Transactivation of the EGF receptor and a PI3 kinase ATF-1 pathway is involved in the upregulation of NOX1, a catalytic subunit of NADPH oxidase

Size: px

Start display at page:

Download "Transactivation of the EGF receptor and a PI3 kinase ATF-1 pathway is involved in the upregulation of NOX1, a catalytic subunit of NADPH oxidase"

June Fleming
5 years ago
Views:

1 FEBS FEBS Letters 579 (2005) Transactivation of the EGF receptor and a PI3 kinase ATF-1 pathway is involved in the upregulation of NOX1, a catalytic subunit of NADPH oxidase ChunYuan Fan 1, Masato Katsuyama 1, Toru Nishinaka, Chihiro Yabe-Nishimura * Department of Pharmacology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-Ku, Kyoto , Japan Received 18 November 2004; revised 24 December 2004; accepted 10 January 2005 Available online 26 January 2005 Edited by Valdimir Skulachev Abstract We previously reported that hypertrophy of vascular smooth muscle cells caused by prostaglandin (PG) F 2a is mediated by the induction of NOX1, a catalytic subunit of NADPH oxidase that generates superoxide. The signal transduction pathway(s) involved in this process, however, remained unresolved. PGF 2a enhanced the phosphorylation of the epidermal growth factor (EGF) receptor, and a selective inhibitor of EGF receptor kinase, tyrphostin AG1478, significantly suppressed PGF 2a -induced NOX1 expression. AG1478 also blunted the PGF 2a -induced phosphorylation of extracellular signal-regulated protein kinase (ERK)1/2 and Akt. Phosphoinositide 3 (PI3) kinase inhibitors not only reduced PGF 2a -induced NOX1 expression, but also suppressed the phosphorylation of ATF-1, a transcription factor previously shown to play a key role in the induction of NOX1. Accordingly, the transactivation of the EGF receptor and the activation of ERK1/2, PI3 kinase, and ATF-1 constitute the signaling pathways involved in the upregulation of NOX1. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: NOX1; NADPH oxidase; Prostaglandin F 2a ; Epidermal growth factor receptor; Phosphoinositide 3 kinase growth factor (PDGF), phorbol ester, and fetal bovine serum (FBS) [4,5]. We previously reported that prostaglandin (PG) F 2a, one of the primary prostanoids generated in the vascular tissue, causes hypertrophy of VSMC by the induction of NOX1 and the subsequent increase in O 2 generation [6]. Recently, we found that ATF-1, a transcription factor of the CREB/ATF family, is essential for the upregulation of NOX1 by PGF 2a, PDGF, and other factors, including phorbol ester and FBS [7]. PGF 2a exerts its biological actions through binding to its specific G-protein-coupled receptor, FP [8]. FP is coupled to phospholipase C and elicits the mobilization of cytosolic Ca 2+. In VSMC isolated from rat aorta, the activation of extracellular signal-regulated kinase (ERK) 2, c-jun-n-terminal kinase (JNK) 1, phosphoinositide 3 (PI3)-kinase, and p70 S6 kinase by PGF 2a has been reported [9]. It is as yet unknown, however, whether these protein kinases take part in the upregulation of NOX1 that leads to the generation of the signaling molecule, O 2. Here, we report that the upregulation of NOX1 by PGF 2a involves the transactivation of the EGF receptor, and ensuing activation of its downstream ERK1/2 and PI3 kinase ATF-1 pathways. 1. Introduction Reactive oxygen species (ROS), including superoxide ðo 2 Þ and hydrogen peroxide (H 2 O 2 ), have been recognized to act as intrinsic signaling molecules in cardiovascular tissues. It has been shown that NADPH oxidases are the major source of O 2 in vascular cells and cardiac myocytes [1 3]. Recently, several homologs of the catalytic subunit of the phagocyte NADPH oxidase (gp91phox; NOX2) were found in vascular smooth muscle cells (VSMC). Among them, NOX1 was implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis after angioplasty, since it mediates the proliferation and hypertrophy of VSMC. Expression of the NOX1 gene is induced by angiotensin II, platelet-derived * Corresponding author. Fax: address: nchihiro@koto.kpu-m.ac.jp (C. Yabe-Nishimura). 1 These authors contributed to this work equally. Abbreviations: PG, prostaglandin; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; MAPK, mitogenactivated protein kinase; MEK, MAPK/ERK kinase; MMP, matrix metalloproteinase; PI3, phosphoinositide 3 2. Materials and methods 2.1. Materials Tyrphostin AG1478 and Wortmannin were purchased from Sigma (St. Louis, MO). PGF 2a was acquired from Nacalai Tesque (Kyoto, Japan). Antibodies against EGF receptor or phosphorylated EGF receptor were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Biosource International (Nivelles, Belgium), respectively. Antibodies against ERK or phosphorylated ERK were purchased from New England Biolabs (Beverly, MA). Antibodies against Akt or phosphorylated Akt and horseradish peroxidase-linked anti-rabbit or anti-mouse antibodies were purchased from Cell Signaling Technology (Beverly, MA). PP2, GM6001, PD98059, SB203580, and SP were purchased from Merck (Tokyo, Japan). LY was purchased from Cayman Chemical (Ann Arbor, MI). [a- 32 P]dCTP and [a- 32 P]UTP were obtained from ICN Biomedicals (Costa Mesa, CA) Cell treatment and Northern blot analysis The rat VSMC line A7r5 obtained from American Type Culture Collection (Rockville, MD) was seeded in 10-cm dishes ( cells/ dish) and cultured for 24 h in DulbeccoÕs modified EagleÕs medium (DMEM) supplemented with 10% FBS. The cells were then cultured for a further 48 h in DMEM containing 0.5% FBS. After pretreatment with or without various inhibitors for 1 h, the cells were subsequently incubated with 100 nm PGF 2a for 24 h. Northern blot analysis was performed as described previously [6]. Representative autoradiographs of the three experiments are shown in figures /$30.00 Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi: /j.febslet

2 1302 C. Fan et al. / FEBS Letters 579 (2005) Western blot analysis A7r5 cells cultured in the absence of FBS for 48 h were pretreated with various inhibitors for 1 h, incubated with 100 nm PGF 2a, washed twice with PBS, and then frozen with liquid nitrogen. The cells were lysed in a buffer containing 1% Triton, 0.5% sodium deoxycholate, 10 mm Tris HCl, 150 mm NaCl, 1 mm EDTA, 20 mm b-glycerophosphate, 1 mm Na 3 VO 4, 1 mm sodium fluoride, and 1 mm phenylmethylsulfonyl fluoride. The lysate was centrifuged and the supernatant was saved for analysis. The aliquots containing equal amounts of protein (10 lg) were subjected to SDS polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). After hybridization with a primary antibody and horseradish-peroxidase-linked secondary antibody, immunoreactive bands were detected using an ECL-Plus System (Amersham Biosciences, Piscataway, NJ). For the detection of ATF- 1, nuclear extracts were prepared and Western blot analyses were performed as described previously [7]. Representative results of the three experiments are shown in figures Statistical analysis Values were expressed as means ± S.E.M. Statistical analysis was performed with StudentÕs t test. For multiple treatment groups, oneway ANOVA followed by BonferroniÕs t test was applied. 3. Results 3.1. PGF 2a transactivates EGF receptor The mitogenic effects of angiotensin II are mediated through AT 1 receptor and subsequent transactivation of the EGF receptor [10]. Similar to the AT 1 receptor, the prostanoid FP receptor is among the Gq-protein-coupled receptors [8]. To examine whether PGF 2a transactivates the EGF receptor in A7r5 cells, phosphorylation of the EGF receptor was analyzed by Western blotting using a phospho-specific antibody. As shown in Fig. 1A, a time-dependent increase in the phosphorylation of the EGF receptor was demonstrated in the cells stimulated with PGF 2a. Increased phosphorylation of the EGF receptor was clearly observed 0.5 min after stimulation with PGF 2a, and the level reached a maximum at 2 min Inhibitors of EGF receptor kinase, Src, and matrix metalloproteinase (MMP) suppress the induction of NOX1 mrna by PGF 2a To examine whether the transactivation of the EGF receptor is involved in NOX1 induction, A7r5 cells were stimulated with PGF 2a for 24 h in the presence or absence of tyrphostin AG1478, a selective inhibitor of EGF receptor kinase. As shown in Fig. 1B, tyrphostin AG1478 significantly suppressed the PGF 2a -induced increase in NOX1 mrna. Transactivation of the EGF receptor by angiotensin II is mediated by the activation of tyrosine kinases Src and Pyk2, and the ADAM family of MMPs, leading to the excision of heparin-binding EGF [10]. Therefore, effects of inhibitors of Src and MMPs were investigated. As shown in Fig. 1C, a Src inhibitor PP2 and a MMP inhibitor GM6001 suppressed the PGF 2a -induced increase in NOX1 mrna. These results suggest that the activation of Src and MMPs is involved in the transactivation of the Fig. 1. Involvement of EGF receptor transactivation in PGF 2a -induced NOX1 expression. (A) Time course of transactivation of the EGF receptor by PGF 2a. Growth-arrested A7r5 cells were incubated with 100 nm PGF 2a for the indicated period and the cell lysates (10 lg) were subjected to Western blot analyses. The phosphorylation status of the EGF receptor was analyzed with anti-phospho-specific EGF receptor antibody. (B) Suppression of PGF 2a -induced NOX1 expression by tyrphostin AG1478. Growth-arrested A7r5 cells were treated with 10 lm tyrphostin AG1478 for 1 h and subsequently incubated with 100 nm PGF 2a for 24 h. (C) Suppression of PGF 2a -induced NOX1 expression by PP2 and GM6001. Growth-arrested A7r5 cells were treated with 50 lm PP2 or 20 lm GM6001 for 1 h and subsequently incubated with 100 nm PGF 2a for 24 h. Bar graphs represent means ± S.E.M. of the expression levels of NOX1 normalized to those of GAPDH obtained from three experiments. *P < 0.05 vs. control; P < 0.05 vs. PGF 2a -treated cells.

3 C. Fan et al. / FEBS Letters 579 (2005) EGF receptor by PGF 2a, which leads to the increased expression of the NOX1 gene PGF 2a -induced activation of ERK1/2 is suppressed by an EGF receptor kinase inhibitor The angiotensin II-induced transactivation of the EGF receptor leads to the activation of ERK [11,12]. We therefore examined whether PGF 2a activates ERK in A7r5 cells. As shown in Fig. 2A, a time-dependent increase in the phosphorylation of ERK1/2 was demonstrated in the cells stimulated with PGF 2a. Increased phosphorylation was clearly observed 2 min after stimulation and the level reached a maximum at 5 min. As shown in Fig. 2B, the phosphorylation of ERK1/2 was significantly suppressed in the presence of tyrphostin AG1478. These results suggest that the activation of ERK1/2 by PGF 2a is mediated by the transactivation of the EGF receptor in A7r5 cells MEK inhibitor suppresses the induction of NOX1 by PGF 2a PGF 2a was found to activate multiple mitogen-activated protein kinase (MAPK) cascades [9]. To examine whether ERK and other MAPK are involved in NOX1 induction, effects of MAP kinase inhibitors were investigated. As shown in Fig. 2C, a MAPK/ERK kinase (MEK) inhibitor, PD98059, significantly suppressed the PGF 2a -induced increase in NOX1 mrna. On the other hand, neither SB nor SP600125, inhibitors of p38 and JNK, respectively, affected the induction of NOX1 mrna by PGF 2a. These results suggest that MEK ERK is the major pathway in the MAPK cascades involved in the upregulation of NOX PGF 2a -induced activation of the PI3 kinase pathway is blocked by an EGF receptor kinase inhibitor It has been reported that the PI3 kinase/akt pathway is also activated by PDGF or angiotensin II to mediate the mitogenesis of VSMC [13,14]. We then examined whether PGF 2a activates Akt, a substrate of PI3 kinase. As shown in Fig. 3A, a time-dependent phosphorylation of Akt was observed in the cells stimulated with PGF 2a, reaching a maximum at 2 min. To clarify whether this PI3 kinase/akt pathway is placed downstream of the EGF receptor, the effect of tyrphostin AG1478 was tested. As shown in Fig. 3B, the phosphorylation of Akt was significantly suppressed by the EGF receptor kinase inhibitor. These results suggest that the activation of the PI3 kinase pathway takes place downstream of the EGF receptor PI3 kinase inhibitors suppress the induction of NOX1 by PGF 2a We next verified effects of PI3 kinase inhibitors on PGF 2a - induced NOX1 expression. As shown in Fig. 3C, PI3 kinase inhibitors Wortmannin and LY almost completely abolished the PGF 2a -induced increase in NOX1 mrna, indicating the involvement of PI3 kinase in the PGF 2a -induced increase in NOX1 expression. Fig. 2. Involvement of ERK1/2 in PGF 2a -induced NOX1 expression. (A) Time course of ERK1/2 activation by PGF 2a. Growth-arrested A7r5 cells were incubated with 100 nm PGF 2a for the indicated period and the cell lysates (10 lg) were subjected to Western blot analyses. (B) Suppression of PGF 2a -induced phosphorylation of ERK1/2 by tyrphostin AG1487. Growth-arrested A7r5 cells were treated with or without 10 lm tyrphostin AG1478 for 1 h, and then incubated with or without 100 nm PGF 2a for 5 min. (C) Effects of MAP kinase inhibitors on PGF 2a -induced NOX1 expression. Growth-arrested A7r5 cells were treated with 10 lm PD98059 (a MEK inhibitor), 10 lm SB (a p38 inhibitor) or 5 lm SP (a JNK inhibitor) for 1 h, and subsequently incubated with 100 nm PGF 2a for 24 h. *P < 0.05 vs. control; P < 0.05 vs. PGF 2a -treated cells.

4 1304 C. Fan et al. / FEBS Letters 579 (2005) Fig. 3. Involvement of PI3 kinase in PGF 2a -induced NOX1 expression. (A) Time course of Akt activation by PGF 2a. Growth-arrested A7r5 cells were incubated with 100 nm PGF 2a for the indicated period and the cell lysates (10 lg) were subjected to Western blot analyses. (B) Suppression of PGF 2a -induced phosphorylation of Akt by tyrphostin AG1487. Growth-arrested A7r5 cells were treated with or without 10 lm tyrphostin AG1478 for 1 h and then incubated with or without 100 nm PGF 2a for 2 min. (C) Suppression of PGF 2a -induced NOX1 expression by PI3 kinase inhibitors. Growth-arrested A7r5 cells were treated with 200 nm Wortmannin or 25 lm LY for 1 h, and subsequently incubated with 100 nm PGF 2a for 24 h. (D) Suppression of PGF 2a -induced phosphorylation of ATF-1 by PI3 kinase inhibitors. Growth-arrested A7r5 cells were pre-incubated with 200 nm Wortmannin or 25 lm LY for 1 h and subsequently stimulated with 100 nm PGF 2a for 10 min. *P < 0.05 vs. control; P < 0.05 vs. PGF 2a -treated cells PI3 kinase inhibitors suppress phosphorylation of ATF-1 by PGF 2a We next examined which pathway is involved in the activation of ATF-1, a transcription factor of the CREB/ATF family previously shown to mediate the upregulation of NOX1 [7]. Tyrphostin AG1478 suppressed phosphorylation of ATF-1 induced by PGF 2a (data not shown). PI3 kinase inhibitors markedly suppressed phosphorylation of ATF-1 (Fig. 3D). On the other hand, a MEK inhibitor, PD98059, did not affect the PGF 2a -induced phosphorylation of ATF-1, even at a concentration of 100 lm (data not shown). Accordingly, the activation of ATF-1 by PGF 2a is mediated by PI3 kinase. 4. Discussion Based on the present findings, transactivation of the EGF receptor by PGF 2a and subsequent activation of ERK1/2, PI3 kinase, and ATF-1 appears to constitute the signaling pathways that lead to the upregulation of NOX1. The major lines of evidence provided in this study are that: (1) PGF 2a enhanced the phosphorylation of the EGF receptor and a selective inhibitor of EGF receptor kinase suppressed the PGF 2a -induced increase in NOX1 mrna; (2) a Src inhibitor, PP2, and a MMP inhibitor, GM6001, suppressed PGF 2a -induced NOX1 expression; (3) an inhibitor of EGF receptor kinase blunted the PGF 2a -induced phosphorylation of ERK1/2, and a MEK inhibitor suppressed PGF 2a -induced NOX1 expression; (4) an inhibitor of EGF receptor kinase abolished the PGF 2a -induced phosphorylation of Akt, a substrate of PI3 kinase; and (5) PI3 kinase inhibitors suppressed PGF 2a -induced NOX1 expression as well as the PGF 2a -induced phosphorylation of ATF-1. PGF 2a exerts its biological actions through binding to its specific G-protein-coupled receptor, FP [8]. FP is coupled to phospholipase C and elicits the mobilization of cytosolic Ca 2+. Some of its effects are, however, reported mediated through the transactivation of the EGF receptor. Proliferation of an endometrial adenocarcinoma cell line induced by PGF 2a was attributed to the transactivation of the EGF receptor and subsequent activation of ERK1/2 [15]. In cultured TMOb oste-

5 C. Fan et al. / FEBS Letters 579 (2005) oblasts, PGF 2a activates ERK1/2 through the transactivation of the EGF receptor to elicit the recruitment and activation of osteoblasts [16]. Our present data clearly indicated that the induction of NOX1 by PGF 2a and ensuing hypertrophy of VSMC [6] is also mediated through the transactivation of the EGF receptor and activation of ERK1/2. There is little information on signaling pathways involved in the induction of NOX1, although various vasoactive factors, including angiotensin II, PDGF, and PGF 2a, were reported to induce NOX1 in VSMC [4 6]. We presently demonstrated that the EGF receptor is transactivated by PGF 2a, and we also observed that PDGF elicits phosphorylation of the EGF receptor in A7r5 cells (data not shown). The transactivation of the EGF receptor therefore seems to be a common event involved in induction of NOX1 by these factors. Recently, the upregulation of NOX1 by the Ras oncogene in NRK cells was reported to be mediated by the MEK ERK pathway, and the increased ROS generation due to NOX1 was required for oncogenic cell transformation [17]. Our present findings in VSMC consequently support the view that the EGF receptor and downstream ERK1/2 may mediate the induction of NOX1 by a wide range of stimuli in different cell lineages. We previously reported that ATF-1, a transcription factor of the CREB/ATF family, is essential for the upregulation of NOX1 by PGF 2a, PDGF, and other factors, and the inhibition of the mitochondrial respiratory chain suppresses the phosphorylation of ATF-1 [7]. However, the question as to which signaling pathway(s) leads to the activation of ATF-1 was left unanswered. Here, we demonstrated that PI3 kinase is the upstream module that activates ATF-1 and induces the expression of the NOX1 gene. PI3 kinase inhibitors, but not a MEK inhibitor, suppressed the PGF 2a -induced phosphorylation of ATF-1. On the other hand, PGF 2a -induced phosphorylation of ERK1/2 was not affected by PI3 kinase inhibitors, or by depletion of ATF-1 with RNAi (data not shown). Thus, the PI3 kinase ATF-1 pathway and the MEK ERK1/2 pathway, both downstream of the EGF receptor, are independently activated to elicit induction of NOX1. Considering the fact that NOX1-derived O 2 is associated with the proliferation and hypertrophy of VSMC, it is reasonable that PI3 kinase and ERK1/2, both known to mediate cell survival and growth, constitute the signal transduction pathways that lead to the up-regulation of NOX1 gene expression. Acknowledgments: This work was supported in part by a Grant-in-Aid for Young Scientists (B) from The Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr. E. Funakoshi of the Faculty of Pharmaceutical Sciences, Setsunan University, and Dr. N. Arakawa of Kyoto Prefectural University of Medicine, for their valuable discussion and advice. References [1] Griendling, K.K., Sorescu, D. and Ushio-Fukai, M. (2000) NAD(P)H oxidase: role in cardiovascular biology and disease. Circ. Res. 86, [2] Irani, K. (2000) Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ. Res. 87, [3] Guzik, T.J., West, N.E., Black, E., McDonald, D., Ratnatunga, C., Pillai, R. and Channon, K.M. (2000) Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors. Circ. Res. 86, E85 E90. [4] Suh, Y.A., Arnold, R.S., Lassègue, B., Shi, J., Xu, X., Sorescu, D., Chung, A.B., Griendling, K.K. and Lambeth, J.D. (1999) Cell transformation by the superoxide-generating oxidase Mox1. Nature 401, [5] Lassègue, B., Sorescu, D., Szöcs, K., Yin, Q., Akers, M., Zhang, Y., Grant, S.L., Lambeth, J.D. and Griendling, K.K. (2001) Novel gp91(phox) homologues in vascular smooth muscle cells: nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ. Res. 88, [6] Katsuyama, M., Fan, C. and Yabe-Nishimura, C. (2002) NADPH oxidase is involved in prostaglandin F 2a -induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF 2a. J. Biol. Chem. 277, [7] Katsuyama, M., Fan, C., Arakawa, N., Nishinaka, T., Miyagishi, M., Taira, K. and Yabe-Nishimura, C. (in press) Essential role of ATF-1 in induction of NOX1, a catalytic subunit of NADPH oxidase: involvement of mitochondrial respiratory chain. Biochem. J. [8] Sugimoto, Y., Hasumoto, K., Namba, T., Irie, A., Katsuyama, M., Negishi, M., Kakizuka, A., Narumiya, S. and Ichikawa, A. (1994) Cloning and expression of a cdna for mouse prostaglandin F receptor. J. Biol. Chem. 269, [9] Rao, G.N., Madamanchi, N.R., Lele, M., Gadiparthi, L., Gingras, A.C., Eling, T.E. and Sonenberg, N. (1999) A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells. J. Biol. Chem. 274, [10] Shah, B.H. and Catt, K.J. (2003) A central role of EGF receptor transactivation in angiotensin II-induced cardiac hypertrophy. Trends Pharmacol. Sci. 24, [11] Shah, B.H. and Catt, K.J. (2002) Calcium-independent activation of extracellularly regulated kinases 1 and 2 by angiotensin II in hepatic C9 cells: roles of protein kinase Cdelta, Src/proline-rich tyrosine kinase 2, and epidermal growth receptor trans-activation. Mol. Pharmacol. 61, [12] Shah, B.H., Yesilkaya, A., Olivares-Reyes, J.A., Chen, H.D., Hunyady, L. and Catt, K.J. (2004) Differential pathways of angiotensin II-induced extracellularly regulated kinase 1/2 phosphorylation in specific cell types: role of heparin-binding epidermal growth factor. Mol. Endocrinol. 18, [13] Ushio-Fukai, M., Alexander, R.W., Akers, M., Yin, Q., Fujio, Y., Walsh, K. and Griendling, K.K. (1999) Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J. Biol. Chem. 274, [14] Goncharova, E.A., Ammit, A.J., Irani, C., Carroll, R.G., Eszterhas, A.J., Panettieri, R.A. and Krymskaya, V.P. (2002) PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 283, L [15] Sales, K.J., Milne, S.A., Williams, A.R., Anderson, R.A. and Jabbour, H.N. (2004) Expression, localization, and signaling of prostaglandin F 2a receptor in human endometrial adenocarcinoma: regulation of proliferation by activation of the epidermal growth factor receptor and mitogen-activated protein kinase signaling pathways. J. Clin. Endocrinol. Metab. 89, [16] Ahmed, I., Gesty-Palmer, D., Drezner, M.K. and Luttrell, L.M. (2003) Transactivation of the epidermal growth factor receptor mediates parathyroid hormone and prostaglandin F2a-stimulated mitogen-activated protein kinase activation in cultured transgenic murine osteoblasts. Mol. Endocrinol. 17, [17] Mitsushita, J., Lambeth, J.D. and Kamata, T. (2004) The superoxide-generating oxidase Nox1 is functionally required for Ras oncogene transformation. Cancer Res. 64,

Electrical Stimulation Control Nerve Regeneration via the p38 Mitogen-activated Protein Kinase and CREB

Electrical Stimulation Control Nerve Regeneration via the p38 Mitogen-activated Protein Kinase and CREB Kenji Kawamura, Yoshio Kano. Kibi International University, Takahashi-city, Japan. Disclosures: K.