Anion exchanger 3 is required for sasanquasaponin to inhibit ischemia/reperfusion-induced elevation of intracellular Cl- concentration and to elicit cardioprotection

Recent studies have shown that the cardioprotection of sasanquasaponin (SQS) against ischemia/reperfusion injury is related to inhibiting ischemia/reperfusion-induced elevation of intracellular Cl(-) concentration ([Cl(-) ](i)). However, the mechanism of inhibition remains unclear. Anion exchanger 3 (AE(3)) is an important regulatory protein for [Cl(-)](i). This study investigated whether AE(3) plays the critical role in the inhibitory effect of SQS on elevation of [Cl(-)](i) induced by ischemia/reperfusion and mediates the cardioprotection of SQS in H9c2 cells. Normal and AE(3) -knockdown H9c2 cells were incubated for 24 h with or without various concentrations of SQS (0.1, 1, or 10 μM) followed by simulated ischemia/reperfusion (sI/R). AE(3) expression was detected by Western blot. Flow cytometer analysis was employed to determine [Cl(-)](i,) [Ca(2+)](i) , reactive oxygen species (ROS) production, and cell apoptosis. The results showed that SQS pretreatment concentration-dependently attenuated sI/R-induced viability loss and lactate dehydrogenase leakage in normal H9c2 cells. Additionally, SQS concentration-dependently up-regulated AE(3) protein expression, and inhibited sI/R-induced the elevation of [Cl(-)](i) followed by the attenuation of Ca(2+) overload, ROS production, and cell apoptosis. However, the dose-dependent cardioprotection induced by SQS was abolished in AE(3) -knockdown H9c2 cells, and the inhibitory effects of SQS on [Cl(-)](i), Ca(2+) overload, ROS production, and cell apoptosis were also reversed. Our data indicate that AE(3) mediates the cardioprotective effect of SQS against sI/R injury. Importantly, AE(3) is required for SQS to inhibit sI/R-induced elevation of [Cl(-)](i), which subsequently inhibited sI/R-induced Ca(2+) overload, ROS production, and cell apoptosis.     

Hypoxic preconditioning up-regulates DJ-1 protein expression in rat heart-derived H9c2 cells through the activation of extracellular-regulated kinase 1/2 pathway

Myocardial preconditioning is a powerful phenomenon that can attenuate ischemia/reperfusion-induced oxidant stress and elicit delayed cardioprotection. Its mechanisms involve activation of intracellular signaling pathways and up-regulation of the protective antioxidant proteins. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in attenuating oxidant stress and promoting cell survival. In the present study, we investigated whether DJ-1 is up-regulated during the late phase of hypoxic preconditioning (HP) and the up-regulation of DJ-1 is mediated by extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway. Rat heart-derived H9c2 cells were exposed to HP. Twenty-four hours later cells were subjected to hypoxia/reoxygenation (H/R) and then cell viability, lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), ERK1/2 phosphorylation, and DJ-1 protein were measured appropriately. The results showed that HP efficiently attenuated H/R-induced viability loss and LDH leakage. In addition, HP promoted ERK1/2 activation, up-regulated DJ-1 protein expression, inhibited H/R induced the elevation of ROS. However, when ERK1/2 phosphorylation was specifically inhibited by U0126, the increase in DJ-1 expression occurring during HP was almost completely abolished and, as a result, the delayed cardioprotection induced by HP was abolished, and the inhibitory effect of HP on H/R-induced oxidant stress was also reversed. Furthermore, knocking down DJ-1 by siRNA attenuated the delayed cardioprotection induced by HP. Our data indicate that HP can up-regulate DJ-1 protein expression through the ERK1/2-dependent signaling pathway. Importantly, DJ-1 might be involved in the delayed cardioprotective effect of HP against H/R injury.     

Anoxic preconditioning up-regulates 14-3-3 protein expression in neonatal rat cardiomyocytes through extracellular signal-regulated protein kinase 1/2

Anoxic preconditioning (APC) attenuates myocardial injury caused by ischemia/reperfusion. The protective mechanisms of APC involve up-regulation of the protective proteins and inhibition of apoptosis. 14-3-3 protein, as a molecular chaperone, plays an important role in regulating cell survival and apoptosis. However, the role of 14-3-3 protein in cardioprotection of APC and the pathways determining 14-3-3 protein expression during APC are not clear. In this work, Western blotting analysis was used to detect the 14-3-3 protein expression and activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in cardiomyocytes subjected to anoxia-reoxygenation injury with and without APC and control. The cardiomyocytes from APC group were more resistant to injury induced by anoxia-reoxygenation and had much stronger phosphorylation of ERK1/2 than the control. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in cardiomyocytes induced by APC. The results indicate that APC up-regulates 14-3-3 protein expression through the ERK1/2 signaling pathways.     

Preconditioning involves selective mitophagy mediated by Parkin and p62/SQSTM1

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia (sI) in vitro and IPC of hearts to investigate the role of Parkin in mediating cardioprotection ex vivo and in vivo. In HL-1 cells, sI induced Parkin translocation to mitochondria and mitochondrial elimination. IPC induced Parkin translocation to mitochondria in Langendorff-perfused rat hearts and in vivo in mice subjected to regional IPC. Mitochondrial depolarization with an uncoupling agent similarly induced Parkin translocation to mitochondria in cells and Langendorff-perfused rat hearts. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports indicating a role for p62/SQSTM1 in mitophagy, we found that depletion of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to sI. While wild type mice showed p62 translocation to mitochondria and an increase in ubiquitination, Parkin knockout mice exhibited attenuated IPC-induced p62 translocation to the mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.     

The Anti-Apoptotic and Cardioprotective Effects of Salvianolic Acid A on Rat Cardiomyocytes following Ischemia/Reperfusion by DUSP-Mediated Regulation of the ERK1/2/JNK Pathway

The purpose of this study was to observe the effects of salvianolic acid A (SAA) pretreatment on the myocardium during ischemia/reperfusion (I/R) and to illuminate the interrelationships among dual specificity protein phosphatase (DUSP) 2/4/16, ERK1/2 and JNK pathways during myocardial I/R, with the ultimate goal of elucidating how SAA exerts cardioprotection against I/R injury (IRI). Wistar rats were divided into the following six groups: control group (CON), I/R group, SAA+I/R group, ERK1/2 inhibitor PD098059+I/R group (PD+I/R), PD+SAA+I/R group, and JNK inhibitor SP600125+I/R group (SP+I/R). The cardioprotective effects of SAA on the myocardium during I/R were investigated with a Langendorff device. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rate of ventricular pressure rise and fall (±dp/dt_max), myocardial infarction areas (MIA), lactate dehydrogenase (LDH), and cardiomyocytes apoptosis were monitored. To determine the crosstalk betwee JNK and ERK1/2 via DUSP2/4/16 with SAA pretreatment, siRNA-DUSP2/4/16 were performed. The expression levels of Bcl-2, Bax, caspase 3, p-JNK, p-ERK1/2 and DUSP2/4/16 in cardiomyocytes were assayed by Western blot. Our results showed that LDH, MIA and cell apoptosis were decreased, and various parameters of heart function were improved by SAA pretreatment and SP application. In the I/R group, the expression levels of p-ERK1/2 and DUSP4/16 were not significantly different compared with the CON group, however, the protein expression levels of p-ERK1/2, Bcl-2 and DUSP4/16 were higher, while p-JNK, Bax, caspase 3 and DUSP2 levels were reduced among the SAA+I/R, PD+SAA+I/R and SP+I/R groups. The above indices were not significantly different between the SAA+I/R and SP+I/R groups. Compared with the SAA+I/R group, p-ERK1/2 was increased and p-JNK was decreased in the SAA+si-DUSP2+I/R, however, p-ERK was downregulated and p-JNK was upregulated in SAA+si-DUSP4+I/R group. SAA exerts an anti-apoptotic role against myocardial IRI by inhibiting DUSP2-mediated JNK dephosphorylation and activating DUSP4/16-mediated ERK1/2 phosphorylation.     

Activation of Ras up-regulates pro-apoptotic BNIP3 in nitric oxide-induced cell death

Nitric oxide (NO) produced by NO synthases causes nitration and nitrosylation of cellular factors. We have shown previously that endogenously produced or exogenously added NO induces expression of BNIP3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3), leading to death of macrophages (Yook, Y.-H., Kang, K.-H., Maeng, O., Kim, T.-R., Lee, J.-O., Kang, K.-i., Kim, Y.-S., Paik, S.-G., and Lee, H. (2004) Biochem. Biophys. Res. Commun. 321, 298-305). We now provide evidence that Ras mediates NO-induced BNIP3 expression via the MEK/ERK/hypoxia-inducible factor (HIF)-1 pathway. (a) ras-Q61L, a constitutively active form of Ras, up-regulated BNIP3 protein expression by enhancing Bnip3 promoter activity, and ras-S17N, a dominant-negative form, and ras-C118S, an S-nitrosylation mutant, blocked NO-induced BNIP3 expression, suggesting that Ras acts downstream of NO and that NO activates Ras by nitrosylation. (b) U0126, a specific MEK inhibitor, completely abolished BNIP3 expression and the stimulation of promoter activity by NO and Ras, whereas 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, SB203580, and wortmannin, specific inhibitors of soluble guanylyl cyclase, p38 MAPK, and phosphatidylinositol 3-kinase, respectively, had no effect. Ras, MEK1/2, and ERK1/2 were sequentially activated by NO treatment of macrophages. (c) Mutation of the HIF-1-binding site (hypoxia-response element) in the Bnip3 promoter abolished BNIP3 induction, and HIF-1alpha was strongly induced by NO. (d) Transient expression of activated Ras promoted macrophage death, as did NO, and this Ras-mediated cell death was inhibited by silencing BNIP3 expression. These results suggest that NO-induced death of macrophages is mediated, at least in part, by BNIP3 induction.     

ERK/PP1a/PLB/SERCA2a and JNK Pathways Are Involved in Luteolin-Mediated Protection of Rat Hearts and Cardiomyocytes following Ischemia/Reperfusion

Luteolin has long been used in traditional Chinese medicine for treatment of various diseases. Recent studies have suggested that administration of luteolin yields cardioprotective effects during ischemia/reperfusion (I/R) in rats. However, the precise mechanisms of this action remain unclear. The aim of this study is to confirm that luteolin-mediated extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways are responsible for their cardioprotective effects during I/R. Wistar rats were divided into the following groups: (i) DMSO group (DMSO); (ii) I/R group (I/R); (iii) luteolin+I/R group (Lut+I/R); (iv) ERK1/2 inhibitor PD98059+I/R group (PD+I/R); (v) PD98059+luteolin+I/R group (PD+Lut+I/R); and (vi) JNK inhibitor SP600125+I/R group (SP+I/R). The following properties were measured: contractile function of isolated heart and cardiomyocytes; infarct size; the release of lactate dehydrogenase (LDH); the percentage of apoptotic cells; the expression levels of Bcl-2 and Bax; and phosphorylation status of ERK1/2, JNK, type 1 protein phosphatase (PP1a), phospholamban (PLB) and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). Our data showed that pretreatment with luteolin or SP600125 significantly improved the contraction of the isolated heart and cardiomyocytes, reduced infarct size and LDH activity, decreased the rate of apoptosis and increased the Bcl-2/Bax ratio. However, pretreatment with PD98059 alone before I/R had no effect on the above indexes. Further, these consequences of luteolin pretreatment were abrogated by co-administration of PD98059. We also found that pretreatment with PD98059 caused a significant increase in JNK expression, and SP600125 could cause ERK1/2 activation during I/R. In addition, we are the first to demonstrate that luteolin affects PP1a expression, which results in the up-regulation of the PLB, thereby relieving its inhibition of SERCA2a. These results showed that luteolin improves cardiomyocyte contractile function after I/R injury by an ERK1/2-PP1a-PLB-SERCA2a-mediated mechanism independent of JNK signaling pathway.     

Protein kinase G-dependent cardioprotective mechanism of phosphodiesterase-5 inhibition involves phosphorylation of ERK and GSK3beta

Sildenafil, a potent inhibitor of phosphodiesterase-5 (PDE-5) induces powerful protection against myocardial ischemia-reperfusion injury. PDE-5 inhibition increases cGMP levels that activate cGMP-dependent protein kinase (PKG). However, the cause and effect relationship of PKG in sildenafil-induced cardioprotection and the downstream targets of PKG remain unclear. Adult ventricular myocytes were treated with sildenafil and subjected to simulated ischemia and reoxygenation. Sildenafil treatment significantly decreased cardiomyocyte necrosis and apoptosis. The PKG inhibitors, KT5823, guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chloro-phenylthio) (R(p)-8-pCPT-cGMPs), or DT-2 blocked the anti-necrotic and anti-apoptotic effect of sildenafil. Selective knockdown of PKG in cardiomyocytes with adenoviral vector containing short hairpin RNA of PKG also abolished sildenafil-induced protection. Furthermore, intra-coronary infusion of sildenafil in Langendorff-isolated mouse hearts prior to ischemia-reperfusion significantly reduced myocardial infarct size after 20 min ischemia and 30 min reperfusion, which was abrogated by KT5823. Sildenafil significantly increased PKG activity in intact hearts and cardiomyocytes. Sildenafil also enhanced the Bcl-2/Bax ratio, phosphorylation of Akt, ERK1/2, and glycogen synthase kinase 3beta. All these changes (except Akt phosphorylation) were significantly blocked by KT5823 and short hairpin RNA of PKG. These studies provide the first evidence for an essential role of PKG in sildenafil-induced cardioprotection. Moreover, our results demonstrate that sildenafil activates a PKG-dependent novel signaling cascade that involves activation of ERK and inhibition of glycogen synthase kinase 3beta leading to cytoprotection.     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.     

Stable overexpression of DJ‐1 protects H9c2 cells against oxidative stress under a hypoxia condition

It has been well accepted that increased reactive oxygen species (ROS) and the subsequent oxidative stress is one of the major causes of ischemia/reperfusion (I/R) injury. DJ‐1 protein, as a multifunctional intracellular protein, plays an important role in regulating cell survival and antioxidant stress. Here, we wondered whether DJ‐1 overexpression attenuates simulated ischemia/reperfusion (sI/R)‐induced oxidative stress. A rat cDNA encoding DJ‐1 was inserted into a mammalian expression vector. After introduction of this construct into H9c2 myocytes, stable clones were obtained. Western blot analysis of the derived clones showed a 2.6‐fold increase in DJ‐1 protein expressing. Subsequently, the DJ‐1 gene‐transfected and control H9c2 cells were subjected to sI/R, and then cell viability, lactate dehydrogenase, malondialdehyde, intracellular ROS and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were measured appropriately. The results showed that stable overexpression of DJ‐1 efficiently attenuated sI/R‐induced viability loss and lactate dehydrogenase leakage. Additionally, stable overexpression of DJ‐1 inhibited sI/R‐induced the elevation of ROS and MDA contents followed by the increase of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities and expression. Our data indicate that overexpression of DJ‐1 attenuates ROS generation, enhances the cellular antioxidant capacity and prevents sI/R‐induced oxidative stress, revealing a novel mechanism of cardioprotection. Importantly, DJ‐1 overexpression may be an important part of a protective strategy against ischemia/reperfusion injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Calcium-sensing receptors induce apoptosis in cultured neonatal rat ventricular cardiomyocytes during simulated ischemia/reperfusion

Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then re-incubated them in a normal culture medium for 24h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase-3, Bcl-2, CaSR, extracellular signal-regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down-regulated Bcl-2 expression, up-regulated caspase-3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl-2, inducing calcium overload and activating the Fas/FasL death receptor pathway.     

Role of ERK1/2 in the anti-apoptotic and cardioprotective effects of nitric oxide after myocardial ischemia and reperfusion

Objective: Experimental results from cultured cells suggest that there is cross-talk between nitric oxide (NO) and extracellular signal-regulated kinase (ERK) in their anti-apoptotic effect. However, the cross-talk between these two molecules in either direction has not been confirmed in the whole organ or whole animal level. The aim of the present study was to determine whether ERK may play a role in the anti-apoptotic and cardioprotective effects of NO in myocardial ischemia/reperfusion (MI/R). Methods: Isolated perfused mouse hearts were subjected to 20 min of global ischemia and 120 min of reperfusion and treated with vehicle or an NO donor (SNAP, 10 μM) during reperfusion. To determine the role of ERK1/2 in the anti-apoptotic and cardioprotective effects of NO, hearts were pre-treated (10 min before ischemia) with U0126, a selective MEK1/2 inhibitor (1 μM). Results: Treatment with SNAP exerted significant cardioprotective effects as evidenced by reduced cardiac apoptosis (TUNEL and caspase 3 activity, p < 0.01), and improved cardiac functional recovery (p < 0.01). In addition, treatment with SNAP resulted in a 2.5-fold increase in ERK activation when compared with heart receiving vehicle. Pre-treatment with U0126 slightly increased post-ischemic myocardial apoptosis but had no significant effect on cardiac functional recovery in this isolated perfused heart model. However, treatment with U0126 completely blocked SNAP-induced ERK activation and markedly, although not completely, inhibited the cardioprotection exerted by SNAP. Conclusion: These results demonstrate that nitric oxide exerts its anti-apoptotic and cardioprotective effects, at least in part, by activation of ERK in ischemic/reperfused heart. The first two authors contribute equally to this study.     

Ischemia/Reperfusion-Induced MKP-3 Impairs Endothelial NO Formation via Inactivation of ERK1/2 Pathway

Mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases. Endothelial cells express multiple MKP family members, such as MKP-3. However, the effects of MKP-3 on endothelial biological processes have not yet been fully elucidated. Here, we address the association between MKP-3 and endothelial Nitric oxide (NO) formation under ischemia/reperfusion (IS/RP) condition. Human umbilical vein endothelial cells (HUVECs) were subjected to IS/RP treatment. The MKP-3 expression and NO formation were examined. IS/RP induced endothelial MKP-3 expression and inhibited eNOS expression and NO formation, accompanied by an increase of endothelial apoptosis. The siRNA experiments showed that MKP-3 was an important mediator in impairing eNOS expression and NO production in endothelial cells. Transfection of HUVECs with constitutively active ERK plasmids suggested that the above mentioned effect of MKP-3 was via inactivation of ERK1/2 pathway. Furthermore, impairment of eNOS expression was restored by treatment of histone deacetylase (HDAC) inhibitor and related to histone deacetylation and recruitment of HDAC1 to the eNOS promoter. Finally, Salvianolic acid A (SalA) markedly attenuated induction of MKP-3 and inhibition of eNOS expression and NO formation under endothelial IS/RP condition. Overall, these results for the first time demonstrated that IS/RP inhibited eNOS expression by inactivation of ERK1/2 and recruitment of HDAC1 to the gene promoter, leading to decreased NO formation through a MKP-3-dependent mechanism in endothelial cells, and SalA has therapeutic significance in protecting endothelial cells from impaired NO formation in response to IS/RP.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

Pathway crosstalk between Ras/Raf and PI3K in promotion of M-CSF-induced MEK/ERK-mediated osteoclast survival

While M-CSF-mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M-CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M-CSF-promoted MEK/ERK activation and osteoclast survival was investigated. M-CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M-CSF-mediated Ras activation. As further support for Ras-mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K-selective or Raf-selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M-CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K-selective Ras-mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK-promoted osteoclast survival induced by M-CSF.