Iron chelator triggers inflammatory signals in human intestinal epithelial cells: involvement of p38 and extracellular signal-regulated kinase signaling pathways

Competition for cellular iron (Fe) is a vital component of the interaction between host and pathogen. Most bacteria have an obligate requirement for Fe to sustain infection, growth, and survival in host. To obtain iron required for growth, many bacteria secrete iron chelators (siderophores). This study was undertaken to test whether a bacterial siderophore, deferoxamine (DFO), could trigger inflammatory signals in human intestinal epithelial cells as a single stimulus. Incubation of human intestinal epithelial HT-29 cells with DFO increased the expression of IL-8 mRNA, as well as the release of IL-8 protein. The signal transduction study revealed that both p38 and extracellular signal-regulated kinase-1/2 were significantly activated in response to DFO. Accordingly, the selective inhibitors for both kinases, either alone or in combination, completely abolished DFO-induced IL-8 secretion, indicating an importance of mitogen-activated protein kinases pathway. These proinflammatory effects of DFO were, in large part, mediated by activation of Na(+)/H(+) exchangers, because selective blockade of Na(+)/H(+) exchangers prevented the DFO-induced IL-8 production. Interestingly, however, DFO neither induced NF-kappaB activation by itself nor affected IL-1beta- or TNF-alpha-mediated NF-kappaB activation, suggesting a NF-kappaB-independent mechanism in DFO-induced IL-8 production. Global gene expression profiling revealed that DFO significantly up-regulates inflammation-related genes including proinflammatory genes, and that many of those genes are down-modulated by the selective mitogen-activated protein kinase inhibitors. Collectively, these results demonstrate that, in addition to bacterial products or cell wall components, direct chelation of host Fe by infected bacteria may also contribute to the evocation of host inflammatory responses.     

Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway

Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

Insulin regulates MAP kinase phosphatase-1 induction in Hirc B cells via activation of both extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK)

Previously, we have reported that insulin induces the expression of the dual-specificity tyrosine phosphatase Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and that this may represent a negative feedback mechanism to regulate insulin-stimulated MAP kinase activity. In this work, the mechanism of regulation of MKP-1 expression by insulin was examined, particularly the role of the MAP kinase superfamily. Inhibition of the ERK pathway attenuated insulin-stimulated MKP-1 mRNA expression. Expression of dominant negative molecules of the JNK pathway also abolished insulin-stimulated MKP-1 expression. However, inhibition of p38^MAPK activity by SB202190 had no effect on insulin-stimulated MKP-1 induction. Simultaneous inhibition of the ERK and JNK pathways abolished the ability of insulin to stimulate MKP-1 expression, however, this combined inhibition was neither additive nor synergistic, suggesting these pathways converge to act on a common final effector. In conclusion, induction of MKP-1 mRNA expression in Hirc B cells by insulin requires activation of both the ERK and JNK pathways, but not p38^MAPK.     

Autocrine extracellular signal-regulated kinase (ERK) activation in normal human keratinocytes: metalloproteinase-mediated release of amphiregulin triggers signaling from ErbB1 to ERK

ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30-60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2-1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin- and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited.     

TLR2 and RIP2 pathways mediate autophagy of Listeria monocytogenes via extracellular signal-regulated kinase (ERK) activation

Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2(-/-) and Nod2(-/-) macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2(-/-) mice. Tlr2(-/-) and Nod2(-/-) cells showed perturbed NF-κB and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes.     

Ectopic B-Raf expression enhances extracellular signal-regulated kinase (ERK) signaling in T cells and prevents antigen-presenting cell-induced anergy

T cells that receive stimulation through the T cell receptor (TCR) in the absence of costimulation become anergic and are refractory to subsequent costimulation. This unresponsiveness is associated with the constitutive activation of the small G protein, Rap1, and the lack of Ras-dependent activation of ERK. Recent studies suggest that Rap1 can activate the MAP kinase kinase kinase B-Raf that is either endogenously or ectopically expressed. Peripheral T cells generally do not express B-Raf; therefore, to test the hypothesis that ectopic expression of B-Raf could permit Rap1 to activate ERK signaling, we generated transgenic mice expressing B-Raf within peripheral T cells. This converted Rap1 into an activator of ERK, to enhance ERK activation and proliferation following TCR engagement in the absence of costimulation. When T cells were incubated with engineered APCs presenting antigen on I-Ek and expressing low levels of B7, they became anergic, displayed constitutive activation of Rap1, and were deficient in Ras and ERK activation. However, when incubated with the same APCs, T cells expressing the B-Raf transgene proliferated upon restimulation and displayed elevated ERK activation. Thus B-Raf expression and enhanced ERK activation is sufficient to prevent anergy in a model of APC-induced T cell anergy. However, studies using anti-TCR antibody-induced anergy showed that the ability of ERKs to reverse T cell anergy is dependent on the anergic model utilized.     

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.     

Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling

Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.     

MicroRNA miR-199a* regulates the MET proto-oncogene and the downstream extracellular signal-regulated kinase 2 (ERK2)

MicroRNAs (miRNAs) constitute a class of small noncoding RNAs that play important roles in a variety of biological processes including development, apoptosis, proliferation, and differentiation. Here we show that the expression of miR-199a and miR-199a* (miR-199a/a*), which are processed from the same precursor, is confined to fibroblast cells among cultured cell lines. The fibroblast-specific expression pattern correlated well with methylation patterns: gene loci on chromosome 1 and 19 were fully methylated in all examined cell lines but unmethylated in fibroblasts. Transfection of miR-199a and/or -199a* mimetics into several cancer cell lines caused prominent apoptosis with miR-199a* being more pro-apoptotic. The mechanism underlying apoptosis induced by miR-199a was caspase-dependent, whereas a caspase-independent pathway was involved in apoptosis induced by miR-199a* in A549 cells. By employing microarray and immunoblotting analyses, we identified the MET proto-oncogene as a target of miR-199a*. Studies with a luciferase reporter fused to the 3'-untranslated region of the MET gene demonstrated miR-199a*-mediated down-regulation of luciferase activity through a binding site of miR-199a*. Interestingly, extracellular signal-regulated kinase 2 (ERK2) was also down-regulated by miR-199a*. Coordinated down-regulation of both MET and its downstream effector ERK2 by miR-199a* may be effective in inhibiting not only cell proliferation but also motility and invasive capabilities of tumor cells.     

Involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in amylin's eating inhibitory effect

Peripheral amylin inhibits eating via the area postrema (AP). Because amylin activates the extracellular-signal regulated kinase 1/2 (ERK) pathway in some tissues, and because ERK1/2 phosphorylation (pERK) leads to acute neuronal responses, we postulated that it may be involved in amylin's eating inhibitory effect. Amylin-induced ERK phosphorylation (pERK) was investigated by immunohistochemistry in brain sections containing the AP. pERK-positive AP neurons were double-stained for the calcitonin 1a/b receptor, which is part of the functional amylin-receptor. AP sections were also phenotyped using dopamine-β-hydroxylase (DBH) as a marker of noradrenergic neurons. The effect of fourth ventricular administration of the ERK cascade blocker U0126 on amylin's eating inhibitory action was tested in feeding trials. The number of pERK-positive neurons in the AP was highest ～10-15 min after amylin treatment; the effect appeared to be dose-dependent (5-20 μg/kg amylin). A portion of pERK-positive neurons in the AP carried the amylin-receptor and 22% of the pERK-positive neurons were noradrenergic. Pretreatment of rats with U0126 decreased the number of pERK-positive neurons in the AP after amylin injection. U0126 also attenuated the ability of amylin to reduce eating, at least when the animals had been fasted 24 h prior to the feeding trial. Overall, our results suggest that amylin directly stimulates pERK in AP neurons in a time- and dose-dependent manner. Part of the AP neurons displaying pERK were noradrenergic. At least under fasting conditions, pERK was shown to be a necessary part in the signaling cascade mediating amylin's anorectic effect.     

Extracellular signal-regulated kinase (ERK) regulates cortactin ubiquitination and degradation in lung epithelial cells

Cortactin, an actin-binding protein, is essential for cell growth and motility. We have shown that cortactin is regulated by reversible phosphorylation, but little is known regarding cortactin protein stability. Here, we show that lipopolysaccharide (LPS)-induced cortactin degradation is mediated by extracellular regulated signal kinase (ERK). LPS induces cortactin serine phosphorylation, ubiquitination, and degradation in mouse lung epithelia, an effect abrogated by ERK inhibition. Serine phosphorylation sites mutant, cortactin(S405A/S418A), enhances its protein stability. Cortactin is polyubiquitinated and degraded within the proteasome, whereas a cortactin(K79R) mutant exhibited proteolytic stability during cyclohexamide (CHX) or LPS treatment. The E3 ligase subunit β-Trcp interacts with cortactin, and its overexpression reduced cortactin protein levels, an effect attenuated by ERK inhibition. Overexpression of β-Trcp was sufficient to reduce the protective effects of exogenous cortactin on epithelial cell barrier integrity, an effect not observed after expression of a cortactin(K79R) mutant. These results provide evidence that LPS modulation of cortactin stability is coordinately regulated by stress kinases and the ubiquitin-proteasomal network.     

Quercetin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells through extracellular signal-regulated kinase.

Studies have shown that intake of quercetin was inversely associated with mortality from coronary heart disease. Since recent studies documented that disruption of atherosclerotic plaques is the key event triggering acute myocardial infarction, and vascular endothelium-derived matrix metalloproteinase-1 (MMP-1) contributes to plaque destabilization, we examined the effect of quercetin on MMP-1 expression in human vascular endothelial cells. Our results showed that quercetin significantly inhibited basal and oxidized LDL (oxLDL)-stimulated MMP-1 expression. Our data also indicated that extracellular signal-regulated kinase (ERK) mediated the basal and oxLDL-stimulated expression of MMP-1, and quercetin is a potent inhibitor of ERK, suggesting that quercetin may inhibit MMP-1 expression by blocking the ERK pathway. Finally, we showed that quercetin stimulated tissue inhibitor of metalloproteinase-1 expression in oxLDL- and PMA-treated cells. In conclusion, the present study demonstrated for the first time that quercetin inhibited MMP-1 expression in vascular endothelial cells, suggesting that quercetin might contribute to plaque stabilization.     

Activation of extracellular signal-regulated kinase (ERK)1/2, but not p38 and c-Jun N-terminal kinase, is involved in signaling of a novel cytokine, ML-1

A novel cytokine, ML-1, was recently discovered, which shares a similar sequence homology with, but is functionally distinct from, IL-17 (Kawaguchi, M., Onuchic, L., Li, X. D., Essayan, D. M., Schroeder, J., Xiao, H. Q., Liu, M. C., Krishnaswamy, G., Germino, G., and Huang, S. K. (2001) J. Immunol. 167, 4430-4435). To determine the signaling mechanisms of ML-1, we investigated activation of mitogen-activated protein (MAP) kinases induced by ML-1. Results show that ML-1 induces in a time-dependent fashion the expression of IL-6 and IL-8 in both primary bronchial epithelial cells (PBECs) and human umbilical vein endothelial cells (HUVECs). ML-1 activated a MAP kinase and an extracellular signal-regulated kinase (ERK)1/2 but not p38 or the c-Jun N-terminal kinase (JNK) in both cell types. Selective MAP kinase kinase (MEK)1/2 inhibitors, PD98059 and U0126, inhibited, in a dose-dependent manner, ML-1-induced expression of IL-6 and IL-8. These findings suggest that ML-1-induced IL-6 and IL-8 production is mediated through the activation of ERK1/2 in both cell types.     

Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-delta19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-delta19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.     

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.     

Activation of extracellular signal-regulated kinase ERK after hypo-osmotic stress in renal epithelial A6 cells

Activation of mitogen-activated protein (MAP) kinases has been reported to occur after a hypo-osmotic cell swelling in various types of cells. In renal epithelial A6 cells, the hypo-osmotic shock induced a rapid increase in the phosphorylation of an extracellular signal-regulated kinase (ERK)-like protein that was maximal 10 min after osmotic stress. Activation of ERK was significantly increased when hypo-osmotic stress was performed in the absence of extracellular Ca²⁺, a condition that inhibits regulatory volume decrease (RVD). Exposure of cells to PD98059, an inhibitor of the MAP kinase kinase MEK, at a concentration that fully cancelled ERK activation, did not inhibit RVD. On the contrary, RVD was abolished when osmotic shock was induced in the presence of SB203580, an inhibitor of stress-activated protein kinases (SAPKs). These results suggest that different MAP kinases are activated after hypo-osmotic stress in A6 cells. SAPKs would be involved in the control of RVD, while ERK would lead to later events, such as gene expression or energy metabolism.     

Activation of extracellular signal-regulated kinase ERK after hypo-osmotic stress in renal epithelial A6 cells

Activation of mitogen-activated protein (MAP) kinases has been reported to occur after a hypo-osmotic cell swelling in various types of cells. In renal epithelial A6 cells, the hypo-osmotic shock induced a rapid increase in the phosphorylation of an extracellular signal-regulated kinase (ERK)-like protein that was maximal 10 min after osmotic stress. Activation of ERK was significantly increased when hypo-osmotic stress was performed in the absence of extracellular Ca2+, a condition that inhibits regulatory volume decrease (RVD). Exposure of cells to PD98059, an inhibitor of the MAP kinase kinase MEK, at a concentration that fully cancelled ERK activation, did not inhibit RVD. On the contrary, RVD was abolished when osmotic shock was induced in the presence of SB203580, an inhibitor of stress-activated protein kinases (SAPKs). These results suggest that different MAP kinases are activated after hypo-osmotic stress in A6 cells. SAPKs would be involved in the control of RVD, while ERK would lead to later events, such as gene expression or energy metabolism.