Zinc interference with Cd‐induced hormetic effect in differentiated Caco‐2 cells: Evidence for inhibition downstream ERK activation

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium represents an effective protective barrier against Cd toxicity, but it is also a target tissue that may accumulate and trap high levels of the ingested metal. Using human enterocytic‐like Caco‐2 cells, we have previously shown that Cd may induce a concentration and time‐dependent increase in 3‐(4,5‐dimethyl‐2‐thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay (MTT)‐reducing activity in differentiated cultures with correlation to ERK1/2 activation. The present study shows that (a) Zn prevents the Cd‐induced hormesis effect on MTT reduction in a concentration‐dependent manner, without inhibiting Cd‐induced ERK1/2 activation; (b) Zn also induces similar hormetic stimulation of MTT‐reducing activity but without ERK1/2 activation. The effect of both metals was sensitive to inhibitors of translation during protein synthesis. There is evidence for the involvement of reactive oxygen species (ROS) in Cd‐induced ERK1/2 activation. In contrast, the Zn effect on the MTT‐reducing activity would not be triggered by ROS but it would be sensitive to the redox state of the cell. Steps downstream ERK1/2 activation by Cd does not involve eIF4E which is rather downregulated by Cd. In conclusion, Cd and Zn both can modify translation processes during protein synthesis via different signaling cascades with crosstalk, and cross‐inhibition may occur. This phenomenon is observed over a small range of metal concentrations and is characterized by a hormesis‐like response. Considering that the hormetic effect on dehydrogenase activity could reflect an adaptive response to the metals whether cross‐inhibition is beneficial is an open question.     

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.     

Role of mitogen‐activated protein kinase in Zn‐BC‐AM PDT‐induced apoptosis in nasopharyngeal carcinoma cells

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn‐BC‐AM was found to effectively induce apoptosis in a well‐differentiated nasopharyngeal carcinoma (NPC) HK‐1 cell line. Sustained activation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal kinase (JNK) as well as a transient increase in activation of extracellular signal‐regulated kinase (ERK) were observed immediately after Zn‐BC‐AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDT‐induced apoptosis of HK‐1 cells. PD169316 also prevented the loss of Bcl‐2 and Bcl‐xL in PDT‐treated HK‐1 cells. However, inhibition of JNK with SP600125 had no effect on Zn‐BC‐AM PDT‐induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn‐BC‐AM PDT‐induced apoptosis. Further study showed that knockdown of the p38β isoform with siRNA also increased Zn‐BC‐AM PDT‐induced apoptosis, indicating that the anti‐apoptotic effect of PD169316 in PDT‐treated HK‐1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38β and ERK may enhance the therapeutic efficacy of Zn‐BC‐AM PDT on NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution. Copyright © 2010 John Wiley & Sons, Ltd.     

Overexpression of fucosyltransferase IV promotes A431 cell proliferation through activating MAPK and PI3K/Akt signaling pathways

Lewis Y (LeY) is a carbohydrate tumor‐asssociated antigen. The majority of cancer cells derived from epithelial tissue express LeY type difucosylated oligosaccharide. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharide. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation, but the mechanism is still largely unknown. Herein, we investigated the role of the mitogen‐activated protein kinases (MAPKs) and phosphoinositide‐3 kinase (PI3K)/Akt signaling pathways on FUT4‐induced cell proliferation. Results show that overexpression of FUT4 increases the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt. Inhibitors of PI3K (LY294002 and Wortmannin) prevented the phosphorylation of ERK1/2, p38 MAPK, and Akt PI3K). Moreover, phosphorylation of Akt is abolished by inhibitors of ERK1/2 (PD98059) and p38 MAPK (SB203580). These data suggested that FUT4 not only activates MAPK and PI3K/Akt signals, but also promotes the crosstalk among these signaling pathways. In addition, FUT4‐induced stimulation of cell proliferation correlates with increased cell cycle progression by promoting cells into S‐phase. The mechanism involves in increased expression of cyclin D1, cyclin E, CDK 2, CDK 4, and pRb, and decreased level of cyclin‐dependent kinases inhibitors p21 and p27, which are blocked by the inhibitors of upstream signal molecules, MAPK and PI3K/Akt. In conclusion, these studies suggest that FUT4 regulates A431 cell growth through controlling cell cycle progression via MAPK and PI3K/Akt signaling pathways. J. Cell. Physiol. 225: 612–619, 2010. © 2010 Wiley‐Liss, Inc.     

Resveratrol enhances perforin expression and NK cell cytotoxicity through NKG2D‐dependent pathways

In a previous report, we showed that the in vivo cytotoxic activity of the natural killer (NK) cells isolated from resveratrol‐pretreated rats is significantly enhanced compared with that of the non‐pretreated rats; however, the underlying mechanism remains unclear. In the present study, we use cultured NK92 cell line to examine the possible signaling pathways underlying the resveratrol‐induced activation. Using cultured K562, HepG2, and A549 cells as targets, we show that resveratrol pretreatment increases NK cell cytotoxicity in a dose‐dependent manner. The enhanced cytotoxic effect is accompanied by increases in JNK and ERK‐1/2 MAP kinase activity and perforin expression. Moreover, the expression of NKG2D, an upstream signaling molecule of the MAP kinases pathway, is also enhanced. Resveratrol‐enhanced perforin expression and cytotoxic activity are effectively inhibited by pretreatment with the inhibitors of JNK (SP600125), ERK‐1/2 (PD98059), or by siRNAs against JNK‐1 and ERK‐2. However, the inhibitors or siRNA to p38 exhibits no effect. Since IL‐2 has been shown to induce NKG2D expression and perforin release, we therefore, examined whether IL‐2 and resveratrol act in parallel. We show that IL‐2 also stimulates perforin expression, however, when treated together with resveratrol, they exhibit no additive effect. The results suggest that in NK92 cells, resveratrol may act via a similar or overlapping pathway as that of IL‐2, to enhance perforin expression and cytotoxic activity. Data presented strongly indicate that resveratrol act via NKG2D‐dependent JNK and ERK‐1/2 pathways. J. Cell. Physiol. 223: 343–351, 2010. © 2010 Wiley‐Liss, Inc.     

ERK regulates strain‐induced migration and proliferation from different subcellular locations

Repetitive deformation like that engendered by peristalsis or villous motility stimulates intestinal epithelial proliferation on collagenous substrates and motility across fibronectin, each requiring ERK. We hypothesized that ERK acts differently at different intracellular sites. We stably transfected Caco‐2 cells with ERK decoy expression vectors that permit ERK activation but interfere with its downstream signaling. Targeting sequences constrained the decoy inside or outside the nucleus. We assayed proliferation by cell counting and migration by circular wound closure with or without 10% repetitive deformation at 10 cycles/min. Confocal microscopy confirmed localization of the fusion proteins. Inhibition of phosphorylation of cytoplasmic RSK or nuclear Elk confirmed functionality. Both the nuclear‐localized and cytosolic‐localized ERK decoys prevented deformation‐induced proliferation on collagen. Deformation‐induced migration on fibronectin was prevented by constraining the decoy in the nucleus but not in the cytosol. Like the nuclear‐localized ERK decoy, a Sef‐overexpressing adenovirus that sequesters ERK in the cytoplasm also blocked the motogenic and mitogenic effects of strain. Inhibiting RSK or reducing Elk ablated both the mitogenic and motogenic effects of strain. RSK isoform reduction revealed isoform specificity. These results suggest that ERK must translocate to the nucleus to stimulate cell motility while ERK must act in both the cytosol and the nucleus to stimulate proliferation in response to strain. Selectively targeting ERK within different subcellular compartments may modulate or replace physical force effects on the intestinal mucosa to maintain the intestinal mucosal barrier in settings when peristalsis or villous motility are altered and fibronectin is deposited into injured tissue. J. Cell. Biochem. 109: 711–725, 2010. Published in 2010 Wiley‐Liss, Inc.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

Heregulin β-1 induces loss of cell-cell contact and enhances expression of MUC1 at the cell surface in HCC2998 and MKN45-1 cells

Signal transduction and cell responses after stimulation with heregulin β-1 (HRG) are examined in HCC2998 and MKN45-1 cells, which have been used for a model system to study the formation of signet ring carcinomas, one of poorly differentiated adenocarcinomas. HRG stimulation causes rounding of the cells, responding to HRG. The adherens junction, which is present in the control cells, is disrupted and cell-cell interaction is lost after stimulation. Inhibition of phosphatidylinositol (PI)-3 kinase or p38 MAP kinase blocked this reaction, which indicates that the PI-3 kinase-p38 MAP kinase pathway is required for this reaction. Inhibition of the p38 MAP kinase pathway resulted in immediate restoration of cell-cell interaction. This result indicates that signaling for adherent molecules is strictly regulated by growth factor signaling. Expression of MUC1 at the cell surface is also observed and found to be expressed only after HRG stimulation. The total amount of MUC1 remains unchanged, suggesting that this amount is not due to induction of gene expression but to translocation of MUC1 from the inner membrane to the plasma membrane. This reaction is independent of the cytohesin pathway but dependent on PI-3 kinase activity. In addition to these reactions, HRG stimulates cell growth of both HCC2998 and MKN45-1 cells, depending on the ERK pathway given that the MEK inhibitor abolishes this effect. Therefore, HRG induces various reactions in HCC2998 and MKN45-1 cells by different pathways. These reactions are all related to characteristics of tumors, which implicates that HRG signaling can contribute to the formation of tumors.     

p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades

One important action of growth factors is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2), but not of JNK (p-JNK1/2), in corneal epithelial cells. Interruption of p38 and ERK1/2 signaling pathways by pretreatment with inhibitors SB203580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/2, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/2, was immediately detectable in epithelium after injury. Inhibition of p38 by SB203580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/2, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/2 or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/2 coordinate the dynamics of wound healing: while growth factor-stimulated p38 induces epithelial migration, ERK1/2 activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

Adiponectin increases BMP‐2 expression in osteoblasts via AdipoR receptor signaling pathway

Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of adiponectin on BMPs expression in cultured osteoblasts are largely unknown. Here we found that adiponectin increased mRNA expression of BMP‐2 but not other BMPs in cultured osteoblastic cells. Stimulation of osteoblasts with adiponectin also increased protein levels of BMP‐2 by Western blot and ELISA assay. Adiponectin‐mediated BMP‐2 expression was attenuated by 5′‐AMP‐activated protein kinase (AMPK) small interference RNA and AMPK inhibitor (araA and compound C). Activations of p38 and NF‐κB pathways after adiponectin treatment were demonstrated, and adiponectin‐induced expression of BMP‐2 was inhibited by the specific inhibitor and mutant of p38 and NF‐κB cascades. Taken together, our results provide evidence that adiponectin enhances BMP‐2 expression in osteoblastic cells, and AdipoR1 receptor, AMPK, p38 and NF‐κB signaling pathways may be involved in increasing BMP‐2 expression by adiponectin. J. Cell. Physiol. 224: 475–483, 2010. © 2010 Wiley‐Liss, Inc.     

Caffeine induces matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 down‐regulation in human leukemia U937 cells via Ca2+/ROS‐mediated suppression of ERK/c‐fos pathway and activation of p38 MAPK/c‐jun pathway

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Down‐regulation of MMP‐2 and MMP‐9 in U937 cells was abrogated by abolishment of caffeine‐elicited increase in intracellular Ca²⁺ concentration and ROS generation. Pretreatment with BAPTA‐AM (Ca²⁺ chelator) and N‐acetylcysteine (ROS scavenger) abolished caffeine‐induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase‐1 (MKP‐1) down‐regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up‐regulation, which were involved in cross‐talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP‐2 and MMP‐9 protein expression in caffeine‐treated cells. Caffeine treatment repressed ERK‐mediated c‐Fos phosphorylation but evoked p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun by siRNA reflected that c‐Fos counteracted the effect of c‐Jun on MMP‐2/MMP‐9 down‐regulation. Taken together, our data indicate that MMP‐2/MMP‐9 down‐regulation in caffeine‐treated U937 cells is elicited by Ca²⁺/ROS‐mediated suppression of ERK/c‐Fos pathway and activation of p38 MAPK/c‐Jun pathway. J. Cell. Physiol. 224: 775–785, 2010. © 2010 Wiley‐Liss, Inc.     

p38 MAPK activity is stimulated by vascular endothelial growth factor receptor 2 activation and is essential for shear stress‐induced angiogenesis

Increased capillary shear stress induces angiogenesis in skeletal muscle, but the signaling mechanisms underlying this response are not known. We hypothesize that shear stress‐dependent activation of vascular endothelial growth factor receptor 2 (VEGFR2) causes p38 and ERK1/2 phosphorylation, which contribute to shear stress‐induced angiogenesis. Skeletal muscle microvascular endothelial cells were sheared (12 dynes/cm², 0.5–24 h). VEGFR2‐Y1214 phosphorylation increased in response to elevated shear stress and VEGF stimulation. p38 and ERK1/2 phosphorylation increased at 2 h of shear stress but only p38 remained phosphorylated at 6 and 24 h of shear stress. VEGFR2 inhibition abrogated p38, but not ERK1/2 phosphorylation. VEGF production was increased in response to shear stress at 6 h, and this increased production was abolished by p38 inhibition. Male Sprague–Dawley rats were administered prazosin (50 mg/L drinking water, 1, 2, 4, or 7 days) to induce chronically elevated capillary shear stress in skeletal muscle. In some experiments, mini‐osmotic pumps were used to dispense p38 inhibitor SB203580 or its inactive analog SB202474, to the extensor digitorum longus (EDL) of control and prazosin‐treated rats. Immunostaining and Western blotting showed increases in p38 phosphorylation in capillaries from rats treated with prazosin for 2 days but returned to basal levels at 4 and 7 days. p38 inhibition abolished the increase in capillary to muscle fiber ratio seen after 7 days of prazosin treatment. Our data suggest that p38 activation is necessary for shear stress‐dependent angiogenesis. J. Cell. Physiol. 222:120–126, 2010. © 2009 Wiley‐Liss, Inc.