to be further investigated.

Fruit-juice concentrate of Asian plum inhibits growth signals of vascular smooth muscle cells induced by angiotensin II.

Bainiku-ekisu, the fruit-juice concentrate of the Oriental plum (Prunus mume) has recently been shown to improve human blood fluidity. We have shown that angiotensin II (AngII) stimulates growth of vascular smooth muscle cells (VSMCs) through epidermal growth factor (EGF) receptor transactivation that involves reactive oxygen species (ROS) production. To better understanding the possible cardiovascular protective effect of Bainiku-ekisu, we have studied whether Bainiku-ekisu inhibits AngII-induced growth promoting signals in VSMCs. Bainiku-ekisu markedly inhibited AngII-induced EGF receptor transactivation. H(2)O(2)-induced EGF receptor transactivation was also inhibited by Bainiku-ekisu. Thus, Bainiku-ekisu markedly inhibited AngII-induced extracellular signal-regulated kinase (ERK) activation. However, EGF-induced ERK activation was not affected by Bainiku-ekisu. AngII stimulated leucine uptake in VSMCs that was significantly inhibited by Bainiku-ekisu. Also, Bainiku-ekisu possesses a potent antioxidant activity. Since the activation of EGF receptor, ERK and the production of ROS play central roles in mediating AngII-induced vascular remodeling, these data suggest that Bainiku-ekisu could exert a powerful cardiovascular protective effect with regard to cardiovascular diseases.

Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor

BACKGROUND Metastin, the final peptide of the KiSS-1 gene, has been proposed to suppress cell motility. OBJECTIVE This study investigated whether renal cell carcinoma (RCC) tissue expresses metastin or its receptor, and clarified whether metastin can suppress migration and/or invasion and/or proliferation of RCC cells in vitro. DESIGN, SETTING, AND PARTICIPANTS Twenty-five RCC samples were submitted. Fresh RCC tissues were prepared for real-time RT-PCR, and formalin-fixed and paraffin-embedded tissues blocks were examined by immunohistochemistry. RCC cell lines Caki-1 and ACHN were supplied for cell migration, invasion, and proliferation assays. MEASUREMENTS Real-time RT-PCR was performed by using Taq Man gene expression system. ENVISION system was used in immunohistochemistry. Wound-healing assay and matrigel assays were used to identify migration and invasion abilities of RCC cell lines. Cell Counting Kit-8 was applied to measure the cell proliferation. Cell morphology was examined under a META system. Statistical analysis was performed with SPSS15.0J. RESULTS AND LIMITATIONS In twenty-five RCC samples, the mRNA level of metastin receptor was identified to be significantly higher than non-neoplastic renal cortex by real-time RT-PCR (p=0.011). Immunohistochemical study also detected metastin receptor protein in all RCC tumors. In vitro, this study showed that metastin inhibited migration and invasion of Caki-1 and ACHN cells. In contrast, it had no effects on cell proliferation. Metastin (10 micromol/l) induced excessive formation of focal adhesions and stress fibers in Caki-1 and ACHN cells; this phenomenon was inhibited by pretreating pharmacological Rho-kinase inhibitor (Y-27632) to those cells. CONCLUSION This is the first report regarding overexpression of the metastin receptor hOT7T175 in human RCC. We demonstrate that metastin can inhibit migration and invasion of the RCC cell line, which is regulated by a Rho-kinase inhibitor. Metastin and its receptor are therefore probable targets for suppressing RCC.