Smad and p38-MAPK signaling mediates apoptotic effects of transforming growth factor-beta1 in human airway epithelial cells

Transforming growth factor-beta1 (TGF-beta1) belongs to a family of multifunctional cytokines that regulate a variety of biological processes, including cell differentiation, proliferation, and apoptosis. The effects of TGF-beta1 are cell context and cell cycle specific and may be signaled through several pathways. We examined the effect of TGF-beta1 on apoptosis of primary human central airway epithelial cells and cell lines. TGF-beta1 protected human airway epithelial cells from apoptosis induced by either activation of the Fas death receptor (CD95) or by corticosteroids. This protective effect was blocked by inhibition of the Smad pathway via overexpression of inhibitory Smad7. The protective effect is associated with an increase in the cyclin-dependent kinase inhibitor p21 and was blocked by the overexpression of key gatekeeper cyclins for the G1/S interface, cyclins D1 and E. Blockade of the Smad pathway by overexpression of the inhibitory Smad7 permitted demonstration of a TGF-beta-mediated proapoptotic pathway. This proapoptotic effect was blocked by inhibition of the p38 MAPK kinase signaling with the inhibitor SB-203580 and was associated with an increase in p38 activity as measured by a kinase assay. Here we demonstrate dual signaling pathways involving TGF-beta1, an antiapoptotic pathway mediated by the Smad pathway involving p21, and an apoptosis-permissive pathway mediated in part by p38 MAPK.     

Differential effects of TGF-beta1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Previously, we demonstrated that hypoxia modulates the proliferation of human peripheral pulmonary artery smooth muscle cells (PASMCs) by induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins. The transforming growth factor (TGF)-beta superfamily plays a critical role in the regulation of pulmonary vascular remodeling, although to date an interaction with hypoxia has not been examined. We therefore investigated the pathways involved in the hypoxic induction of COX-2 in peripheral PASMCs and the contribution of TGF-beta1 and bone morphogenetic protein (BMP)-4 in this response. In the present study, we demonstrate that hypoxia induces activation of p38MAPK, ERK1/2, and Akt in PASMCs and that these pathways are involved in the hypoxic regulation of COX-2. Whereas inhibition of p38(MAPK) or ERK1/2 activity suppressed hypoxic induction of COX-2, inhibition of the phosphoinositide 3-kinase pathway enhanced hypoxic induction of COX-2. Furthermore, exogenous TGF-beta1 induced COX-2 mRNA and protein expression, and our findings demonstrate that release of TGF-beta1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-beta superfamily is part of an autocrine/paracrine system involved in the regulation of COX-2 expression in the distal pulmonary circulation, and this modulates hypoxia-induced pulmonary vascular cell proliferation.     

KRAS(G12V) enhances proliferation and initiates myelomonocytic differentiation in human stem/progenitor cells via intrinsic and extrinsic pathways

In human hematopoietic malignancies, RAS mutations are frequently observed. Yet, little is known about signal transduction pathways that mediate KRAS-induced phenotypes in human CD34(+) stem/progenitor cells. When cultured on bone marrow stroma, we observed that KRAS(G12V)-transduced cord blood (CB) CD34(+) cells displayed a strong proliferative advantage over control cells, which coincided with increased early cobblestone (CAFC) formation and induction of myelomonocytic differentiation. However, the KRAS(G12V)-induced proliferative advantage was transient. By week three no progenitors remained in KRAS(G12V)-transduced cultures and cells were all terminally differentiated into monocytes/macrophages. In line with these results, LTC-IC frequencies were strongly reduced. Both the ERK and p38 MAPK pathways, but not JNK, were activated by KRAS(G12V) and we observed that proliferation and CAFC formation were mediated via ERK, while differentiation was predominantly mediated via p38. Interestingly, we observed that KRAS(G12V)-induced proliferation and CAFC formation, but not differentiation, were largely mediated via secreted factors, since these phenotypes could be recapitulated by treating non-transduced cells with conditioned medium harvested from KRAS(G12V)-transduced cultures. Multiplex cytokine arrays and genome-wide gene expression profiling were performed to gain further insight into the mechanisms by which oncogenic KRAS(G12V) can contribute to the process of leukemic transformation. Thus, angiopoietin-like 6 (ANGPTL6) was identified as an important factor in the KRAS(G12V) secretome that enhanced proliferation of human CB CD34(+) cells.     

Superoxide activates mTOR–eIF4E–Bax route to induce enhanced apoptosis in leukemic cells

Mammalian target of rapamycin (mTOR) is a central kinase that regulates cell survival, proliferation and translation. Reactive oxygen species (ROS) are second messengers with potential in manipulating cellular signaling. Here we report that two ROS generating phytochemicals, hydroxychavicol and curcumin synergize in leukemic cells in inducing enhanced apoptosis by independently activating both mitogen activated protein kinase (MAPK) (JNK and P³⁸) and mTOR pathways. Low level transient ROS generated after co-treatment with these phytochemicals led to activation of these two pathways. Both mTOR and MAPK pathways played important roles in co-treatment-induced apoptosis, by knocking down either mTOR or MAPKs inhibited apoptosis. Activation of mTOR, as evident from phosphorylation of its downstream effector eukaryotic translation initiation factor 4E-binding protein 1, led to release of eukaryotic translation initiation factor 4E (eIF4E) which was subsequently phosphorylated by JNK leading to translation of pro-apoptotic proteins Bax and Bad without affecting the expression of anti-apoptotic protein Bcl-xl. Our data suggest that mTOR and MAPK pathways converge at eIF4E in co-treatment-induced enhanced apoptosis and provide mechanistic insight for the role of mTOR activation in apoptosis.     

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.     

Hepatocyte growth factor installs a survival platform for colorectal cancer cell invasive growth and overcomes p38 MAPK-mediated apoptosis

Hepatocyte growth factor (HGF) induces invasive growth, a biological program that confers tumor cells the capability to invade and metastasize by integrating cell proliferation, motility, morphogenesis, and survival. We here demonstrate that HGFR activation promotes survival of colorectal carcinoma (CRC) cells exposed to conditions that mimic those met during tumor progression, i.e. nutrient deprivation or substrate detachment, and following chemotherapeutic treatment. In all these conditions, a sustained activation of p38 MAPK delivers a main death signal that is overcome by cell treatment with HGF. HGF-driven survival requires the engagement of the PI3K/Akt/mTOR/p70S6K and ERK MAPK transduction pathways. Abrogation of p38 MAPK activity prevents CRC cell apoptosis also when these transduction pathways are inhibited, and treatment with HGF further increases survival. Engagement of these signaling cascades is also needed for HGF to induce CRC cell scattering, morphogenesis, motility and invasion. Activation of p38 MAPK signaling is therefore a main apoptotic switch for CRC cells in the stressful conditions encountered during tumor progression. Conversely, HGF orchestrates several biochemical pathways, which allow cell survival in these same conditions and promote the biological responses required for tumor invasive growth. Both p38 MAPK and HGF/HGFR signaling constitute potential molecular targets for inhibiting colorectal carcinogenesis.     

Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.     

Activation of pro-apoptotic p38-MAPK pathway in the prostate cancer cell line M12 expressing a truncated IGF-IR.

The type I insulin-like growth factor receptor (IGF-IR) plays a critical role in signaling survival and proliferation in many cell types. Activation of IGF-IR by its ligands promotes cell proliferation via mitogen-activated protein kinase (MAPK) cascade and cell survival via phosphoinositide 3-kinase (PI3K) cascade. The IGF-IR emerges as a powerful growth factor for many tumor cells. A truncated IGF-IR 486/STOP, described as a dominant negative IGF-IR mutant, was shown to induce apoptosis and inhibit tumor growth in vivo while endogenous IGF-IR was activated. To investigate the mechanism(s) of the action of 486/STOP, we have introduced 486/STOP into the prostate tumor model cell line M12 and its derivative M12lisn that expresses high levels of wild type IGF-IR. We have found that 486/STOP induces apoptosis in M12 and M12lisn cells in culture and that 486/STOP acts through activation of the pro-apoptotic p38-MAPK without interfering with wild type IGF-IR activation. In addition, our results have indicated that 486/STOP induced activation of p38-MAPK increases through activation of endogenous IGF-IR. These data suggest that activation of the IGF-IR by 486/STOP can selectively enhance the previously reported IGF-IR pro-apoptotic signaling pathways.     

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.     

Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-beta is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-beta1 mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-beta1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-beta1 on ASM cells was inhibited by selective inhibitors of TGF-beta receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-kappaB (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-beta1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-beta1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-beta1-induced proliferation. Our findings suggest that TGF-beta1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-kappaB is also involved in the TGF-beta1 mitogenic signaling, but Smad pathway does not appear important.     

Differential activation of Smads in HeLa and SiHa cells that differ in their response to transforming growth factor-beta

We assessed the responsiveness of six human cervical cancer cell lines to transforming growth factor (TGF)-beta with p3TP-lux reporter assay and found that HeLa and SiHa cells were highly responsive to TGF-beta. However, when pSBE4-BV/Luc reporter with four Smad binding elements was used, only the SiHa, not the HeLa, cells showed Smad activation. Smad DNA binding activity was relatively more in SiHa than in HeLa cells upon TGF-beta treatment, and the active complex contained Smad 2 and Smad 4. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, HeLa cells treated with 5 ng/ml of TGF-beta for 24 h showed proliferation, whereas SiHa cells showed growth inhibition under the same conditions. TGF-beta treatment resulted in G(0)/G(1) arrest with a reduction in S-phase only in SiHa cells. A chemical inhibitor of Smad activation (SB203580) blocked the growth inhibitory effect of TGF-beta in SiHa, whereas the proliferative response in HeLa was unaffected. TGF-beta-induced translocation of phospho-Smad 2 was relatively less in HeLa than in SiHa cells. MAPK activation occurred within 5 min and persisted up to 15 min upon TGF-beta treatment in HeLa but was negligible in SiHa cells. TGF-beta activated JNK in HeLa, but SiHa cells showed a down-regulation of its activity. When an inhibitor of MAPK (U0126) was used, the TGF-beta-mediated proliferative response in HeLa cells was completely abolished. SB203580 did not affect MAPK activation induced by TGF-beta in HeLa cells. We report for the first time an activation, presumably independent of Smad activation, of TGF-beta-dependent MAPK within 5 min of treatment that resulted in cell cycle progression in a cervical adenocarcinoma cell line, HeLa.     

Endoglin modulation of TGF-beta1-induced collagen synthesis is dependent on ERK1/2 MAPK activation.

BACKGROUND/AIMS: Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the extracellular matrix accumulation observed in fibrotic diseases. Endoglin is an important component of the TGF-beta receptor complex highly expressed in tissues undergoing fibrotic processes. Endoglin expression regulates the effect of TGF-beta on extracellular matrix synthesis. The purpose of our study has been to understand the molecular mechanism by which endoglin exerts its effects on fibrosis and the possible role of MAP kinases in these effects. METHODS: We have assessed in mock and in endoglin-transfected L6E9 myoblasts the effect of TGF-beta1 on collagen mRNA by Northern blot and effect of TGF-beta1 on collagen content in the cultured medium by [(3)H]-Proline incorporation into collagen proteins. Total and activated MAPK and their role on collagen synthesis were assessed by Western blot. RESULTS: TGF-beta1 induced an increase on alpha(2) (I) collagen mRNA expression and collagen accumulation in mock-transfected myoblasts, whereas the response was much lower in endoglintransfected cells. TGF-beta1 activated the ERK1/2 and p38 MAPK pathways but not the JNK pathway in L6E9 myoblasts. TGF-beta1-induced alpha(2) (I) collagen mRNA expression and collagen accumulation were completely inhibited by SB203580, in either mock or endoglintransfected myoblasts. PD98059 increased TGF-beta1 induced-collagen synthesis and accumulation in endoglin-transfected myoblasts but not in mock cells. CONCLUSION: Our studies demonstrate that TGF-beta1- induced collagen synthesis is mediated by p38 MAPK activation in L6E9 myoblasts. Furthermore, endoglin expression reduces basal and TGF-beta1 induced collagen synthesis when ERK1/2 pathway is operating.     

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-beta1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-beta1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-beta1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-beta receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-beta1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-beta1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-beta1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-beta1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-beta1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-beta1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects.     

Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells

Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signaling via a Gα_q/11/PLCβ/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gα_q/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analyses. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in Gα_q/11 double knockout MEF cells. Although CTGF expression is regulated by TGFβ, rPMT did not activate TGFβ pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT.     

TGF-beta1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production

Serum deprivation induces apoptosis in NIH3T3 cells, which is associated with increased intracellular ceramide generation and with the activation of p38 mitogen-activated protein (MAP) kinase. Treatment of cells with transforming growth factor-beta1 (TGF-beta1) activated the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2), inhibited the serum deprivation-induced p38 activation and the increase in intracellular ceramide formation, leading to the stimulation of cell proliferation and the suppression of apoptosis. Inhibition of p38 MAP kinase by SB203580 significantly reduced the serum-deprivation-induced apoptosis. Overexpression of p38 increased the cell apoptosis and reduced the antiapoptotic effect of TGF-beta1. Inhibition of ERK1/ERK2 by PD98059 completely inhibited the TGF-beta1-stimulated proliferation and partially inhibited the antiapoptotic effects of TGF-beta1. Neither SB203580 nor PD98059 has obvious effect on TGF-beta1-mediated inhibition of the increased ceramide generation. Serum-deprivation-induced apoptosis in NIH3T3 cells can also be blocked by broad-spectrum caspase inhibitor. TGF-beta1 treatment has an inhibitory effect on caspase activities. Our results indicate that ceramide, p38, and ERK1/ERK2 play critical but differential roles in cell proliferation and stress-induced apoptosis. TGF-beta1 suppresses the serum-deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 activation and increased ceramide generation.     

Zinc depletion induces JNK/p38 phosphorylation and suppresses Akt/mTOR expression in acute promyelocytic NB4 cells

BackgroundMyeloid leukemia is associated with reduced serum zinc and increased intracellular zinc. Our previous studies found that zinc depletion by TPEN induced apoptosis with PML-RARα oncoprotein degradation in acute promyelocytic NB4 cells. The effect of zinc homeostasis on intracellular signaling pathways in myeloid leukemia cells remains unclear.ObjectiveThis study examined how zinc homeostasis affected MAPK and Akt/mTOR pathways in NB4 cells.MethodsWe used western blotting to detect the activation of p38 MAPK, JNK, ERK1/2, and Akt/mTOR pathways in NB4 cells stimulated with the zinc chelator TPEN. Whether the effects of TPEN on these pathways could be reversed by zinc or the nitric oxide donor sodium nitroprusside (SNP) was further explored by western blotting. We used Zinpyr-1 staining to assess the role of SNP on labile zinc levels in NB4 cells treated with TPEN. In additional, we evaluated expressional correlations between the zinc-binding protein Metallothionein-2A (MT2A) and genes related to MAPKs and Akt/mTOR pathways in acute myeloid leukemia (AML) based on the TCGA database.ResultsZinc depletion by TPEN activated p38 and JNK phosphorylation in NB4 cells, whereas ERK1/2 phosphorylation was increased first and then decreased. The protein expression levels of Akt and mTOR were downregulated by TPEN. The nitric oxide donor SNP promotes zinc release in NB4 cells under zinc depletion conditions. We further found that the effects of zinc depletion on MAPK and Akt/mTOR pathways in NB4 cells can be reversed by exogenous zinc supplementation or treatment with the nitric oxide donor SNP. By bioinformatics analyses based on the TCGA database, we demonstrated that MT2A expression was negatively correlated with the expression of JNK, and was positively correlated with the expression of ERK1 and Akt in AML.ConclusionOur findings indicate that zinc plays a critical role in leukemia cells and help understanding how zinc depletion induces apoptosis.