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Dive into the research topics where Tomohiko Wakayama is active.

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Featured researches published by Tomohiko Wakayama.


Pharmaceutical Research | 2010

Gene Knockout and Metabolome Analysis of Carnitine/Organic Cation Transporter OCTN1

Yukio Kato; Yoshiyuki Kubo; Daisuke Iwata; Sayaka Kato; Tomohisa Sudo; Tomoko Sugiura; Takashi Kagaya; Tomohiko Wakayama; Akiyoshi Hirayama; Masahiro Sugimoto; Kazushi Sugihara; Shuichi Kaneko; Tomoyoshi Soga; Masahide Asano; Masaru Tomita; Toshiyuki Matsui; Morimasa Wada; Akira Tsuji

ABSTRACTPurposeSolute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1.MethodsWe first constructed octn1 gene knockout (octn1−/−) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined.ResultsThe metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1−/− mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [3H]ergothioneine, both of which were much reduced in octn1−/− mice. The octn1−/− mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn’s disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis.ConclusionsThese results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.


Nature Medicine | 2012

Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function

Kazuaki Yoshioka; Kotaro Yoshida; Hong Cui; Tomohiko Wakayama; Noriko Takuwa; Yasuo Okamoto; Wa Du; Xun Qi; Ken Asanuma; Kazushi Sugihara; Sho Aki; Hidekazu Miyazawa; Kuntal Biswas; Chisa Nagakura; Masaya Ueno; Shoichi Iseki; Robert J. Schwartz; Hiroshi Okamoto; Takehiko Sasaki; Osamu Matsui; Masahide Asano; Ralf H. Adams; Nobuyuki Takakura; Yoh Takuwa

The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell–specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate–enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.


Biology of Reproduction | 2003

Expression and Functional Characterization of the Adhesion Molecule Spermatogenic Immunoglobulin Superfamily in the Mouse Testis

Tomohiko Wakayama; Hiroyuki Koami; Hiroyuki Ariga; Daisuke Kobayashi; Yoshimichi Sai; Akira Tsuji; Miyuki Yamamoto; Shoichi Iseki

Abstract Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80–130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 spermatids and was present in the elongating spermatids until spermiation. The immunoreactivity was localized primarily on the cell membrane. Consistent with the findings in adult testes, the analysis of the developing testes revealed that SgIGSF was expressed separately in the spermatogenic cells in earlier and later phases. Sertoli cells had no expression of SgIGSF, whereas both SgIGSF immunoprecipitated from the testis lysate and produced in COS-7 cells was shown to bind to the surface of Sertoli cells in primary culture. These results suggested that SgIGSF on the surface of spermatogenic cells binds to some membrane molecules on Sertoli cells in a heterophilic manner and thereby may play diverse roles in the spermatogenesis.


Molecular Pharmacology | 2006

Organic Cation/Carnitine Transporter OCTN2 (Slc22a5) Is Responsible for Carnitine Transport across Apical Membranes of Small Intestinal Epithelial Cells in Mouse

Yukio Kato; Mikihiro Sugiura; Tomoko Sugiura; Tomohiko Wakayama; Yoshiyuki Kubo; Daisuke Kobayashi; Yoshimichi Sai; Ikumi Tamai; Shoichi Iseki; Akira Tsuji

The organic cation/carnitine transporter OCTN2 is responsible for renal tubular reabsorption of its endogenous substrate, carnitine, although its physiological role in small intestine remains controversial. Here we present direct evidence for a predominant role of OCTN2 in small intestinal absorption of carnitine based on experiments with juvenile visceral steatosis (jvs) mice, which have a hereditary deficiency of the octn2 gene. Uptake of carnitine, assessed with an Ussing-type chamber system, from the apical surface of the small intestine was saturable and higher than that from the basal surface in wild-type mice, whereas carnitine uptake having these characteristics was almost absent in jvs mice. Saturable uptake of carnitine was also confirmed in isolated enterocytes obtained from wild-type mice, and the Km value obtained (∼20 μM) was close to that reported for carnitine uptake by human embryonic kidney 293 cells stably expressing mouse OCTN2 (Slc22a5). The carnitine uptake by enterocytes was decreased in the presence of various types of organic cations, and this inhibition profile was similar to that of mouse OCTN2, whereas uptake of carnitine was quite small and unsaturable in enterocytes obtained from jvs mice. Immunohistochemical and immunoprecipitation analyses suggested colocalization of OCTN2 with PDZK1, an adaptor protein that functionally regulates OCTN2. Immunoelectron microscopy visualized both OCTN2 and PDZK1 in microvilli of absorptive epithelial cells. These findings indicate that OCTN2 is predominantly responsible for the uptake of carnitine from the apical surface of mouse small intestinal epithelial cells, and it may therefore be a promising target for oral delivery of therapeutic agents that are OCTN2 substrates.


Laboratory Investigation | 2003

Expression of the TSLC1 adhesion molecule in pulmonary epithelium and its down-regulation in pulmonary adenocarcinoma other than bronchioloalveolar carcinoma

Akihiko Ito; Morihito Okada; Kazuya Uchino; Tomohiko Wakayama; Yu-ichiro Koma; Shoichi Iseki; Noriaki Tsubota; Yutaka Okita; Yukihiko Kitamura

TSLC1 (tumor suppressor in lung cancer-1) is an adhesion molecule of the Ig superfamily that binds homophilically and mediates cell-cell interactions. Originally, TSLC1 was cloned as a candidate tumor suppressor from the genomic region that frequently exhibits loss of heterogeneity in human non–small-cell lung cancer (NSCLC). However, there have been no studies on TSLC1 expression in normal lungs or NSCLC. Here we show that pulmonary epithelial cells express TSLC1 and its expression levels are often decreased or lost in primary pulmonary adenocarcinoma, a major histologic type of NSCLC. Immunohistochemistry revealed that TSLC1 was localized at cell-cell boundaries of all columnar epithelial cells in mouse embryonic lungs of 10.5 and 13 days postcoitus. Similar staining patterns were observed in bronchiolar and alveolar epithelial cells of adult human lungs, suggesting a physiologic role for TSLC1 in interactions of these cells. Next we performed Western blot analyses of TSLC1 in 47 primary pulmonary adenocarcinomas and judged each tumor as either decreased or nondecreased by comparing TSLC1 expression levels of the tumor with the levels of normal lungs. The expression profiles had a significant relation to histologic subtypes but not to other clinicopathologic parameters. Sixteen bronchioloalveolar carcinomas (BACs) were all judged nondecreased, while 19 of 31 (63%) adenocarcinomas other than BAC were judged decreased (p < 0.0001). Immunohistochemistry of tumors judged nondecreased revealed that not only BAC cells but also tumor cells in lepidic growth components of adenocarcinomas other than BAC expressed TSLC1 on their lateral plasma membranes. These tumor cells are considered less invasive because they proliferate in a lepidic growth pattern along alveolar walls. Thus, the present results not only support the hypothesis that TSLC1 is a tumor suppressor of NSCLC but also suggest that preserved integrity of TSLC1 may contribute to less invasive phenotypes of lepidic growth tumor cells.


Drug Metabolism and Disposition | 2008

PDZK1 Regulates Two Intestinal Solute Carriers (Slc15a1 and Slc22a5) in Mice

Tomoko Sugiura; Yukio Kato; Tomohiko Wakayama; David L. Silver; Yoshiyuki Kubo; Shoichi Iseki; Akira Tsuji

Gastrointestinal (GI) absorption of certain therapeutic agents is thought to be mediated by solute carrier (SLC) transporters, although minimal in vivo evidence has been reported. Here, we show key roles of postsynaptic density 95/disk-large/ZO-1 (PDZ) domain-containing protein, PDZK1, as a regulatory mechanism of two solute carriers, Slc15a1 (oligopeptide transporter PEPT1) and Slc22a5 (carnitine/organic cation transporter OCTN2) in mouse small intestine by using pdzk1 gene knockout (pdzk1–/–) mice. GI absorption of cephalexin, a substrate of PEPT1, after p.o. administration was delayed in pdzk1–/– mice compared with wild-type mice. Absorption of carnitine, a substrate of OCTN2, was also decreased in pdzk1–/– mice. Immunohistochemical analysis revealed the localization of both PEPT1 and OCTN2 at apical membrane of small intestinal epithelial cells in wild-type mice, whereas such apical localization was reduced in pdzk1–/– mice, with a concomitant decrease in their protein levels assessed by Western blotting in intestinal brush-border membranes. Electron microscopy revealed localization of PEPT1 in intracellular vesicular structures in pdzk1–/– mice. In addition, we first identified interaction between PEPT1 and PDZK1 in mouse small intestine and found that PDZK1 stimulates transport activity of PEPT1 by increasing its expression level in human embryonic kidney 293 cells. Taken together, the present findings provide direct evidence that PDZK1 regulates two intestinal SLC transporters in vivo as an adaptor protein for these transporters and affects oral absorption of their substrates. These findings also raise the possibility that intestinal absorption of the substrate drugs for PEPT1 and OCTN2 is governed by the protein network of these transporters and their adaptor PDZK1.


Biology of Reproduction | 2007

Heterophilic Binding of the Adhesion Molecules Poliovirus Receptor and Immunoglobulin Superfamily 4A in the Interaction Between Mouse Spermatogenic and Sertoli Cells

Tomohiko Wakayama; Yoshimichi Sai; Akihiko Ito; Yukio Kato; Miho Kurobo; Yoshinori Murakami; Emi Nakashima; Akira Tsuji; Yukihiko Kitamura; Shoichi Iseki

Abstract The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.


Biochemical Pharmacology | 2012

Enhanced expression of organic anion transporting polypeptides (OATPs) in androgen receptor-positive prostate cancer cells: Possible role of OATP1A2 in adaptive cell growth under androgen-depleted conditions

Hiroshi Arakawa; Takeo Nakanishi; Chihiro Yanagihara; Tomohiro Nishimoto; Tomohiko Wakayama; Atsushi Mizokami; Mikio Namiki; Keiichi Kawai; Ikumi Tamai

The biological mechanisms underlying castration resistance of prostate cancer are not fully understood. In the present study, we examined the role of organic anion transporting polypeptides (OATPs) as importers of dehydroepiandrosterone sulfate (DHEAS) into cells to support growth under androgen-depleted conditions. Cell growth and mRNA expression of OATP genes were studied in human prostate cancer LNCaP and 22Rv1 cells under androgen-depleted conditions. The stimulatory effect of DHEAS on cell growth was investigated in LNCaP cells in which OATP1A2 had been silenced. Growth of both cell lines was stimulated by DHEAS and the effect was attenuated by STX64, an inhibitor of steroid sulfatase which can covert DHEAS to DHEA. OATP1A2 mRNA expression was increased most prominently among various genes tested in LNCaP cells grown in androgen-depleted medium. Similar results were obtained with 22Rv1 cells. Furthermore, the characteristics of [(3)H]DHEAS uptake by LNCaP cells were consistent with those of OATP-mediated transport. Knockdown of OATP1A2 in LNCaP cells resulted in loss of the DHEAS sensitivity of cell growth. Our results suggest that enhanced OATP1A2 expression is associated with adaptive cell growth of prostate cancer cells under androgen-depleted conditions. Thus, OATP1A2 may be a pharmacological target for prostate cancer treatment.


International Journal of Oncology | 2012

Angiotensin II enhances epithelial-to-mesenchymal transition through the interaction between activated hepatic stellate cells and the stromal cell-derived factor-1/CXCR4 axis in intrahepatic cholangiocarcinoma

Koichi Okamoto; Hidehiro Tajima; Shinichi Nakanuma; Seisho Sakai; Isamu Makino; Jun Kinoshita; Hironori Hayashi; Keishi Nakamura; Katsunobu Oyama; Hisatoshi Nakagawara; Hideto Fujita; Hiroyuki Takamura; Itasu Ninomiya; Hirohisa Kitagawa; Sachio Fushida; Takashi Fujimura; Shinichi Harada; Tomohiko Wakayama; Shoichi Iseki; Tetsuo Ohta

We previously reported that hepatic stellate cells (HSCs) activated by angiotensin II (AngII) facilitate stromal fibrosis and tumor progression in intrahepatic cholangiocarcinoma (ICC). AngII has been known as a growth factor which can promote epithelial-to-mesenchymal transition (EMT) in renal epithelial cells, alveolar epithelial cells and peritoneal mesothelial cells. However, in the past, the relationship between AngII and stromal cell-derived factor-1 (SDF-1) in the microenvironment around cancer and the role of AngII on EMT of cancer cells has not been reported in detail. SDF-1 and its specific receptor, CXCR4, are now receiving attention as a mechanism of cell progression and metastasis. In this study, we examined whether activated HSCs promote tumor fibrogenesis, tumor progression and distant metastasis by mediating EMT via the AngII/AngII type 1 receptor (AT-1) and the SDF-1/CXCR4 axis. Two human ICC cell lines and a human HSC line, LI-90, express CXCR4. Significantly higher concentration of SDF-1α was released into the supernatant of LI-90 cells to which AngII had been added. SDF-1α increased the proliferative activity of HSCs and enhanced the activation of HSCs as a growth factor. Furthermore, addition of SDF-1α and AngII enhanced the increase of the migratory capability and vimentin expression, reduced E-cadherin expression, and translocated the expression of β-catenin into the nucleus and cytoplasm in ICC cells. Co-culture with HSCs also enhanced the migratory capability of ICC cells. These findings suggest that SDF-1α, released from activated HSCs and AngII, play important roles in cancer progression, tumor fibrogenesis, and migration in autocrine and paracrine fashion by mediating EMT. Our mechanistic findings may provide pivotal insights into the molecular mechanism of the AngII and SDF-1α-initiated signaling pathway that regulates fibrogenesis in cancerous stroma, tumor progression and meta-stasis of tumor cells expressing AT-1 and CXCR4.


Reproduction | 2007

Transport of carnitine and acetylcarnitine by carnitine/organic cation transporter (OCTN) 2 and OCTN3 into epididymal spermatozoa

Daisuke Kobayashi; Ikumi Tamai; Yoshimichi Sai; Kazuhiro Yoshida; Tomohiko Wakayama; Yasuto Kido; Jun Ichi Nezu; Shoichi Iseki; Akira Tsuji

Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na(+)-dependent. Kinetic analyses revealed the presence of a high-affinity transport system in the spermatozoa, with K(m) values of 23.6 and 6.57 muM for carnitine and acetylcarnitine respectively in the presence of Na(+). Expression of OCTN2 and OCTN3 in epididymal spermatozoa was confirmed by immunofluorescence analysis. The involvement of these two transporters in carnitine and acetylcarnitine transport was supported by a selective inhibition study. We conclude that both Na(+)-dependent and -independent carnitine transporters, OCTN2 and OCTN3, mediate the supply of carnitine and acetylcarnitine to epididymal spermatozoa in mice.

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