-
[show abstract]
[hide abstract]
ABSTRACT: Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) which belongs to the MAP kinase superfamily regulates many cellular events. We previously reported that interleukin 1 (IL-1) stimulates the synthesis of interleukin 6 (IL-6) through activation of ERK and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that AMP-activated protein kinase (AMPK) negatively regulates the IL-1-induced IL-6 synthesis through IκB/NF-κB pathway. In the present study, we investigated the role of SAPK/JNK in the IL-1-stimulated IL-6 synthesis in these cells. IL-1 induced the phosphorylation of SAPK/JNK. SP600125, an inhibitor of SAPK/JNK, increased the release and the mRNA expression levels of IL-6 induced by IL-1. IL-1-stimulated IL-6 release was significantly up-regulated in SAPK/JNK-knocked down cells. SP600125 remarkably suppressed the IL-1-induced phosphorylation of both IκB and NF-κB, whereas SP600125 failed to affect the IL-1-induced phosphorylation of AMPK, STAT3 or Src. Compound C, an AMPK inhibitor, attenuated the IL-1-induced phosphorylation of SAPK/JNK. SP600125 enhanced IL-1-stimulated IL-6 release also in normal human osteoblasts. These results strongly suggest that SAPK/JNK negatively regulates IL-1-stimulated IL-6 synthesis and acts at the point between AMPK and IκB/NF-κB in osteoblasts.
Archives of Biochemistry and Biophysics 04/2013; · 2.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: AMP-activated protein kinase (AMPK) is recognized as a main regulator of energy homeostasis. Osteocalcin (OC), which is produced specifically by mature osteoblasts, is stored in bone matrix, strongly binds to hydroxyapatite and is released into the circulation, has been recognized as a marker of bone metabolism. It has recently been shown that OC released from osteoblasts influences energy metabolism as a hormone. We previously reported that triiodothyronine (T3) stimulates the synthesis of OC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether AMPK participates in T3-stimulated OC synthesis in osteoblasts. T3 time-dependently induced the phosphorylation of the AMPKα-subunit (Thr-172), whereas T3 failed to induce the phosphorylation of AMPKα-subunit (Ser-485), AMPKβ-subunit (Ser-108) and AMPKβ-subunit (Ser-182). Both the release and the mRNA expression of OC induced by T3 were significantly inhibited by compound C, an AMPK inhibitor. Compound C suppressed the T3-induced phosphorylation of acetyl-CoA carboxylase, a direct substrate of AMPK. T3-stimulated OC release was significantly reduced in AMPK-knockdown cells using AMPK-siRNA. These results strongly suggest that AMPK positively regulates T3-stimulated OC synthesis in osteoblasts.
International Journal of Molecular Medicine 04/2013; · 1.98 Impact Factor
-
Nguyen The Cuong,
Tomoaki Doi,
Rie Matsushima-Nishiwaki,
Shigeru Akamatsu,
Gen Kuroyanagi,
Akira Kondo,
Jun Mizutani,
Ikuo Wada,
Takanobu Otsuka, Haruhiko Tokuda,
Osamu Kozawa,
Shinji Ogura
[show abstract]
[hide abstract]
ABSTRACT: It has been shown that thrombopoietin (TPO) amplifies agonist-induced platelet activation. However, the precise mechanism of action of TPO has not yet been fully elucidated. We have previously reported that the adenosine diphosphate (ADP)‑induced phosphorylation of heat shock protein 27 (HSP27) via the p38 mitogen-activated protein (MAP) kinase pathway correlates with the ADP-induced platelet-derived growth factor (PDGF)-AB secretion and the release of soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of TPO on platelet activation induced by ADP. We examined the effects of TPO on ADP-induced platelet activation under different treatments: TPO was administered 15 min prior to stimulation with ADP (pre-treatment); TPO and ADP were simultaneously administered (simultaneous treatment); and TPO was administered 2 min following stimulation with ADP (post-treatment). TPO, which alone had no effect on platelet aggregation, synergistically enhanced the ADP (1 mM)-induced platelet aggregation only when it was administered prior to stimulation with ADP. Pre-treatment with TPO significantly increased the secretion of PDGF-AB and the release of sCD40L, and markedly enhanced the ADP-induced phosphorylation of p38 MAP kinase and HSP27 in the platelets. However, simultaneous treatment with TPO or TPO post-treatment failed to affect the ADP-induced platelet aggregation, the secretion of PDGF-AB, the release of sCD40L and the phosphorylation p38 MAP kinase or HSP27. These results strongly suggest that pre-treatment with TPO significantly amplifies ADP-induced HSP27 phosphorylation via the p38 MAP kinase pathway in human platelets.
International Journal of Molecular Medicine 04/2013; · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Evidence is accumulating that Rho-associated kinase (Rho-kinase) plays important roles not only in vascular smooth muscle cell contraction, but also in a variety of cellular functions, including bone metabolism. In the present study, we investigated the involvement of Rho-kinase in the osteocalcin synthesis induced by triiodothyronine (T(3)) in osteoblast-like MC3T3-E1 cells. T(3) time-dependently induced phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, attenuated the MYPT-1 phosphorylation induced by T(3). T(3)-stimulated osteocalcin release was significantly enhanced by Y27632. Fasudil, another Rho-kinase inhibitor, amplified the osteocalcin release induced by T(3). T(3)-stimulated osteocalcin release was significantly augmented in Rho-knockdown cells with Rho A-siRNA. Y27632 and fasudil also increased the mRNA expression level of osteocalcin induced by T(3). These results strongly suggest that T(3) stimulates the activation of Rho-kinase in osteoblasts, which functions as a negative regulator of T(3)-stimulated osteocalcin synthesis.
Biochimie 11/2012; · 3.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously showed that prostaglandin F2α (PGF2α) 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 PGF2α-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF2α time-dependently induced the phosphorylation of the AMPK α-subunit. Compound C, an inhibitor of AMPK, dose-dependently suppressed PGF2α-stimulated IL-6 release. Compound C reduced the PGF2α-induced acetyl-CoA carboxylase phosphorylation. In addition, PGF2α-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The IL-6 mRNA expression induced by PGF2α was markedly reduced by compound C. Downregulation of the AMPK α1-subunit by short interfering RNA (siRNA) significantly suppressed the PGF2α-stimulated IL-6 release. PGF2α-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 PGF2α-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts.
International Journal of Molecular Medicine 10/2012; · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To clarify the mechanism underlying a high risk of thrombotic complications in diabetic patients, we investigated the relationship between HSP27 phosphorylation and the platelet activation induced by adenosine diphosphate (ADP) in diabetic patients. Platelet-rich plasma was prepared from the blood of type 2 diabetes mellitus (DM) patients. By measuring the dose response of platelet aggregation to ADP, an individual ED50 was determined. Based on the normal range identified in non-DM controls, the subjects were divided into a hyper-aggregate (Group 1) and a normo- or hypo-aggregate group (Group 2). The protein phosphorylation was analyzed by western blotting. The release of PDGF-AB and sCD40 ligand (sCD40L) was measured by ELISA. In both groups, ADP induced HSP27 phosphorylation at Ser-78 and Ser-82. The phosphorylation at Ser-78 and the release of both PDGF-AB and sCD40L induced by a low dose of ADP (1 µM) in Group 1 were significantly higher than these values in Group 2. There was a significant relationship between the ADP-induced HSP27 phosphorylation level at Ser-78 and the ADP ED50 value of platelet aggregation. The ADP (1 µM)-induced phosphorylation of HSP at Ser-78 observed in the platelets from Group 1 was inhibited by PD98059 or SB203580. The use of aspirin ameliorated the accelerated microaggregation of platelets in Group 1, and the low-dose ADP-induced phosphorylation of HSP27 at Ser-78 was no longer observed. These results strongly suggest that the phosphorylation of HSP27 at Ser-78 is correlated with the acceleration of platelet aggregation induced by ADP in type 2 DM patients.
International Journal of Molecular Medicine 10/2012; · 1.98 Impact Factor
-
Tomoaki Doi, Haruhiko Tokuda,
Rie Matsushima-Nishiwaki,
Nguyen The Cuong,
Yasunari Kageyama,
Yuko Iida,
Akira Kondo,
Shigeru Akamatsu,
Takanobu Otsuka,
Hiroki Iida,
Osamu Kozawa,
Shinji Ogura
[show abstract]
[hide abstract]
ABSTRACT: We have previously shown that ristocetin, an activator of glycoprotein Ib/IX/V, induces release of soluble CD40 (sCD40) ligand via thromboxane (TX) A(2) production from human platelets. In the present study, we investigated the effect of antithrombin-III (AT-III), an anticoagulant, on the ristocetin-induced glycoprotein Ib/IX/V activation in human platelets. AT-III inhibited ristocetin-stimulated platelet aggregation. The ristocetin-induced production of 11-dehydro-TXB(2), a stable metabolite of TXA(2), and the release of sCD40 ligand were suppressed by AT-III. AT-III also reduced the ristocetin-stimulated secretion of platelet-derived growth factor (PDGF)-AB. AT-III failed to affect U46619-, a TXA(2) receptor agonist, induced levels of p38 mitogen-activated protein kinase phosphorylation or sCD40 ligand release. AT-III reduced the binding of SZ2, a monoclonal antibody to the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that AT-III reduced ristocetin-stimulated release of sCD40 ligand due to inhibiting TXA(2) production in human platelets.
Prostaglandins Leukotrienes and Essential Fatty Acids 07/2012; 87(2-3):57-62. · 3.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression of IL6 mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. The IL6 mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.
Journal of Molecular Endocrinology 05/2012; 49(1):47-55. · 3.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is known that Wnt3a affects bone metabolism via the canonical Wnt/β-catenin signaling pathway. We have previously shown that prostaglandin F2α (PGF2α) stimulates the synthesis of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) via mitogen-activated protein (MAP) kinases, including p44/p42 MAP kinase, p38 MAP kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of Wnt3a on the synthesis of VEGF or IL-6 stimulated by PGF2α in MC3T3-E1 cells using an ELISA kit and various antibodies. Cells were cultured and pretreated with various doses of Wnt3a or SB216763, an inhibitor of glycogen synthase kinase 3β, prior to western blotting. Wnt3a significantly enhanced the PGF2α-stimulated VEGF release but had little effect on the PGF2α-stimulated IL-6 release. SB216763 markedly amplified the PGF2α-stimulated VEGF release without affecting the IL-6 release, similar to Wnt3a. Wnt3a failed to affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by PGF2α via activation of the canonical pathway in osteoblasts without affecting IL-6 synthesis.
Molecular Medicine Reports 05/2012; 6(2):421-5. · 0.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: AMP-activated protein kinase (AMPK) is currently known to act as a key regulator of metabolic homeostasis. Several biosynthetic enzymes for fatty acid or glycogen are recognized as the targets of AMPK. In the present study, we investigated the role of AMPK in the interleukin-1 (IL-1)-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. IL-1 induced phosphorylation of AMPK-α (Thr-172), which regulates AMPK activities, and acetyl-CoA carboxylase, a direct substrate of AMPK. Compound C, an inhibitor of AMPK, which suppressed the IL-1-induced phosphorylation of acetyl-CoA carboxylase, increased the release and the mRNA level of IL-6 stimulated by IL-1. Transfection of AMPK siRNA-α also amplified the IL-1-stimulated IL-6 release compared to the control cells. On the other hand, IL-1 elicited the phosphorylation of IκB, which caused subsequent decrease of total level of IκB. Wedelolactone, an inhibitor of IκB kinase, which reduced the phosphorylation both of IκB and NF-κB, significantly enhanced the IL-1-stimulated IL-6 synthesis. Compound C remarkably suppressed the IL-1-induced phosphorylation of IκB. These results strongly suggest that AMPK negatively regulates IL-1-stimulated IL-6 synthesis through the IκB/NF-κB pathway in osteoblasts.
Cellular signalling 04/2012; 24(8):1706-12. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is generally recognized that AMP-activated protein kinase (AMPK) acts as a key regulator of energy homeostasis. We have previously shown that transforming growth factor-β (TGF-β) stimulates synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether AMPK is involved in the TGF-β-stimulated VEGF synthesis in osteoblast-like MC3T3-E1 cells. TGF-β time-dependently induced the phosphorylation of the AMPK α-subunit (Thr172) and the AMPK β-subunit (Ser108). Compound C, an AMPK inhibitor, significantly reduced the TGF-β-stimulated VEGF release. The inhibitory effect of compound C was also observed in normal human osteoblasts (NHOst). Although compound C failed to affect the TGF-β-induced phosphorylation of SAPK/JNK, p38 MAP kinase or Smad2, it markedly suppressed the TGF-β-induced phosphorylation of both MEK1/2 and p44/p42 MAP kinase. In addition, compound C significantly suppressed the VEGF mRNA expression induced by TGF-β. Taken together, our results strongly suggest that AMPK is involved in TGF-β-stimulated VEGF synthesis, and that it functions at a point upstream of MEK1/2.
International Journal of Molecular Medicine 04/2012; 29(4):550-6. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To determine the approximate percentage of women in nursing homes who have vitamin D deficiency and to investigate whether, in assessing vitamin D status in elderly women, there are problems with measuring only 25 hydroxy-vitamin D(3) (25(OH)D(3) ) and whether decreased vitamin D activation as a result of poor renal function needs to be considered.
Cross-sectional study.
Forty-eight nursing homes in Japan.
Four hundred three women with a mean age of 86.5 living in nursing homes who had participated in a clinical trial for hip protectors and were not bedridden.
At the start of the trial, in addition to general biochemical data, 25(OH)D(3) , 1,25-dihydroxy-vitamin D(3) (1,25(OH)(2) D(3) ), intact parathyroid hormone (intact PTH), calcium (Ca), phosphorus (P), bone alkaline phosphate (BAP), cross-linked N-telopeptide of type I collagen (NTx), and osteocalcin were measured in participants' blood, and statistical analysis was performed.
25(OH)D(3) , which is thought to reflect vitamin D status in the body, was surveyed and found to have a mean value of 16.7 ng/mL. 25(OH)D(3) was less than 16 ng/mL in 49.1% of all participants. Creatinine clearance (CCr) was less than 30 mL/min in 20.1% of participants. Participants with serum 25(OH)D(3) less than 16 ng/mL and CCr less than 30 mL/min had significantly higher levels of intact PTH and serum NTx. Participants with a CCr less than 30 mL/min had significantly lower levels of 1,25(OH)(2) D(3) .
Frail elderly adults living in nursing homes with poor renal function had lower 1,25(OH)(2) D(3) and higher intact PTH levels and were thus thought to have poorer vitamin D activating capacity. Supplementation with cholecalciferol may be insufficient in people who have poor renal function.
Journal of the American Geriatrics Society 02/2012; 60(2):251-5. · 3.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells.
The levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR.
PDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C.
These results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.
Life sciences 11/2011; 90(1-2):71-6. · 2.56 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously reported that thrombin stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of thrombin in the thrombin-stimulated IL-6 synthesis and the involvement of Rho-kinase in MC3T3-E1 cells. Thrombin time-dependently induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and myosin phosphatase targeting subunit-1 (MYPT-1), a Rho-kinase substrate. While SP600125, an inhibitor of SAPK/JNK, failed to reduce IL-6 synthesis, PD98059, a specific inhibitor of MEK, and SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by thrombin. Y27632, a specific Rho-kinase inhibitor, significantly reduced thrombin-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed thrombin-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect thrombin-induced phosphorylation of p44/p42 MAP kinase. Y27632 as well as fasudil attenuated thrombin-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates thrombin-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.
International Journal of Molecular Medicine 10/2011; 28(4):653-8. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heat shock protein 27 (HSP27) is known to act as a molecular chaperone. We have recently reported that HSP27 regulates osteocalcin synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the role of HSP27 in tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 (IL-6) synthesis in MC3T3-E1 cells. The levels of IL-6 release and IL-6 mRNA stimulated by TNF-α in MC3T3-E1 cells transfected with HSP27 was significantly higher than those in the control cells. In addition, the levels of secreted IL-6 and IL-6 mRNA in the phospho-mimic HSP27-overexpressing cells were significantly higher than those in the non-phosphoryl-atable HSP27-overexpressing cells. Furthermore, we observed no significant differences in the phosphorylation levels of IκB/NFκB, Akt, and p44/p42 mitogen-activated protein kinase among the 4 types of transfected cells. Therefore, these results strongly suggest that HSP27 enhances TNF-α-stimulated IL-6 synthesis, and that the phosphorylation status of HSP27 is related to IL-6 synthesis in osteoblasts.
International Journal of Molecular Medicine 08/2011; 28(5):887-93. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is recognized that Wnt3a affects bone metabolism via the canonical Wnt/β-catenin signalling pathway. We have previously shown that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TGF-β-stimulated VEGF synthesis in these cells. Wnt3a, which alone had little effect on the VEGF levels, significantly enhanced the TGF-β-stimulated VEGF release. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3β, markedly amplified the TGF-β-stimulated VEGF release. Wnt3a failed to affect the TGF-β-induced phosphorylation of Smad2, p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. Wnt3a and lithium chloride strengthened the VEGF mRNA expression induced by TGF-β. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by TGF-β via activation of the canonical pathway in osteoblasts.
Cell Biochemistry and Function 04/2011; 29(5):371-7. · 1.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is recognized that catechin possesses beneficial properties for bone metabolism. We previously revealed that triiodothyronine (T₃)-activated p38 mitogen-activated protein (MAP) kinase, but not p44/p42 MAP kinase, is involved in the synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of (-)-epigallocatechin gallate (EGCG), the predominant green tea polyphenol, on the synthesis of osteocalcin in MC3T3-E1 cells. EGCG significantly suppressed T₃-stimulated osteocalcin synthesis. The inhibitory effect of EGCG was dose-dependent in the range of 3 to 30 µM. On the other hand, T₃-induced phosphorylation of p38 MAP kinase was not affected by EGCG. EGCG profoundly inhibited T₃-stimulated transcriptional activity. These results strongly suggest that EGCG suppresses T3-stimulated osteocalcin synthesis upstream of the transcriptional level in osteoblast-like MC3T3-E1 cells.
Molecular Medicine Reports 03/2011; 4(2):297-300. · 0.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously reported that transforming growth factor-β (TGF-β) stimulates heat shock protein 27 (HSP27) induction via p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase in osteoblast-like MC3T3-E1 cells, and that the release of vascular endothelial growth factor (VEGF) is induced by TGF-β in these cells. In the present study, we investigated the effect of HSP27 knockdown on the TGF-β-stimulated VEGF release in these cells. Gene silencing using short interfering RNA against HSP27 (HSP27-siRNA) significantly suppressed the TGF-β-induced VEGF release. Immunofluorescence microscopy also revealed that HSP27-siRNA suppressed the TGF-β-stimulated VEGF induction as well as the reduction of HSP27 induction in these cells. However, the mRNA expression of VEGF stimulated by TGF-β was not reduced even in cells transfected with HSP27-siRNA. These results strongly suggest that HSP27 induction is critical for TGF-β-induced VEGF release in osteoblasts.
International Journal of Molecular Medicine 03/2011; 27(3):423-8. · 1.98 Impact Factor
-
Yukiko Enomoto,
Seiji Adachi,
Tomoaki Doi,
Hideo Natsume,
Kenji Kato,
Rie Matsushima-Nishiwaki,
Shigeru Akamatsu, Haruhiko Tokuda,
Shinichi Yoshimura,
Takanobu Otsuka,
Shinji Ogura,
Osamu Kozawa,
Toru Iwama
[show abstract]
[hide abstract]
ABSTRACT: Elevation of cAMP in platelets is recognized to play a suppressive role in platelet functions. We have previously shown that adenosine diphosphate (ADP)-induced phosphorylation of heat shock protein 27 (HSP27) via p38 mitogen-activated protein (MAP) kinase is correlated with platelet-derived growth factor (PDGF)-AB secretion and soluble CD40 ligand (sCD40L) release. In the present study, we investigated the relationship between cAMP and HSP27 phosphorylation in platelet function. 8-Bromoadenosine-3',5'-cyclic monophosphate (8-bromo-cAMP), a plasma membrane-permeable cAMP analogue, or cilostazol, an inhibitor of cAMP phosphodiesterase, markedly attenuated the ADP-induced phosphorylation levels of p38 MAP kinase. In addition, the ADP-induced HSP27 phosphorylation was suppressed by 8-bromo-cAMP or cilostazol. 8-Bromo-cAMP, forskolin and cilostazol remarkably reduced the ADP-stimulated PDGF-AB secretion and sCD40L release. These results strongly suggest that cAMP regulates ADP-stimulated platelet activation due to inhibition of HSP27 phosphorylation via p38 MAP kinase.
International Journal of Molecular Medicine 03/2011; 27(5):695-700. · 1.98 Impact Factor
-
Kenji Kato,
Seiji Adachi,
Rie Matsushima-Nishiwaki,
Chiho Minamitani,
Hideo Natsume,
Yasuo Katagiri,
Yoshinobu Hirose,
Jun Mizutani, Haruhiko Tokuda,
Osamu Kozawa,
Takanobu Otsuka
[show abstract]
[hide abstract]
ABSTRACT: We have previously reported that various stimuli, including sphingosine 1-phosphate, are able to induce heat shock protein (HSP) 27 in osteoblast-like MC3T3-E1 cells. However, the precise role of HSP27 in bone metabolism has not been satisfactory clarified. In this study, we investigated the effect of HSP27 on osteocalcin synthesis induced by bone morphogenetic protein (BMP)-4 or T₃ in these cells. In MC3T3-E1 cells, pretreatment with sphingosine 1-phosphate, sodium arsenite, or heat stress caused the attenuation of osteocalcin synthesis induced by BMP-4 or T₃ with concurrent HSP27 induction. To further investigate the effect of HSP27, we established stable HSP27-transfected cells. The osteocalcin synthesis was significantly reduced in the stable HSP27-transfected MC3T3-E1 cells and normal human osteoblasts compared with empty-vector transfected cells. On the other hand, anisomycin, a p38 MAPK activator, caused the phosphorylation of HSP27 in both sphingosine 1-phosphate-stimulated untransfected MC3T3-E1 cells and HSP27-transfected MC3T3-E1 cells. An immunofluorescence microscopy study showed that the phosphorylated HSP27 induced by anisomycin concentrated perinuclearly in these cells, in which it colocalized with the endoplasmic reticulum. We also established stable mutant-HSP27-transfected cells. Osteocalcin synthesis induced by either BMP-4 or T₃ was markedly suppressed in the nonphosphorylatable HSP27-overexpressing MC3T3-E1 cells compared with the phosphomimic HSP27-overexpressing cells. In contrast, the matrix mineralization was more obvious in nonphosphorylatable HSP27-overexpressing cells than that in phosphomimic HSP27-overexpressing cells. Taken together, these results strongly suggest that unphosphorylated HSP27 has an inhibitory effect on osteocalcin synthesis, but has a stimulatory effect on mineralization, in osteoblasts.
Endocrinology 03/2011; 152(5):1872-82. · 4.46 Impact Factor
