Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK,p44/42 MAPK and PDGFRβ

Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor β (PDGFRβ) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRβ phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRβ phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.     

TGF-β1 induces endothelial cell apoptosis by shifting VEGF activation of p38(MAPK) from the prosurvival p38β to proapoptotic p38α

TGF-β1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-β1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-β1 induces endothelial cell expression of VEGF, which mediates TGF-β1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-β1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, β, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-β1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, β, and γ. VEGF activates p38β, whereas TGF-β1 activates p38α. TGF-β1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38β is activated, and the surviving cells become refractory to TGF-β1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-β1 induction of apoptosis, whereas downregulation of p38β or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38β and p38γ transduce survival signaling. TGF-β1 activation of p38α is mediated by VEGF, which in the absence of TGF-β1 activates p38β. Therefore, these results show that TGF-β1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38β to the proapoptotic p38α.     

A Computer‐Based Model for the Regulation of Mitogen Activated Protein Kinase (MAPK) Activation

Abstract

Computer simulations and mathematical modeling of biological processes are becoming increasingly popular, and yet the complexity of the biochemical systems or the differences between experimental setups make it very difficult to establish a standard formula for these modeling projects. Before we can start using computer‐based models for predictions or targeted experiment designs, it is very important to establish a reliable model on which those predictions can be based and experimentally tested. Here we attempt to present a computer model for the mitogen‐activated protein kinase (MAPK) signaling cascade which is consistent with previously published experimental results. In this study we have focused our attention to a generic MAPK ERK (extracellular signal‐regulated kinase) pathway activated by epidermal growth factor (EGF) in an attempt to understand how receptors may achieve different activation kinetics of the MAPK signaling. We successfully show that the level of receptor expression is one key determinant in this regulation, and that the binding affinity of the active receptor to adaptor proteins can have a small but albeit direct effect on the downstream activation.     

Activity of Ligustrum vulgare L extracts against acute pancreatitis in murine models by regulation of p38 MAPK and NF-κB signaling pathways

Pancreatitis is a fatal disease associated with significant mortality and morbidity. At present, no specific treatment is available for pancreatitis and the patients are mainly treated with supportive medication. The need for specific antipancreatitic chemotherapy is an urgent medical obligation. In the current study, protective effects of the methanolic extract of the Ligustrum vulgare berries were investigated in the rat model of acute pancreatitis. Acute pancreatitis (AP) was induced in the male Sprague-Dawley (SD) rats by cerulein injection. Fruit extract of L. vulgare L extract was prepared using the methanol. Treatment effects of L. vulgare were evaluated in AP rats. Serum levels of lipase, amylase, proinflamatory cytokines (TNF-α, IL-6, TL-1β), lipid peroxidase (LPO), myeloperoxidase (MPO) were determined. Histological changes in the pancreas were assessed. L. vulgare treatment prevented the increase in serum amylase and lipase levels, reduced the disease progression in pancreas, and reduced the serum levels of tumour necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in AP rats. Moreover, L. vulgare significantly suppressed pancreatic edema, inhibited oxidative damage (MPO activity and SOD activity), and inhibited the expression of NF-κB/p65 and activation (phosphorylation) of the inhibitor of NF-κB (IκBα) and p38 MAPKs. Histological examinations showed that L. vulgare significantly reduced the inflammatory and fibrotic changes. The results indicated the potent pancreato-protective effects of L. vulgare in AP.     

Contribution of p38 MAPK, NF-κB and glucocorticoid signaling pathways to ER stress-induced increase in retinal endothelial permeability

Diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. Endoplasmic reticulum (ER) stress is known to play a pathogenic role in vascular impairment in DR. The present study demonstrated that the treatment of human retinal endothelial cells with ER stress inducers such as thapsigargin (Tg) and tunicamycin (Tm) significantly increased the permeability of exogenously added FITC-dextran, accompanied by a decrease of transendothelial electrical resistance (TEER). The expression of claudin-5 among tight junction proteins was significantly decreased by the treatment with Tg or Tm. A p38 MAPK inhibitor, SB203580, and an NF-κB inhibitor, dexamethasone, significantly suppressed the Tg-induced down-regulation of claudin-5, decrease of TEER and leakage of added FITC-dextran. The translocation of NF-κB p65 subunit to the nucleus was also inhibited by the addition of SB203580 or dexamethasone. The effects of dexamethasone are thought to be due to the transrepression of the above signaling and direct regulation of claudin-5 gene.     

Human menstrual blood–derived stem cells promote the repair of impaired endometrial stromal cells by activating the p38 MAPK and AKT signaling pathways

Multiple studies have confirmed that human menstrual blood–derived stem cells (MenSCs) have potential applications in regenerative medicine or cell therapy. However, the contribution of MenSCs to endometrial repair is currently unknown. We evaluated the protective effects of MenSCs on impaired endometrial stromal cells (ESCs), as well as the signaling pathways involved in this process. Mifepristone was used to damage human ESCs, which were subsequently cocultured with MenSCs. The proliferation, apoptosis, and migration of ESCs were assessed, together with the expression of related signaling proteins including total p38 mitogen-activated protein kinase, P-p38, total protein kinase B (AKT), P-AKT, β-catenin, and vascular endothelial growth factor (VEGF). MenSCs significantly recovered the proliferation and migration ability of impaired ESCs, inhibited ESC apoptosis, and upregulated protein expression of P-AKT, P-p38, VEGF, and β-catenin. Our findings suggest that MenSC-based therapies could be promising strategies for the treatment of endometrial injury, and that AKT and p38 signaling pathways may be involved in this process.     

TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages

Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2^−/− macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NF-kappaB pathways was blocked in TNFR1^−/− macrophages. In contrast, although TNFR2^−/− macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB pro-inflammatory signalling pathways.     

Combinatorial treatment with anacardic acid followed by TRAIL augments induction of apoptosis in TRAIL resistant cancer cells by the regulation of p53, MAPK and NFκβ pathways

TRAIL, an apoptosis inducing cytokine currently in phase II clinical trial, was investigated for its capability to induce apoptosis in six different human tumor cell lines out of which three cell lines showed resistance to TRAIL induced apoptosis. To investigate whether Anacardic acid (A1) an active component of Anacardium occidentale can sensitize the resistant cell lines to TRAIL induced apoptosis, we treated the resistant cells with suboptimal concentration of A1 and showed that it is a potent enhancer of TRAIL induced apoptosis which up-regulates the expression of both DR4 and DR5 receptors, which has been observed in the cellular, protein and mRNA levels. The death receptors upregulation consequent to A1 treatment was corroborated by the activation of p53 as well as phosphorylation of p38 and JNK MAP kinases and concomitant inactivation of NFκβ and ERK signaling cascades. Also, A1 modulated the expression of key apoptotic players like Bax, Bcl-2 and CAD along with the abatement of tumor angiogenesis in vivo in EAT mouse model. Thus, post A1 treatment the TRAIL resistant cells turned into TRAIL sensitive cells. Hence our results demonstrate that A1 can synergize TRAIL induced apoptosis through the upregulation of death receptors and downregulation of anti-apoptotic proteins in cancer context.     

A Chemical Genetic Approach Reveals That p38�� MAPK Activation by Diphosphorylation Aggravates Myocardial Infarction and Is Prevented by the Direct Binding of SB203580

The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical “gatekeeper” threonine residue for binding. We addressed these controversies by using mice in which the p38α alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wild-type hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38α kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38α (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.     

Hemokinin-1 gene expression is upregulated in microglia activated by lipopolysaccharide through NF-κB and p38 MAPK signaling pathways

The mammalian tachykinins, substance P (SP) and hemokinin-1 (HK-1), are widely distributed throughout the nervous system and/or peripheral organs, and function as neurotransmitters or chemical modulators by activating their cognate receptor NK(1). The TAC1 gene encoding SP is highly expressed in the nervous system, while the TAC4 gene encoding HK-1 is uniformly expressed throughout the body, including a variety of peripheral immune cells. Since TAC4 mRNA is also expressed in microglia, the resident immune cells in the central nervous system, HK-1 may be involved in the inflammatory processes mediated by these cells. In the present study, we found that TAC4, rather than TAC1, was the predominant tachykinin gene expressed in primary cultured microglia. TAC4 mRNA expression was upregulated in the microglia upon their activation by lipopolysaccharide, a well-characterized Toll-like receptor 4 agonist, while TAC1 mRNA expression was downregulated. Furthermore, both nuclear factor-κB and p38 mitogen-activated protein kinase intracellular signaling pathways were required for the upregulation of TAC4 mRNA expression, but not for the downregulation of TAC1 mRNA expression. These findings suggest that HK-1, rather than SP, plays dominant roles in the pathological conditions associated with microglial activation, such as neurodegenerative and neuroinflammatory disorders.     

The p38 MAPK Regulates IL-24 Expression by Stabilization of the 3′ UTR of IL-24 mRNA

Background

IL-24 (melanoma differentiation-associated gene-7 (mda-7)), a member of the IL-10 cytokine family, possesses the properties of a classical cytokine as well as tumor suppressor effects. The exact role of IL-24 in the immune system has not been defined but studies have indicated a role for IL-24 in inflammatory conditions such as psoriasis. The tumor suppressor effects of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis. Current knowledge on the regulation of IL-24 expression is sparse. Previous studies have suggested that mRNA stabilization is of major importance to IL-24 expression. Yet, the mechanisms responsible for the regulation of IL-24 mRNA stability remain unidentified. As p38 MAPK is known to regulate gene expression by interfering with mRNA degradation we examined the role of p38 MAPK in the regulation of IL-24 gene expression in cultured normal human keratinocytes.

Methodology/Principal Findings

In the present study we show that anisomycin- and IL-1β- induced IL-24 expression is strongly dependent on p38 MAPK activation. Studies of IL-24 mRNA stability in anisomycin-treated keratinocytes reveal that the p38 MAPK inhibitor SB 202190 accelerates IL-24 mRNA decay suggesting p38 MAPK to regulate IL-24 expression by mRNA-stabilizing mechanisms. The insertion of the 3′ untranslated region (UTR) of IL-24 mRNA in a tet-off reporter construct induces degradation of the reporter mRNA. The observed mRNA degradation is markedly reduced when a constitutively active mutant of MAPK kinase 6 (MKK6), which selectively activates p38 MAPK, is co-expressed.

Conclusions/Significance

Taken together, we here report p38 MAPK as a regulator of IL-24 expression and determine interference with destabilization mediated by the 3′ UTR of IL-24 mRNA as mode of action. As discussed in the present work these findings have important implications for our understanding of IL-24 as a tumor suppressor protein as well as an immune modulating cytokine.     

Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gα_q/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca²⁺ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca²⁺-dependent early phase p38 MAPK phosphorylation and extracellular Ca²⁺-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca²⁺ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.     

Effects of Preconditioning with Sevoflurane on TNF-α-Induced Permeability and Activation of p38 MAPK in Rat Pulmonary Microvascular Endothelial Cells

Preconditioning with sevoflurane (SPC) diminishes effusion of rat alveolar membrane during inflammation. It is not clear whether this preconditioning directly inhibits permeability of pulmonary microvascular endothelial cell (PMVEC) monolayer. In this article, we evaluated effects of SPC on permeability of PMVEC monolayer and identified signaling pathways involved in these effects. PMVEC monolayer was exposed to different conditions (5-hydroxydecanoate (5-HD), TNF-α, SPC, SPC with subsequent exposure to TNF-α and 5-HD, and SPC with subsequent exposure to TNF-α alone), and the permeability of PMVEC monolayer was assessed using FITC-bovine serum albumin (ELISA). Expression of ICAM-1 (Western blot and RT-PCR) and activation of p38 MAPK (Western blot) were also assessed. Compared to the TNF-α group, permeability of PMVEC monolayer in the SPC + TNF-α group was significantly lower. Activation of p38 MAPK was also diminished in the TNF-α group. Pre-treatment with 5-HD reverted beneficial effects of SPC. Expression of ICAM-1 was not modulated by any of the tested experimental exposures. The results of this study demonstrate that SPC is capable of diminishing the TNF-α-induced increase of permeability of PMVEC monolayer, and that this beneficial effect is partly reversed by 5-HD. Further, SPC suppresses activation of p38 MAPK.     

IL-32γ Delays Spontaneous Apoptosis of Human Neutrophils through MCL-1, Regulated Primarily by the p38 MAPK Pathway

IL-32γ is a multifunctional cytokine involved in various inflammatory and auto-immune diseases in which neutrophils can affect the evolution of these diseases. To persist at inflammatory sites, neutrophils require inhibition of their rapid and constitutive apoptosis, an inhibitory effect that phlogogenic cytokines support. To date, the effects of IL-32γ on neutrophils remain unknown. We demonstrate that IL-32γ delays, in a dose-dependent manner, the spontaneous apoptosis of human blood neutrophils by activating mainly p38 MAPK through rapid p38 phosphorylation. PI3-K and ERK1/2 MAPK are also involved, but to a lesser extent. Most of cytokines that induce retardation of neutrophil apoptosis activate the expression of MCL-1 at both mRNA and protein levels. IL-32γ added to human blood neutrophils in vitro is associated with sustained levels of MCL-1 protein. This effect in neutrophils corresponds to a decrease of MCL-1 protein degradation without any effect on MCL-1 mRNA levels. The sustained levels of MCL-1 induced by IL-32γ are only abrogated by the p38β MAPK inhibitor SB202190. Additionally, IL-32γ induces a reduction in caspase 3 activity in neutrophils. In conclusion, IL-32γ affects human blood neutrophils in vitro by increasing their survival, suggesting that this cytokine could have profound effects on the deleterious functions of neutrophils in several diseases.     

Involvement of MAPK/NF-κB Signaling in the Activation of the Cholinergic Anti-Inflammatory Pathway in Experimental Colitis by Chronic Vagus Nerve Stimulation

Background

Autonomic nervous system dysfunction is implicated in the etiopathogenesis of inflammatory bowel diseases (IBD). Therapies that increase cardiovagal activity, such as Mind-Body interventions, are currently confirmed to be effective in clinical trials in IBD. However, a poor understanding of pathophysiological mechanisms limits the popularization of therapies in clinical practice. The aim of the present study was to explore the mechanisms of these therapies against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats using a chronic vagus nerve stimulation model in vivo, as well as the lipopolysaccharide (LPS)-induced inflammatory response in human epithelial colorectal adenocarcinoma cells (Caco-2) by acetylcholine in vitro.

Methods and Results

Colitis was induced in rats with rectal instillation of TNBS, and the effect of chronic VNS (0.25 mA, 20 Hz, 500 ms) on colonic inflammation was evaluated. Inflammatory responses were assessed by disease activity index (DAI), histological scores, myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS), TNF-α and IL-6 production. The expression of Mitogen-activated protein kinases (MAPK) family members, IκB-α, and nuclear NF-κB p65 were studied by immunoblotting. Heart rate variability (HRV) analysis was also applied to assess the sympathetic-vagal balance. DAI, histological scores, MPO activity, iNOS, TNF-α and IL-6 levels were significantly decreased by chronic VNS. Moreover, both VNS and acetylcholine reduced the phosphorylation of MAPKs and prevented the nuclear translocation of NF-κB p65. Methyllycaconitine (MLA) only reversed the inhibitory effect on p-ERK and intranuclear NF-κB p65 expression by ACh in vitro, no significant change was observed in the expression of p-p38 MAPK or p-JNK by MLA.

Conclusion

Vagal activity modification contributes to the beneficial effects of the cholinergic anti-inflammatory pathway in IBD-related inflamed colonic mucosa based on the activation of MAPKs and nuclear translocation of NF-κB. Our work may provide key pathophysiological mechanistic evidence for novel therapeutic strategies that increase the cardiovagal activity in IBD patients.     

O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β-N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins (O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase (O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr(308) and Ser(473) phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.