Pirh2 mediates the sensitivity of myeloma cells to bortezomib via canonical NF-κB signaling pathway

Clinical success of the proteasome inhibitor established bortezomib as one of the most effective drugs in treatment of multiple myeloma (MM). While survival benefit of bortezomib generated new treatment strategies, the primary and secondary resistance of MM cells to bortezomib remains a clinical concern. This study aimed to highlight the role of p53-induced RING-H2 (Pirh2) in the acquisition of bortezomib resistance in MM and to clarify the function and mechanism of action of Pirh2 in MM cell growth and resistance, thereby providing the basis for new therapeutic targets for MM. The proteasome inhibitor bortezomib has been established as one of the most effective drugs for treating MM. We demonstrated that bortezomib resistance in MM cells resulted from a reduction in Pirh2 protein levels. Pirh2 overexpression overcame bortezomib resistance and restored the sensitivity of myeloma cells to bortezomib, while a reduction in Pirh2 levels was correlated with bortezomib resistance. The levels of nuclear factorkappaB (NF-κB) p65, pp65, pIKBa, and IKKa were higher in bortezomib-resistant cells than those in parental cells. Pirh2 overexpression reduced the levels of pIKBa and IKKa, while the knockdown of Pirh2 via short hairpin RNAs increased the expression of NF-κB p65, pIKBa, and IKKa. Therefore, Pirh2 suppressed the canonical NF-κB signaling pathway by inhibiting the phosphorylation and subsequent degradation of IKBa to overcome acquired bortezomib resistance in MM cells.     

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.     

Disruption of a single copy of the p38alpha MAP kinase gene leads to cardioprotection against ischemia-reperfusion

The p38 mitogen-activated protein kinase (p38) is activated in the heart during ischemia-reperfusion. However, it is not clear whether the activation of p38 is the protective response or the kinase mediates the cellular damage by ischemia-reperfusion. We examined the role of p38alpha in ischemia-reperfusion injury by studying p38alpha(+/-) mice. The p38alpha protein level in the p38alpha(+/-) heart was 50+/-8.7% compared with that in the p38alpha(+/+) heart. Upon reperfusion following ischemia for 25min, p38alpha activity was transiently increased. The maximum level of p38 activity in p38alpha(+/-) was 60+/-10.5% compared with that in p38alpha(+/+). In the p38alpha(+/+) heart, 25min ischemia and 2h reperfusion resulted in necrotic injury (37.1+/-2.7% of the area at risk), whereas infarct size was drastically reduced to 7.2+/-0.7% in the p38alpha(+/-) heart. These suggested that p38alpha plays a pivotal role in the signal transduction pathway mediating myocardial cell death caused by ischemia-reperfusion.     

The binding of actin to p38 MAPK and inhibiting its kinase activity in vitro

Ineukaryocyte,themitogen-activatedproteinkinases(MAPKs)playcriticalrolesinmanysignaltransductionprocesses[1].p38signalpathwayisanimportantbranchoftheMAPKs[2].Oneoftheprimaryfunctionsofp38medicatestheinflammatorysignalbyphosphorylatingATF[3].Itisknownthatinhibitingthep38activitycanblockthesignaltransductionofinflammationandsub-sequentlyalleviateinflammatoryresponse[4].Inrecentyears,severalresearchgroupshavetriedtousesomespecificinhibitorsofp38forclinictrial[5—7].IthasbeendemonstratedthatP…     

Activation of MKK6, an upstream activator of p38, in Alzheimer's disease

Mitogen-activated protein kinase (MAPK) p38 has been implicated in the pathogenesis of Alzheimer's disease, but the upstream cascade leading to p38 activation has not been elucidated in the disease. In the present study, we focused on mitogen-activated protein kinase kinase 6 (MKK6), one of the upstream activators of p38 MAPK. We found that MKK6 was not only increased but also specifically associated with granular structures in the susceptible neurons in the hippocampus and cortex of Alzheimer's disease patients, but was only weakly diffuse in the cytoplasm in neurons in control cases. Immunoblot analysis demonstrated a significant increase of MKK6 level in Alzheimer's disease compared with age-matched controls. In this regard, in hippocampal and cortical regions of individuals with Alzheimer's disease, the activated phospho-MKK6 was localized exclusively in association with pathological alterations including neurofibrillary tangles, senile plaques, neuropil threads and granular structures, overlapping with activated p38 MAPK suggesting both a functional and mechanic link. By immunoblot analysis, phospho-MKK6 is also significantly increased in AD compared with control cases. Together, these findings lend further credence to the notion that the p38 MAPK pathway is dysregulated in Alzheimer's disease and also indicates an active role for this pathway in disease pathogenesis.     

脊髓p38丝裂原活化蛋白激酶激活参与坐骨神经压迫性损伤所致神经病理性痛

本研究旨在观察脊髓p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38 MAPK）在坐骨神经压迫性损伤所致神经病理性痛中的作用。雄性Sprague-Dawley大鼠鞘内置管后,4-0丝线松结扎左侧坐骨神经制作慢性压迫性损伤（chronic constriction injury,CCI）模型。CCI后第5天,鞘内注射不同剂量的p38 MAPK特异性抑制剂SB203580,并在给药前及给药后不同时间点,分别用von Frey机械痛敏监测仪和热辐射刺激仪监测大鼠损伤侧后爪机械和热刺激反应闽值,用免疫印迹技术（Western blot）观察给药前后脊髓磷酸化p38 MAPK（p-p38 MAPK）和磷酸化环磷酸腺苷反应元件结合蛋白（phosphorylated cAMP response element binding protein,pCREB）表达变化。结果发现：坐骨神经压迫性损伤引起脊髓p-p38 MAPK蛋白表达明显增加;鞘内注射SB203580能剂量依赖性逆转CCI引起的机械性痛觉异常和热痛觉过敏及脊髓水平p-p38 MAPK表达的增加,也明显抑制CCI引起的脊髓pCREB表达的增加。结果提示,脊髓水平p38 MAPK激活参与坐骨神经压迫性损伤所致神经病理性痛的发展,其作用可能通过pCREB介导。     

Endogenous δ-Opioid and ORL1 Receptors Couple to Phosphorylation and Activation of p38 MAPK in NG108-15 Cells and This Is Regulated by Protein Kinase A and Protein Kinase C

The p38 mitogen-activated protein kinase (MAPK) cascade transduces multiple extracellular signals from cell surface to nucleus and is employed in cellular responses to cellular stresses and apoptotic regulation. The involvement of the p38 MAPK cascade in opioid- and opioid receptor-like receptor-1 (ORL1) receptor-mediated signal transduction was examined in NG108-15 neuroblastoma x glioma hybrid cells. Stimulation of endogenous delta-opioid receptor (DOR) or ORL1 resulted in activation of p38 MAPK. It also induced the activation of extracellular signal-regulated kinases (ERKs), another member of the MAPK family, with slower kinetics. Activation of p38 MAPK was abolished by selective antagonists of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant effect on opioid-induced ERK activation, indicating that p38 MAPK activity was not required for ERK activation, though its stimulation preceded ERK activation. Inhibition of protein kinase A (PKA) strongly diminished p38 activation mediated by DOR or ORL1 but had no significant effect on ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation of p38 while inhibiting activation of ERKs. Taken together, our results provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor-mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.     

The binding of actin to p38 MAPK and inhibiting its kinase activity<Emphasis Type="Italic">in vitro</Emphasis>

p38 MAP kinase mediates a signal pathway that is involved in many physiological and pathological processes such as inflammation, cellular stress, apoptosis, cell cycle and growth, ischemia/re-perfusion, and myocardium hypertrophy. To determine the molecular and regulative mechanism of p38 signal pathway, we usedin vitro binding methods to screen the proteins that interact with p38. Here we report two proteins from mouse macrophage RAW264.7 strain treated with lipopolysaccharide (LPS) or ultraviolet radiation (UV), binding directly to p38. One of them is β-actin identified by peptide mass spectrum and ProFound program. Actin can inhibit the autophosphorylation of p38 and the phosphorylation of ATF by p38. It suggests that the binding of actin to p38in vitro may represent a negative feedback to the kinase activity of p38, which leads to the regulation of p38 pathway and cellular function.     

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.     

大鼠脑内远位触液神经元p38丝裂原活化蛋白激酶的分布及在噪声应激时的表达

目的：观察脑内远位触液神经元内p-p38丝裂原活化蛋白激酶（MAPK）的分布及其在噪声应激时的表达。方法：用霍乱毒素亚单位B与辣根过氧化物酶复合物（CB-HRP）标记和免疫组织化学相结合的双重标记技术．观察SD大鼠脑实质内远位触液神经元中p-p38MAPK的分布：进一步制作噪声应激动物模型,观察噪声应激后该类神经元中p-p38MAPK的表达变化。结果：在脑干的特定部位恒定出现被CB-HRP标记的两组神经细胞簇,其他脑区未见CB-HRP标记神经细胞簇。不予应激刺激,该细胞簇内仅有个别神经元见有CB-HRP／p—p38MAPK;噪声应激刺激1d时,上述特定部位细胞簇的CB-HRP／p-p38MAPK双重标记神经元数目没有明显变化;噪音应激刺激5d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激10d时CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激20d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．01）：结论：在脑干特定部位恒定存在的两组被CBHRP标记的细胞团为远位触液神经元,其中少数触液神经元有p-p38MAPK表达,且当给予动物噪声应激刺激时,p-p38MAPK免疫阳性神经元和CB-HRP／p—p38MAPK双重标记神经元数量显著增加,提示脑实质内的这种远位触液神经元中的P—p38MAPK可能参与了机体对噪声应激的信息传递或调控,其作用随应激天数增加而日趋增强．     

Cell type-specific activation of p38 MAPK in the brain regions of hypoxic preconditioned mice

Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly (p < 0.05) in the regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6, n = 6 for each group) when compared to values of the control normoxic group (H0, n = 6) using Western blot analysis. Similar results were also confirmed by an immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38 MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute to the development of cerebral HPC mechanism in mice.