SUMO-Modification of Human Nrf2 at K110 and K533 Regulates Its Nucleocytoplasmic Localization, Stability and Transcriptional Activity.

BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress. METHODS: The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell. RESULTS: Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one 532LKDE535 of human (h) and its homologous 524LKDE527 of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K110, in the motifs 109PKSD112 and 109PKQD112 of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m)K110, hK533 and mK525) showed that these residues are needed for SUMO-2 conjugation, nuclear localization and ARE driven transcription of reporter genes and the endogenous HO-1 expression by Nrf2. These residues also stabilized Nrf2, as evident from shorter half-lives of the mutant protein compared to wild-type Nrf2. CONCLUSION: Our findings indicate that SUMO-2mediated sumoylation of K110 and K533 in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.

Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632, suggesting protease-activated receptor coupling to Gq and G12/13, respectively. These data suggest a vascular bed specific differential coupling of protease-activated receptors to the signaling pathways that regulate endothelial nitric oxide synthase and nitric oxide production that may be responsible for endothelial dysfunction associated with cardiovascular disease.

Distinct roles of protease-activated receptors in signal transduction regulation of endothelial nitric oxide synthase.

Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.

Role of protease-activated receptor-1 in endothelial nitric oxide synthase-Thr495 phosphorylation.

Protease activated receptors (PARs) are G protein-coupled receptors that are known to regulate endothelial nitric oxide synthase (eNOS) activity in part by phosphorylating the enzyme at various sites. Ser1177 is a positive regulatory site, which leads to the enhanced production of nitric oxide (NO), a vasodilator of arteries. Thr495 is a negative regulatory site, which inhibits NO production. We have shown that thrombin, a PAR agonist, mediates eNOS-Ser1177 phosphorylation through Gq and a calcium and protein kinase C (PKC)-delta sensitive, but phosphatidylinositol 3-kinase (PI3K)/Akt-independent pathway. However, the mechanism for eNOS-Thr495 phosphorylation by PAR agonists is unknown. We used a specific synthetic PAR-1 activating peptide, TFLLR, and thrombin to assess the role of PAR-1 involvement in the phosphorylation of eNOS-Thr495 in human umbilical vein endothelial cells (HUVECs). Using Western blot analysis and the Griess Reagent assay, we found that both agonists phosphorylated Thr495 in a time- and dose-dependent manner and significantly decreased nitrite production, respectively. Pretreatment of cells with the PAR-1 inhibitor, SCH-79797, resulted in a significant decrease in thrombin- and TFLLR-induced phosphorylation of eNOS-Thr495 and an increase in nitrite production. We further demonstrated that inhibition of Rho with C3 exoenzyme or dominant negative (dn) RhoA, and inhibition of Rho-Kinase (ROCK) with Y-27632 caused a significant decrease in thrombin and TFLLR-induced Thr495 phosphorylation. Blockade of the Rho/ROCK pathway also caused an increase in nitrite production. This suggests that PAR-1 regulates eNOS activity via phosphorylation of eNOS-Thr495, which is dependent upon activation of the Rho/ROCK pathway. These findings will be beneficial in further understanding the signaling pathways that regulate eNOS-induced NO production, which plays an important role in endothelial dysfunction associated with cardiovascular disease.

Mechanism of endothelial nitric oxide synthase phosphorylation and activation by thrombin.

Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca2+/calmodulin, eNOS has several regulatory sites. Ser1179 phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser1179 in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser1179 was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser473 at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca2+ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca2+ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC) and PKC or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser1179 phosphorylation-dependent eNOS activation through a Ca2+-dependent, PKC-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway.

Activation of endothelial nitric oxide synthase by the angiotensin II type 1 receptor.

Enhanced angiotensin II (AngII) action has been implicated in endothelial dysfunction that is characterized as decreased nitric oxide availability. Although endothelial cells have been reported to express AngII type 1 (AT1) receptors, the exact role of AT1 in regulating endothelial NO synthase (eNOS) activity remains unclear. We investigated the possible regulation of eNOS through AT1 in bovine aortic endothelial cells (BAECs) and its functional significance in rat aortic vascular smooth muscle cells (VSMCs). In BAECs infected with adenovirus encoding AT1 and in VSMCs infected with adenovirus encoding eNOS, AngII rapidly stimulated phosphorylation of eNOS at Ser1179. This was accompanied with increased cGMP production. These effects were blocked by an AT1 antagonist. The cGMP production was abolished by a NOS inhibitor as well. To explore the importance of eNOS phosphorylation, VSMCs were also infected with adenovirus encoding S1179A-eNOS. AngII did not stimulate cGMP production in VSMCs expressing S1179A. However, S1179A was able to enhance basal NO production as confirmed with cGMP production and enhanced vasodilator-stimulated phosphoprotein phosphorylation. Interestingly, S1179A prevented the hypertrophic response similar to wild type in VSMCs. From these data, we conclude that the AngII/AT1 system positively couples to eNOS via Ser1179 phosphorylation in ECs and VSMCs if eNOS and AT1 coexist. However, basal level NO production may be sufficient for prevention of AngII-induced hypertrophy by eNOS expression. These data demonstrate a novel molecular mechanism of eNOS regulation and function and thus provide useful information for eNOS gene therapy under endothelial dysfunction.

Recent progress in signal transduction research of the angiotensin II type-1 receptor: protein kinases, vascular dysfunction and structural requirement.

Accumulating evidence strongly implicates the critical roles of intracellular signaling of angiotensin II (AngII) in mediating cardiovascular diseases such as hypertension, atherosclerosis, and restenosis after vascular injury. The importance of AngII signals has also been reported in endothelial dysfunction and insulin resistance, two strong predictors of cardiovascular disease. Through its G protein-coupled AngII type-1 receptor (AT1), AngII activates various intracellular protein kinases, such as receptor or non-receptor tyrosine kinases and serine/threonine kinases. Activation of these kinases requires both G protein-dependent and independent pathways, reactive oxygen species and a metalloprotease, and each kinase could be involved specifically in mediating pathophysiological function of the AT1 receptor target organs. In fact, some of the kinases are indispensable for AngII-induced hypertrophy and migration. The role of these AT1-activated kinases in mediating vascular remodeling, vascular contractility, endothelial dysfunction, and insulin resistance will be discussed in this review. In addition, the AT1 receptor undergoes rapid phosphorylation, desensitization, and internalization upon AngII stimulation. Recent studies with site-directed mutagenesis of the AT1 receptor not only elucidated a G protein interaction and desensitization of the receptor, but also demonstrated a structural requirement of the receptor for downstream signal transduction. Thus, AT1 mutants have provided an excellent means to examine the mechanism of signal transduction and their significance in mediating AngII function. Taken together, in this review, we will focus our discussion on the recent findings of the signal transduction research elucidating novel signaling mechanisms of the AT1 receptor that are relevant to the vascular pathophysiology of AngII.

Angiotensin II-induced activation of p21-activated kinase 1 requires Ca2+ and protein kinase C{delta} in vascular smooth muscle cells.

ANG II promotes remodeling of vascular smooth muscle cells (VSMCs) in cardiovascular diseases. It has been shown to activate p21-activated kinase (PAK)1, a critical component of signaling pathways implicated in growth and migration. However, the detailed signaling mechanism by which ANG II induces PAK1 activation in VSMCs remains unclear. Therefore, we have examined the mechanism required for activation of PAK1 by ANG II in VSMCs. ANG II, through activation of the ANG II type 1 receptor, rapidly promotes phosphorylation of PAK1 in VSMCs via a pathway independent of transactivation of the epidermal growth factor receptor. Using selective agonists and inhibitors, we demonstrated that mobilization of intracellular Ca(2+) and PKCdelta activation are required for ANG II-induced PAK1 phosphorylation. Rottlerin, a PKCdelta inhibitor, significantly blocked ANG II-induced PAK1 phosphorylation. Further support for this notion was provided through infection of VSMCs with adenovirus encoding a dominant-negative (dn)PKCdelta, which also markedly reduced phosphorylation of PAK1 by ANG II. In this pathway, Ca(2+) acts upstream of PKCdelta because a Ca(2+) ionophore rapidly induced PKCdelta phosphorylation at Tyr311 and Ca(2+)-dependent PAK1 phosphorylation was blocked by rottlerin. In addition, dnPYK-2, dnRac, and antioxidants inhibited ANG II-induced PAK1 phosphorylation, suggesting that PYK-2, Rac, and reactive oxygen species are involved in the upstream signaling. Finally, dnPAK1 markedly inhibited ANG II-induced protein synthesis in VSMCs. These data provide a novel signaling pathway by which ANG II may contribute to vascular remodeling.

The role of reactive oxygen species in insulin signaling in the vasculature.

Although there is an abundance of evidence suggesting that insulin resistance plays a significant role in the vasculature, the precise mechanistic role involved still remains unclear. In this review, we discuss the current background of insulin resistance in the context of insulin signaling and action in the vasculature. Also, studies suggest that insulin resistance, diabetes, and cardiovascular disease all share a common involvement with oxidative stress. Recently, we reported that lysophosphatidylcholine, a major bioactive product of oxidized low-density lipoprotein, and angiotensin II, a vasoactive hormone and a potent inducer of reactive oxygen species (ROS), negatively regulate insulin signaling in vascular smooth muscle cells (VSMCs). In endothelial cells, insulin stimulates the release of nitric oxide, which results in VSMC relaxation and inhibition of atherosclerosis. Other data suggest that angiotensin II inhibits the vasodilator effects of insulin through insulin receptor substrate-1 phosphorylation at Ser312 and Ser616. Moreover, ROS impair insulin-induced vasorelaxation by neutralizing nitric oxide to form peroxynitrite. Thus, evidence is growing to enable us to better understand mechanistically the relationship between insulin/insulin resistance and ROS in the vasculature, and the impact they have on cardiovascular disease.

Signal-crosstalk between Rho/ROCK and c-Jun NH2-terminal kinase mediates migration of vascular smooth muscle cells stimulated by angiotensin II.

BACKGROUND: Rho and its effector Rho-kinase/ROCK mediate cytoskeletal reorganization as well as smooth muscle contraction. Recent studies indicate that Rho and ROCK are critically involved in vascular remodeling. Here, we tested the hypothesis that Rho/ROCK are critically involved in angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs) by mediating a specific signal cross-talk. METHODS AND RESULTS: Immunoblotting demonstrated that Ang II stimulated phosphorylation of a ROCK substrate, regulatory myosin phosphatase targeting subunit (MYPT)-1. Phosphorylation of MYPT-1 as well as migration of VSMCs induced by Ang II was inhibited by dominant-negative Rho (dnRho) or ROCK inhibitor, Y27632. Ang II-induced c-Jun NH2-terminal kinase (JNK) activation, but extracellular signal-regulated kinase (ERK) activation was not mediated through Rho/ROCK. Thus, infection of adenovirus encoding dnJNK inhibited VSMC migration by Ang II. We have further demonstrated that the Rho/ROCK activation by Ang II requires protein kinase C-delta (PKCdelta) and proline-rich tyrosine kinase 2 (PYK2) activation, but not epidermal growth factor receptor transactivation. Also, VSMCs express PDZ-Rho guanine nucleotide exchange factor (GEF) and Ang II stimulated PYK2 association with tyrosine phosphorylated PDZ-RhoGEF. CONCLUSIONS: PKCdelta/PYK2-dependent Rho/ROCK activation through PDZ-RhoGEF mediates Ang II-induced VSMC migration via JNK activation in VSMCs, providing a novel mechanistic role of the Rho/ROCK cascade that is involved in vascular remodeling.

Overexpression of Cu/Zn-superoxide dismutase and/or catalase in mice inhibits aorta smooth muscle cell proliferation.

BACKGROUND: Increasing evidence demonstrates that reactive oxygen species, for example, superoxide (O(2)(-.)) and hydrogen peroxide (H(2)O(2)), promote vascular smooth muscle cell (VSMC) proliferation, and that superoxide dismutase (SOD) and catalase work in concert to scavenge O(2)(-.) and H(2)O(2). This report examined the effect of overexpressing Cu/Zn-SOD or catalase on epidermal growth factor (EGF)-induced proliferation and mitogen-activated protein kinase (MAPK) phosphorylation in VSMCs. METHODS: The VSMCs were obtained from the aorta of wild-type mice and transgenic mice overexpressing Cu/Zn-SOD and catalase in combination or overexpressing Cu/Zn-SOD or catalase alone. The VSMC proliferation was measured by cell counting and bromodeoxyuridine incorporation assay. The MAPK phosphorylation was determined with Western blotting. RESULTS: Treatment of wild-type VSMCs with EGF significantly increased proliferation and phosphorylation of extracellular signal-regulated kinases (ERK1/2) and p38 MAPK. Overexpression of Cu/Zn-SOD or catalase attenuated EGF-induced phosphorylation of ERK1/2 and p38 MAPK and suppressed EGF-induced proliferation in VSMCs. For example, the EGF-induced phosphorylation of ERK1/2 and p38 MAPK and EGF-induced proliferation in VSMCs overexpressing Cu/Zn-SOD or catalase were significantly less than in wild-type VSMCs. Moreover, VSMCs overexpressing Cu/Zn-SOD and catalase in combination showed significantly less proliferation and less phosphorylation of the MAPKs than those overexpressing Cu/Zn-SOD or catalase alone. CONCLUSIONS: Overexpression of Cu/Zn-SOD and catalase in combination is more efficient in inhibiting VSMC proliferation and MAPK phosphorylation than overexpression of Cu/Zn-SOD or catalase alone.

Hydrogen peroxide inhibits insulin signaling in vascular smooth muscle cells.

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 microM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 microM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.

Insulin-induced Akt activation is inhibited by angiotensin II in the vasculature through protein kinase C-alpha.

Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, the specific role of insulin resistance in the etiology of these diseases is poorly understood. Angiotensin (Ang) II is a potent vasculotrophic and vasoconstricting factor. We hypothesize that in vascular smooth muscle cells (VSMCs), Ang II interferes with insulin action by inhibiting Akt, a major signaling molecule implicated in the biological actions of insulin. By immunoblotting with a phospho-specific antibody for Akt, we found that Ang II inhibits insulin-induced Akt phosphorylation in a time- and concentration-dependent manner. The inhibitory effect of Ang II was blocked by a Ang II type 1 receptor antagonist, RNH6270. A protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, also inhibited insulin-induced Akt phosphorylation. PKC inhibitors, including Go6976 (specific for alpha- and beta-isoforms), blocked the Ang II- and PMA-induced inhibition of Akt phosphorylation by insulin. Moreover, overexpression of PKC-alpha but not PKC-beta isoform by adenovirus inhibited insulin-induced Akt phosphorylation. By contrast, an epidermal growth factor receptor inhibitor (AG1478), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 598,059), and a p38 MAPK inhibitor (SB 203,580) did not block the Ang II-induced inhibition of Akt phosphorylation. From these data, we conclude that Ang II negatively regulates the insulin signal, Akt, in the vasculature specifically through PKC-alpha activation, providing an alternative molecular mechanism that may explain the association of hyperinsulinemia with cardiovascular diseases.

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

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

Ligand-independent trans-activation of the platelet-derived growth factor receptor by reactive oxygen species requires protein kinase C-delta and c-Src.

Reactive oxygen species are involved in the mitogenic signal transduction cascades initiated by several growth factors and play a critical role in mediating cardiovascular diseases. Interestingly, H(2)O(2) induces tyrosine phosphorylation and trans-activation of the platelet-derived growth factor receptor and the epidermal growth factor receptor in many cell lines including vascular smooth muscle cells. To investigate the molecular mechanism by which reactive oxygen species contribute to vascular diseases, we have examined a signal transduction cascade involved in H(2)O(2)-induced platelet-derived growth factor receptor activation in vascular smooth muscle cells. We found that H(2)O(2) induced a ligand-independent phosphorylation of the platelet-derived growth factor-beta receptor at Tyr(1021), a phospholipase C-gamma binding site, involving the requirement of protein kinase C-delta and c-Src that is distinct from a ligand-dependent autophosphorylation. Also, H(2)O(2) induced the association of protein kinase C-delta with the platelet-derived growth factor-beta receptor and c-Src in vascular smooth muscle cells. These findings will provide new mechanistic insights by which enhanced reactive oxygen species production in vascular smooth muscle cells induces unique alleys of signal transduction distinct from those induced by endogenous ligands leading to an abnormal vascular remodeling process.

Cyclosporin A inhibits angiotensin II-induced c-Jun NH(2)-terminal kinase activation but not protein synthesis in vascular smooth muscle cells.

Angiotensin II activates three major mitogen-activated protein kinases (MAPK) in vascular smooth muscle cells. Although other angiotensin II-induced MAPKs activation require transactivation of a growth factor receptor, the detailed mechanism by which angiotensin II activates c-Jun NH(2)-terminal kinase (JNK) remains unclear. Here, an immunosuppressant, cyclosporin A but not FK506, selectively inhibited angiotensin II-induced JNK activation in vascular smooth muscle cells. However, cyclosporin A had no inhibitory effect on angiotensin II-induced protein synthesis. Thus, angiotensin II-induced JNK activation but not protein synthesis is mediated by a mechanism sensitive to cyclosporin A, which is independent from calcineurin in vascular smooth muscle cells.

Metalloprotease inhibitor blocks angiotensin II-induced migration through inhibition of epidermal growth factor receptor transactivation.

In vascular smooth muscle cells (VSMCs), angiotensin II (AngII) induces transactivation of the EGF receptor (EGFR) which involves a metalloprotease that stimulates processing of heparin-binding EGF from its precursor. However, the identity and pharmacological sensitivity of the metalloprotease remain unclear. Here, we screened the effects of several metalloprotease inhibitors on AngII-induced EGFR transactivation in VSMCs. We found that an N-phenylsulfonyl-hydroxamic acid derivative [2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide] (BiPS), previously known as matrix metalloprotease (MMP)-2/9 inhibitor, markedly inhibited AngII-induced EGFR transactivation, whereas the MMP-2 or -9 inhibition by other MMP inhibitors failed to block the transactivation. BiPS markedly inhibited AngII-induced ERK activation and protein synthesis without affecting AngII-induced intracellular Ca2+ elevation. VSMC migration induced by AngII was also inhibited not only by an EGFR inhibitor but also by BiPS. Thus, BiPS is a specific candidate to block AngII-induced EGFR transactivation and subsequent growth and migration of VSMCs, suggesting its potency to prevent vascular remodeling.

Lysophosphatidylcholine activates extracellular signal-regulated kinases 1/2 through reactive oxygen species in rat vascular smooth muscle cells.

Lysophosphatidylcholine (lysoPC) acts on vascular smooth muscle cells (VSMCs) to produce a mitogenic response through the activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we examined the importance of reactive oxygen species (ROS) in lysoPC-stimulated ERK1/2 activation in cultured rat VSMCs. Treatment with lysoPC for 3 minutes caused a 2-fold increase in intracellular ROS that was blocked by the NADH/NADPH oxidase inhibitor, diphenylene iodonium (DPI). Antioxidants, N-acetyl-L-cysteine, glutathione monoester, or alpha -tocopherol, inhibited ERK1/2 activation by lysoPC. Almost identical results were obtained in the VSMC line A10. Pretreatment of VSMCs with DPI but not allopurinol or potassium cyanide (KCN) abrogated the activation of ERK1/2. The Flag-tagged p47phox expressed in A10 cells was translocated from the cytosol to the membrane after 2 minutes of stimulation with lysoPC. The overexpression of dominant-negative p47phox in A10 cells suppressed lysoPC-induced ERK activation. The ROS-dependent ERK activation by lysoPC seems to involve protein kinase C- and Ras-dependent raf-1 activation. Induction of c-fos expression and enhanced AP-1 binding activity by lysoPC were also inhibited by DPI and NAC. Taken together, these data suggest that ROS generated by NADH/NADPH oxidase contribute to lysoPC-induced activation of ERK1/2 and subsequent growth promotion in VSMCs.

Lysophosphatidylcholine inhibits insulin-induced Akt activation through protein kinase C-alpha in vascular smooth muscle cells.

To better understand the intracellular signaling mechanism that causes the association of insulin resistance and hyperlipidemia with cardiovascular diseases, we specifically looked at the ability of lysophosphatidylcholine (lysoPC) to inhibit the Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells. LysoPC inhibited the insulin-induced phosphorylation of Akt at Ser473, and the inhibition was concentration dependent. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, inhibited the insulin-induced phosphorylation of Akt. LysoPC stimulated PKC phosphorylation at Ser660, which was inhibited by the PKC inhibitor GF109203X. The PKC-alpha/beta-selective inhibitor Go6976 also blocked the PMA- and lysoPC-induced inhibition of Akt phosphorylation by insulin. PKC-alpha, but not PKC-beta, is expressed in vascular smooth muscle cells, and overexpression of PKC-alpha, but not PKC-beta or PKC-delta, inhibited insulin-induced Akt activation. LysoPC rapidly stimulated PKC-alpha translocation to the membrane. In contrast, pretreatment with the p42/44 mitogen-activated protein kinase kinase inhibitor PD98059 or the p38 mitogen-activated protein kinase inhibitor SB203580 did not block the lysoPC-induced inhibition of Akt phosphorylation by insulin. In addition, lysoPC inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 but not that of the insulin receptor beta subunit or insulin binding. PMA treatment or PKC-alpha overexpression also inhibited the tyrosine phosphorylation of IRS-1. From these data, we conclude that lysoPC negatively regulates the insulin signal at the point of IRS-1 through PKC-alpha in the vasculature, which may explain the association of hyperlipidemia with hyperinsulinemia in cardiovascular diseases.