Targeting neuronal MAPK14/p38α activity to modulate autophagy in the Alzheimer disease brain

Dysregulated autophagic-lysosomal degradation of proteins has been linked to the most common genetic defect in familial Alzheimer disease, and has been correlated with disease progression in both human disease and in animal models. Recently, it was demonstrated that the expression of MAPK14/p38α protein is upregulated in the brain of APP-PS1 transgenic Alzheimer mouse and further that genetic deficiency of Mapk14 in the APP-PS1 mouse stimulates macroautophagy/autophagy, which then leads to reduced amyloid pathology via increasing autophagic-lysosomal degradation of BACE1. The findings resolve at least in the context of the APP-PS1 mouse, prior conflicting in vitro observations that have implicated MAPK14 in autophagic processes, and indicate that inhibition of MAPK14 enzyme activity has potential as a therapeutic approach to mitigate a critical physiological defect within neurons of the Alzheimer disease brain. Moreover, the findings suggest that biomarkers of BACE1 activity could be utilized to evaluate the effects of MAPK14 inhibition and other autophagy-inducing therapeutic approaches in human clinical studies, thereby potentially facilitating the clinical development of such agents.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.     

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.     

Caveolin-1 and integrin β1 regulate embryonic stem cell proliferation via p38 MAPK and FAK in high glucose

The involvement of caveolin-1 (Cav-1) and integrin β1 (IN β1) in regulation of embryonic stem (ES) cell growth by high glucose is by no means clear cut. Therefore, the aim of this study was to examine the influence of high glucose on Cav-1 and IN β1 expression in mouse ES cells and their signaling pathways to modulate proliferation. High glucose significantly increased Cav-1 and IN β1 expression. In addition, increased IN β1 expression was inhibited by Cav-1 small interfering RNA (siRNA). High glucose caused reactive oxygen species generation and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Inhibition of p38 MAPK blocked high glucose-induced Cav-1 and fibronectin (FN) expression. Moreover, phosphorylation of both Src and focal adhesion kinase (FAK) were increased by high glucose, which were inhibited by IN β1 antibody. In addition, high glucose increased the expression levels of PINCH1/2, integrin-linked kinase (ILK), and α-parvin [PIP] complex proteins, which were all inhibited by the FAK siRNA and Src specific inhibitor (PP2, 10(-7) M). High glucose also increased F-actin expression, which was inhibited by ILK, PINCH1/2, and α-parvin siRNAs. Finally, high glucose-induced increase of ES cell proliferation was inhibited by TRIO and F-actin binding protein (TRIOBP) siRNA. The results demonstrate that high glucose-induced Cav-1 and IN β1 activation can stimulate ES cell proliferation through the modification of focal adhesion signaling pathways.     

The effect of glucose and manganese on adenosine-3′, 5′-monophosphate levels during growth and differentiation of Aspergillus nidulans

The role of cyclic adenosine monophosphate (cAMP) during growth and development of Aspergillus nidulans was investigated. In normal cultures the highest amount of cAMP, expressed on a dry weight basis, was found after 24 h of growth when still more than 5% glucose was present in the medium. After depletion of the medium even a slight fall in cAMP was noted. Glucose concentrations ranging from 0.5–12% resulted in a slight decrease in the amount of cAMP as measured after 24 h of growth.Cultures with manganese deficiency resulted in a low cAMP level after 24 h of growth. However, the exhaustion of glucose in the absence of manganese was connected with a sharp increase in cAMP. This indicates that manganese shortage was not a direct cause of the low cAMP level after 24 h. The amount of cAMP rose with increasing concentration of manganese in the medium until a maximum at 0.25 M. It is tempting to speculate that this rise in cAMP in the manganese deficient culture is explained by the absence of glucose, that in the control culture is derived from the breakdown of the reserve material -1,3-glucan.Addition of manganese after glucose exhaustion to a manganese deficient culture induced cleistothecium formation. However, they contained only a few ascospores indicating the importance of -1,3 glucan as a carbon and energy source for ascospore formation. The regulation of the level of cAMP by the transport of glucose into the cell or its intracellular concentration is discussed.     

Visfatin induces MUC8 and MUC5B expression via p38 MAPK/ROS/NF-κB in human airway epithelial cells

Background

Among a variety of inflammatory mediators, visfatin is a proinflammatory adipocytokine associated with inflammatory reactions in obesity, metabolic syndrome, chronic inflammatory disease, and autoimmune disease. However, the biological role of visfatin in secretion of major mucins in human airway epithelial cells has not been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of visfatin on MUC8 and MUC5B expression in human airway epithelial cells.

Results

Visfatin significantly induced MUC8 and MUC5B expression. Visfatin significantly activated phosphorylation of p38 MAPK. Treatment with SB203580 (p38 MAPK inhibitor) and knockdown of p38 MAPK by siRNA significantly blocked visfatin-induced MUC8 and MUC5B expression.Visfatin significantly increased ROS formation. Treatment with SB203580 significantly attenuated visfatin-induced ROS formation. Treatment with NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression. However, treatment with NAC and DPI did not attenuate visfatin-activated phosphorylation of p38 MAPK. Visfatin significantly activated the phosphorylation of NF-κB. Treatment with PDTC (NF-κB inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression.

Conclusions

These results suggest that visfatin induces MUC8 and MUC5B expression through p38 MAPK/ROS/NF-κB signaling pathway in human airway epithelial cells.     

Differential activation of MAPK during Müllerian duct growth and apoptosis: JNK and p38 stimulation by DES blocks tissue death

By a dual approach, using electron microscopy and biochemical techniques, we investigated the topology of the somatostatin receptor sst2 with its inhibitory G protein Gialpha after ligand-induced stimulation and internalization in human glioma cells. On intact cells, the sst2 was labeled at 8 degrees C by an antibody directed to its extracellular sequence followed by a 15-nm gold-labeled secondary antibody. In the presence of the ligand, internalization was induced by exposure to 37 degrees C for 5-10 min. Then, cells were either fixed for immunoelectron-microscopic analysis or homogenized for density gradient separation. After post-embedding staining of the sst2-labeled sections with anti-Gialpha1- 3 or anti-caveolin, a co-localization of sst2, Gialpha and caveolin was detected in endosomal vesicles after 5 min of internalization, but not after 10 min. Furthermore, the gold-labeled organelles containing the internalised receptor were separated from the non-labeled ones on sucrose gradients (density shift separation) and analyzed by Western blotting. Also here, in fractions with higher densities, sst2 could be costained with Gialpha and caveolin after 5 min. From these congruent results from both methods, it can be concluded that, in human glioma cells, the receptor sst2 (1) is internalised in caveolin-positive vesicles and (2) is neighboured to its Gialpha proteins at the plasma membrane and early endosomes.     

Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK,p44/42 MAPK and PDGFRβ

Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor β (PDGFRβ) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRβ phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRβ phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.     

α-倒捻子素通过激活ROS/p38 MAPK/Bax级联反应诱导B淋巴瘤Ramos细胞凋亡的机制研究CSCD

本文以B淋巴瘤Ramos细胞为研究对象,探讨α-倒捻子素(α-mangostin,α-Ma)对B淋巴瘤的增殖抑制作用并初步阐释其分子机制。首先通过CCK-8法探究α-Ma对Ramos细胞的增殖抑制作用;再利用倒置显微镜成像法观察α-Ma对Ramos细胞形态的影响;随后利用DCFH-DA、JC-1、Annexin V-FITC/PI荧光染色和流式细胞术检测α-Ma对Ramos细胞活性氧水平、线粒体膜电位、凋亡的影响;并通过蛋白免疫印迹技术测定α-Ma作用Ramos后细胞凋亡相关蛋白及信号通路蛋白的表达情况。CCK-8分析结果显示,α-Ma以药物浓度依赖和时间依赖的方式抑制Ramos细胞增殖,其24 h和48 h的IC值分别为14.84μmol/L和8.087μmol/L;倒置显微镜成像法发现α-Ma能减少Ramos细胞数量并诱导细胞形态发生凋亡样改变;流式细胞术检测发现,α-Ma能增加Ramos细胞内活性氧水平、降低细胞线粒体膜电位,同时诱导细胞凋亡;蛋白免疫印迹结果显示,α-Ma下调caspase-3/9的表达,上调cleaved caspase-3/9、cleaved PARP、Bax、Bim和p-p38 MAPK的表达。综上所述,α-Ma对B淋巴瘤Ramos细胞具有增殖抑制作用,其可能机制是α-Ma活化ROS/p38 MAPK/Bax级联反应诱导B淋巴瘤细胞凋亡。     

Effects of Preconditioning with Sevoflurane on TNF-α-Induced Permeability and Activation of p38 MAPK in Rat Pulmonary Microvascular Endothelial Cells

Preconditioning with sevoflurane (SPC) diminishes effusion of rat alveolar membrane during inflammation. It is not clear whether this preconditioning directly inhibits permeability of pulmonary microvascular endothelial cell (PMVEC) monolayer. In this article, we evaluated effects of SPC on permeability of PMVEC monolayer and identified signaling pathways involved in these effects. PMVEC monolayer was exposed to different conditions (5-hydroxydecanoate (5-HD), TNF-α, SPC, SPC with subsequent exposure to TNF-α and 5-HD, and SPC with subsequent exposure to TNF-α alone), and the permeability of PMVEC monolayer was assessed using FITC-bovine serum albumin (ELISA). Expression of ICAM-1 (Western blot and RT-PCR) and activation of p38 MAPK (Western blot) were also assessed. Compared to the TNF-α group, permeability of PMVEC monolayer in the SPC + TNF-α group was significantly lower. Activation of p38 MAPK was also diminished in the TNF-α group. Pre-treatment with 5-HD reverted beneficial effects of SPC. Expression of ICAM-1 was not modulated by any of the tested experimental exposures. The results of this study demonstrate that SPC is capable of diminishing the TNF-α-induced increase of permeability of PMVEC monolayer, and that this beneficial effect is partly reversed by 5-HD. Further, SPC suppresses activation of p38 MAPK.     

TGF-β1 induces endothelial cell apoptosis by shifting VEGF activation of p38(MAPK) from the prosurvival p38β to proapoptotic p38α

TGF-β1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-β1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-β1 induces endothelial cell expression of VEGF, which mediates TGF-β1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-β1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, β, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-β1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, β, and γ. VEGF activates p38β, whereas TGF-β1 activates p38α. TGF-β1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38β is activated, and the surviving cells become refractory to TGF-β1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-β1 induction of apoptosis, whereas downregulation of p38β or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38β and p38γ transduce survival signaling. TGF-β1 activation of p38α is mediated by VEGF, which in the absence of TGF-β1 activates p38β. Therefore, these results show that TGF-β1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38β to the proapoptotic p38α.     

Skeletal muscle expression of p43, a truncated thyroid hormone receptor α, affects lipid composition and metabolism

Thyroid hormone is a major regulator of metabolism and mitochondrial function. Thyroid hormone also affects reactions in almost all pathways of lipids metabolism and as such is considered as the main hormonal regulator of lipid biogenesis. The aim of this study was to explore the possible involvement of p43, a 43 Kda truncated form of the nuclear thyroid hormone receptor TRα1 which stimulates mitochondrial activity. Therefore, using mouse models overexpressing p43 in skeletal muscle (p43-Tg) or lacking p43 (p43?/?), we have investigated the lipid composition in quadriceps muscle and in mitochondria. Here, we reported in the quadriceps muscle of p43?/? mice, a fall in triglycerides, an inhibition of monounsaturated fatty acids (MUFA) synthesis, an increase in elongase index and an decrease in desaturase index. However, in mitochondria from p43?/? mice, fatty acid profile was barely modified. In the quadriceps muscle of p43-Tg mice, MUFA content was decreased whereas the unsaturation index was increased. In addition, in quadriceps mitochondria of p43-Tg mice, we found an increase of linoleic acid level and unsaturation index. Last, we showed that cardiolipin content, a key phospholipid for mitochondrial function, remained unchanged both in quadriceps muscle and in its mitochondria whatever the mice genotype. In conclusion, this study shows that muscle lipid content and fatty acid profile are strongly affected in skeletal muscle by p43 levels. We also demonstrate that regulation of cardiolipin biosynthesis by the thyroid hormone does not imply p43.     

CD14/TLR1-TLR2/p38MAPK信号通路在莱姆病发病机制中的作用

莱姆病的致病源是伯氏疏螺旋体,机体感染伯氏疏螺旋体后主要表现为神经系统病变、皮肤病变和关节损伤。炎症作为机体受到感染后的一种重要反应,对疾病的发生发展有着重要意义。CD14/TLR1-TLR2/p38 MAPK信号通路通过CD14对伯氏疏螺旋体表面脂蛋白的识别,引起TLR1-TLR2、p38 MAPK等下游信号的激活,同时,机体分泌促炎因子,引起炎症反应。本文就CD14/TLR1-TLR2/p38 MAPK信号通路引起的炎症反应在莱姆病发病机制中的研究进行综述。