Researcher Database


Freeword

KUROYANAGI Gen

FacultyGraduate School of Medical Sciences Core Laboratory
PositionAssistant Professor
Mail
HomepageURL
Birthday
Last Updated :2020/07/02

Researcher Profile and Settings

Profile & Settings

    Research funding number:80790831

Association Memberships

  • JAPAN COLLEGE OF RHEUMATOLOGY
  • THE JAPANESE ORTHOPAEDIC ASSOCIATION

Research Activities

Research Areas

  • Life sciences, Orthopedics

Research Interests

    osteonecrosis, interleukin-6, osteoblast, ischemic osteonecrosis

Published Papers

  • Interleukin-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis in a murine model., Kuroyanagi G, Adapala NS, Yamaguchi R, Kamiya N, Deng Z, Aruwajoye O, Kutschke M, Chen E, Jo C, Ren Y, Kim HKW, Bone, 116, 221 - 231, 08 , Refereed
  • Treatment of Lateral Tibial Condylar Fractures Using Bioactive, Bioresorbable Forged Composites of Raw Particulate Unsintered Hydroxyapatite/Poly-L-Lactide Screws., Kuroyanagi G, Yoshihara H, Yamamoto N, Suzuki H, Yamada K, Yoshida Y, Otsuka T, Takada N, Orthopedics, 41, (3) e365 - e368, 05 , Refereed
  • Ristocetin induces phosphorylated-HSP27 (HSPB1) release from the platelets of type 2 DM patients: Anti-platelet agent-effect on the release., Tokuda H, Kuroyanagi G, Onuma T, Enomoto Y, Doi T, Iida H, Otsuka T, Ogura S, Iwama T, Kojima K, Kozawa O, Biomedical reports, 8, (4) 365 - 372, 04 , Refereed
  • Suppression by HSP90 inhibitors of BMP-4-stimulated osteoprotegerin synthesis in osteoblasts: Attenuation of p70 S6 kinase, Tetsu Kawabata, Takanobu Otsuka, Kazuhiko Fujita, Shingo Kainuma, Naohiro Yamamoto, Gen Kuroyanagi, Go Sakai, Rie Matsushima-Nishiwaki, Osamu Kozawa, Haruhiko Tokuda, MOLECULAR MEDICINE REPORTS, 16, (6) 8507 - 8512, 12 , Refereed, Heat shock protein 90 (HSP90) is an ATP-dependent ubiquitous molecular chaperon which is important in cell homeostasis. The authors previously demonstrated that bone morphogenetic protein (BMP)-4 stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates the OPG synthesis by BMP-4. The present study investigated the involvement of HSP90 in the BMP-4-stimulated OPG synthesis and the mechanism in MC3T3-E1 cells. HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG), 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG) and geldanamycin significantly suppressed the BMP-4-stimulated OPG release. Geldanamycin markedly reduced the BMP-4-induced mRNA expression of OPG. 17-AAG and 17-DMAG significantly attenuated the phosphorylation of p70 S6 kinase induced by BMP-4 without affecting the BMP-4-induced phosphorylation of mothers against decapentaplegic homolog 1/5. The results suggest that HSP90 inhibitors suppress the BMP-4-stimulated OPG synthesis in osteoblasts, and that their suppressive effects are exerted through downregulating p70 S6 kinase.
  • Amplification by (-)-epigallocatechin gallate of prostaglandin F-2 alpha-stimulated synthesis of osteoprotegerin in osteoblasts, Go Sakai, Takanobu Otsuka, Kazuhiko Fujita, Shingo Kainuma, Gen Kuroyanagi, Tetsu Kawabata, Rie Matsushima-Nishiwaki, Osamu Kozawa, Haruhiko Tokuda, MOLECULAR MEDICINE REPORTS, 16, (5) 6376 - 6381, 11 , Refereed, (-)-Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), major flavonoids in green tea, and coffee, respectively, are recognized as possessing potential benefits in a multitude of human health conditions, including bone disorders. We have previously demonstrated that prostaglandin F-2 alpha (PGF(2 alpha)), a potent bone remodeling mediator, stimulates the synthesis of osteoprotegerin (OPG) through the activation of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK and stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of EGCG and CGA on PGF(2 alpha)-stimulated OPG synthesis in MC3T3-E1 cells were investigated. EGCG significantly upregulated PGF(2 alpha)-stimulated OPG release, whereas CGA did not affect OPG release. The PGF(2 alpha)-induced expression level of OPG mRNA was enhanced by EGCG. Regarding the intracellular signaling underlying the effect of EGCG, EGCG failed to affect PGF(2 alpha)-stimulated phosphorylation of p44/p42 MAPK, p38 MAPK or SAPK/JNK. EGCG by itself markedly induced the phosphorylation of p44/p42 MAP kinase for up to 10 min and the status decreased subsequently, whereas EGCG did not significantly affect the phosphorylation status of p38 MAPK or SAPK/JNK within 60 min. These results indicated that EGCG, but not CGA amplifies the PGF(2 alpha)-stimulated OPG synthesis in osteoblasts.
  • Resveratrol suppresses thyroid hormone-induced osteocalcin synthesis in osteoblasts, Kazuhiko Fujita, Haruhiko Tokuda, Shingo Kainuma, Gen Kuroyanagi, Naohiro Yamamoto, Rie Matsushima-Nishiwaki, Atsushi Harada, Osamu Kozawa, Takanobu Otsuka, MOLECULAR MEDICINE REPORTS, 16, (3) 2881 - 2886, 09 , Refereed, Resveratrol, a polyphenolic compound that is present in grape skins, berries and red wine, may be beneficial for human health through its anti-inflammatory and anti-oxidant effects. It has been previously demonstrated that resveratrol exerts its biological effects primarily via sirtuin 1 (SIRT1) activation. We previously reported that triiodothyronine (T-3) induces osteocalcin synthesis in osteoblast-like MC3T3-E1 cells, and that p38 mitogen-activated protein (MAP) kinase mediates the T-3-stimulated synthesis of osteocalcin. The present study investigated the effect of resveratrol on T-3-induced osteocalcin synthesis and its underlying mechanism in MC3T3-E1 cells. Cultured cells were stimulated with T-3, and osteocalcin release from MC3T3-E1 cells was measured by ELISA and phosphorylation of p38 MAP kinase was analyzed by western blotting. Resveratrol significantly suppressed the release of osteocalcin stimulated by T-3, and SRT1720, a SIRT1 activator, significantly reduced T-3-induced osteocalcin release. The expression level of osteocalcin mRNA stimulated by T-3 was significantly attenuated by resveratrol and T-3-induced transactivation activity of the thyroid hormone-responsive element was significantly diminished by resveratrol. However, only limited effects of resveratrol on the T-3-induced phosphorylation of p38 MAP kinase were observed. The results of the present study demonstrated that resveratrol suppresses T-3-stimulated osteocalcin synthesis at a point upstream of transcription in osteoblasts, and that the inhibitory effect of resveratrol is mediated, at least partially, through SIRT1 activation. These results indicate that there may be a novel role for the polyphenol in the modulation of bone metabolism.
  • HSP90 inhibitors potentiate PGF(2 alpha)-induced IL-6 synthesis via p38 MAP kinase in osteoblasts, Kazuhiko Fujita, Haruhiko Tokuda, Gen Kuroyanagi, Naohiro Yamamoto, Shingo Kainuma, Tetsu Kawabata, Go Sakai, Rie Matsushima-Nishiwaki, Osamu Kozawa, Takanobu Otsuka, PLOS ONE, 12, (5) , 05 , Refereed, Heat shock protein 90 (HSP90) that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F-2 alpha(PGF(2 alpha)), a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6) through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF(2 alpha)-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF(2 alpha)-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG) and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG) and onalespib, enhanced the PGF(2 alpha)-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF(2 alpha)-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF(2 alpha)-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF(2 alpha)-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF(2 alpha)-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF(2 alpha)-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.
  • Incretins amplify TNF-alpha-stimulated IL-6 synthesis in osteoblasts: Suppression of the IB/NF-kappa B pathway, Kazuhiko Fujita, Haruhiko Tokuda, Naohiro Yamamoto, Shingo Kainuma, Tetsu Kawabata, Go Sakai, Gen Kuroyanagi, Rie Matsushima-Nishiwaki, Atsushi Harada, Osamu Kozawa, Takanobu Otsuka, INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, 39, (4) 1053 - 1060, 04 , Refereed, Incretins including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) secreted from the small intestine after oral food ingestion are currently recognized to stimulate insulin secretion from pancreatic cells. We previously reported that p70 S6 kinase limits the tumor necrosis factor- (TNF-)-stimulated interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of incretins on the TNF--induced IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. GLP-1 and GIP significantly upregulated both TNF--stimulated IL-6 release and mRNA levels. Wedelolactone, an inhibitor of IB kinase, amplified the TNF--induced IL-6 release. GLP-1 significantly attenuated the TNF--induced phosphorylation of IB without affecting the phosphorylation of p70 S6 kinase. On the other hand, GLP-1 markedly induced the phosphorylation of cAMP response element-binding protein (CREB). H-89, an inhibitor of protein kinase A, significantly suppressed the enhancement by GLP-1 of TNF--stimulated IL-6 release. Dibutyryl cAMP, a permeable analogue of cAMP, which suppressed the TNF--induced IB phosphorylation, amplified the IL-6 release. These results strongly suggest that incretins upregulate the TNF--stimulated IL-6 synthesis in osteoblasts, and that the amplifying effect of incretin is exerted via reducing the IB/NF-B pathway through the adenylyl cyclase-cAMP system.
  • Attenuation of prostaglandin E-1-induced osteoprotegerin synthesis in osteoblasts by normoxic HIF inducers, Gen Kuroyanagi, Haruhiko Tokuda, Naohiro Yamamoto, Shingo Kainuma, Kazuhiko Fujita, Reou Ohguchi, Rie Matsushima-Nishiwaki, Osamu Kozawa, Takanobu Otsuka, MOLECULAR MEDICINE REPORTS, 15, (4) 1847 - 1852, 04 , Refereed, Mimosine, which is a natural plant amino acid present in the Leucaena genus, is able to induce hypoxia-inducible factors (HIFs). Previous evidence has indicated that HIF regulates angiogenesis-osteogenesis coupling in bone metabolism, and it has previously been reported that mimosine inhibits prostaglandin (PG)F-2 alpha-induced osteoprotegerin (OPG) synthesis without affecting interleukin-6 (IL-6) production in osteoblast-like MC3T3-E1 cells. In addition, PGE(1) has been demonstrated to induce OPG synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in these cells, and PGE(1) stimulates IL-6 production via the activation of protein kinase A. In the present study, the effects of mimosine on the PGE(1)-stimulated synthesis of OPG and IL-6 were investigated in osteoblast-like MC3T3-E1 cells. The concentrations of OPG and IL-6 were measured using relevant ELISA kits. OPG mRNA was measured by semi-quantitative reverse transcription polymerase chain reaction. The phosphorylation of p38 MAP kinase and SAPK/JNK was analyzed by western blotting. Mimosine significantly reduced PGE(1)-induced release of OPG and OPG mRNA expression levels without affecting the release of IL-6. In addition, deferoxamine, which is also a normoxic HIF inducer, significantly inhibited PGE(1)-induced OPG release and OPG mRNA expression levels; however, it had little effect on IL-6 release. Furthermore, mimosine and deferoxamine failed to affect PGE(1)-stimulated phosphorylation of p38 MAP kinase or SAPK/JNK. These results strongly suggest that normoxic HIF inducers attenuate PGE(1)-stimulated OPG synthesis without affecting IL-6 production in osteoblasts.
  • Heat shock protein 22 (HSPB8) limits TGF-beta-stimulated migration of osteoblasts, Naohiro Yamamoto, Haruhiko Tokuda, Gen Kuroyanagi, Shingo Kainuma, Rie Matsushima-Nishiwaki, Kazuhiko Fujita, Osamu Kozawa, Takanobu Otsuka, MOLECULAR AND CELLULAR ENDOCRINOLOGY, 436, (C) 1 - 9, 11 , Refereed, Heat shock proteins (HSPs) are induced in response to various physiological and environmental conditions such as chemical and heat stress, and recognized to function as molecular chaperones. HSP22 (HSPB8), a low-molecular weight HSP, is ubiquitously expressed in many cell types. However, the precise role of HSP22 in bone metabolism remains to be clarified. In the present study, we investigated whether HSP22 is implicated in the transforming growth factor-beta (TGF-beta)-stimulated migration of osteoblast-like MC3T3-E1 cells. Although protein levels of HSP22 were clearly detected in unstimulated MC3T3-E1 cells, TGF-beta failed to induce the protein levels. The TGF-beta-stimulated migration was significantly up-regulated by knockdown of HSP22 expression. The cell migration stimulated by platelet-derived growth factor-BB was also enhanced by HSP22 knockdown. SB203580, an inhibitor of p38 mitogen-activated protein kinase, PD98059, an inhibitor of MEK1/2, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase had no effects on the TGF-beta-induced migration. SIS3, a specific inhibitor of TGF-beta-dependent Smad3 phosphorylation, significantly reduced the migration with or without TGF-beta stimulation. Smad2, Smad3, Smad4 or Smad7 was not coimmunoprecipitated with HSP22. On the other hand, the TGF-beta-induced Smad2 phosphorylation was enhanced by HSP22 down-regulation. The protein levels of TGF-beta type II receptor (TGF-beta RII) but not TGF-beta type I receptor (TGF-beta RI) was significantly up-regulated in HSP22 knockdown cells compared with those in the control cells. However, the levels of TGF-beta RII mRNA in HSP22 knockdown cells were little different from those of the control cells. Neither TGF-beta RI nor TGF-beta RII was coimmunoprecipitated with HSP22. SIS3 reduced the amplification by HSP22 knockdown of the TGF-beta-stimulated cell migration almost to the basal level. Our results strongly suggest that HSP22 functions as a negative regulator in the TGF-beta-stimulated migration of osteoblasts via suppression of the Smad-dependent pathway, resulting from modulating the protein levels of TGF-beta RII. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
  • Mimosine suppresses the PGF(2 alpha)-induced synthesis of osteoprotegerin but not interleukin-6 in osteoblasts, Gen Kuroyanagi, Takanobu Otsuka, Naohiro Yamamoto, Shingo Kainuma, Reou Ohguchi, Kazuhiko Fujita, Rie Matsushima-Nishiwaki, Osamu Kozawa, Haruhiko Tokuda, INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, 37, (2) 533 - 541, 02 , Refereed, Mimosine, a plant amino acid, is known to act as a normoxic inducer of hypoxia-inducible factor (HIF). Previous research has suggested that HIF plays important roles in angiogenesis-osteogenesis coupling and bone metabolism. We previously reported that prostaglandin F-2 alpha (PGF(2 alpha)) induced osteoprotegerin synthesis through p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. We have also demonstrated that PGF(2 alpha) induced the synthesis of interleukin-6 (IL-6) via p38 MAP kinase and p44/p42 MAP kinase but not SAPK/JNK in these cells. In the present study, we investigated the effects of mimosine on the PGF(2 alpha)-induced synthesis of osteoprotegerin or IL-6 in MC3T3-E1 cells. We found that deferoxamine, another inducer of HIF, as well as mimosine, upregulated the protein levels of HIF-1 alpha. Both mimosine and deferoxamine significantly suppressed the PGF(2 alpha)-induced release of osteoprotegerin, and the mRNA expression level, without markedly affecting PGF(2 alpha)-induced IL-6 release. Both mimosine and deferoxamine, by themselves, induced the release of vascular endothelial growth factor. The phosphorylation of p38 MAP kinase, p44/p42 MAP kinase or SAPK/JNK induced by PGF(2 alpha) was not markedly affected by either mimosine or deferoxamine. Thus, the results of the present study strongly suggest that mimosine, a normoxic inducer of HIF, inhibits the PGF(2 alpha)-induced osteoprotegerin synthesis without affecting the IL-6 synthesis in osteoblasts.
  • Amplification by (-)-epigallocatechin gallate and chlorogenic acid of TNF-alpha-stimulated interleukin-6 synthesis in osteoblasts, Naohiro Yamamoto, Haruhiko Tokuda, Gen Kuroyanagi, Shingo Kainuma, Reou Ohguchi, Kazuhiko Fujita, Rie Matsushima-Nishiwaki, Osamu Kozawa, Takanobu Otsuka, INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, 36, (6) 1707 - 1712, 12 , Refereed, Polyphenolic compounds in foods and beverages have beneficial effects on human health. (-)-Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), a major flavonoid in green tea and a major phenolic acid in coffee, respectively, have potent properties, including antioxidative effects. Our previous study demonstrated that p70 S6 kinase acts as a negative regulator in tumor necrosis factor-alpha (TNF-alpha)-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, the effects of EGCG and CGA on the TNF-alpha-stimulated interleukin-6 synthesis were investigated in MC3T3-E1 cells. EGCG and CGA significantly enhanced TNF-alpha-stimulated interleukin-6 release. In addition, the interleukin-6 mRNA expression levels induced by TNF-alpha were supported by EGCG, as well as CGA. EGCG markedly attenuated the TNF-alpha-induced phosphorylation of p70 S6 kinase whereas CGA failed to affect the phosphorylation. These results strongly suggest that EGCG and CGA enhance the TNF-alpha-stimulated interleukin-6 synthesis in osteoblasts, and that the amplifying effect of EGCG, but not CGA, is exerted via inhibiting p70 S6 kinase.
  • alpha B-crystallin reduces ristocetin-induced soluble CD40 ligand release in human platelets: Suppression of thromboxane A(2) generation, Masanori Tsujimoto, Tomoaki Doi, Gen Kuroyanagi, Naohiro Yamamoto, Rie Matsushima-Nishiwaki, Yuko Iida, Yukiko Enomoto, Hiroki Iida, Shinji Ogura, Takanobu Otsuka, Haruhiko Tokuda, Osamu Kozawa, Toru Iwama, MOLECULAR MEDICINE REPORTS, 12, (1) 357 - 362, 07 , Refereed, Our group has previously shown that B-crystallin (HSPB5), a small heat shock protein, inhibits human platelet aggregation by ristocetin, an activator of glycoprotein Ib/IX/V. In addition, it was demonstrated that glycoprotein Ib/IX/V activation induces soluble CD40 (sCD40) ligand release via thromboxane (TX) A(2). In the present study, the effect of B-crystallin on the ristocetin-induced sCD40 ligand release in human platelets was investigated. The ristocetin-induced release of sCD40 ligand was suppressed by B-crystallin. In addition, B-crystallin reduced the ristocetin-stimulated production of 11-dehydro-TX B-2, a stable metabolite of TXA(2). B-crystallin did not suppress the platelet aggregation induced by U46619, a TXA(2) receptor agonist. B-crystallin had little effect on the U46619-induced phosphorylation of p38 mitogen-activated protein kinase or sCD40 ligand release. In addition, B-crystallin failed to reduce the binding of SZ2, a monoclonal antibody against the sulfated sequence in the -chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that B-crystallin extracellularly suppresses ristocetin-stimulated release of sCD40 ligand by inhibiting the TXA(2) production in human platelets.
  • Resveratrol suppresses TGF-beta-induced VEGF synthesis in osteoblasts: Inhibition of the p44/p42 MAPK and SAPK/JNK pathways, Gen Kuroyanagi, Takanobu Otsuka, Naohiro Yamamoto, Rie Matsushima-Nishiwaki, Osamu Kozawa, Haruhiko Tokuda, EXPERIMENTAL AND THERAPEUTIC MEDICINE, 9, (6) 2303 - 2310, 06 , Refereed, Resveratrol, which is found in grape and berry skins and red wine, is generally known to be beneficial for human health due to its anti-inflammation and antioxidant effects. We have recently reported that transforming growth factor-beta (TGF-beta) stimulates vascular endothelial growth factor (VEGF) synthesis through Smad-independent pathways, such as the p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways, in osteoblast-like MC3T3-E1 cells. The aim of the present study was to investigate the effect of resveratrol on the TGF-beta-induced VEGF synthesis and the mechanism in osteoblast-like MC3T3-E1 cells. Resveratrol significantly suppressed the TGF-beta-stimulated release of VEGF and the VEGF mRNA expression levels. SRT1720, a synthetic sirtuin 1 (SIRT1) activator, also reduced the VEGF release and the mRNA levels. With regard to the intracellular signaling in the TGF-beta-stimulated VEGF synthesis, resveratrol and SRT1720 significantly attenuated the phosphorylation of p44/p42 MAP kinase and SAPK/JNK stimulated by TGF-beta; however, the TGF-beta-induced phosphorylation of Smad2 and p38 MAP kinase was hardly affected by resveratrol or SRT1720. These results strongly suggest that the TGF-beta-stimulated VEGF synthesis is suppressed by resveratrol through the inhibition of p44/p42 MAP kinase and SAPK/JNK in osteoblasts, and that the suppressive effect is mediated, at least in part, via SIRT1 activation.
  • Rac limits TGF-beta-induced VEGF synthesis in osteoblasts, Naohiro Yamamoto, Takanobu Otsuka, Akira Kondo, Rie Matsushima-Nishiwaki, Gen Kuroyanagi, Osamu Kozawa, Haruhiko Tokuda, MOLECULAR AND CELLULAR ENDOCRINOLOGY, 405, (C) 35 - 41, 04 , Refereed, We previously showed that transforming growth factor-beta (TGF-beta) stimulates vascular endothelial growth factor (VEGF) synthesis via p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rac, which is a member of the Rho family of small GTPases, in the TGF-beta-stimulated VEGF synthesis in MC3T3-E1 cells. TGF-beta markedly increased the levels of GTP-bound Rac. NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, significantly increased both the release of VEGF and the mRNA expression levels induced by TGF-beta. In addition, the release of VEGF stimulated by TGF-beta was amplified in Rac-knock down cells. Meanwhile, SIS3, a specific inhibitor of TGF-beta-dependent Smad3 phosphorylation, significantly reduced the TGF-beta-stimulated VEGF release. However, the phosphorylation of Smad2 or Smad3 induced by TGF-beta was hardly affected by NSC23766. On the other hand, NSC23766 enhanced the TGF-beta-induced phosphorylation of p38 MAP kinase without affecting the phosphorylation of p44/p42 MAP kinase or SAPK/JNK. Furthermore, the phosphorylation of p38 MAP kinase induced by TGF-beta was markedly upregulated in the Racknock down cells. These results strongly suggest that Rac negatively regulates the TGF-beta-stimulated VEGF synthesis via the inhibition of p38 MAP kinase in osteoblasts. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
  • Regulation by heat shock protein 22 (HSPB8) of transforming growth factor-alpha-induced ovary cancer cell migration, Mariko Suzuki, Rie Matsushima-Nishiwaki, Gen Kuroyanagi, Noriko Suzuki, Reika Takamatsu, Tatsuro Furui, Naoki Yoshimi, Osamu Kozawa, Ken-ichirou Morishige, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 571, 40 - 49, 04 , Refereed, Accumulating evidence suggests that heat shock proteins (HSPs) are implicated in progression of cancer. HSP22 (HSPB8), a small HSP, is recognized to be ubiquitously expressed in various tissues. However, the expression and the role of HSP22 in ovarian cancer remain to be clarified. In the present study, we investigated the involvement of HSP22 in transforming growth factor (TGF)-alpha-induced migration of ovarian cancer cells. The expression of HSP22 was detected in a serous ovarian cancer cell line, SKOV3.ip1. The migration was reduced by down-regulation of HSP22 expression. The TGF-alpha-induced migration was reduced by SB203580 (a p38 MAP kinase inhibitor), SP600125 (a SAPK/JNK inhibitor) and Y27632 (a Rho-kinase inhibitor). However, down-regulation of HSP22 had little effect on the TGF-alpha-induced phosphorylation of p38 MAP kinase, SAPK/JNK and MYPT, a target protein of Rho-kinase. The HSP22 expression was further analyzed in 20 resected specimens of human ovarian serous carcinoma. The expression of HSP22 was detected in-all the twenty tissues (8.24-109.22 pg/mg protein), and the cases with highly expression of HSP22 showed a tendency to acquire the progressive ability. Our results strongly suggest that HSP22 acts as a positive regulator in TGF-alpha-induced migration of ovarian cancer cells, subsequently directing ovarian cancer toward progression. (C) 2015 Elsevier Inc. All rights reserved.
  • Involvement of Rac in thromboxane A(2)-induced human platelet activation: Regulation of sCD40 ligand release and PDGF-AB secretion, Yasunari Kageyama, Tomoaki Doi, Rie Matsushima-Nishiwaki, Yuko Iida, Shigeru Akamatsu, Akira Kondo, Gen Kuroyanagi, Naohiro Yamamoto, Jun Mizutani, Takanobu Otsuka, Haruhiko Tokuda, Hiroki Iida, Osamu Kozawa, Shinji Ogura, MOLECULAR MEDICINE REPORTS, 10, (1) 107 - 112, 07 , Refereed, We have previously shown that glycoprotein Ib/IX/V activation stimulates the release of the soluble CD40 ligand (sCD40L) via the generation of thromboxane A(2) from human platelets. In the present study, the role of Rac, which is a member of the Rho family, was investigated in the thromboxane A(2)-stimulated release of platelet-derived growth factor (PDGF)-AB and sCD40L in human platelets. U46619, a thromboxane receptor agonist, stimulated the activation of Rac time-dependently in human platelets, and NSC23766, a selective inhibitor of the Rac-guanine nucleotide exchange factor interaction, reduced the U46619-induced platelet aggregation. NSC23766 markedly suppressed the U46619-induced p38 mitogen-activated protein (MAP) kinase phosphorylation. The thromboxane A(2)-induced release of PDGF-AB and sCD40L was significantly suppressed by NSC23766 in a dose-dependent manner. In addition, NSC23766 reduced the sCD40L release stimulated by ristocetin, a glycoprotein Ib/IX/V activator. These results indicate that Rac regulates the thromboxane A(2)-induced stimulation of PDGF-AB secretion and sCD40L release via the p38 MAP kinase in human platelets.
  • Resveratrol suppresses prostaglandin F-2 alpha-induced osteoprotegerin synthesis in osteoblasts: Inhibition of the MAP kinase signaling, Gen Kuroyanagi, Haruhiko Tokuda, Rie Matsushima-Nishiwaki, Akira Kondo, Jun Mizutani, Osamu Kozawa, Takanobu Otsuka, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 542, 39 - 45, 01 , Refereed, Resveratrol, a natural polyphenol abundantly found in grape skins and red wine, possesses various beneficial properties for human health. In the present study, we investigated the mechanism underlying the effects of prostaglandin F-2 alpha (PGF(2 alpha)) on osteoprotegerin (OPG) synthesis and of resveratrol on the OPG synthesis in osteoblast-like MC3T3-E1 cells. PGF(2 alpha) stimulated both the release of the OPG protein and the expression of OPG mRNA. Treatment with PD98059, SB203580 and SP600125, specific inhibitors of MEK1/2, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) all suppressed the OPG release induced by PGF(2 alpha). Resveratrol also significantly reduced the PGF(2 alpha)-stimulated OPG release and the mRNA levels of OPG. Similarly, treatment with SRT1720, an activator of SIRT1, also suppressed the PGF(2 alpha)-stimulated OPG release. Resveratrol and SRT1720 both attenuated the phosphorylation of p44/p42 MAP kinase, MEK1/2, Raf-1, p38 MAP kinase and SAPK/JNK induced by PGF(2 alpha). These findings strongly suggest that resveratrol suppresses PGF(2 alpha)-stimulated OPG synthesis by inhibiting the MAP kinase pathways in osteoblasts, and that the effect is mediated via SIRT1 activation. (C) 2013 Elsevier Inc. All rights reserved.
  • Unphosphorylated heat shock protein 27 suppresses fibroblast growth factor-2-stimulated vascular endothelial growth factor release in osteoblasts, Akira Kondo, Haruhiko Tokuda, Rie Matsushima-Nishiwaki, Kenji Kato, Gen Kuroyanagi, Jun Mizutani, Muneyoshi Fukuoka, Ikuo Wada, Osamu Kozawa, Takanobu Otsuka, MOLECULAR MEDICINE REPORTS, 8, (2) 691 - 695, 08 , Refereed, Heat shock protein 27 (HSP27) also known as heat shock protein beta 1 (HSPB1) is a member of the family of small heat shock proteins ubiquitously expressed in all tissues. It has previously been demonstrated that HSP27 regulated the synthesis of osteocalcin and interleukin-6 in osteoblast-like MC3T3-E1 cells. In the present study, the effect of HSP27 on basic fibroblast growth factor (FGF-2) -stimulated vascular endothelial growth factor (VEGF) synthesis in MC3T3-E1 cells, was observed. The levels of VEGF release stimulated by FGF-2 in the HSP27-overexpressing MC3T3-E1 cells were significantly lower compared with those in the control cells. In addition, the levels of VEGF release stimulated by FGF-2 in the phosphomimic HSP27-overexpressing cells were significantly higher compared with those in the non-phosphorylatable HSP27-overexpressing cells. Furthermore, no significant differences were observed in the FGF-2-induced phosphorylation levels of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) or p70 S6 kinase among the four types of transfected cells. These results suggested that unphosphorylated HSP27 attenuated the FGF-2-stimulated VEGF synthesis in osteoblasts.
  • Inhibition of SAPK/JNK leads to enhanced IL-1-induced IL-6 synthesis in osteoblasts., Kondo A, Otsuka T, Matsushima-Nishiwaki R, Kuroyanagi G, Mizutani J, Wada I, Kozawa O, Tokuda H, Archives of biochemistry and biophysics, 535, (2) 227 - 233, 07 , Refereed
  • Thrombopoietin amplifies ADP-induced HSP27 phosphorylation in human platelets: importance of pre-treatment., Cuong NT, Doi T, Matsushima-Nishiwaki R, Akamatsu S, Kuroyanagi G, Kondo A, Mizutani J, Wada I, Otsuka T, Tokuda H, Kozawa O, Ogura S, International journal of molecular medicine, 31, (6) 1291 - 1297, 06 , Refereed
  • Rho-kinase negatively regulates thyroid hormone-stimulated osteocalcin synthesis in osteoblasts., Kondo A, Tokuda H, Kato K, Matsushima-Nishiwaki R, Kuroyanagi G, Mizutani J, Kozawa O, Otsuka T, Biochimie, 95, (4) 719 - 724, 04 , Refereed
  • AMP-activated protein kinase inhibitor decreases prostaglandin F-2 alpha-stimulated interleukin-6 synthesis through p38 MAP kinase in osteoblasts, Akira Kondo, Takanobu Otsuka, Kenji Kato, Hideo Natsume, Gen Kuroyanaci, Jun Mizutani, Yoshiki Ito, Rie Matsushima-Nishiwaki, Osamu Kozawa, Haruhiko Tokuda, INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, 30, (6) 1487 - 1492, 12 , Refereed, We previously showed that prostaglandin F-2 alpha (PGF(2 alpha)) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in part via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of AMP-activated protein kinase (AMPK), an intracellular energy sensor, in PGF(2 alpha)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2 alpha) time-dependently induced the phosphorylation of the AMPK alpha-subunit. Compound C. an inhibitor of AMPK, dose-dependently suppressed PGF(2 alpha)-stimulated IL-6 release. Compound C reduced the PGF(2 alpha)-induced acetyl-CoA carhoxylase phosphorylation. In addition, PGF(2 alpha)-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The IL-6 mRNA expression induced by PGF(2 alpha) was markedly reduced by compound C. Downregulation of the AMPK alpha 1-subunit by short interfering RNA (siRNA) significantly suppressed the PGF(2 alpha)-stimulated IL-6 release. PGF(2 alpha)-induced phosphorylation of p38 MAP kinase was inhibited by compound C, which failed to affect the p44/p42 MAP kinase phosphorylation. These results strongly suggest that AMPK regulates PGF(2 alpha)-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts.

Awards & Honors

  •   2017 09 , American Society for Bone and Mineral Research, 2017 ASBMR Young Investigator Award


Copyright (c) MEDIA FUSION Co.,Ltd. All rights reserved.