Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation

We previously reported that suppression of the MEK/ERK pathway increases drug resistance of SiHa cells. In this study, we further characterized the underlying mechanism of this phenomenon. Pretreatment of SiHa cells with MEK/ERK inhibitor enhanced cisplatin-induced NF-kappaB activation. However, results of immunoblotting analysis showed that neither cisplatin nor MEK/ERK inhibitors induced marked IkappaBalpha degradation, suggesting that suppression of the MEK/ERK signaling pathway may enhance cisplatin-induced NF-kappaB activation via mechanisms other than the conventional pathway. Previous findings that protein phosphatase 4 (PP4), a nuclear serine/threonine phosphatase, directly interacts with and activates NF-kappaB led us to examine the phosphorylation status of NF-kappaB p65. Coincident with activation of NF-kappaB, cisplatin induced Ser phosphorylation but decreased Thr phosphorylation of NF-kappaB p65. Suppression of the MEK/ERK pathway further enhanced cisplatin-induced Thr dephosphorylation but did not affect cisplatin-induced Ser phosphorylation of NF-kappaB p65. Further, in parallel with Thr dephosphorylation, the protein level of nuclear PP4 was increased in cisplatin-treated cells and was further increased by suppression of the MEK/ERK pathway. SiHa cells were then transfected by a sense or an antisense PP4 gene. PP4-overexpressing cells showed a decrease in Thr phosphorylation of NF-kappaB p65 to nearly undetectable levels, and both basal and cisplatin-induced NF-kappaB activities were higher than those in parental cells. By contrast, cisplatin, either alone or with MEK/ERK inhibitors, induced little NF-kappaB activation in antisense PP4-transfected cells. Coprecipitated complex kinase assay revealed a fragment of NF-kappaB p65 (amino acids 279-444) to contain potential phosphorylation sites that directly interact with PP4. Further studies by site-directed mutagenesis suggested that Thr(435) was the major phosphorylation site.     

Integrin β4 attenuates SHP‐2 and MAPK signaling and reduces human lung endothelial inflammatory responses

We previously identified the marked upregulation of integrin β4 in human lung endothelial cells (EC) treated with simvastatin, an HMG coA‐reductase inhibitor with vascular‐protective and anti‐inflammatory properties in murine models of acute lung injury (ALI). We now investigate the role of integrin β4 as a novel mediator of vascular inflammatory responses with a focus on mitogen‐activated protein kinases (MAPK) signaling and the downstream expression of the inflammatory cytokines (IL‐6 and IL‐8) essential for the full elaboration of inflammatory lung injury. Silencing of integrin β4 (siITGB4) in human lung EC resulted in significant increases in both basal and LPS‐induced phosphorylation of ERK 1/2, JNK, and p38 MAPK, consistent with robust integrin β4 regulation of MAPK activation. In addition, siITB4 increased both basal and LPS‐induced expression of IL‐6 and IL‐8 mRNA and protein secretion into the media. We next observed that integrin β4 silencing increased basal and LPS‐induced phosphorylation of SHP‐2, a protein tyrosine phosphatase known to modulate MAPK signaling. In contrast, inhibition of SHP‐2 enzymatic activity (sodium stibogluconate) abrogated the increased ERK phosphorylation associated with integrin β4 silencing in LPS‐treated EC and attenuated the increases in levels of IL‐6 and IL‐8 in integrin‐β4‐silenced EC. These findings highlight a novel negative regulatory role for integrin β4 in EC inflammatory responses involving SHP‐2‐mediated MAPK signaling. Upregulation of integrin β4 may represent an important element of the anti‐inflammatory and vascular‐protective properties of statins and provides a novel strategy to limit inflammatory vascular syndromes. J. Cell. Biochem. 110: 718–724, 2010. © 2010 Wiley‐Liss, Inc.     

T‐cadherin modulates endothelial barrier function

T‐cadherin is an atypical member of the cadherin family, which lacks the transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. Unlike canonical cadherins, it is believed to function primarily as a signaling molecule. T‐cadherin is highly expressed in endothelium. Using transendothelial electrical resistance measurements and siRNA‐mediated depletion of T‐cadherin in human umbilical vein endothelial cells, we examined its involvement in regulation of endothelial barrier. We found that in resting confluent monolayers adjusted either to 1% or 10% serum, T‐cadherin depletion modestly, but consistently reduced transendothelial resistance. This was accompanied by increased phosphorylation of Akt and LIM kinase, reduced phosphorylation of p38 MAP kinase, but no difference in tubulin acetylation and in phosphorylation of an actin filament severing protein cofilin and myosin light chain kinase. Serum stimulation elicited a biphasic increase in resistance with peaks at 0.5 and 4–5 h, which was suppressed by a PI3 kinase/Akt inhibitor wortmannin and a p38 inhibitor SB 239063. T‐cadherin depletion increased transendothelial resistance between the two peaks and reduced the amplitude of the second peak. T‐cadherin depletion abrogated serum‐induced Akt phosphorylation at Thr308 and reduced phosphorylation at Ser473, reduced phosphorylation of cofilin, and accelerated tubulin deacetylation. Adiponectin slightly improved transendothelial resistance irrespectively of T‐cadherin depletion. T‐cadherin depletion also resulted in a reduced sensitivity and delayed responses to thrombin. These data implicate T‐cadherin in regulation of endothelial barrier function, and suggest a complex signaling network that links T‐cadherin and regulation of barrier function. J. Cell. Physiol. 223: 94–102, 2010. © 2009 Wiley‐Liss, Inc.     

Cross‐talk between MAP kinase pathways is involved in IGF‐independent,IGFBP‐6‐induced Rh30 rhabdomyosarcoma cell migration

Insulin‐like growth factor binding protein‐6 (IGFBP‐6) inhibits the tumorigenic properties of IGF‐II‐dependent cancer cells by directly inhibiting IGF‐II actions. However, in some cases, IGFBP‐6 is associated with increased cancer cell tumorigenicity, which is unlikely to be due to IGF‐II inhibition. The mechanisms underlying the contradictory actions of IGFBP‐6 remain unclear. We recently generated an IGFBP‐6 mutant that does not bind IGFs (mIGFBP‐6) to address this issue. Although RD rhabdomyosarcoma cells express IGF‐II, we previously showed that mIGFBP‐6 promoted migration through an IGF‐independent, p38‐dependent pathway. We further studied the role of MAP kinases in IGFBP‐6‐induced migration of Rh30 rhabdomyosarcoma cells, which also express IGF‐II. In these cells, mIGFBP‐6 induced chemotaxis rather than chemokinesis. Both wild‐type (wt) and mIGFBP‐6 transiently induced phosphorylation of ERK1/2 and JNK1, but not p38. Inhibition of ERK1/2 phosphorylation completely prevented mIGFBP‐6‐induced ERK1/2 activation and cell migration, whereas a JNK inhibitor partially prevented migration. Interestingly, p38 pathway inhibition completely prevented mIGFBP‐6‐induced ERK1/2 and JNK1 activation and migration despite mIGFBP‐6 not activating p38. Furthermore, blocking the ERK1/2 pathway also inhibited mIGFBP‐6‐induced JNK1 activation. In contrast, IGFBP‐6 had no effect on Akt phosphorylation and an Akt inhibitor had no effect on migration. These results indicate that IGFBP‐6 promotes Rh30 rhabdomyosarcoma chemotaxis in an IGF‐independent manner, and that MAPK signaling pathways and their cross‐talk play an important role in this process. Therefore, besides decreasing Rh30 cell proliferation by inhibiting IGF‐II, IGFBP‐6 promotes their migration via a distinct pathway. Understanding these disparate actions of IGFBP‐6 may lead to the development of novel cancer therapeutics. J. Cell. Physiol. 224: 636–643, 2010. © 2010 Wiley‐Liss, Inc.     

12‐O‐tetradecanoylphorbol‐13‐acetate‐induced invasion/migration of glioblastoma cells through activating PKCα/ERK/NF‐κB‐dependent MMP‐9 expression

An increase in MMP‐9 gene expression and enzyme activity with stimulating the migration of GBM8401 glioma cells via wound healing assay by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was detected in glioblastoma cells GBM8401. TPA‐induced translocation of protein kinase C (PKC)α from the cytosol to membranes, and migration of GBM8401 elicited by TPA was suppressed by adding the PKCα inhibitors, GF109203X and H7. Activation of extracellular signal‐regulated kinase (ERK) and c‐Jun‐N‐terminal kinase (JNK) by TPA was identified, and TPA‐induced migration and MMP‐9 activity was significantly blocked by ERK inhibitor PD98059 and U0126, but not JNK inhibitor SP600125. Activation of NF‐κB protein p65 nuclear translocation and IκBα protein phosphorylation with increased NF‐κB‐directed luciferase activity by TPA were observed, and these were blocked by the PD98059 and IkB inhibitor BAY117082 accompanied by reducing migration and MMP‐9 activity induced by TPA in GBM8401 cells. Transfection of GBM8401 cells with PKCα siRNA specifically reduced PKCα protein expression with blocking TPA‐induced MMP‐9 activation and migration. Additionally, suppression of TPA‐induced PKCα/ERK/NK‐κB activation, migration, and MMP‐9 activation by flavonoids including kaempferol (Kae; 3,5,7,4′‐tetrahydroxyflavone), luteolin (Lut; 5,7,3′4′‐tetrahydroxyflavone), and wogonin (Wog; 5,7‐dihydroxy‐8‐methoxyflavone) was demonstrated, and structure–activity relationship (SAR) studies showed that hydroxyl (OH) groups at C4′ and C8 are critical for flavonoids' action against MMP‐9 enzyme activation and migration/invasion of glioblastoma cells elicited by TPA. Application of flavonoids to prevent the migration/invasion of glioblastoma cells through blocking PKCα/ERK/NF‐κB activation is first demonstrated herein. J. Cell. Physiol. 225: 472–481, 2010. © 2010 Wiley‐Liss, Inc.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

Cadmium‐induced hormetic effect in differentiated Caco‐2 cells: ERK and p38 activation without cell proliferation stimulation

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium may in part protect against Cd toxicity but is also a target tissue. Using human enterocytic‐like Caco‐2 cells, we have previously shown differences in sensitivity to Cd according to the differentiation status. The present study focuses on Cd effects on differentiated cells. Concentration and time‐dependent increases in MTT (3‐[4,5‐dimethyl‐2‐thiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide assay) activity were observed in post‐confluent cultures exclusively, with a twofold maximal stimulation in 21‐day‐old cells exposed to 10 µM Cd for 24 h. No concomitant increase in [methyl‐³H] thymidine incorporation was noted and Cd did not modify cell distribution in the cell‐cycle phases. However, Cd‐induced increase in MTT activity was inhibited by cycloheximine as well as by inhibitors of ERK1/2 and p38, but not by that of JNK. Consistently, Cd increased the levels of ERK1/2 and p38 phosphorylation. Inhibition of Ras‐GTP or PI3K enhanced the stimulatory effect of Cd, whereas mTOR inhibition had no effect. Inhibition of G protein‐phospholipase and PKC decreased MTT stimulation. These results show a hormesis‐like stimulation of Cd on MTT activity in differentiated intestinal cells exclusively. This effect is not related to cell proliferation but more likely to increased protein synthesis which involves ERK1/2 and p38 cascades and possibly PLC‐β signaling pathways. Because growth‐related differentiation of intestinal cells is linked to the selective and sequential activation of MAPKs, the impacts that these Cd‐induced perturbations in signaling pathways may have on intestinal functions clearly deserve to be investigated. J. Cell. Physiol. 224:250–261, 2010 © 2010 Wiley‐Liss, Inc.     

MEK/ERK pathway mediates PKC activation‐induced recruitment of PKCζ and MMP‐9 to podosomes

Podosomes are adhesive structures on the ventral surface of cells that invade and degrade the extracellular matrix. Recently, we reported that phorbol 12,13‐dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in normal human bronchial epithelial (NHBE) cells, and atypical PKCζ regulated MMP‐9 recruitment to podosomes for its release and activation. The objective of this study was to explore signaling pathways that are involved in PKC activation‐induced podosome formation and matrix degradation. Herein, we found that PDBu increased phosphorylation of PI3K p85, Akt, Src, ERK1/2, and JNK. Inhibitors for PI3K, Akt, and Src suppressed PDBu‐induced podosome formation and matrix degradation. In contrast, blockers for MEK/ERK or JNK did not inhibit podosome formation but reduced proteolytic activity of podosomes. Inhibition of PKCζ activity with its pseudosubstrate peptide (PS)‐inhibited PDBu‐induced phosphorylation of MEK/ERK and JNK. On the other hand, inhibition of MEK/ERK or JNK pathway did not affect PKCζ phosphorylation, but reduced the recruitment of PKCζ and MMP‐9 to podosomes. We conclude that PKCζ may regulate MEK/ERK and JNK phosphorylation and in turn activated MEK/ERK and JNK may regulate the proteolytic activity of PDBu‐induced podosomes by influencing the recruitment of PKCζ and MMP‐9 to podosomes. J. Cell. Physiol. 228: 416–427, 2013. © 2012 Wiley Periodicals, Inc.     

Changes in contraction‐induced phosphorylation of AMP‐activated protein kinase and mitogen‐activated protein kinases in skeletal muscle after ovariectomy

Recent evidence suggests that ovarian hormones contribute to altered function of skeletal muscle, however the signaling processes thought to regulate muscle function remain undefined in females. Thus, the purpose of this investigation is to determine if ovarian hormone status is critical for contraction‐induced activation of AMPK or MAPK in skeletal muscle. Female mice were divided into two groups, ovariectomy (OVX) and SHAM, which were then subjected to in situ isometric contractile protocols. AMPK, ERK 1/2, p38, and JNK phosphorylation were measured in the control and contracting limb. In the in situ protocol, OVX muscles were significantly more resistant to fatigue compared to the SHAM animals. In addition, the muscles from OVX mice demonstrated significantly lower levels of normalized AMPK phosphorylation at rest. AMPK phosphorylation was not increased in the muscles from SHAM mice after the in situ contractile protocol, while the OVX demonstrated significant increases in AMPK phosphorylation. After contraction, normalized ERK2 phosphorylation was significantly higher in the OVX group compared to the SHAM group. Both p38 and JNK phosphorylation increased in response to contraction; but no group differences were detected. A second set of SHAM and OVX animals were subjected to fatigue stimulated under in vitro conditions. Significant increases in AMPK and ERK2 phosphorylation were detected, but no differences were found between groups. In conclusion, removal of the ovaries results in different responses to contraction‐induced changes in phosphorylation of AMPK and ERK2 in female mice and suggests hormones secreted from the ovaries significantly impacts cellular signaling in skeletal muscle. J. Cell. Biochem. 107: 171–178, 2009. © 2009 Wiley‐Liss, Inc.     

Activation of GABA‐A receptor ameliorates homocysteine‐induced MMP‐9 activation by ERK pathway

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase‐9 (MMP‐9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ‐aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA‐A receptor in Hcy‐induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP‐9 activity via the extracellular signal‐regulated kinase (ERK) signaling pathway and by inhibition of GABA‐A receptors, thus behaving as an inhibitory neurotransmitter. Hcy‐induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′‐dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate‐oxidase‐4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP‐9, measured by gelatin zymography. The GABA‐A receptor agonist, muscimol ameliorated the Hcy‐mediated MMP‐9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase‐4 (TIMP‐4). Treatment of the endothelial cell with muscimol restored the levels of TIMP‐4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy‐induced MMP‐9 via ERK signaling pathway. These results suggest that Hcy competes with GABA‐A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. J. Cell. Physiol. 220: 257–266, 2009. © 2009 Wiley‐Liss, Inc.