Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

Desmosomal cadherins are transmembrane adhesion molecules that provide cell adhesion by interacting in the intercellular space of adjacent cells. In keratinocytes, several desmoglein (Dsg1–4) and desmocollin (Dsc1–3) isoforms are coexpressed. We have shown previously that Dsg2 is less important for keratinocyte cohesion compared with Dsg3 and that the latter forms a complex with p38 MAPK. In this study, we compared the involvement of Dsg2 and Dsg3 in the p38 MAPK-dependent regulation of keratinocyte cohesion. We show that loss of cell adhesion and keratin filament retraction induced by Dsg3 depletion is ameliorated by specific p38 MAPK inhibition. Furthermore, in contrast to depletion of Dsg2, siRNA-mediated silencing of Dsg3 induced p38 MAPK activation, which is in line with immunoprecipitation experiments demonstrating the interaction of activated p38 MAPK with Dsg3 but not with Dsg2. Cell fractionation into a cytoskeleton-unbound and a cytoskeleton-anchored desmosome-containing pool revealed that Dsg3, in contrast to Dsg2, is present in relevant amounts in the unbound pool in which activated p38 MAPK is predominantly detectable. Moreover, because loss of cell adhesion by Dsg3 depletion was partially rescued by p38 MAPK inhibition, we conclude that, besides its function as an adhesion molecule, Dsg3 is strengthening cell cohesion via modulation of p38 MAPK-dependent keratin filament reorganization. Nevertheless, because subsequent targeting of Dsg3 in Dsg2-depleted cells led to drastically enhanced keratinocyte dissociation and Dsg2 was enhanced at the membrane in Dsg3 knockout cells, we conclude that Dsg2 compensates for Dsg3 loss of function.     

CD137 stimulation and p38 MAPK inhibition improve reactivity in an in vitro model of glioblastoma immunotherapy

Dendritic cell vaccination has become an interesting option for cancer immunotherapy. Tumor-lysate-pulsed dendritic cells (DC) can prime naïve T cells and induce the regression of established tumors including gliomas as shown in various animal models. Despite hopeful results even in clinical studies, the outcome for many patients is still unsatisfying. In the present study, we tested the combination of tumor-lysate-pulsed dendritic cells (TPDC) with a monoclonal antibody against CD137, a monoclonal antibody against CD25 (daclizumab) and a specific p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for improving immunostimulation in an in vitro model of immunotherapy for human gliomas. We observed a higher secretion of interferon gamma by TPDC-primed peripheral blood mononuclear cells (PBMC) that were incubated with an antibody against CD137 or the p38 MAPK inhibitor. In addition, we observed higher specific lysis of tumor cells after incubation of PBMC with the p38 MAPK inhibitor or the anti-CD137 antibody. In contrast, incubation of TPDC-primed PBMC with the anti-CD25 antibody did enhance neither interferon gamma secretion nor cellular cytotoxicity. Cell depletion experiments demonstrated that the immune reaction induced by TPDC is strongly dependent on CD4-positive and CD8-positive cells. Incubation of DC during maturation and antigen loading with the anti-CD137 antibody did not enhance cytotoxicity and interferon gamma secretion in comparison with application of the anti-CD137 antibody during priming. In conclusion, our data suggest that p38 MAPK inhibition and anti-CD137 antibodies can enhance the immune response against glioblastoma cells.     

Role of p38γ MAPK in regulation of EMT and cancer stem cells

p38γ is a member of p38 MAPK family which contains four isoforms p38α, p38β, p38γ, and p38δ. p38γ MAPK has unique function and is less investigated. Recent studies revealed that p38γ MAPK may be involved in tumorigenesis and cancer aggressiveness. However, the underlying cellular/molecular mechanisms remain unclear. Epithelial-mesenchymal transition (EMT) is a process that epithelial cancer cells transform to facilitate the loss of epithelial features and gain of mesenchymal phenotype. EMT promotes cancer cell progression and metastasis, and is involved in the regulation of cancer stem cells (CSCs) which have self-renewal capacity and are resistant to chemotherapy and target therapy. We showed that p38γ MAPK significantly increased EMT in breast cancer cells; over-expression of p38γ MAPK enhanced EMT while its down-regulation inhibited EMT. Meanwhile, p38γ MAPK augmented CSC population while knock down of p38γ MAPK decreased CSC ratio in breast cancer cells. MicroRNA-200b (miR-200b) was down-stream of p38γ MAPK and inhibited by p38γ MAPK; miR-200b mimics blocked p38γ MAPK-induced EMT while miR-200b inhibitors promoted EMT. p38γ MAPK regulated miR-200b through inhibiting GATA3. p38γ MAPK induced GATA3 ubiquitination, leading to its proteasome-dependent degradation. Suz12, a Polycomb group protein, was down-stream of miR-200b and involved in miR-200b regulation of EMT. Thus, our study established an important role of p38γ MAPK in EMT and identified a novel signaling pathway for p38γ MAPK–mediated tumor promotion.     

Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.     

The stress kinase MRK contributes to regulation of DNA damage checkpoints through a p38gamma-independent pathway

DNA damage induced by ionizing radiation (IR) activates a complex cellular response that includes checkpoints leading to cell cycle arrest. The stress-activated mitogen-activated protein kinase (MAPK) p38gamma has been implicated in the G(2) phase checkpoint induced by IR. We recently discovered MRK as a member of the MAPK kinase kinase family that activates p38gamma. Here we investigated the role of MRK in the checkpoint response to IR. We identified autophosphorylation sites on MRK that are important for its kinase activity. A phosphospecific antibody that recognizes these sites showed that MRK is activated upon IR in a rapid and sustained manner. MRK depletion by RNA interference resulted in defective S and G(2) checkpoints induced by IR that were accompanied by reduced Chk2 phosphorylation and delayed Cdc25A degradation. We also showed that Chk2 is a substrate for MRK in vitro and is phosphorylated at Thr(68) by active MRK in cells. MRK depletion also increased sensitivity to the killing effects of IR. In addition, MRK depletion reduced IR-induced activation of p38gamma but had no effect on p38alpha activation, indicating that MRK is a specific activator of p38gamma after IR. Inhibition of p38gamma by RNA interference, however, did not impair IR-induced checkpoints. Thus, in response to IR MRK controls two independent pathways: the Chk2-Cdc25A pathway leading to cell cycle arrest and the p38gamma MAPK pathway.     

Rescue of CH31 B cells from antigen receptor-induced apoptosis by inhibition of p38 MAPK

CH31 B lymphomas represent a model for antigen-induced deletional tolerance of immature B lymphocytes, because cross-linking the B cell antigen receptor (BCR) induces G(1) phase arrest and apoptosis. We have recently demonstrated that BCR cross-linking leads to a transient activation of p38 mitogen-activated protein kinase (MAPK) in CH31 B cells. In this paper, we functionally characterize the role of p38 MAPK in BCR-induced apoptosis as well as evaluate the regulation of additional MAPKs by the BCR. We demonstrate that JNK and ERK activities are not affected by BCR cross-linking, suggesting that these MAPKs are not directly involved in initiating the apoptotic cascade. By contrast, we show that pretreatment of CH31 B cells with the highly specific p38 MAPK inhibitor SB203580 ablated both BCR-induced p38 MAPK activity and apoptosis. Pretreatment of CH31 cells with an inactive SB203580 analog, SB202474, did not prevent apoptosis. These findings establish a key role for p38 MAPK in antigen receptor-mediated apoptosis of CH31 B cells.     

Mechanism of p53-dependent apoptosis induced by 3-methylcholanthrene: involvement of p53 phosphorylation and p38 MAPK.

Polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (MC) cause untoward effects including carcinogenesis. Here we investigated the effect of MC on apoptosis. MC induced apoptosis, preceded by serine 15 phosphorylation and accumulation of p53. MC failed to cause apoptosis in p53-deficient MG63 cells, whereas ectopic expression of p53 in MG63 cells restored the response to MC. Therefore, MC-induced apoptosis was dependent on p53. MC also activated p38 mitogen-activated protein kinase (MAPK) at 16-24 h. Accumulation of p53 and p53 phosphorylated at serine 15 was not changed by SB203580, a specific inhibitor of p38 MAPK or overexpression of a dominant negative mutant of p38 MAPK at 8 h after MC treatment, whereas the accumulation was suppressed at 24 h. These results suggest that MC induces accumulation and phosphorylation of p53 via a p38 MAPK-independent (early) and p38 MAPK-dependent (late) pathway. SB203580 repressed MC-induced apoptosis. MC induced p38 MAPK activation in p53 expressing cells but not in p53-deficient cells, indicating that the p38 MAPK activation was dependent on early p53 activation. The current study shows that both p53 and p38 MAPK activation are required for MC-induced apoptosis and provides a novel model of a functional regulation between p53 and p38 MAPK in chemical stress-induced apoptosis.     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

p38 MAP kinase negatively regulates angiotensin II-mediated effects on cell cycle molecules in human coronary smooth muscle cells

Many of the signaling events in VSMC stimulated by angiotensin II (AngII) are mediated by members of the mitogen-activated protein kinase (MAPK) family, including p38 MAPK. The role of p38 MAPK in AngII-mediated cell cycle regulation is poorly understood. Therefore, we examined the involvement of p38 MAPK signaling in AngII-stimulated DNA synthesis, phosphorylation of the retinoblastoma protein (Rb), and expression of the G1-phase cyclin D1 in human coronary artery smooth muscle cells (CASMC). AngII (1 microM) stimulated p38 MAPK and ERK1/2 activation. Pretreatment with the p38 MAPK inhibitors SB203580 (10 microM) (SB) or SKF-86002 (10 microM) (SKF) potently inhibited AngII-induced p38 MAPK activation, but enhanced AngII-mediated ERK1/2 activation. AngII-induced-phosphorylation of Rb (Ser 795 and Ser 807/811), -cyclin D1 expression, and -DNA synthesis was also markedly enhanced by pharmacological inhibition of the p38 MAPK pathway. The present study demonstrates that p38 MAPK negatively regulates AngII-induced ERK1/2 activity, Rb phosphorylation, cyclin D1 expression, and DNA-synthesis in human CASMC. These findings support an important role for p38 MAPK in modulating AngII-mediated VSMC hyperplasia.     

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.     

Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway

Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-β-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2′,7′-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.     

Contribution of circulating leukocytes to cytokine production in pancreatic duct obstruction-induced acute pancreatitis in rats

p38 MAPK was originally characterized as a stress-induced kinase, along with JNK. Subsequently, p38 MAPK was found to be activated by stimuli other than cellular stress, such as growth factors and mitogens, like interleukin (IL)-2, IL-7 and IL-3. A notable exception was IL-4, as studies in mast cells showed no activation of p38 MAPK by this cytokine. In this study we show that the regulation of p38 MAPK is cell type dependent. Like other cytokines that signal through the gamma (gamma)(c), IL-4 can activate p38 MAPK in the CT6 T-cell line and BA/F3 pro-B-cells. However, IL-4 was unable to activate p38 MAPK in the murine macrophage cell line, RAW 264.7 and, indeed, prolonged exposure of cells to IL-4 results in suppression of LPS-induced MAPK activation. This result correlates with the well defined inhibitory effect of IL-4 on tumour necrosis factor alpha (TNFalpha) production. In contrast, studies in primary human monocytes showed that prolonged exposure to IL-4 resulted in enhanced activation of LPS-stimulated p38 MAPK; this correlated with an enhanced TNFalpha production. These data highlight the complexity of IL-4 signalling mechanisms, the diversity that can exist in the regulation of a given signalling pathway by a given cytokine and, furthermore, indicate the problems that can arise from extrapolation between different cell systems.     

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.