Cancer-Induced Muscle Wasting Requires p38ß MAPK Activation of p300.

Cancer-associated cachexia, characterized by muscle wasting, is a lethal metabolic syndrome without defined etiology or established treatment. We previously found that p300 mediates cancer-induced muscle wasting by activating C/EBPß, which then upregulates key catabolic genes. However, the signaling mechanism that activates p300 in response to cancer is unknown. Here, we show that upon cancer-induced activation of Toll-like receptor 4 in skeletal muscle, p38ß MAPK phosphorylates Ser-12 on p300 to stimulate C/EBPß acetylation, which is necessary and sufficient to cause muscle wasting. Thus, p38ß MAPK is a central mediator and therapeutic target of cancer-induced muscle wasting. In addition, nilotinib, an FDA-approved kinase inhibitor that preferentially binds p38ß MAPK, inhibited p300 activation 20-fold more potently than the p38α/ß MAPK inhibitor, SB202190, and abrogated cancer cell-induced muscle protein loss in C2C12 myotubes without suppressing p38α MAPK-dependent myogenesis. Systemic administration of nilotinib at a low dose (0.5 mg/kg/day, i.p.) in tumor-bearing mice not only alleviated muscle wasting, but also prolonged survival. Therefore, nilotinib appears to be a promising treatment for human cancer cachexia due to its selective inhibition of p38ß MAPK. SIGNIFICANCE: These findings demonstrate that prevention of p38ß MAPK-mediated activation of p300 by the FDA-approved kinase inhibitor, nilotinib, ameliorates cancer cachexia, representing a potential therapeutic strategy against this syndrome.

Optimal linker length for small molecule PROTACs that selectively target p38α and p38ß for degradation.

We report the design of hetero-bifunctional small molecules that selectively target p38α and p38ß for degradation. These proteolysis targeted chimeras (PROTACs) are based on an ATP competitive inhibitor of p38α and p38ß, which is linked to thalidomide analogues to recruit the Cereblon E3 ubiquitin ligase complex. Compound synthesis was facilitated by the use of a copper catalyzed "click" reaction. We show that optimization of the linker length and composition is crucial for the degradation-inducing activity of these PROTACs. We provide evidence that these chemical compounds can induce degradation of p38α and p38ß but no other related kinases at nanomolar concentrations in several mammalian cell lines. Accordingly, the PROTACs inhibit stress and cytokine-induced p38α signaling. Our compounds contribute to understanding the development of PROTACs, and provide a useful tool to investigate functions of the p38 MAPK pathway and its involvement in diseases.

p38 MAPK regulates the Wnt inhibitor Dickkopf-1 in osteotropic prostate cancer cells.

The Wnt inhibitor Dickkopf-1 (DKK-1) has been associated with the occurrence of bone metastases in osteotropic prostate cancer by inhibiting osteoblastogenesis. P38 mitogen-activated protein kinase (MAPK) activity is also dysregulated in advanced prostate cancer. However, the impact of p38 MAPK signaling on DKK-1 remains unknown. Inhibition of p38 MAPK signaling in osteolytic PC3 cells by small molecule inhibitors (doramapimod, LY2228820 and SB202190) suppressed DKK-1 expression, whereas activation of p38 MAPK by anisomycin increased DKK-1. Further dissection by targeting individual p38 MAPK isoforms with siRNA revealed a stronger role for MAPK11 than MAPK14 and MAPK12 in the regulation of DKK-1. Moreover, prostate cancer cells with a predominantly osteolytic phenotype produced sufficient amounts of DKK-1 to inhibit Wnt3a-induced osteoblastic differentiation in C2C12 cells. This inhibition was blocked directly by neutralizing DKK-1 using a specific antibody and also indirectly by blocking p38 MAPK. Furthermore, tissue expression in human prostate cancer revealed a correlation between p38 MAPK and DKK-1 expression with higher expression in tumor compared with normal tissues. These results reveal that p38 MAPK regulates DKK-1 in prostate cancer and may present a potential target in osteolytic prostate cancers.

R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling.

Identification of p38ß as a therapeutic target for the treatment of Sézary syndrome.

Cutaneous T-cell lymphomas (CTCLs) represent a group of hematopoietic malignancies that home to the skin and have no known molecular basis for disease pathogenesis. Sézary syndrome (SS) is the leukemic variant of CTCL. Currently, CTCL is incurable, highlighting the need for new therapeutic modalities. We have previously observed that combined small-molecule inhibition of protein kinase C-ß (PKCß) and glycogen synthase kinase 3 (GSK3) causes synergistic apoptosis in CTCL cell lines and patient cells. Through microarray analysis of a SS cell line, we surveyed global gene expression following combined PKCß-GSK3 treatment to elucidate therapeutic targets responsible for cell death. Clinically relevant targets were defined as genes differentially expressed in SS patients that were modulated by combination-drug treatment of SS cells. Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways. Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment. This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells. These data establish p38 as a SS biomarker and a potential therapeutic target for the treatment of CTCL.

Selective regulation of p38ß protein and signaling by integrin-linked kinase mediates bladder cancer cell migration.

Integrin-linked kinase (ILK) and p38(MAPK) are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38(MAPK) is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38(MAPK) is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38ß, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38ß or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38ß cellular protein level, without inhibiting p38ß messenger RNA (mRNA) expression. The loss of p38ß protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38ß. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK-p38ß complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38ß implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK-p38ß signaling complexes, playing a key role in bladder cancer cell migration.

p38ß, A novel regulatory target of Pokemon in hepatic cells.

Pokemon is an important proto-oncogene involved in various biological processes and cancer development, such as cell differentiation, tumorigenesis and metastasis. Pokemon is recognized as a transcription factor localized upstream of several oncogenes, regulating their expression. p38MAPKs act as key regulatory factors in cellular signaling pathways associated with inflammatory responses, cell proliferation, differentiation and survival. p38ß, a member of p38MAPK family, is closely correlated with tumorigenesis, but the mechanism of activation remains unclear. In this study, we found overexpression of Pokemon promoted the growth, migration and invasion of HepG2 cells. However, a p38 inhibitor SB202190 efficiently attenuated the promoting effect of Pokemon in the HepG2 cells. Targeted expression or silencing of Pokemon changed cellular p38ß protein level and phosphorylation of downstream ATF2 in the p38 signaling pathway. Both dual luciferase report assay and ChIP assay suggested that p38ß is a novel regulatory target of the transcription factor Pokemon and positively regulated by Pokemon in hepatic cells.

Thymoquinone induces apoptosis in oral cancer cells through p38ß inhibition.

Oral cancer is a common malignancy associated with high morbidity and mortality. While p38 MAPK is reported to be involved in different cellular activities such as proliferation and differentiation, reports rarely define the roles of the individual members of the p38 MAPK family in cancer. We used two unique cell lines developed by our lab representing chemically induced oral cancer cells (T28) and non-tumor cells (N28) obtained from tissues surrounding the induced cancer as a model to screen out whether p38 MAPK is involved in the malignant transformation processes. The results suggest an association between p38ß not p38α and oral cancer development. Additionally, the anti-cancer activity of thymoquinone (TQ) was screened out and we found evidences suggesting that the anti-tumor activity of TQ may be attributed to the downregulation of p38ß MAPK.

Signaling mechanism of tumor cell-induced up-regulation of E3 ubiquitin ligase UBR2.

The N-end rule pathway contributes significantly to accelerated muscle proteolysis mediated by the ubiquitin-proteasome pathway in various catabolic conditions. UBR2 (aka E3α-II) is the only known E3 ubiquitin ligase of the N-end rule pathway that is up-regulated by cachectic stimuli including proinflammatory cytokines and tumors. However, the signaling mechanism through which UBR2 is up-regulated remains undetermined. Here we identify a signaling pathway that mediates tumor cell-induced up-regulation of UBR2. UBR2 expression in C2C12 myotubes was up-regulated by conditioned medium from Lewis lung carcinoma cells or C26 colon adenocarcinoma cells, which was blocked by a pharmacological inhibitor of p38α/ß mitogen-activated protein kinase (MAPK), SB202190. Similarly, SB202190 administration (i.p.) abolished UBR2 up-regulation in the tibialis anterior of LLC tumor-bearing mice. Genetic gain and loss of function assays in C2C12 myotubes indicated that tumor-induced activation of the p38ß isoform is sufficient and necessary for UBR2 up-regulation. In addition, UBR2 up-regulation required p38ß-mediated phosphorylation of CCAAT/enhancer binding protein (C/EBP)-ß Thr-188, which was critical to C/EBPß binding to the UBR2 promoter. Furthermore, luciferase reporter assay revealed that the C/EBPß binding motif in the UBR2 promoter is a functional C/EBPß-responsive cis-element that enhances the promoter activity on activation by p38ß. Finally, genetic ablation of C/EBPß blocked UBR2 up-regulation in LLC tumor-bearing mice. These results suggest that UBR2 up-regulation in cachectic muscle is mediated by the p38ß-C/EBPß signaling pathway responsible for the bulk of tumor-induced muscle proteolysis.

Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells.

As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 µM) or therapeutic (5, 10 or 20 µM). Retinol at 10 and 20 µM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 µM), SB203580 (10 µM) or siRNA to either p38α (MAPK14) or p38ß (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 µg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by retinol is mediated by the redox-dependent activation of p38/NF-kB signaling pathway.

Intratracheal administration of p38α short-hairpin RNA plasmid ameliorates lung ischemia-reperfusion injury in rats.

BACKGROUND: Lung ischemia-reperfusion injury (LIRI) remains a significant problem after lung transplantation. A crucial signaling enzyme involved in inflammation and apoptosis during LIRI is p38 mitogen-activated protein kinase (MAPK). Gene silencing of p38α by short hairpin RNA (shRNA) can downregulate p38α expression. The lungs have an extremely large surface area, which makes the absorption of shRNA highly effective. Therefore, we evaluated the therapeutic efficacy of p38α shRNA plasmids in a rat model of lung transplantation. METHODS: The delivery of p38α shRNA plasmid was performed by intratracheal administration 48 hours before transplantation into donor rats. Control animals received scrambled shRNA plasmids. Reverse-transcription polymerase chain reaction and Western blots were used to assess gene silencing efficacy. The therapeutic effects of shRNA plasmids were evaluated by lung function tests. We determined the levels of inflammatory cytokines, the level of intercellular adhesion molecule 1 (ICAM-1), c-Myc mRNA expression, and ICAM-1 protein expression, and the presence of cell apoptosis. RESULTS: Rats administered p38α shRNA plasmids showed a significant downregulation in lung expression of p38α transcripts and protein levels. Compared with the control group, the p38α shRNA group showed a higher pulmonary vein oxygen level, lower wet weight-to-dry weight ratio, lower lung injury score, and lower serum levels of tumor necrosis factor-α, interleukin-6, and interleukin-8. Messenger RNA levels of ICAM-1 and c-Myc in the p38α shRNA group were dramatically lower than in the control group. Levels of ICAM-1 protein expression exhibited a similar trend. Cell apoptosis decreased in the p38α shRNA group vs the control group. CONCLUSION: Intratracheal administration of p38α shRNA plasmids provided therapeutic effects in a rat model of lung transplantation.

p38ß MAP kinase as a therapeutic target for pancreatic cancer.

Pancreatic cancer is very difficult to diagnose in its early stage. Molecular marker and imaging have not proven to be accurate modalities for screening of pancreatic cancer. This study aims to develop p38ß as a protein marker for pancreatic cancer and to design peptide inhibitor against the same. The serum p38ß level of pancreatic cancer (n = 35; 5.06 µg/mL) was twofold higher compared to that of the chronic pancreatitis (n = 10; 2.92 µg/mL) and matched normal control (n = 10; 2.86 µg/ml) (p < 0.0005). Peptide inhibitors were designed to inhibit the activity of p38ß and the kinetic assay had shown the dissociation constant, (K(D)) to be 3.16 × 10(-8) M and IC(50), 25 nM by Surface Plasmon Resonance (SPR) and Enzyme-Linked Immunosorbent Assay (ELISA), respectively. The peptide inhibitor also significantly reduced viability and induced cytotoxicity in Human Pancreatic carcinoma epithelial-like cell line (PANC-1) cells.

Transforming growth factor beta 1 induces tight junction disruptions and loss of transepithelial resistance across porcine vas deferens epithelial cells.

Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%-99% decrease in basal transepithelial electrical resistance (R(TE), a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on R(TE) significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.

Inhibition of SAPK2/p38 enhances sensitivity to mTORC1 inhibition by blocking IRES-mediated translation initiation in glioblastoma.

A variety of mechanisms confer hypersensitivity of tumor cells to the macrolide rapamycin, the prototypic mTORC1 inhibitor. Several studies have shown that the status of the AKT kinase plays a critical role in determining hypersensitivity. Cancer cells in which AKT activity is elevated are exquisitely sensitive to mTORC1 inhibitors while cells in which the kinase is quiescent are relatively resistant. Our previous work has shown that a transcript-specific protein synthesis salvage pathway is operative in cells with quiescent AKT levels, maintaining the translation of crucial mRNAs involved in cell-cycle progression in the face of global eIF-4E-mediated translation inhibition. The activation of this salvage pathway is dependent on SAPK2/p38-mediated activation of IRES-dependent initiation of the cyclin D1 and c-MYC mRNAs, resulting in the maintenance of their protein expression levels. Here, we show that both genetic and pharmacologic inhibition of SAPK2/p38 in glioblastoma multiforme cells significantly reduces rapamycin-induced IRES-mediated translation initiation of cyclin D1 and c-MYC, resulting in increased G(1) arrest in vitro and inhibition of tumor growth in xenografts. Moreover, we observed that the AKT-dependent signaling alterations seen in vitro are also displayed in engrafted tumors cells and were able to show that combined inhibitor treatments markedly reduced the mRNA translational state of cyclin D1 and c-MYC transcripts in tumors isolated from mice. These data support the combined use of SAPK2/p38 and mTORC1 inhibitors to achieve a synergistic antitumor therapeutic response, particularly in rapamycin-resistant quiescent AKT-containing cells.

Role of p38 mitogen-activated protein kinase isoforms in murine skin inflammation induced by 12-O-tetradecanoylphorbol 13-acetate.

p38 mitogen-activated protein kinase plays a pivotal role in skin inflammation. The purpose of this study was to investigate the role of the various p38 isoforms. p38ß/δ-knockout-C57BL/6 mice were generated, studied in a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin inflammation model and compared with wild-type mice. The inflammatory response was determined by ear thickness, myeloperoxidase activity and histology. mRNA and protein expression of interleukin (IL)-1ß and IL-6 was determined by quantitative real-time reverse transcription PCR and enzyme-linked immunoassay. In both groups application of TPA resulted in a significant increase in inflammation, and pretreatment with the p38α/ß inhibitor, SB202190 resulted in a significant inhibition. A significantly slower onset but prolonged duration of the response was seen in p38ß/δ knockout mice. This was paralleled by a significant, but transient, lower IL-1ß and IL-6 protein expression in p38ß/δ knockout mice. Although the p38α isoform is important, our data also demonstrate an important role of the p38ß and/or δ isoforms in the regulation of TPA-induced skin inflammation.

Oestrogen prevents cardiomyocyte apoptosis by suppressing p38α-mediated activation of p53 and by down-regulating p53 inhibition on p38ß.

AIMS: we have previously shown that 17-ß-estradiol (E2) protects cardiomyocytes exposed to simulated ischaemia-reperfusion (I/R) by differentially regulating pro-apoptotic p38α mitogen-activated protein kinase (p38α MAPK) and pro-survival p38ß. However, little is known about how E2 modulation of these kinases alters apoptotic signalling. An attractive downstream target is p53, a well-known mediator of apoptosis and a substrate of p38α MAPK. The aim of this study was to determine whether the cytoprotective actions of oestrogen involve regulation of p53 via cardiac p38 MAPKs. METHODS AND RESULTS: cultured rat cardiomyocytes underwent hypoxia followed by reoxygenation (H/R) to simulate I/R. We found that inhibiting p53 significantly reduced apoptosis. Phosphorylation of p53 at serine 15 [p-p53(S15)] increased after H/R in a p38α MAPK- and reactive oxygen species (ROS)-dependent manner. E2 at 10 nM effectively inhibited p-p53(S15) and mitochondrial translocation of p53. Blocking p53 led to augmented p38ß activity and attenuated ROS, suggesting suppression of this antioxidant kinase by p53. The use of a specific agonist for each oestrogen receptor (ER) isoform, ERα and ERß, demonstrated that both isoforms participate in preventing cell death by inhibiting p53 in the mitochondria-centred apoptotic processes. CONCLUSION: our results demonstrate that during H/R stress, cardiomyocytes undergo p53-dependent apoptosis following phosphorylation of p53 by p38α MAPK, leading to p38ß suppression. E2 protects cardiomyocytes by inhibiting p38α-p53 signalling in apoptosis.

p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting.

Oxidative stress is a primary trigger of cachectic muscle wasting, but the signaling pathway(s) that links it to the muscle wasting processes remains to be defined. Here, we report that activation of p38 mitogen-activated protein kinase (MAPK) (phosphorylation) and increased oxidative stress (trans-4-hydroxy-2-nonenal protein modification) in skeletal muscle occur as early as 8 h after lipopolysaccharide (1 mg/kg) and 24 h after dexamethasone (25 mg/kg) injection (intraperitoneal) in mice, concurrent with upregulation of autophagy-related genes, Atg6, Atg7, and Atg12. Treating cultured C2C12 myotubes with oxidant hydrogen peroxide (4 h) resulted in increased p38 phosphorylation and reduced FoxO3 phosphorylation along with induced Atg7 mRNA expression without activation of NF-kappaB or FoxO3a transcriptional activities. Furthermore, inhibition of p38alpha/beta by SB202190 blocked hydrogen peroxide-induced atrophy with diminished upregulation of Atg7 and atrogenes [muscle atrophy F-box protein (MAFbx/Atrogin-1), muscle ring finger protein 1 (MuRF-1), and Nedd4]. These findings provide direct evidence for p38alpha/beta MAPK in mediating oxidative stress-induced autophagy-related genes, suggesting that p38alpha/beta MAPK regulates both the ubiquitin-proteasome and the autophagy-lysosome systems in muscle wasting.

p38 MAPK contributes to CD54 expression and the enhancement of phagocytic activity during macrophage development.

p38 is a subfamily of the mitogen-activated protein kinase (MAPK) superfamily with four isoforms. It has been well established that p38 plays a central role in the production of inflammatory molecules and is therefore required for the activation of macrophages in response to inflammatory stimuli. However, little is known about the roles of p38 in macrophage development. The difficulty to get mice deficient in multiple p38 isoforms complicates the study of p38 in macrophage development. With the model of bone marrow-derived murine macrophages and highly selective p38alpha/beta inhibitors SB203580 and SB239063, here we report that macrophage colony-stimulating factor (M-CSF) induces p38 activation during macrophage development. Inhibition of p38 activity showed minor effects on macrophage proliferation or survival, and did not block CD14, F4/80 expression. However, p38 inhibitors resulted in a significant reduction in CD54 expression and impaired phagocytic activity. Taken together, our data suggest that p38 contributes to macrophage development.

p38 MAPK-dependent eNOS upregulation is critical for 17beta-estradiol-mediated cardioprotection following trauma-hemorrhage.

Studies have shown that p38 MAPK and nitric oxide (NO), generated by endothelial NO synthase (eNOS), play key roles under physiological and pathophysiological conditions. Although administration of 17beta-estradiol (E2) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the E2-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35-40 mmHg for 90 min), followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB-203580 (SB; 2 mg/kg), and nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 microg/kg) treatment, followed by resuscitation, and were killed 2 h thereafter. Cardiovascular performance and other parameters were measured. E2 administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression and phosphorylation at Ser(1177), and nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, E2 also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-alpha), chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-1), and ICAM-1, which was reversed by l-NAME administration. Administration of E2 following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity, and nitrotyrosine level, which were reversed by treatment with SB and l-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated, in part, through activation of p38 MAPK and subsequent eNOS expression and phosphorylation.

Involvement of the p38 mitogen-activated protein kinase alpha, beta, and gamma isoforms in myogenic differentiation.

We and others previously showed that p38 mitogen-activated protein kinase is indispensable for myogenic differentiation. However, it is less clear which of the four p38 isoforms in the mouse genome participates in this process. Using C2C12 myogenic cells as a model, we showed here that p38alpha, beta, and gamma are expressed with distinct expression patterns during differentiation. Knockdown of any of them by small interfering RNA inhibits myogenic differentiation, which suggests that the functions of the three p38 isoforms are not completely redundant. To further elucidate the unique role of each p38 isoform in myogenic differentiation, we individually knocked down one p38 isoform at a time in C2C12 cells, and we compared the whole-genome gene expression profiles by microarrays. We found that some genes are coregulated by all three p38 isoforms, whereas others are uniquely regulated by one particular p38 isoform. Furthermore, several novel p38 target genes (i.e., E2F2, cyclin D3, and WISP1) are found to be required for myogenin expression, which provides a molecular basis to explain why different p38 isoforms are required for myogenic differentiation.