Activation of p38MAPK contributes to expanded polyglutamine-induced cytotoxicity

Background

The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood.

Methodologies/Principal Findings

Herein we demonstrate that expanded polyglutamine protein cytotoxicity is mediated primarily through activation of p38MAPK and that the atypical PKC iota (PKCι) enzyme antagonizes polyglutamine-induced cell death through induction of the ERK signaling pathway. We show that pharmacological blockade of p38MAPK rescues cells from polyglutamine-induced cell death whereas inhibition of ERK recapitulates the sensitivity observed in cells depleted of PKCι by RNA interference. We provide evidence that two unrelated proteins with expanded polyglutamine repeats induce p38MAPK in cultured cells, and demonstrate induction of p38MAPK in an in vivo model of neurodegeneration (spinocerebellar ataxia 1, or SCA-1).

Conclusions/Significance

Taken together, our data implicate activated p38MAPK in disease progression and suggest that its inhibition may represent a rational strategy for therapeutic intervention in the polyglutamine disorders.     

ERK/MAPK is essential for endogenous neuroprotection in SCN2.2 cells

Background

Glutamate (Glu) is essential to central nervous system function; however excessive Glu release leads to neurodegenerative disease. Strategies to protect neurons are underdeveloped, in part due to a limited understanding of natural neuroprotective mechanisms, such as those present in the suprachiasmatic nucleus (SCN). This study tests the hypothesis that activation of ERK/MAPK provides essential protection to the SCN after exposure to excessive Glu using the SCN2.2 cells as a model.

Methodology

Immortalized SCN2.2 cells (derived from SCN) and GT1-7 cells (neurons from the neighboring hypothalamus) were treated with 10 mM Glu in the presence or absence of the ERK/MAPK inhibitor PD98059. Cell death was assessed by Live/Dead assay, MTS assay and TUNEL. Caspase 3 activity was also measured. Activation of MAPK family members was determined by immunoblot. Bcl2, neuritin and Bid mRNA (by quantitative-PCR) and protein levels (by immunoblot) were also measured.

Principal Findings

As expected Glu treatment increased caspase 3 activity and cell death in the GT1-7 cells, but Glu alone did not induce cell death or affect caspase 3 activity in the SCN2.2 cells. However, pretreatment with PD98059 increased caspase 3 activity and resulted in cell death after Glu treatment in SCN2.2 cells. This effect was dependent on NMDA receptor activation. Glu treatment in the SCN2.2 cells resulted in sustained activation of the anti-apoptotic pERK/MAPK, without affecting the pro-apoptotic p-p38/MAPK. In contrast, Glu exposure in GT1-7 cells caused an increase in p-p38/MAPK and a decrease in pERK/MAPK. Bcl2-protein increased in SCN2.2 cells following Glu treatment, but not in GT1-7 cells; bid mRNA and cleaved-Bid protein increased in GT1-7, but not SCN2.2 cells.

Conclusions

Facilitation of ERK activation and inhibition of caspase activation promotes resistance to Glu excitotoxicity in SCN2.2 cells.

Significance

Further research will explore ERK/MAPK as a key molecule in the prevention of neurodegenerative processes.     

Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells

Background

Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that has been implicated in the regulation of mitosis. In addition, the activation of mitogen-activated protein kinase (MAPK) is a key event in the early stage of the growth factor response. The role of Plk1 in MAPK phosphorylation in cells has not been investigated.

Methods

Immunoblot analysis was used to evaluate Plk1 and MAPK phosphorylation in cells upon stimulation with platelet-derived growth factor (PDGF). We also generated stable Plk1 knockdown (KD) cells to assess the role of Plk1 in MAPK activation and cell proliferation. Furthermore, we used a non-phosphorylatable Plk1 mutant to determine the function of Plk1 phosphorylation in these processes.

Results

Treatment with PDGF increased Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in human airway smooth muscle cells. Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 as well as cell proliferation. However, phosphorylation of Raf-1 and AKT upon stimulation with PDGF was not reduced in Plk1 KD cells. Furthermore, the expression of T210A Plk1 (alanine substitution at Thr-210) inhibited the PDGF-stimulated MEK1/2 phosphorylation, ERK1/2 phosphorylation and cell proliferation.

Conclusions

Together, these findings suggest that Plk1 is activated upon growth factor stimulation, which may control the activation of MEK1/2 and ERK1/2, and smooth muscle cell proliferation.     

Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia

Background

T cells play a dominant role in the pathogenesis of asthma. Costimulation of T cells is necessary to fully activate them. An inducible costimulator (ICOS) of T cells is predominantly expressed on Th2 cells. Therefore, interference of signaling pathways precipitated by ICOS may present new therapeutic options for Th2 dominated diseases such as asthma. However, these signaling pathways are poorly characterized in vitro and in vivo.

Methods

Human primary CD4⁺ T cells from blood were activated by beads with defined combinations of surface receptor stimulating antibodies and costimulatory receptor ligands. Real-time RT-PCR was used for measuring the production of cytokines from activated T cells. Activation of mitogen activated protein kinase (MAPK) signaling pathways leading to cytokine synthesis were investigated by western blot analysis and by specific inhibitors. The effect of inhibitors in vivo was tested in a murine asthma model of late phase eosinophilia. Lung inflammation was assessed by differential cell count of the bronchoalveolar lavage, determination of serum IgE and lung histology.

Results

We showed in vitro that ICOS and CD28 are stimulatory members of an expanding family of co-receptors, whereas PD1 ligands failed to co-stimulate T cells. ICOS and CD28 activated different MAPK signaling cascades necessary for cytokine activation. By means of specific inhibitors we showed that p38 and ERK act downstream of CD28 and that ERK and JNK act downstream of ICOS leading to the induction of various T cell derived cytokines. Using a murine asthma model of late phase eosinophilia, we demonstrated that the ERK inhibitor U0126 and the JNK inhibitor SP600125 inhibited lung inflammation in vivo. This inhibition correlated with the inhibition of Th2 cytokines in the BAL fluid. Despite acting on different signaling cascades, we could not detect synergistic action of any combination of MAPK inhibitors. In contrast, we found that the p38 inhibitor SB203580 antagonizes the action of the ERK inhibitor U0126 in vitro and in vivo.

Conclusion

These results demonstrate that the MAPKs ERK and JNK may be suitable targets for anti-inflammatory therapy of asthma, whereas inhibition of p38 seems to be an unlikely target.     

Rosiglitazone inhibits transforming growth factor-β1 mediated fibrogenesis in ADPKD cyst-lining epithelial cells

Background

Interstitial fibrosis plays an important role in progressive renal dysfunction in autosomal dominant polycystic kidney disease (ADPKD). In our previous studies, we confirmed that PPAR-γ agonist, rosiglitazone could protect renal function and prolong the survival of a slowly progressive ADPKD animal model by reducing renal fibrosis. However, the mechanism remains unknown.

Methods

Primary culture epithelial cells pretreated with TGF-β1 were incubated with rosiglitazone. Extracellular matrix proteins were detected using real-time PCR and Western blotting. MAPK and Smad2 phosphorylation were measured with western blot. ERK1/2 pathway and P38 pathway were inhibited with the specific inhibitors PD98059 and SB203580. The Smad2 pathway was blocked with the siRNA. To address whether PPAR-γ agonist-mediated inhibition of TGF-β1–induced collagen type I expression was mediated through a PPAR-γ dependent mechanism, genetic and pharmaceutical approaches were used to block the activity of endogenous PPARγ.

Results

TGF-β1-stimulated collagen type I and fibronectin expression of ADPKD cyst-lining epithelia were inhibited by rosiglitazone in a dosage-dependent manner. Smad2, ERK1/2 and P38 pathways were activated in response to TGF-β1; however, TGF-β1 had little effect on JNK pathway. Rosiglitazone suppressed TGF-β1 induced Smad2 activation, while ERK1/2 and P38MAPK signals remained unaffected. Rosiglitazone could also attenuate TGF-β1-stimulated collagen type I and fibronectin expression in primary renal tubular epithelial cells, but had no effect on TGF-β1–induced activation of Smad2, ERK1/2 and P38 pathways. There was no crosstalk between the Smad2 and MAPK pathways in ADPKD cyst-lining epithelial cells. These inhibitory effects of rosiglitazone were reversed by the PPARγ specific antagonist GW9662 and PPARγ siRNA.

Conclusion

ADPKD cyst-lining epithelial cells participate in TGF-β1 mediated fibrogenesis. Rosiglitazone could suppress TGF-β1–induced collagen type I and fibronectin expression in ADPKD cyst-lining epithelia through modulation of the Smad2 pathway. Our study may provide therapeutic basis for clinical applications of rosiglitazone in retarding the progression of ADPKD.     

Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling

Background

Changes in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established.

Methodology/Principal Findings

Here, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels.

Conclusions

Together, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.     

An Inhibition of p38 Mitogen Activated Protein Kinase Delays the Platelet Storage Lesion

Background and Objectives

Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK). Another MAPK, extracellular signal-related kinase (ERK), is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors.

Materials and Methods

A single Trima apheresis platelet unit (n = 12) was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20–24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters.

Results

Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties.

Conclusion

Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.     

Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells

Background

To demonstrate the involvement of tobacco smoking in the pathophysiology of lung disease, the responses of pulmonary epithelial cells to cigarette smoke condensate (CSC) — the particulate fraction of tobacco smoke — were examined.

Methods

The human alveolar epithelial cell line A549 and normal human bronchial epithelial cells (NHBEs) were exposed to 0.4 μg/ml CSC, a concentration that resulted in >90% cell survival and <5% apoptosis. Changes in gene expression and signaling responses were determined by RT-PCR, western blotting and immunocytofluorescence.

Results

NHBEs exposed to CSC showed increased expression of the inflammatory mediators sICAM-1, IL-1β, IL-8 and GM-CSF, as determined by RT-PCR. CSC-induced IL-1β expression was reduced by PD98059, a blocker of mitogen-actived protein kinase (MAPK) kinase (MEK), and by PDTC, a NFκB inhibitor. Analysis of intracellular signaling pathways, using antibodies specific for phosphorylated MAPKs (extracellular signal-regulated kinase [ERK]-1/2), demonstrated an increased level of phosphorylated ERK1/2 with increasing CSC concentration. Nuclear localization of phosphorylated ERK1/2 was seen within 30 min of CSC exposure and was inhibited by PD98059. Increased phosphorylation and nuclear translocation of IκB was also seen after CSC exposure. A549 cells transfected with a luciferase reporter plasmid containing a NFκB-inducible promoter sequence and exposed to CSC (0.4 μg/ml) or TNF-α (50 ng/ml) had an increased reporter activity of approximately 2-fold for CSC and 3.5-fold for TNF-α relative to untreated controls.

Conclusion

The acute phase response of NHBEs to cigarette smoke involves activation of both MAPK and NFκB.     

Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway

Background

Mesenchymal Stem Cells (MSC) are important candidates for therapeutic applications due to their ex vivo proliferation and differentiation capacity. MSC differentiation is controlled by both intrinsic and extrinsic factors and actin cytoskeleton plays a major role in the event. In the current study, we tried to understand the initial molecular mechanisms and pathways that regulate the differentiation of MSC into osteocytes or adipocytes.

Results

We observed that actin modification was important during differentiation and differentially regulated during adipogenesis and osteogenesis. Initial disruption of actin polymerization reduced further differentiation of MSC into osteocytes and osteogenic differentiation was accompanied by increase in ERK1/2 and p38 MAPK phosphorylation. However, only p38 MAPK phosphorylation was down regulated upon inhibition of actin polymerization which as accompanied by decreased CD49E expression.

Conclusion

Taken together, our results show that actin modification is a pre-requisite for MSC differentiation into osteocytes and adipocytes and osteogenic differentiation is regulated through p38 MAPK phosphorylation. Thus by modifying their cytoskeleton the differentiation potential of MSC could be controlled which might have important implications for tissue repair and regeneration.     

SHP-2 promotes the maturation of oligodendrocyte precursor cells through Akt and ERK1/2 signaling in vitro

Background

Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs), which are responsible for myelination. Myelin is essential for saltatory nerve conduction in the vertebrate nervous system. However, the molecular mechanisms of maturation and myelination by oligodendrocytes remain elusive.

Methods and Findings

In the present study, we showed that maturation of oligodendrocytes was attenuated by sodium orthovanadate (a comprehensive inhibitor of tyrosine phosphatases) and PTPi IV (a specific inhibitor of SHP-2). It is also found that SHP-2 was persistently expressed during maturation process of OPCs. Down-regulation of endogenous SHP-2 led to impairment of oligodendrocytes maturation and this effect was triiodo-L-thyronine (T3) dependent. Furthermore, over-expression of SHP-2 was shown to promote maturation of oligodendrocytes. Finally, it has been identified that SHP-2 was involved in activation of Akt and extracellular-regulated kinases 1 and 2 (ERK1/2) induced by T3 in oligodendrocytes.

Conclusions

SHP-2 promotes oligodendrocytes maturation via Akt and ERK1/2 signaling in vitro.     

Flt-1 in colorectal cancer cells is required for the tumor invasive effect of placental growth factor through a p38-MMP9 pathway

Background

Placenta growth factor (PlGF), a dimeric glycoprotein with 53% homology to VEGF, binds to VEGF receptor-1 (Flt-1), but not to VEGF receptor-2 (Flk-1), and may function by modulating VEGF activity. We previously have showed that PlGF displays prognostic value in colorectal cancer (CRC) but the mechanism remains elucidated.

Results

Overexpression of PlGF increased the invasive/migration ability and decreased apoptosis in CRC cells showing Flt-1 expression. Increased migration was associated with increasing MMP9 via p38 MAPK activation. Tumors grew faster, larger; with higher vascularity from PlGF over-expression cells in xenograft assay. In two independent human CRC tissue cohorts, PlGF, MMP9, and Flt-1 expressions were higher in the advanced than the localized disease group. PlGF expression correlated with MMP9, and Flt-1 expression. CRC patients with high PlGF and high Flt-1 expression in tissue had poor prognosis.

Conclusion

PlGF/Flt-1 signaling plays an important role in CRC progression, blocking PlGF/Flt-1 signaling maybe an alternative therapy for CRC.     

The Interaction of Endothelin-1 and TGF-β1 Mediates Vascular Cell Remodeling

Background

Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor.

Objective

To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC.

Methods

PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580.

Results

ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27^(Kip). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.

Conclusion and Clinical Relevance

ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.     

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate,but Not FAK

Background

Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.

Principal Findings

Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.

Conclusions

These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.     

Supernatant from bifidobacterium differentially modulates transduction signaling pathways for biological functions of human dendritic cells

Background

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, the signaling pathways engaged by probiotics are poorly understood. We have previously reported that a fermentation product from Bifidobacterium breve C50 (BbC50sn) could induce maturation, high IL-10 production and prolonged survival of DCs via a TLR2 pathway. We therefore studied the roles of mitogen-activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and phosphatidylinositol 3-kinase (PI3K) pathways on biological functions of human monocyte-derived DCs treated with BbC50sn.

Methodology/Principal Findings

DCs were differentiated from human monocytes with IL-4 and GM-CSF for 5 days and cultured with BbC50sn, lipopolysaccharide (LPS) or Zymosan, with or without specific inhibitors of p38MAPK (SB203580), ERK (PD98059), PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and GSK3 (SB216763). We found that 1) the PI3K pathway was positively involved in the prolonged DC survival induced by BbC50sn, LPS and Zymosan in contrast to p38MAPK and GSK3 which negatively regulated DC survival; 2) p38MAPK and PI3K were positively involved in DC maturation, in contrast to ERK and GSK3 which negatively regulated DC maturation; 3) ERK and PI3K were positively involved in DC-IL-10 production, in contrast to GSK3 that was positively involved in DC-IL-12 production whereas p38MAPK was positively involved in both; 4) BbC50sn induced a PI3K/Akt phosphorylation similar to Zymosan and a p38MAPK phosphorylation similar to LPS.

Conclusion/Significance

We report for the first time that a fermentation product of a bifidobacteria can differentially activate MAPK, GSK3 and PI3K in order to modulate DC biological functions. These results give new insights on the fine-tuned balance between the maintenance of normal mucosal homeostasis to commensal and probiotic bacteria and the specific inflammatory immune responses to pathogen bacteria.     

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

Background

RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation.

Methodology/Principal Findings

Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment.

Conclusions/Significance

A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.