The role of the cyclic GMP-inhibited cyclic AMP-specific phosphodiesterase (PDE3) in regulating clonal BRIN-BD11 insulin secreting cell survival

We report here that the cyclic GMP-inhibited cyclic AMP specific phosphodiesterase (PDE3B) is expressed as a membrane-bound protein in clonal insulin-secreting BRIN-BD11 cells. This was shown using SKF94836 (PDE3 inhibitor) which maximally inhibited membrane-bound cyclic AMP PDE activity by approximately 25-30% and by RT-PCR. We also demonstrated that insulin growth factor-1 (IGF-1) activates PDE3B in BRIN-BD11 cells. We therefore evaluated the role of phosphoinositide 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathways in regulating this enzyme. We report here that the PI3K inhibitor, wortmannin, prevented the IGF-1-dependent stimulation of PDE3B activity. In contrast, the inhibitor of MEK-1 activation, PD098059 (which reduced IGF-1-stimulated p42/p44 MAPK phosphorylation), had no effect on PDE3B activation. Furthermore, IGF-1-dependent stimulation of p42/p44 MAPK and PDE3B was abolished in serum-deprived cells and this was associated with apoptosis. We propose that the deregulation of the PI3K/PDE3B pathway might result in increased intracellular cyclic AMP accumulation, which promotes apoptosis. This was supported by the finding that the adenylyl cyclase activator, forskolin, also induced apoptosis. Finally, we found that orthovanadate (a phosphotyrosine phosphatase inhibitor) fully restored the activation of p42/p44 MAPK in serum-deprived cells, but had only a small effect on PDE activity. This confirmed that p42/p44 MAPK is on a separate pathway to PDE3B. Therefore, IGF-1-dependent regulation of PDE3B may be linked to cell survival through PI3K and not p42/p44 MAPK.     

STAT-1 mediates the stimulatory effect of IL-10 on CD14 expression in human monocytic cells

IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 up-regulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of PI3K and p42/44 ERK MAPK. By using specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT-1 and consequently CD14 expression. However, IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Finally, STAT-1 interfering RNA inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 up-regulation in human monocytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK.     

Inhibition of p42 and p44 Mitogen-Activated Protein Kinase Activity by PD98059 Does Not Suppress Nerve Growth Factor-Induced Survival of Sympathetic Neurones

Abstract: Nerve growth factor (NGF) induces persistent p42 and p44 mitogen-activated protein kinase (MAPK) activity in sympathetic neurones in parallel to its survival-promoting activity. To investigate whether these MAPK activities are necessary for NGF-induced survival, we have inhibited NGF-stimulated p42/p44 MAPK activity over extended periods using the compound 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059). Despite attaining up to 95% inhibition of p42/p44 MAPK activity in cultures treated with NGF and PD98059, neuronal survival is maintained undiminished, although a decrease in the density of the neuritic network is observed. Because p21Ras activity is essential for NGF-induced survival, we conclude that p21Ras-linked activities other than p42 and p44 MAPKs are responsible for mediating NGF-dependent survival of rat sympathetic neurones.     

Evidence that inhibition of p44/42 mitogen-activated protein kinase signaling is a factor in proteasome inhibitor-mediated apoptosis

The proteasome is emerging as a target for cancer therapy because small molecule inhibitors of its catalytic activity induce apoptosis in both in vitro and in vivo models of human malignancies and are proving to have efficacy in early clinical trials. To further elucidate the mechanism of action of these inhibitors, their impact on signaling through the p44/42 mitogen-activated protein kinase (MAPK) pathway was studied. Proteasome inhibition with either carbobenzoxy-leucyl-leucyl-phenylalaninal or lactacystin led to a loss of dually phosphorylated, activated p44/42 MAPK in A1N4-myc human mammary and MDA-MB-231 breast carcinoma cells in a dose- and time-dependent fashion. This correlated with an induction of the dual specificity MAPK phosphatases (MKP)-1 and -2, and blockade of MKP induction using either actinomycin D or Ro-31-8220 significantly decreased loss of activated p44/42 MAPK. Inhibition of p44/42 MAPK signaling by use of the MAPK kinase inhibitors PD 98059 or U0126, or by use of a dominant negative MAPK construct, enhanced proteasome inhibitor-mediated apoptosis. Conversely, activation of MAPK by epidermal growth factor, or use of a mutant MAPK resistant to MKP-mediated dephosphorylation, inhibited apoptosis. These studies support a role for inactivation of signaling through the p44/42 MAPK pathway in proteasome inhibitor-mediated apoptosis.     

Human leptin activates PI3K and MAPK pathways in human peripheral blood mononuclear cells: possible role of Sam68.

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. Thus, we have recently found that human leptin promotes stimulation and proliferation of human peripheral blood mononuclear cells. In the present work, we sought to study the mechanisms underlying these effects. First, we have assessed the presence of the long isoform of the human leptin receptor by RT-PCR. Next, we have studied tyrosine phosphorylation of cell proteins in response to leptin stimulation. We have found that leptin receptor, IRS-1 and the RNA-binding protein Sam68 are tyrosine phosphorylated upon leptin challenge in a dose-dependent manner. Moreover, tyrosine phosphorylation of IRS-1 and Sam68 promotes their association with p85, the regulatory subunit of PI3K, and this association leads to the stimulation of PI3K activity. On the other hand, the leptin-stimulated tyrosine phosphorylation of Sam68 mediates the dissociation from RNA as assessed by Sepharose-conjugated poly(U) binding. Finally, leptin receptor activation also triggers MAPK signaling pathway. Thus, leptin dose-dependently stimulates tyrosine and threonine phosphorylation of MAPK in mononuclear cells. In summary, the present work demonstrates the presence of the long isoform of the human leptin receptor in peripheral blood mononuclear cells and the activation of two signaling pathways, PI3K and MAPK. The effects on Sam68 phosphorylation may modulate its binding to RNA, although the physiological implications remain to be studied. These signal transduction pathways may mediate the described effects of human leptin on human peripheral blood mononuclear cells.     

MAPK and PI3K activities are required for leptin stimulation of protein synthesis in human trophoblastic cells

Leptin, the LEP gene product, is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy. Thus, we have recently described the antiapoptotic and trophic effect of leptin on choriocarcinoma cell line JEG-3, stimulating DNA and protein synthesis. We have also demonstrated the presence of leptin receptor and leptin signaling in normal human trophoblastic cells, activating JAK-STAT, PI3K and MAPK pathways. In the present work we have employed dominant negative forms of MAPK and PKB constructs to find out the signaling pathways that specifically mediates the effect of leptin on protein synthesis. As previously shown, leptin stimulates protein synthesis as assessed by ³H-leucine incorporation. However, both dominant negative forms of MAPK and PKB inhibited protein synthesis in JEG-3 choriocarcinoma cells. The inhibition of PKB and MAPK activity by transfection with the dominant negative kinases prevented the leptin stimulation of p70 S6K, which is known to be an important kinase in the regulation of protein synthesis. Moreover, leptin stimulation of phosphorylation of EIF4EBP1 and EIF4E, which allows the initiation of translation was also prevented by MAPK and PI3K dominant negative constructs. Therefore, these results demonstrate that both PI3K and MAPK are necessary to observe the effect of leptin signaling that mediates protein synthesis in choriocarcinoma cells JEG-3.     

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44(ERK1/2) and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44(ERK1/2) MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44(ERK1/2) MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44(ERK1/2), but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.     

VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes endothelial cell survival and angiogenesis. We recently showed that VEGF can support the growth of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells in serum-free medium. Reasoning that VEGF might be modulating apoptotic signal transduction pathways, we examined mechanisms involved in the anti-apoptotic effect of VEGF on starvation- and ceramide-induced apoptosis in HDMEC. We observed that VEGF ameliorated the time-dependent increase in apoptosis, as demonstrated by morphologic observations, TUNEL assay, and DNA fragmentation. On the other hand, basic fibroblast growth factor only partially prevented apoptosis in serum-starved HDMEC; platelet-derived growth factor-BB was completely ineffective. VEGF activated the phosphorylation of extracellular signal regulated kinase (ERK)1 (p44 mitogen-activated protein kinase; MAPK) and ERK2 (p42 MAPK) in a time- and concentration-dependent manner. Both the VEGF-induced activation and its anti-apoptotic effect were prevented by the specific MAPK/ERK inhibitor PD98059. The presence of VEGF also inhibited the sustained activation of stress-activated protein kinase/c-jun-NH2-kinase (SAPK/JNK) caused by serum starvation and ceramide treatment. Activation of the MAPK pathway together with inhibition of SAPK/JNK activity by VEGF appears to be a key event in determining whether an endothelial cell survives or undergoes programmed cell death.     

Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-alpha signaling

Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor beta1 (TGF-beta1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-beta1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-beta1-induced angiogenesis mainly by compromising cell survival. We established that TGF-beta1 stimulated the expression of TGF-alpha mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-beta1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-beta1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-alpha alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-beta1. We therefore propose that TGF-beta1 promotes angiogenesis at least in part via the autocrine secretion of TGF-alpha, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.     

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.     

Leptin Inhibits Glucose Intestinal Absorption via PKC,p38MAPK,PI3K and MEK/ERK

The role of leptin in controlling food intake and body weight is well recognized, but whether this is achieved by modulating nutrient absorption is still a controversial issue. The aim of this work was to investigate the direct effect of luminal leptin on glucose intestinal absorption and elucidate for the first time its signaling pathway. Fully differentiated Caco-2 cells grown on transwell filters were used for glucose transport studies. Leptin caused a significant reduction in glucose absorption. Individual and simultaneous inhibition of ERK, p38MAPK, PI3K or PKC abrogated completely the inhibitory effect of leptin. Activating PKC, lead to a stimulatory effect that appeared only when ERK, p38MAPK, or PI3K was inactive. Moreover, leptin increased the phosphorylation of ERK, Akt and p38MAPK. This increase changed into a decrease when p38MAPK and PKC were inactivated individually. Inhibiting ERK maintained the leptin-induced up-regulation of p-Akt and p-p38MAPK while inhibiting PI3K reduced the level of p-ERK and p-Akt but maintained the increase in p-p38MAPK. These results suggest that leptin reduces glucose absorption by activating PKC. Although the latter modulates glucose absorption via a stimulatory and an inhibitory pathway, only the latter is involved in leptin’s action. Active PKC leads to a sequential activation of p38MAPK, PI3K and ERK which exerts an inhibitory effect on glucose absorption. The results reveal a modulatory role of leptin in nutrient absorption in addition to its known satiety inducing effect.     

Globular adiponectin decreases leptin-induced tumor necrosis factor-alpha expression by murine macrophages: involvement of cAMP-PKA and MAPK pathways

Several lines of evidence have supported a link between obesity and inflammation. The present study investigated the capacity of leptin and globular adiponectin to affect tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. Leptin stimulated TNF-alpha production at mRNA as well as protein levels in a dose- and time-dependent manner. Intracellular cAMP concentration was increased and protein kinase A (PKA) was activated with the treatment of leptin, subsequently downstream MAPK signal proteins, ERK1/2 and p38, were phosphorylated. Specific inhibitors for the signal proteins, Rp cAMPS, H89, PD98059, and U0126, or SB203580, suppressed the signaling pathway and TNF-alpha expression. Although gAd partially increased cAMP concentration and PKA activity, it directly reduced leptin-induced ERK1/2 and p38 MAPK phosphorylation thus inhibiting TNF-alpha production. In conclusion, leptin promotes inflammation by stimulating TNF-alpha production, which is mediated by cAMP-PKA-ERK1/2 and p38 MAPK pathways. gAd inhibited leptin-induced TNF-alpha production through suppressing phosphorylation of ERK1/2 and p38 pathways.     

Downregulation of angiotensin converting enzyme by TNF-alpha in differentiating human macrophages

OBJECTIVE: To investigate regulation of angiotensin converting enzyme (ACE) by tumour necrosis factor alpha (TNF-alpha) in differentiating human peripheral blood monocytes (PBM). METHODS: Human PBM were allowed to differentiate to macrophages for 0-7 days and ACE amount was measured during differentiation. Experiments with TNF-alpha were performed after 2 days of differentiation. Cell cultures were incubated with TNF-alpha (0.5-10ng/ml) without or with SB 202190 (5microM), or PD 98059 (40microM). ACE amounts were measured by an inhibitor binding assay (IBA) and ACE mRNA levels by RNase protection assay (RPA). Activated p44/42 and p38 MAP kinases were measured by Western Blot analysis using phospho-p44/42 and -p38 MAPK antibodies. RESULTS: ACE amount increased by 40-fold along with macrophage differentiation. TNF-alpha caused dose dependent suppression of the amount of ACE and decreased levels of ACE mRNA. TNF-alpha activated p44/42 and p38 MAP kinases, which was inhibited by the specific inhibitors of these kinases, PD98059 or SB202190, respectively. Pretreatment of the cells with SB 202190, or PD 98059 both partly reversed TNF-alpha induced ACE suppression. CONCLUSIONS: TNF-alpha downregulated ACE, which effect was probably mediated by both p44/42 and p38 MAPK pathways. Local downregulation of ACE by TNF-alpha may be a counterbalancing mechanism in inflammatory processes.     

cFLIPL prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis

Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. However, it is unknown if TRAIL cytotoxic signaling engages the lysosomal pathway of cell death. Our aim, therefore, was to ascertain if TRAIL killing involves lysosomal permeabilization. TRAIL-induced apoptosis of hepatocellular carcinoma cells (HuH-7, Hep3B) was associated with lysosomal permeabilization, as demonstrated by redistribution of the lysosomal protease cathepsin B into the cytosol. Pharmacological and short hairpin RNA-targeted inhibition of cathepsin B reduced apoptosis. Because cellular FLICE-inhibitory protein (cFLIP) inhibits TRAIL-induced cell death and is frequently overexpressed by human cancers, the ability of cFLIP to prevent lysosomal permeabilization during TRAIL treatment was examined. Enforced long-form cFLIP (cFLIP(L)) expression reduced release of cathepsin B from lysosomes and attenuated apoptosis. cFLIP(L) overexpression was also associated with robust p42/44 MAPK activation following exposure to TRAIL. In contrast, cFLIP(L) overexpression attenuated p38 MAPK activation and had no significant effect on JNK and NF-kappaB activation. Inhibition of p42/44 MAPK by PD98059 restored TRAIL-mediated lysosomal permeabilization and apoptosis in cFLIP-overexpressing cells. In conclusion, these results demonstrate that lysosomal permeabilization contributes to TRAIL-induced apoptosis of hepatocellular carcinoma cells and suggest that cFLIP(L) cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown.     

Phosphatidylinositol 3-Kinase/Akt Plays a Part in Tumor Necrosis Factor-alpha-induced Interleukin-6 Synthesis in Osteoblasts.

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl- CHIRO-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.     

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.     

Cbl-b promotes chemotherapy-induced apoptosis in rat basophilic leukemia cells by suppressing PI3K/Akt activation and enhancing MEK/ERK activation

The ubiquitin ligase Cbl-b is a negative regulator of the PI3K/Akt pathway, the survival pathway implicated in chemotherapy resistance. However, it remains unclear whether Cbl-b can regulate chemosensitivity through modulating Akt activation. In this study, VP-16-induced RBL-2H3 cells apoptosis was accompanied by the activation of Akt and ERK. The PI3K inhibitor LY294002, not the ERK inhibitor PD98059, enhanced the apoptosis. In addition, down-regulation of Cbl-b was also detected. Over expression of Cbl-b significantly enhanced VP-16-induced cell apoptosis with inhibition of Akt activity, while a dominant negative (DN) RING Finger domain mutation completely abolished this enhancement. On the other hand, ERK activity was enhanced by Cbl-b, and the ERK inhibitor PD98059 reversed Cbl-b-enhanced apoptosis. The consistent results were also showed in the process of Ara-c treatment. These observations indicate that Cbl-b promotes RBL-2H3 apoptosis induced by VP-16 or Ara-c, probably through inhibition of Akt and activation of ERK.