Role of ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction

The treatment of endothelial cell monolayers with phorbol 12-myristate 13-acetate (PMA), a direct protein kinase C (PKC) activator, leads to disruption of endothelial cell monolayer integrity and intercellular gap formation. Selective inhibition of PKC (with bisindolylmaleimide) and extracellular signal-regulated kinases (ERKs; with PD-98059, olomoucine, or ERK antisense oligonucleotides) significantly attenuated PMA-induced reductions in transmonolayer electrical resistance consistent with PKC- and ERK-mediated endothelial cell barrier regulation. An inhibitor of the dual-specificity ERK kinase (MEK), PD-98059, completely abolished PMA-induced ERK activation. PMA also produced significant time-dependent increases in the activity of Raf-1, a Ser/Thr kinase known to activate MEK ( approximately 6-fold increase over basal level). Similarly, PMA increased the activity of Ras, which binds and activates Raf-1 ( approximately 80% increase over basal level). The Ras inhibitor farnesyltransferase inhibitor III (100 microM for 3 h) completely abolished PMA-induced Raf-1 activation. Taken together, these data suggest that the sequential activation of Ras, Raf-1, and MEK are involved in PKC-dependent endothelial cell barrier regulation.     

A Role for the MEK/MAPK Pathway in PMA-Induced Cell Cycle Arrest: Modulation of Megakaryocytic Differentiation of K562 Cells

In vitromegakaryocytic differentiation of the pluripotent K562 human leukemia cell line is induced by PMA. Treatment of K562 cells with PMA results in growth arrest, polyploidy, morphological changes, and increased cell–cell and cell–substrate adhesion. These PMA-induced changes in K562 cells are preceded by a rapid rise in the activity of MEK (MAP kinase/extracellular regulated kinases) that leads to a sustained activation of ERK2 (extracellular regulated kinase; MAPK). Blockade of MEK1 activation by PD098059, a recently described specific MEK inhibitor [D. T. Dudleyet al.(1995).Proc. Natl. Acad. Sci. USA92, 7686–7689], reverses both the growth arrest and the morphological changes of K562 cells induced by PMA treatment. These changes are not associated with a disruption of PMA-induced down-regulation of BCR-ABL kinase or early integrin signaling events but are associated with a block of the cell-surface expression of the gpIIb/IIIa (CD41) integrin, a cell marker of megakaryocytic differentiation. These results demonstrate that the PMA-induced signaling cascade initiated by protein kinase C activation requires the activity of the MEK/ERK signaling complex to regulate cell cycle arrest, thus regulating the program that leads to the cell-surface expression of markers associated with megakaryocytic differentiation.     

Non-redundant function of the MEK5-ERK5 pathway in thymocyte apoptosis

The mitogen-activated protein kinases (MAPKs) ERK1/2, p38, and JNK are thought to determine survival-versus-death fate in developing thymocytes. However, this view was challenged by studies using 'MEK1-ERK1/2-specific' pharmacological inhibitors, which block both positive and negative selection. Recently, these inhibitors were also shown to affect MEK5, an upstream activator of ERK5, another class of MAPK with homology to ERK1/2. To define the contribution of the MEK5-ERK5 pathway in T-cell development, we retrovirally expressed dominant-negative or constitutively activated form of MEK5 to inhibit or activate the MEK5-ERK5 pathway. We demonstrate that MEK5 regulates apoptosis of developing thymocytes but has no function in positive selection. ERK5 activity correlates with the levels of Nur77 family members but not that of Bim, two effector pathways of thymocyte apoptosis. These results illustrate the critical involvement of the MEK5-ERK5 pathway in thymocyte development distinct from that of ERK1/2 and highlight the importance of the MAPK network in mediating differential effects pertaining to T-cell differentiation and apoptosis.     

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.     

Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ERK2) signaling in human T cells.

This study was conducted on human Jurkat T cell lines to elucidate the role of EPA and DHA, n-3 PUFA, in the modulation of two mitogen-activated protein (MAP) kinases, that is, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). The n-3 PUFA alone failed to induce phosphorylation of ERK1/ERK2. We stimulated the MAP kinase pathway with anti-CD3 antibodies and phorbol 12-myristate 13-acetate (PMA), which act upstream of the MAP kinase (MAPK)/ERK kinase (MEK) as U0126, an MEK inhibitor, abolished the actions of these two agents on MAP kinase activation. EPA and DHA diminished the PMA- and anti-CD3-induced phosphorylation of ERK1/ERK2 in Jurkat T cells. In the present study, PMA acts mainly via protein kinase C (PKC) whereas anti-CD3 antibodies act via PKC-dependent and -independent mechanisms. Furthermore, DHA and EPA inhibited PMA-stimulated PKC enzyme activity. EPA and DHA also significantly curtailed PMA- and ionomycin-stimulated T cell blastogenesis. Together these results suggest that EPA and DHA modulate ERK1/ERK2 activation upstream of MEK via PKC-dependent and -independent pathways and that these actions may be implicated in n-3 PUFA-induced immunosuppression.     

A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis.

Resting thymocytes undergoing apoptosis in response to specific stimuli degrade the cdk inhibitor p27(Kip1) and upregulate Cdk2 kinase activity. Inhibition of Cdk2 kinase activity efficiently blocks cell death via certain apoptosis pathways whereas overexpression of Cdk2 accelerates such cell death, suggesting its involvement in the signal transduction pathways activated by certain apoptotic stimuli. We found that Cdk2 activation during thymocyte apoptosis can be regulated by p53, Bax and Bcl-2. The highly elevated Cdk2 kinase activity in the apoptosing thymocytes is not associated with its canonical cyclins, cyclin E and cyclin A, and requires de novo synthesis of proteins for activation to take place. We therefore propose Cdk2 activation to be a crucial event in distinct pathways of apoptosis and the point at which the cell cycle and cell death pathways interact.     

Integrin alphav-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen

Integrin alphav is required for melanoma cell survival and tumor growth in various models. To elucidate integrin alphav-mediated melanoma cell survival mechanisms, we used a three-dimensional (3D) collagen gel model mimicking the pathophysiological microenvironment of malignant melanoma in the dermis. We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo. This indicates that integrin alphav-mediated inactivation of p53 functionally controls melanoma cell survival. Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis. Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis. This suggests that integrin alphav controls melanoma cell survival in 3D-collagen through a pathway involving p53 regulation of MEK1 signaling.     

CD45 negatively regulates monocytic cell differentiation by inhibiting phorbol 12-myristate 13-acetate-dependent activation and tyrosine phosphorylation of protein kinase Cdelta

The protein-tyrosine phosphatase CD45 is expressed on all monocytic cells, but its function in these cells is not well defined. Here we report that CD45 negatively regulates monocyte differentiation by inhibiting phorbol 12-myristate 13-acetate (PMA)-dependent activation of protein kinase C (PKC) delta. We found that antisense reduction of CD45 in U937 monocytic cells (CD45as cells) increased by 100% the ability of PMA to enlarge cell size, increase cell cytoplasmic process width and length, and induce surface expression of CD11b. In addition, reduction in CD45 expression caused the duration of peak PMA-induced MEK and extracellular signal-regulated kinase (ERK) 1/2 activity to increase from 5 min to 30 min while leading to a 4-fold increase in PMA-dependent PKCdelta activation. Importantly, PMA-dependent tyrosine phosphorylation of PKCdelta was also increased 4-fold in CD45as cells. Finally, inhibitors of MEK (PD98059) and PKCdelta (rottlerin) completely blocked PMA-induced monocytic cell differentiation. Taken together, these data indicate that CD45 inhibits PMA-dependent PKCdelta activation by impeding PMA-dependent PKCdelta tyrosine phosphorylation. Furthermore, this blunting of PKCdelta activation leads to an inhibition of PKCdelta-dependent activation of ERK1/2 and ERK1/2-dependent monocyte differentiation. These findings suggest that CD45 is a critical regulator of monocytic cell development.     

Variable apoptotic response of NSCLC cells to inhibition of the MEK/ERK pathway by small molecules or dominant negative mutants

To evaluate the role of the MEK/ERK pathway in NSCLC survival, we analyzed NSCLC cell lines that differed in tumor histology and status of p53, Rb, and K-ras. Constitutive ERK1/2 activity was demonstrated in 17 of 19 cell lines by maintenance of ERK1/2 phosphorylation with serum deprivation. Phosphorylation of ERK1/2 correlated with phosphorylation of MEK1/2 and p90RSK, but was inversely correlated with phosphorylation of c-Raf at S259. With serum deprivation, the MEK inhibitors, PD98059 and U0126, inhibited ERK1/2 activity but did not increase apoptosis. PD98059 and U0126 induced cell cycle arrest in G(0)/G(i) in cells with the highest levels of ERK1/2 activity, which correlated with induction of p27 but not p21. To confirm the cytostatic response to MEK inhibitors, we performed transient transfections with dominant negative forms of MEK or ERK. Surprisingly, dominant negative MEK and ERK mutants increased apoptosis without affecting cell cycle or p27 levels. When combined with paclitaxel, MEK inhibitors had no effect on apoptosis. In contrast, dominant negative ERK2 potentiated paclitaxel-induced apoptosis. Our studies show that constitutive ERK1/2 activity in NSCLC cells promotes cellular survival and chemotherapeutic resistance. Moreover, our data are the first to demonstrate divergent cellular responses to inhibition of the MEK/ERK pathway by small molecule inhibitors or dominant negative mutants.     

Inhibition of ligand-independent ERK1/2 activity in kidney proximal tubular cells deprived of soluble survival factors up-regulates Akt and prevents apoptosis

Mouse kidney proximal tubular epithelial (MK-PT) cells die by apoptosis over 7-10 days when deprived of all survival factors. We show here that withdrawal of all survival factors from MK-PT cells is associated with a progressive increase in the activity of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and a progressive decrease in phosphorylated Akt, a kinase critical to cell survival. Pharmacological inhibition of MEK1/2, the immediate upstream kinase for ERK1/2, not only prevented the decrease in phosphorylated Akt, but also prolonged MK-PT cell survival. Inhibition of ERK1/2, by itself, in the absence of any other known survival factors, was as potent as epidermal growth factor in maintaining MK-PT cell viability. ERK1/2 co-immunoprecipitated with Akt in a multimolecular assembly of signaling molecules, containing at a minimum ERK1/2, Akt, Rsk, and 3-phosphoinositide dependent kinase 1 (PDK1). We hypothesize that the kinase Rsk, whose activation requires phosphorylation by both ERK1/2 and PDK1, acts as a bridge bringing ERK1/2 into proximity with PDK1-associated Akt. Although a number of interactions between the Raf-MEK-ERK and PI3K-Akt signaling pathways have been described, our results are the first to show modulation of Akt activity by signaling events originating with ERK1/2. Spontaneous activation of ERK1/2 occurs via MEK1/2 and appears to depend on oxidant stress, accompanying induction of the default pathway of apoptosis. Together, these data suggest that the spontaneous activation of ERK1/2, in the absence of known extracellular stimuli, represents a previously unrecognized major regulatory pathway determining the fate of cells destined to die by the default pathway of apoptosis.     

Duration and strength of extracellular signal-regulated kinase signals are altered during positive versus negative thymocyte selection.

During thymocyte development, high-affinity/avidity TCR engagement leads to the induction of negative selection and apoptosis, while lower TCR affinity-avidity interactions lead to positive selection and survival. To elucidate how these extracellular interactions are translated into intracellular signals that distinguish between positive and negative selection, we developed a culture system in which naive double-positive thymocytes were either induced to differentiate along the CD8(+) lineage pathway or were triggered for clonal deletion. Using this system, we show that sustained low level activation of extracellular signal-regulated kinases (ERKs) promotes positive selection, whereas strong but transient ERK activation is coupled with negatively selecting stimuli. Importantly, similar ERK activation profiles were demonstrated during positive selection for strong agonist ligands presented at low concentrations or weak agonist ligands. This is consistent with the affinity/avidity model and a role for strong or weak agonists during positive selection. Surprisingly, the addition of a pharmacological inhibitor which blocks ERK activation prevented the induction of negative selection. These data suggest that the duration and strength of the TCR signal is involved in discriminating between positive and negative selection.     

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.     

Molecular regulation of NADPH oxidase 5 via the MAPK pathway

The mechanisms controlling the activity of NADPH oxidase 5 (Nox5) are unique in that they are independent of the protein: protein interactions that coordinate the activation of other Nox isoforms. Instead, the primary driving force for Nox5 activity is calcium. However, in a previous study we reported that the protein kinase C (PKC)-agonist PMA could induce a sustained activation of Nox5 that was independent of calcium changes. This apparent calcium-independent activation was found to be mediated by the PKC-dependent phosphorylation of specific serine and threonine residues on Nox5 which increased the calcium sensitivity of the enzyme and enabled activation at resting levels of calcium. However, the specific kinase(s) mediating the phosphorylation and activation of Nox5 are not known. As PKC can activate the MEK/ERK1/2 signaling pathway, we hypothesized that Nox5 is activated by the coordinated phosphorylation of both MAPK and PKC pathways. The inhibition of MEK1 using PD-98059 and U-0126 significantly reduced the phosphorylation and activity of Nox5 in response to PMA but not to the calcium-mobilizing stimulus ionomycin. Dominant negative MEK1 and knockdown of endogenous MEK1/2 using a specific small interfering RNA also inhibited Nox5 activity in response to PMA. The mutation of S498 to a nonphosphorylatable residue and to a lesser degree T494 blocked the ability of ERK to stimulate Nox5 activity. However, a constitutively active form of MEK1 failed to increase Nox5 activity in the absence of PMA stimulation. These results suggest that the MEK/ERK1/2 pathway is necessary but not sufficient to regulate the PMA-dependent activation of Nox5.     

Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways.

To define the signaling pathways during NO-induced apoptotic events and their possible modulation by two protein kinase systems, we explored the involvement of three structurally related mitogen-activated protein kinase subfamilies. Exposure of HL-60 cells to sodium nitroprusside (SNP) strongly activated p38 kinase, but did not activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, SNP-induced apoptosis was markedly blocked by the selective p38 kinase inhibitor (SB203580) but not by MEK1 kinase inhibitor (PD098059), indicating that p38 kinase serves as a mediator of NO-induced apoptosis. In contrast, treatment of cells with phorbol 12-myristate 13-acetate (PMA) strongly activated not only JNK but also ERK, while not affecting p38 kinase. However, although SNP by itself weakly activated CPP32-like protease, SNP in combination with PMA markedly increased the extent of CPP32-like protease activation. Interestingly, N6,O2-dibutylyl cAMP (DB-cAMP) significantly blocked SNP- or SNP plus PMA-induced activation of CPP32-like protease and the resulting induction of apoptosis. DB-cAMP also blocked PMA-induced JNK activation. Collectively, these findings demonstrate the presence of specific up- or down-modulatory mechanisms of cell death pathway by NO in which (1) p38 kinase serves as a mediator of NO-induced apoptosis, (2) PKC acts at the point and/or upstream of JNK and provides signals to potentiate NO-induced CPP32-like protease activation, and (3) PKA lies upstream of either JNK or CPP32-like protease to protect NO- or NO plus PMA-induced apoptotic cell death in HL-60 cells.     

Epidermal growth factor receptor (EGFR)-mediated positive feedback of protein-tyrosine phosphatase epsilon (PTPepsilon) on ERK1/2 and AKT protein pathways is required for survival of human breast cancer cells

Increased tyrosine phosphorylation has been correlated with human cancer, including breast cancer. In general, the activation of tyrosine kinases (TKs) can be antagonized by the action of protein-tyrosine phosphatases (PTPs). However, in some cases PTPs can potentiate the activation of TKs. In this study, we have investigated the functional role of PTPε in human breast cancer cell lines. We found the up-regulation and activation of receptor PTPε (RPTPε) in MCF-7 cells and MDA-MB-231 upon PMA, FGF, and serum stimulation, which depended on EGFR and ERK1/2 activity. Diminishing the expression of PTPε in human breast cancer cells abolished ERK1/2 and AKT activation, and decreased the viability and anchorage-independent growth of the cells. Conversely, stable MCF-7 cell lines expressing inducible high levels of ectopic PTPε displayed higher activation of ERK1/2 and anchorage-independent growth. Our results demonstrate that expression of PTPε is up-regulated and activated in breast cancer cell lines, through EGFR, by sustained activation of the ERK1/2 pathway, generating a positive feedback regulatory loop required for survival of human breast cancer cells.     

Modulation of CREB and NF-kappaB signal transduction by cannabinol in activated thymocytes

Cannabinoid compounds inhibit the cAMP signalling cascade in leukocytes. One of these compounds, cannabinol (CBN) has been shown to inhibit interleukin-2 (IL-2) expression and the activation of cAMP response element binding protein (CREB) and nuclear factor for immunoglobulin kappa chain in B cells (NF-kappaB) following phorbol-12-myristate-13 acetate (PMA) plus ionomycin (Io) treatment of thymocytes. Therefore, the objective of the present studies was to determine the role of cAMP and protein kinase A (PKA) in the CBN-mediated inhibition of IL-2, CREB, and NF-kappaB in PMA/Io-activated thymocytes. The inhibition of CREB/ATF-1 phosphorylation, or cAMP response element (CRE) or kappaB DNA binding activity produced by CBN in PMA/Io-activated thymocytes, could not be reversed by DBcAMP costimulation. Furthermore, DBcAMP failed to reverse the concentration-dependent inhibition of IL-2 protein secretion by CBN. Pretreatment of thymocytes with H89 produced a modest inhibition of PMA/Io-induced CREB/ATF-1 phosphorylation and CRE DNA binding activity but H89 had no effect on protein binding to a kappaB motif. Additionally, H89 modestly inhibited PMA/Io-induced IL-2 secretion. In light of the modest involvement of the cAMP pathway in CBN-mediated inhibition of CREB and IL-2 in PMA/Io-activated thymocytes, PD098059 (PD), the MEK inhibitor, was utilized to determine the role of ERK MAP kinases in thymocytes. ERKs play a critical role in IL-2 production but not for CREB phsophorylation. Collectively, these findings suggest that CBN may modulate several signalling pathways in activated T cells.     

Calcium-activated RAF/MEK/ERK signaling pathway mediates p53-dependent apoptosis and is abrogated by alpha B-crystallin through inhibition of RAS activation

The ocular lens is the only organ that does not develop spontaneous tumor. The molecular mechanism for this phenomenon remains unknown. Through examination of the signaling pathways mediating stress-induced apoptosis, here we presented evidence to show that different from most other tissues in which the extracellular signal-regulated kinases (ERKs) pathway is generally implicated in mediation of survival signals activated by different factors, the RAF/MEK/ERK signaling pathway alone plays a key role in stress-activated apoptosis of lens epithelial cells. Treatment of N/N1003A cells with calcimycin, a calcium mobilizer, activates the RAF/MEK/ERK pathway through RAS, which is indispensable for the induced apoptosis because inhibition of this pathway by either pharmacological drug or dominant negative mutants greatly attenuates the induced apoptosis. Calcimycin also activates p38 kinase and JNK2, which are not involved in calcium-induced apoptosis. Downstream of ERK activation, p53 is essential. Activation of RAF/MEK/ERK pathway by calcimycin leads to distinct up-regulation of p53. Moreover, overexpression of p53 enhances calcimycin-induced apoptosis, whereas inhibition of p53 expression attenuates calcimycin-induced apoptosis. Up-regulation of p53 directly promotes Bax expression, which changes the integrity of mitochondria, leading to release of cytochrome c, activation of caspase-3 and eventually execution of apoptosis. Overexpression of alphaB-crystallin, a member of the small heat-shock protein family, blocks activation of RAS to inhibit ERK1/2 activation, and greatly attenuates calcimycin-induced apoptosis. Together, our results provide 1) a partial explanation for the lack of spontaneous tumor in the lens, 2) a novel signaling pathway for calcium-induced apoptosis, and 3) a novel antiapoptotic mechanism for alphaB-crystallin.     

Bromelain, from pineapple stems, proteolytically blocks activation of extracellular regulated kinase-2 in T cells.

Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3epsilon mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-gamma, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways.     

Functional role of extracellular signal-regulated kinase activation and c-Jun induction in phorbol ester-induced promoter activation of human 12(S)-lipoxygenase gene

The functional role of mitogen-activated protein kinase (MAPK) signaling and c-Jun induction in phorbol 12-myristate 13-acetate (PMA)-induced human 12(S)-lipoxygenase gene expression was studied in human epidermoid carcinoma A431 cells. Among the family of MAPK, PMA only increased the activity of extracellular signal-regulated kinase (ERK). Treatment of cells with PD98059, which is an inhibitor of mitogen-activated protein kinase kinase (MEK), decreased the PMA-induced expression of 12(S)-lipoxygenase. Transfection of cells with Ras, Raf and ERK2 dominant negative mutants inhibited the PMA-induced promoter activation of the 12(S)-lipoxygenase gene in all cases. PMA-induced expression of c-Jun was inhibited by pretreatment with PD98059. Following treatment with PMA, the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in cells increased with time. Enhancement of binding between the c-Jun-Sp1 complex and the Sp1 oligonucleotide was observed in cells treated with PMA, suggesting the possible interaction of c-Jun-Sp1 with GC-rich binding sites in the gene promoter. These results indicate that PMA treatment induced ERK activation mainly through the Raf-MEK-ERK signaling pathway following induction of c-Jun expression, and the formation of the c-Jun-Sp1 complex. Finally, PMA activated the promoter activity of the 12(S)-lipoxygenase gene in cells overexpressing protein kinase C (PKC)delta but not PKCalpha, indicating that PKCdelta played the functional role in mediating the gene activation of 12(S)-lipoxygenase induced by PMA.     

Pharmacological and genetic evaluation of proposed roles of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p90(RSK) in the control of mTORC1 protein signaling by phorbol esters

The mammalian target of rapamycin complex 1 (mTORC1) links the control of mRNA translation, cell growth, and metabolism to diverse stimuli. Inappropriate activation of mTORC1 can lead to cancer. Phorbol esters are naturally occurring products that act as potent tumor promoters. They activate isoforms of protein kinase C (PKCs) and stimulate the oncogenic MEK/ERK signaling cascade. They also activate mTORC1 signaling. Previous work indicated that mTORC1 activation by the phorbol ester PMA (phorbol 12-myristate 13-acetate) depends upon PKCs and may involve MEK. However, the precise mechanism(s) through which they activate mTORC1 remains unclear. Recent studies have implicated both the ERKs and the ERK-activated 90-kDa ribosomal S6 kinases (p90(RSK)) in activating mTORC1 signaling via phosphorylation of TSC2 (a regulator of mTORC1) and/or the mTORC1 component raptor. However, the relative importance of each of these kinases and phosphorylation events for the activation of mTORC1 signaling is unknown. The recent availability of MEK (PD184352) and p90(RSK) (BI-D1870) inhibitors of improved specificity allowed us to address the roles of these protein kinases in controlling mTORC1 in a variety of human and rodent cell types. In parallel, we used specific shRNAs against p90(RSK1) and p90(RSK2) to further test their roles in regulating mTORC1 signaling. Our data indicate that p90(RSKs) are dispensable for the activation of mTORC1 signaling by phorbol esters in all cell types tested. Our data also reveal striking diversity in the requirements for MEK/ERK in the control of mTORC1 between different cell types, pointing to additional signaling connections between phorbol esters and mTORC1, which do not involve MEK/ERK. This study provides important information for the design of efficient strategies to combat the hyperactivation of mTORC1 signaling by oncogenic pathways.