Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways

The human cardiovascular system has adapted to function optimally in Earth''s 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47^phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47^phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47^phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47^phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.     

Amelogenin promotes odontoblast-like MDPC-23 cell differentiation via activation of ERK1/2 and p38 MAPK

Amelogenin (AMG) is a highly conserved protein secreted by ameloblasts. Some research indicates that AMG might induce the differentiation and maturation of odontoblasts. The aim of this study was to clarify the function of AMG during the differentiation of odontoblast-like MDPC-23 cells. The results revealed that the alkaline phosphatase activity and the number of mineralized nodules were significantly enhanced in AMG-overexpressing MDPC-23 cells during the mineralization process. Tissue-specific markers such as dentin matrix protein 1 and dentin sialophosphoprotein also elevated significantly, indicating the cell differentiation and maturation process. Furthermore, AMG could upregulate the phosphorylation levels of ERK1/2 and p38 MAPK. However, JNK, another MAPK pathway molecule, didn't change the activity at all. And the differentiation induced by AMG was abrogated when the MDPC-23 cells were treated with U0126 and SB203580, the inhibitors of ERK1/2 and p38, respectively. Taken together, our present results showed that AMG could promote the differentiation of odontoblast-like MDPC-23 cells via ERK1/2 and p38 MAPK pathways.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

Syndecan-1 downregulates syndecan-4 expression by suppressing the ERK1/2 and p38 MAPK signaling pathways in cultured vascular endothelial cells

Syndecan-1 and syndecan-4 are members of the syndecan family of transmembrane heparan sulfate proteoglycans. Vascular endothelial cells synthesize both species of proteoglycans and use them to regulate the blood coagulation-fibrinolytic system and their proliferation via their heparin-like activity and FGF-2 binding activity, respectively. However, little is known about the crosstalk between the expressions of the proteoglycan species. Previously, we reported that biglycan, a small leucine-rich dermatan sulfate proteoglycan, intensifies ALK5–Smad2/3 signaling by TGF-β₁ and downregulates syndecan-4 expression in vascular endothelial cells. In the present study, we investigated the crosstalk between the expressions of syndecan-1 and other proteoglycan species (syndecan-4, perlecan, glypican-1, and biglycan) in bovine aortic endothelial cells in a culture system. These data suggested that syndecan-1 downregulated syndecan-4 expression by suppressing the endogenous FGF-2-dependent ERK1/2 pathway and FGF-2-independent p38 MAPK pathway in the cells. Moreover, this crosstalk was a one-way communication from syndecan-1 to syndecan-4, suggesting that syndecan-4 compensated for the reduced activity in the regulation of vascular endothelial cell functions caused by the decreased expression of syndecan-1 under certain conditions.     

Thrombin stimulates dissociation and induction of HSP27 via p38 MAPK in vascular smooth muscle cells

We investigated the effects of thrombin on the induction of heat shock proteins (HSP) 70 and 27, and the mechanism behind the induction in aortic smooth muscle A10 cells. Thrombin increased the level of HSP27 but had little effect on the level of HSP70. Thrombin stimulated the accumulation of HSP27 dose dependently between 0.01 and 1 U/ml and cycloheximide reduced the accumulation. Thrombin stimulated an increase in the level of HSP27 mRNA and actinomycin D suppressed the thrombin-increased mRNA level. Thrombin induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The HSP27 accumulation by thrombin was reduced by SB-203580 and PD-169316 but not by SB-202474. SB-203580 and PD-169316 suppressed the thrombin-induced phosphorylation of p38 MAPK. SB-203580 reduced the thrombin-increased level of HSP27 mRNA. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by thrombin. Dissociation was inhibited by SB-203580. Thrombin induced the phosphorylation of HSP27 and the phosphorylation was suppressed by SB-203580. These results indicate that thrombin stimulates not only the dissociation of HSP27 but also the induction of HSP27 via p38 MAPK activation in aortic smooth muscle cells.     

Hispolon from Phellinus linteus possesses mediate caspases activation and induces human nasopharyngeal carcinomas cells apoptosis through ERK1/2, JNK1/2 and p38 MAPK pathway

Hispolon, a phenol compound isolated from Phellinus linteus (PL), possesses anti-inflammatory, antiproliferative, and antioxidant effects. However, the effects of hispolon on human nasopharyngeal carcinomas have yet to be evaluated. Here, the molecular mechanism by which hispolon anticancer effects in human nasopharyngeal carcinomas cells was investigated. The results showed that hispolon significantly inhibited cell proliferation of HONE-1 and NP-039 cell lines. Furthermore, hispolon induced apoptosis through caspases-3, -8, and -9 activations and PARP cleavage in dose- and time-dependent manner in HONE-1 and NP-039 cells. Moreover, hispolon also showed that increase phosphorylation of ERK1/2, p38 MAPK and JNK1/2 in dose- and time-dependent manner by western blot analysis. However, hispolon-induced activation of the caspase-3, -8 and -9 significantly abolished by inhibition of p38 MAPK and JNK1/2 specific inhibitors. In this study, we determine that the effects of hispolon on the apoptosis and related regulation mechanism in HONE-1 and NPC-039 cells takes place. Our findings revealed that hispolon may be a useful candidate as a chemotherapeutic agent for NPC therapy.     

IL-27 induces Th1 differentiation via p38 MAPK/T-bet- and intercellular adhesion molecule-1/LFA-1/ERK1/2-dependent pathways

IL-27, a novel member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in positive and negative regulations of immune responses. We recently demonstrated that IL-27 induces Th1 differentiation through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-independent mechanism. In this study, we further investigated the molecular mechanisms by focusing on p38 MAPK and ERK1/2. IL-27-induced Th1 differentiation was partially inhibited by lack of T-bet expression or by blocking ICAM-1/LFA-1 interaction with anti-ICAM-1 and/or anti-LFA-1, and further inhibited by both. Similarly, the p38 MAPK inhibitor, SB203580, or the inhibitor of ERK1/2 phosphorylation, PD98059, partially suppressed IL-27-induced Th1 differentiation and the combined treatment completely suppressed it. p38 MAPK was then revealed to be located upstream of T-bet, and SB203580, but not PD98059, inhibited T-bet-dependent Th1 differentiation. In contrast, ERK1/2 was shown to be located downstream of ICAM-1/LFA-1, and PD98059, but not SB203580, inhibited ICAM-1/LFA-1-dependent Th1 differentiation. Furthermore, it was demonstrated that STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK, and that IL-27 directly induces mRNA expression of growth arrest and DNA damage-inducible 45gamma, which is known to mediate activation of p38 MAPK. Finally, IL-12Rbeta2 expression was shown to be up-regulated by IL-27 in both T-bet- and ICAM-1/LFA-1-dependent mechanisms. Taken together, these results suggest that IL-27 induces Th1 differentiation via two distinct pathways, p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways. This is in contrast to IL-12, which induces it via only p38 MAPK/T-bet-dependent pathway.     

p38MAPK suppresses chronic pancreatitis by regulating HSP27 and BAD expression

Mitogen-activated protein kinases (MAPKs) are ubiquitous proteins that function in both normal and stress-related pathophysiological states of the cell. This study aimed to analyze the importance of p38MAPK in pancreatic injury using WBN/Kob rats with spontaneous chronic pancreatitis. Male WBN/Kob rats were injected with the p38MAPK inhibitor SB203580, starting at the age of 4 weeks, and sacrificed 6 weeks later. Compared with vehicle-treated rats, p38 inhibitor-treated rats exhibited a significant increase in pancreatic cell death and inflammation as assessed by histologic examination and myeloperoxidase activity, respectively. p38 inhibition decreased the expression of heat shock protein 27 (HSP27), an antioxidant protein, and enhanced accumulation of reactive oxygen species (ROS). In addition, the proapoptotic protein BAD was increased in the pancreas of rats treated with p38 inhibitor. In a pancreatic cell line (PANC-1), HSP27 knockdown augmented reactive oxygen species accumulation and cell death induced by tumor necrosis factor-α plus actinomycin D. In conclusion, p38MAPK suppresses chronic pancreatitis by upregulating HSP27 expression and downregulating BAD expression.     

Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain

We have previously reported that cyclic strain results in rapid phosphorylation of p38 mitogen activated protein kinase (MAPKs). The aim of this study was to examine the role of protein phosphatase type 2A (PP2A) in regulating p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain. In this study, we demonstrate that the catalytic subunit of PP2A is tyrosine phosphorylated by cyclic strain, resulting in inhibition of phosphatase activity. Okadaic acid, an inhibitor of PP2A at lower concentrations increased phosphorylation of p-38. Phospho-p38 MAPK physically associated with the catalytic subunit, PP2Ac. Phospho-p38 MAPK was dephosphorylated by purified PP2Ac in cell lysates, but if pretreated with okadaic acid, phospho-p38 MAPK was maintained. Taken together, our result suggests that PP2A plays a regulatory role in p38 MAPK activation in endothelial cells exposed to cyclic strain.     

Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs’ proliferation and migration. Over-expression of Bcl-2 increased HAECs’ tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation.Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.     

Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells

Angiotensin II activates a variety of signaling pathways in vascular smooth muscle cells (VSMCs), including the MAPKs and Akt, both of which are required for hypertrophy. However, little is known about the relationship between these kinases or about the upstream activators of Akt. In this study, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive kinase p38 MAPK and its substrate MAPKAPK-2 mediate Akt activation in VSMCs. In unstimulated VSMCs, Akt and p38 MAPK are constitutively associated and remain so after angiotensin II stimulation. Inhibition of p38 MAPK activity with SB-203580 dose-dependently inhibits Akt phosphorylation on Ser⁴⁷³, but not Thr³⁰⁸. Angiotensin II-induced phosphorylation of MAPKAPK-2 is also attenuated by SB-203580, as well as by inhibitors of ROS. In addition, angiotensin II stimulates the association of MAPKAPK-2 with the Akt-p38 MAPK complex, and an in vitro kinase assay shows that MAPKAPK-2 immunoprecipitates of VSMC lysates phosphorylate recombinant Akt in an angiotensin II-inducible manner. Finally, intracellular delivery of a MAPKAPK-2 peptide inhibitor blocks Akt phosphorylation on Ser⁴⁷³. These results suggest that the p38 MAPK-MAPKAPK-2 pathway mediates Akt activation by angiotensin II in these cells by recruiting active MAPKAPK-2 to a signaling complex that includes both Akt and p38 MAPK. Through this mechanism, p38 MAPK confers ROS sensitivity to Akt and facilitates downstream signaling. These results provide evidence for a novel signaling complex that may help to spatially organize hypertrophy-related, ROS-sensitive signaling in VSMCs. mitogen-activated protein kinase; reactive oxygen species     

Dextran sulfate sodium enhances interleukin-1 beta release via activation of p38 MAPK and ERK1/2 pathways in murine peritoneal macrophages

Interleukin (IL)-1 beta is a pro-inflammatory cytokine that has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD), however, the molecular mechanisms underlying the production of IL-1 beta in IBD are not fully understood. We investigated dextran sulfate sodium (DSS)-induced IL-1 beta production and caspase-1 activities in murine peritoneal macrophages (pM phi). Further, the activation status of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun NH(2)-terminal kinase (JNK1/2), as well as their upstream target kinases, were examined by Western blotting. In addition, mRNA expression was assessed by RT-PCR and CXC chemokine ligand 16 (CXCL16) protein was detected by immunocytochemistry. DSS-treated pM phi released IL-1 beta protein in a time-dependent manner without affecting mRNA levels during 3-24 h, and caspase-1 activity peaked at 5 min (29-fold). IL-1 beta release and caspase-1 activity induced by DSS were significantly inhibited by a MAPK kinase 1/2 inhibitor, a p38 MAPK inhibitor, and NAC, however, not by JNK1/2 or a protein kinase C inhibitor. In addition, DSS strikingly induced the phosphorylation of p38 MAPK and ERK1/2 within 2 and 10 min, respectively. DSS also induced intracellular generation of reactive oxygen species (ROS). Pre-treatment with anti-CXCL16 for 24 h, but not anti-scavenger receptor-A, anti-CD36, or anti-CD68 antibodies, significantly suppressed DSS-induced IL-1 beta production. Our results suggest that DSS triggers the release of IL-1 beta protein from murine pM phi at a post-translational level through binding with CXCL16, ROS generation, and resultant activation of both p38 MAPK and ERK1/2 pathways, and finally caspase-1 activation.