P38 MAPK Inhibitor SB203580 Research Articles

Dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathways in lung squamous cell carcinoma.

Lung squamous cell carcinoma (SCC) is one of the deadliest cancers both in China and worldwide. To date, the efficacy of lung SCC treatments is limited. Recent studies have elucidated the powerful anti-tumour role of dioscin in different human cancers. Here, our study aims to investigate the effect of dioscin on lung SCC and its underlying mechanism. First, we found that dioscin not only inhibited cell proliferation and cell migration and induced cell apoptosis in lung SCC cells but also suppressed tumour growth in tumour-bearing mice. Furthermore, we noted that the accumulation of intracellular reactive oxygen species (ROS) was triggered by dioscin in lung SCC cells, leading to the phosphorylation of HSP27 through p38-MAPK and consequent cell apoptosis. The activation of p38-MAPK/HSP27 induced by the p38-MAPK activator Anisomycin enhanced the apoptosis of lung SCC cells, while the ROS inhibitor N-acetyl-L-cysteine (NAC) and the p38-MAPK inhibitor SB203580 both attenuated dioscin-mediated cell apoptosis. Moreover, NAC suppressed the activation of p38-MAPK/HSP27 that induced by dioscin. In conclusion, these results confirm that dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathway in lung SCC.

Effect of p38 mitogen-activated protein kinase inhibitor on liver function and tissue in rats with hepatic hydatidosis

Objective To investigate the effect of p38 mitogen-activated protein kinase (p38 MAPK) inhibitor on liver function and tissue in rats with hepatic hydatidosis. Methods A model of liver echinococcosis was established in 100 female Wistar rats, 60 of 100 were, randomly divided into three groups, Control group (0.3 ml normal saline), Low dose group (50 μmol/L p38MAPK inhibitor SB-202190), High dose group (100 μmol/L SB-202190B). The reagents were given via the hepatic artery 1, 3, 7, 14 and 42 days after the rat model was generated. Rats were sacrificed 42 days after the intervention, liver tissue and blood samples were collected for liver function study. Results Alanine aminotransferase levels were (49.58±2.38) U/L, (38.35±1.34) U/L and (30.93±1.51) U/L and aspartic aminotransferase levels were (67.45±5.14) U/L, (54.86±1.09) U/L and (45.76±1.04) U/L in the Control group, the Low-dose group and High-dose group, showing a decreasing trend, with statistically significant differences (all P<0.05). Triglycerides in the Low-dose group were higher than those in Control group and the High-dose group, with statistically significant differences (all P<0.05). In the Control group, the hepatocytes were severely injured, with almost no normal hepatocytes left, and the normal hepatocyte boundaries were also disrupted, he normal hepatic lobule was replaced by the pseudolobules. In the Low-dose group, there were more inflammatory cells, and less replacement of normal liver cells by pseudolobules. High dose group of a small amount of inflammatory cells infiltration, roughly normal liver cells, normal liver cell line is clear, visible central vein of liver cells. Conclusion p38MAPK inhibitor SB-202190 improved liver function and reduced liver tissue damage in rats with hepatic hydatidosis. Key words: Echinococcosis, hepatic; P38 mitogen-activated protein kinases; Inhibitors; Liver function

Exogenous FABP4 interferes with differentiation, promotes lipolysis and inflammation in adipocytes.

Fatty acid binding protein 4 (FABP4) has been demonstrated to be secreted from adipocytes in an unconventional pathway associated with lipolysis. Circulating FABP4 is elevated in metabolic disorders and has been shown to affect various peripheral cells such as pancreatic β-cells, hepatocytes and macrophages, but its effects on adipocytes remains unclear. The aim of this study was to investigate the effects of exogenous FABP4 (eFABP4) on adipocyte differentiation and function. 3T3-L1 pre-adipocytes or mature adipocytes were treated with recombinant FABP4 in the absence or presence of FABP4 inhibitor I-9/p38 MAPK inhibitor SB203580; Meanwhile male C57BL/6J mice were subcutaneously injected twice a day with recombinant FABP4 (0.35 mg/kg) with or without I-9 (50 mg/kg) for 2 weeks. The effects of eFABP4 on differentiation, lipolysis and inflammation were determined by triglyceride measurement or lipolysis assay, western blotting, or RT-qPCR analysis. eFABP4 treatment significantly reduced intracellular triglyceride content and decreased expression of adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), intracellular FABP4, and adiponectin in 3T3-L1 cells. Besides, eFABP4 promoted lipolysis and inflammation in differentiated 3T3-L1 adipocytes as well as in adipose tissue of eFABP4-treated C57BL/6J mice, with elevated gene expression of monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and elevated protein expression of adipose triglyceride lipase (ATGL), phosphorylation of hormone-sensitive lipase (HSL) (Ser-660), p38, and nuclear factor-kappa B (NF-κB). The pro-inflammatory and pro-lipolytic effects of eFABP4 could be reversed by SB203580/I-9. These findings indicate that eFABP4 interferes with adipocyte differentiation, induces p38/HSL mediated lipolysis and p38/NF-κB mediated inflammation in adipocytes in vitro and in vivo.

Pomegranate peel extract ameliorates liver fibrosis induced by carbon tetrachloride in rats through suppressing p38MAPK/Nrf2 pathway

Abstract Hepatic fibrosis is a chronic process characterized by excessive production of extracellular matrix in response to liver injury, which eventually leads to cirrhosis and even death. Growing evidence has demonstrated that pomegranate could efficiently reduce liver fibrosis in rats. Previous studies have also shown that Nrf2 activation alleviated liver fibrosis. However, whether the MAPK-Nrf2 pathway were involved in the protective effect of extracts from pomegranate peels (EPP) on carbon tetrachloride (CCl4)-induced hepatic fibrosis remains elusive. The hepatic fibrosis model was established by injection with 50% CCl4 subcutaneously in male SD rats twice a week for four weeks. After four weeks, EPP and/or signaling inhibitors were administered every day for 4 weeks, respectively. The protective effects of EPP on liver function, oxidative markers, and liver fibrosis were determined. The results showed that EPP treatment decreased MDA levels, enhanced GSH-Px activities, inhibited the development of hepatic fibrosis evidenced by reduced TGF-β and collagen1-α2 levels as well as fibrosis areas, improved liver function in CCl4 injury rats. Similarly, p38MAPK inhibitor SB203580 produced the protective effect as EPP in the setting of rat model. Moreover, Nrf2 inhibitor ML385 did not affect the inhibitory effect of EPP on p38MAPK activity, which indicates that Nrf2 might be a downstream molecule of p38MAPK in the protective mechanism of EPP against hepatic fibrosis. In conclusion, EPP displays a protective effect against liver fibrosis induced by CCl4, and its underlying mechanisms might be associated with suppressing p38MAPK-Nrf2 signaling pathway.

Yes-Associated Protein 1 Plays Major Roles in Pancreatic Stellate Cell Activation and Fibroinflammatory Responses.

Background: Yes-associated protein 1 (YAP), a transcriptional co-activator and major effector of the Hippo pathway, regulates cell differentiation and morphology in many cell types and supports aberrant tumor growth. Recent studies showed that YAP is expressed in pancreas tissues in pancreatic ductal adenocarcinoma (PDAC) patients and experimental models of PDAC, with YAP largely found in cancer cells and pancreatic stellate cells (PaSC) in the stroma.Methods and Results: We studied here the role of YAP in the activated phenotype of PaSC. We found that YAP is expressed at low levels in normal mouse pancreas, but protein levels significantly increased after pancreas inflammatory damage induced by repeated cerulein administration in wild-type mice or upon initiation of neoplastic transformation of the pancreas parenchyma in Ptf1-Cre;LSL-KrasG12D/+ (KC) mice. In these animal models, YAP upregulation occurred in parallel with activation and proliferation of PaSC. Consistent with these findings, we found robust YAP expression in culture-activated mouse and human PaSC but not in quiescent, freshly isolated cells. Fully activated PaSC isolated from KC mice or PDAC patient tissues exhibited robust nuclear YAP suggesting YAP transcriptional activity. Agents that induce quiescence such as the Bromodomain and Extra-Terminal (BET) inhibitor iBET151 and the p38 MAPK inhibitor SB203580 reduced YAP levels in PaSC. Stimulation of PaSC with the potent mitogen PDGF elicited marked YAP Ser127 phosphorylation. However, unexpectedly, this effect did not diminish YAP nuclear localization, suggesting that YAP phosphorylation at this site does not govern YAP cellular localization in PaSC. siRNA-mediated knockdown of YAP reduced PDGF-induced PaSC expansion in culture and blunted the persistent activation of Akt and ERK elicited by PDGF stimulation, supporting a role for YAP in PDGF-induced cell growth. YAP knockdown also blunted fibroinflammatory gene expression responses both in unstimulated and transforming growth factor beta 1 (TGFβ1)-stimulated PaSC.Conclusion: Our data suggest a central role for YAP in sustaining the activated phenotype and fibroinflammatory responses in PaSC. Moreover, our findings indicate that a complex crosstalk between YAP, TGFβ1, and PDGF pathways regulates PaSC activity and growth.

Inhibition of Heme Oxygenase-1 Attenuates Bortezomib Resistance in Multiple Myeloma Via Down-Regulating Gas6 Production

Background Multiple myeloma (MM), one of the most prevalent hematological malignancies, is characterized by the proliferation of abnormal plasma cells. The proteasome inhibitor bortezomib is a frontline drug in the treatment of MM; however, most patients will develop acquired resistance to bortezomib, which has been a critical challenge in clinic. Our previous studies have indicated that inhibition of heme oxygenase-1 (HO-1) can attenuate drug resistance to MM. Moveover, emerging evidence suggests that growth arrest-specific gene 6 (Gas6) may regulate tumor microenvironment and serve as a novel target for cancer therapy. However, whether HO-1 mediated-Gas6 production involved in the development of bortezomib resistance in MM remains unclear. Aims The aims to perform the present study were to: 1) assess the association between intracellular HO-1 and Gas6 expression in the bone marrow of CD138+ cells of patients with MM; 2) test the role of HO-1 and Gas6 in the development of bortezomib resistance in MM; 3) explore whether HO-1 mediate Gas6 production and the potential mechanism. Methods Patients with different stages of MM (n=18) and healthy donors (n=18) at Affiliated Hospital of Guizhou Medical University were included in this study. Baseline clinical characteristic data and bone marrow samples were obtained from study participants. CD138+ plasma cells were purified from bone marrow samples. The recombinant lentiviral vector of HO-1 was constructed and subsequent transfected into U266 and RPMI8226 cells. The expressions of mRNA and protein levels were determined by real-time PCR and western blot, respectively. Secreted Gas6 was determined by ELISA. Flow cytometry was used to assess cell apoptosis. Results In human study, the HO-1 mRNA and protein level in CD138+ cells of patients with MM showed a significant increase compared with those in healthy group, respectively (P&lt;0.01). With the development of MM, HO-1 mRNA and protein level were gradually increased from stage I to III. The similar findings were also observed in the expression of Gas6 mRNA and protein level (P&lt;0.01). Interestingly, spearman correlation analysis showed that higher HO-1 mRNA and protein level were associated with increased Gas6 mRNA and protein level, respectively (P&lt;0.05). To test whether HO-1 mediate Gas6 production, recombinant lentivirus-HO-1 vector and HO-1 siRNA were transfected into U266 and RPMI8226 cells to up/down-regulate HO-1 expression. Compared with control group, Gas6 mRNA and protein expression were significantly increased when U266 and RPMI8226 cells were up-regulated by recombinant lentivirus-HO-1 vector. Additionally, Gas6 mRNA and protein expression were also increased by the use of hemin (HO-1 inducer). In contrast, Gas6 expression presented a decrease in mRNA and protein level with the stimulation of HO-1 siRNA and HO-1 inhibitor ZnPPIX. Similar significantly trend was also observed in the culture medium. Moreover, the effect of HO-1 on bortezomib resistance in MM cells was markedly inhibited by the stimulation with Gas6 siRNA and Gas6 neutralizing antibody (the apoptosis rate in MM cells treated with PBS, Gas6 siRNA and Gas6 neutralizing antibody is 28%, 56% and 42%, respectively; P&lt;0.01). These results indicated that HO-1 up-regulated Gas6 production in the development of bortezomib resistance in MM. Furthermore, to explore the potential mechanism how HO-1 regulates Gas6 production, MAPK, an important pathway in drug resistance, was investigated. U266 or RPMI8226 cells transfected with recombinant lentiviral vector of HO-1 were stimulated with p38MAPK inhibitor SB203580, the MEK inhibitor PD0325901, the JNK inhibitor SP600125, the PI3K inhibitor LY294002, and the STAT3 inhibitor S3I-201, and the HO-1 level of cells and Gas6 levels of cells and culture medium were determined. Findings showed that HO-1 level did not significantly change, while Gas6 expression in cells and Gas6 level in culture medium showed markedly decrease by the treatment with MAPK pathway inhibitor compared with control group. These results indicated that HO-1 mediated Gas6 production via MAPK pathway. Conclusion These results demonstrate that inhibition of HO-1 attenuates bortezomib resistance in MM via down-regulating Gas6 production. Targeting these pathological interactions holds promise for improve MM treatment. Disclosures No relevant conflicts of interest to declare.

Mechanism of delayed apoptosis of peripheral blood neutrophils in severely burned patients

To observe the changes of peripheral blood neutrophil apoptosis rate and the effect of p38 mitogen-activated protein kinase (p38MAPK) inhibitor on peripheral blood neutrophil apoptosis in severely burned patients. Severe burn patients [burn area ≥ 30% total body surface area (TBSA), deep II to III degrees, burn index ≥ 20%, age > 20 years old] admitted to the department of burn and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from January to May in 2019 and health examination volunteers during the same period were enrolled. The peripheral blood neutrophils were isolated by Ficoll gradient centrifugation. The neutrophils of severely burned patients were divided into burn group and p38MAPK inhibitor SB203580 group (after 30 minutes incubation at room temperature and 24 hours incubation in incubator); the neutrophils of healthy volunteers were used as control group. The apoptosis of neutrophils was detected by flow cytometry; the level of reactive oxygen species (ROS) in neutrophils was detected by fluorescence probe; the expression of p38MAPK protein and its phosphorylation (p-p38MAPK) level were detected by Western Blot. Compared with the control group, the apoptosis rate of neutrophils in burn group was significantly decreased [(37.42±3.14)% vs. (50.20±9.87)%, P < 0.05], and the ROS production level in neutrophils was significantly increased (fluorescence intensity: 83.28±0.29 vs. 75.23±0.34, P < 0.05). The apoptosis rate of neutrophils in SB203580 group was further lower than that in burn group [(25.51±1.56)% vs. (37.42±3.14)%, P < 0.05], and the level of ROS production was further higher than that in burn group (fluorescence intensity: 95.56±3.67 vs. 83.28±0.29, P < 0.05). There was no significant difference in the protein expression of p38MAPK among the three groups. The p-p38MAPK protein level in burn group was significantly lower than that in control group (p-p38MAPK/p38MAPK: 0.45±0.06 vs. 0.91±0.09, P < 0.05), while the expression level of p-p38MAPK in SB203580 group was further lower than that in burn group (p-p38MAPK/p38MAPK: 0.22±0.04 vs. 0.45±0.06, P < 0.05). Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.

Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells

Although we recently reported that sudachitin (5,7,4′-trihydroxy-6,8,3′-trimethoxyflavone), a polymethoxyflavone isolated from the peel of Citrus sudachi, can induce apoptosis in human keratinocyte HaCaT cells, the mechanism underlying its action remains unclear. In this study, we explored the mechanisms underlying sudachitin-induced apoptosis in HaCaT cells. Sudachitin activated p38MAPK and inhibited ERK1/2, whereas another polymethoxyflavone, nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone), activated ERK1/2. The p38MAPK inhibitor SB203580 significantly attenuated sudachitin-induced heat shock protein 27 phosphorylation, downstream of p38MAPK, and subsequent apoptosis, indicating that sudachitin induces apoptosis via the p38MAPK pathway. Additionally, sudachitin inhibited serum- and EGF-stimulated Raf-1-ERK1/2 activation, and blocked EGF-induced cell migration and proliferation in HaCaT cells. These results suggest that small structural differences in polymethoxyflavones can induce different cellular responses by altering the regulation of MAPK activities and that sudachitin may be a potential candidate for developing new drugs for skin diseases such as psoriasis.

Resveratrol induces apoptosis of benign prostatic hyperplasia epithelial cell line (BPH-1) through p38 MAPK-FOXO3a pathway

BackgroundResveratrol is reported to inhibit the growth of prostate, which is characteristic of benign prostatic hyperplasia (BPH) condition. However, the mechanism remains unclear. This study aimed to identify the effects and probable mechanism of resveratrol on BPH.MethodsWe used the BPH epithelial cell line BPH-1 to investigate the effect of resveratrol. Cells were treated with various concentrations of resveratrol, and its effects on cells viability, apoptosis, ROS accumulation, and cell cycle were assessed. Western blot was used to examine activation of p38 MAPK and protein levels of FOXO3a, Bcl2, Bcl-XL, and caspase3. Cells were also co-treated with the p38 MAPK inhibitor SB203580 or ROS scavenger N-Acetyl-L-cysteine (NAC) to further investigate the mechanism.ResultsResveratrol treatment inhibited the growth of BPH-1 and increased apoptosis of cells. In addition, levels of phosphorylated p38 MAPK level was elevated and FOXO3a repression was observed. Concomitantly, ROS was accumulated. All of these resveratrol-mediated effects were suppressed by additional treatment with SB203580 or NAC. Resveratrol was also found to induce cell cycle arrest at S phase.ConclusionsResveratrol can activate p38 MAPK and repress FOXO3a, thereby causing repression of SOD2, catalase, and increase of ROS accumulation, leading to apoptosis in BPH-1 cells.

Dysregulated expression of ACTN4 contributes to endothelial cell injury via the activation of the p38-MAPK/p53 apoptosis pathway in preeclampsia.

Preeclampsia (PE) is a hypertensive disease associated with increased endothelial cell dysfunction caused by systemic oxidative stress. Alpha-actinin-4 (ACTN4) is a member of the α-actinin family of actin crosslinking proteins that are upregulated in several types of cancer. However, its role in PE remains unclear. In this study, we found that ACTN4 was localized in placenta vascular endothelial cells (ECs), and its expression was downregulated in primary human umbilical vein endothelial cells (HUVECs) from severe preeclamptic patients compared to that in HUVECs from normotensive pregnant women. ACTN4 expression was also decreased in normotensive HUVECs treated with H2O2. Downregulation of ACTN4 by siRNA or H2O2 treatment promoted normotensive HUVEC apoptosis and increased p38-MAPK phosphorylation along with elevated levels of p53 phosphorylation, caspase cascade proteins, and bax and repressed expression of bcl-2. Conversely, upregulation of ACTN4 in PE HUVECs significantly inhibited apoptosis and decreased p38-MAPK phosphorylation compared to that of the PE HUVEC controls. In addition, overexpression of ACTN4 in normotensive HUVECs attenuated H2O2 treatment-induced apoptosis with decreased p53 phosphorylation, caspase cascade, and bax expression levels and increased expression of bcl-2 compared to that of only H2O2 treatment. Moreover, the suppression of ACTN4 induced apoptosis, which could be blocked by the p38-MAPK inhibitor SB202190. Collectively, these results demonstrate that dysregulated ACTN4 expression may be associated with PE due to its effects on endothelial cell apoptosis via the p38-MAPK/p53 apoptosis pathway.

Dehydroepiandrosterone alleviates E. Coli O157:H7-induced inflammation by preventing the activation of p38 MAPK and NF-κB pathways in mice peritoneal macrophages

As an important metabolite in cholesterol metabolism, dehydroepiandrosterone (DHEA) can modulate the immune function in animals and humans, but the underlying mechanism is still unclear. The present study investigated the effect and mechanism of DHEA’s anti-inflammatory action in primary mice peritoneal macrophages infected with E. coli O157:H7. The finding showed that DHEA improved the phagocytic ability in E. coli O157:H7-infected macrophages. DHEA inhibited the cytokines (including tumor necrosis factor-α, interleukin-1β and interleukin-6) secretion in E. coli O157:H7-infected macrophages. The inducible nitric oxide synthase and cyclooxygenase-2 protein level were significantly decreased in E. coli O157:H7-infected macrophages treated with DHEA. In addition, DHEA markedly decreased the phospho (p)-p38 MAPK protein level in E. coli O157:H7-infected macrophages. Furthermore, DHEA prevented the nuclear translocation of NF-κB by decreasing of p-IκB-α protein level in E. coli O157:H7-infected macrophage; and these effects of DHEA were heightened when the cells were pre-treated with p38 MAPK inhibitor SB203580. Our data indicated that DHEA alleviates the pro-inflammatory mediator production in E. coli O157:H7-infected mice peritoneal macrophages; and this beneficial action associated with it prevents the activation of p38 MAPK and NF-κB signaling pathway.

Lobaplatin-Induced Apoptosis Requires p53-Mediated p38MAPK Activation Through ROS Generation in Non-Small-Cell Lung Cancer.

Platinum-based chemotherapy is recommended as the first-line treatment regimen for patients with advanced non-small-cell lung cancer (NSCLC). Lobaplatin (LBP), a third-generation platinum anti-neoplastic agent, has shown an improved efficacy. This study is aimed to investigate the mechanisms of LBP-induced apoptosis in the A549 p53 wild-type cell line. The Cell Counting Kit-8 assay (CCK-8), flow cytometry (FCM), Western blot, xenograft tumor models, terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), and RNA interference were used in this study. Our results showed that the proliferation of A549 cells could be inhibited by LBP. At lower concentrations, LBP triggered cell cycle arrest at the G1 phase in A549 cells. LBP could also induce apoptosis of A549 cells. LBP also increased the expression of PARP and Bax and the cleavage of caspase-3, caspase-8, and caspase-9 and reduced Bcl-2 expression. In vivo experiment confirmed that LBP could inhibit tumor growth in the A549 xenograft models and induce apoptosis. Apoptosis of A549 cells was decreased after transfected with p53 shRNA or treated with reactive oxygen species inhibitor NAC and p38MAPK inhibitor SB203580, suggesting that the p53/ROS/p38MAPK pathway appeared to mediate the LBP-induced apoptosis of A549 cells. Our data demonstrate that LBP could be a promising candidate for the treatment of NSCLC with wild-type p53.

Hesperetin Induces Apoptosis in Human Glioblastoma Cells via p38 MAPK Activation

Glioblastoma (GBM) is the most common and aggressive form of malignant brain tumor, with poor prognosis and a lack of effective treatment. Hesperetin, a natural product found in citrus fruits, displayed bioactivities including antioxidant, anti-inflammatory, and anticancer, while its effects on GBM cells were largely unknown. Here, we explored the anticancer effect of hesperetin on human GBM cells in vitro, as well as the underlying signaling mechanisms. By CCK-8 assay and live/dead assay, hesperetin presented significant inhibitory effect on human GBM U-251 and U-87 cell viability. By DAPI staining and Annexin V-FITC/PI assay, apoptotic death was proved to contribute to the cell viability reduction, and it was verified by the increased Bax/Bcl-2 ratio in western blotting results. Furthermore, by cell cycle analysis and western blotting for cyclin B1, CDK1, and p21, hesperetin was found to induce cell-cycle arrest at G2/M phase. For signaling mechanism, the western blotting results showed elevated p38 MAPK activation, and the reduced Bcl-2 and enhanced Bax upon hesperetin treatment were partly reversed by p38 MAPK inhibitor SB203580. In summary, we have discovered hesperetin as a natural product candidate for the treatment of GBM, and that it could induce GBM cell apoptosis via p38 MAPK activation.

Retinoic acid-induced expression of CD103 in human DCs is dependent on p38 MAPK signaling and NFAT1c

Abstract Mucosal CD103+ (αE integrin+) DCs are critically involved in driving a T regulatory response and enabling mucosal homeostasis. However, the regulation of CD103 expression in human DCs is not fully understood. We previously found that retinoic acid (RA) induces CD103 expression in human monocyte-derived DCs (MoDCs). Here, we investigated the molecular mechanisms leading to RA-induced CD103 expression on human DCs. We used a phospho-protein array to screen RA-treated MoDCs and found multiple potential targets. We focused on p38 MAPK based on previous studies that showed reduced CD103+ DCs in p38 MAPK KO mice. Western blots confirmed increased phosphorylation of p38 MAPK in RA-treated MoDCs. To determine whether p38 MAPK signaling influences CD103 expression, we next used an inhibitor of p38 MAPK and evaluated CD103 expression on MoDCs. The p38 MAPK inhibitor SB202190 significantly decreased expression of CD103 protein and mRNA in RA treated, but not in control MoDCs (p=0.037 and p=0.047 respectively). The transcription factor NFAT1c is a downstream target of p38 MAPK that has been shown to bind to the promotor region of CD103 in T cells. To determine whether NFAT1c regulates RA-induced CD103 expression, we next exposed RA-treated MoDCs to an NFAT1c peptide inhibitor. We demonstrated a significant dose-dependent decrease in CD103 protein expression of RA-treated MoDCs with increasing concentrations of the NFAT1c inhibitor (p=0.03). These data indicate that RA-induced expression of CD103 is dependent on p38 MAPK signaling and NFAT1c in human DCs and give insight into molecular mechanisms of DC signaling.

Regulation of p38MAPK-mediated dendritic cell functions by the deubiquitylase otubain 1.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that represent the essential link between innate and acquired immunity. Otubain (OTUB) 1 is shown to deubiquitinate TRAFs to suppress virus-induced inflammatory response. MAPK, a downstream molecule of TRAFs, is involved in regulating LPS-induced immune reactions and its activation is sensitive to the presence of OTUB1. Little is known about contributions of OTUB1 to changes in biological properties of DCs. The present study, therefore, explored whether DC functions are influenced by OTUB1. To this end, DCs were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs) and followed by treatment with lipopolysaccharide (LPS) in the presence or absence of OTUB1 siRNA. Expression of markers of cellular maturation and proliferation were analyzed by flow cytometry, and secretion of inflammatory cytokines and ability to stimulate CD4+ T-cells in allogenic mixed leukocyte reaction (allo-MLR) by ELISA, cell migration by a transwell migration assay and phagocytic capacity by FITC-dextran uptake measurement. As a result, treatment of the cells with OTUB1 siRNA prolonged activation of p38MAPK, increased CD54 expression and IL-6 release and reduced FITC-dextran uptake. Moreover, cytokine release produced from CD4+ T-cells in allo-MLR was different. The enhanced level of IFN-γ, but not other cytokine production was observed in the presence of siRNA OTUB1. All the effects were completely abolished when the cells were exposed with p38MAPK inhibitor SB203580. In conclusion, OTUB1 prevents the prolonged activation of p38MAPK, which in turn compromises DC functions.

Porcine epidemic diarrhea virus infections induce apoptosis in Vero cells via a reactive oxygen species (ROS)/p53, but not p38 MAPK and SAPK/JNK signalling pathways

Porcine epidemic diarrhea virus (PEDV) is a member of Coronavirus, which causes severe watery diarrhea in piglets with high morbidity and mortality. ROS and p53 play key roles in regulating many kinds of cell process during viral infection, however, the exact function in PEDV-induced apoptosis remains unclear. In this study, the pro-apoptotic effect of PEDV was examined in Vero cells and we observed that PEDV infection increased MDM2 and CBP, promoted p53 phosphorylation at serine 20 and, promoted p53 nuclear translocation, leading to p53 activation in Vero cells. Treatment with the p53 inhibitor PFT-α could significantly inhibit PEDV-induced apoptosis. We also observed PEDV infection induced time-dependent ROS accumulation. Treatment with antioxidants, such as pyrrolidine dithiocarbamate (PDTC) or N-acetylcysteine (NAC), significantly inhibited PEDV-induced apoptosis.Moreover, further inhibition tests were established to prove that p53 was regulated by ROS in PEDV-induced apoptosis. In addition, we also found that p38 MAPK and SAPK/JNK were activated in PEDV-infected Vero cells. However, treatment with the p38 MAPK inhibitor SB203580, and the SAPK/JNK inhibitor SP600125 reversed PEDV-induced apoptosis. Taken together, the results of this study demonstrate that activated p53 and accumulated ROS participated in PEDV-induced apoptosis and p53 could be regulated by ROS during PEDV infection. Activated p38 MAPK and SAPK/JNK exerted no influence on PEDV-induced apoptosis. These findings provide new insights into the function of p53 and ROS in the interaction of PEDV with Vero cells.

Aβ1-42 increases the expression of neural KATP subunits Kir6.2/SUR1 via the NF-κB, p38 MAPK and PKC signal pathways in rat primary cholinergic neurons.

ATP-sensitive potassium channels (KATP) may mediate a potential neuroprotective role in Alzheimer's disease (AD). Given that exposure to Aβ1-42 in cultured primary cholinergic neurons for 72 h significantly upregulates the expression of KATP subunits Kir6.2/SUR1, we aim to study the underlying signal transduction mechanisms that are involved in Aβ1-42-induced upregulation of KATP subunits Kir6.2/SUR1. In the present study, we first identified the primary cultured rat cortical and hippocampal neurons using immunocytochemistry. 0.5 μM NF-κB inhibitor SN-50, 2 μM p38MAPK inhibitor SB203580 or 2 μM PKC inhibitor Chelerythrine chloride (CTC) were then added in three separate groups, followed by 2 μM Aβ1-42 30 min later in all 3 groups. Western Blot was performed 72 h later to detect the expression of KATP subunits Kir6.2/SUR1. We found that Aβ1-42 significantly increased the level of KATP subunits Kir6.2/SUR1 expression at 72 h when compared with the control group ( p < 0.05). However, when compared with the Aβ1-42 group, the level of KATP subunits Kir6.2/SUR1 expression at 72 h significantly decreased in the SN50 + Aβ1-42 group, SB203580 + Aβ1-42 group, and the CTC + Aβ1-42 group ( p < 0.05). Our findings suggest that the NF-κB, p38 MAPK, and PKC signal pathways are partially involved in the upregulation of KATP subunits Kir6.2/SUR1 expression induced by Aβ1-42 cytotoxicity in neurons, which supports a potential theoretical basis of targeting these signal pathways in the treatment of AD.

Plastin 3 down-regulation augments the sensitivity of MDA-MB-231 cells to paclitaxel via the p38 MAPK signalling pathway

Plastin 3 (PLS3) overexpression may serve as a marker for predicting chemotherapeutic outcomes in drug-resistant cancer cells, but the mechanism is unclear. Herein, we show that the down-regulation of PLS3 by PLS3 gene silencing augments the sensitivity of MDA-MB-231 triple-negative breast cancer cells to paclitaxel. Interestingly, a low concentration of paclitaxel was able to induce strong apoptosis in the PLS3-silenced cells. Further study revealed that p38 MAPK signalling was responsible for the increased sensitivity to paclitaxel in these cells, as the p38 MAPK inhibitor SB203580 impaired the changes mediated by PLS3 down-regulation in response to paclitaxel. Therefore, our study identifies PLS3 as a potential target for enhancing the p38 MAPK-mediated apoptosis induced by paclitaxel. Unlike paclitaxel, Abraxane was unable to induce strong apoptosis in the PLS3-silenced cells. As PLS3 was found to be involved in the process of endocytosis in breast cancer cells, the reliance of cellular Abraxane uptake on this process may render it not as efficient as paclitaxel in PLS3-depleted tumour cells. The finding that PLS3 could be a critical regulator of paclitaxel sensitivity may have important implications for breast cancer chemotherapy.

P38-MAP Kinase Negatively Regulates the Slow Force Response to Stretch in Rat Myocardium through the Up-Regulation of Dual Specificity Phosphatase 6 (DUSP6).

Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na⁺/H⁺ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na⁺-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation.

The mechanism of GLT-1 mediating cerebral ischemic injury depends on the activation of p38 MAPK

The previous studies have shown that glial glutamate transporter-1 (GLT-1) participates in cerebral ischemic injury in rats. However, the mechanism involved remains to be elucidated. This study was undertaken to investigate whether p38 MAPK was involved in regulating GLT-1 in the process. At first, it was observed that global brain ischemia for 8 min led to obvious delayed neuronal death, GLT-1 down-regulation and p-p38 MAPK up-regulation in CA1 hippocampus in rats. Then, whether p-p38 MAPK was involved in regulating GLT-1 during cerebral ischemic injury was studied in vitro. Astrocyte-neuron co-cultures exposed to oxygen and glucose deprivation (OGD) were used to mimic brain ischemia. It was observed that lethal OGD (4-h OGD) decreased GLT-1 expression and increased p-p38 MAPK expression in astrocytes. The p-p38 MAPK protein rised from 0 min to 48 h that is the end time of the observation, and the peak value was at 12 h, which was 12.45 times of the control group. Moreover, pre-administration of p38 MAPK inhibitor SB203580 or its siRNA dose-dependently increased GLT-1 expression, and meanwhile alleviated the neuronal death induced by lethal OGD. The above results indicated that p38 MAPK signaling pathway participated in regulating GLT-1 during OGD injury in vitro. Finally, back to in vivo experiment, it was found that pre-administration of SB203580 by intracerebroventricular injection dose-dependently reversed the down-regulation of GLT-1 expression and attenuated the delayed neuronal death normally induced by global brain ischemia in CA1 hippocampus in rats. Taken together, it can be concluded that the mechanism of GLT-1 mediating cerebral ischemic injury depends on the activation of p38 MAPK.