The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

Role of nuclear factor-kappaB activation in cytokine- and sphingomyelinase-stimulated inducible nitric oxide synthase gene expression in vascular smooth muscle cells

Inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF alpha), are known to activate sphingomyelinase (SMase) and nuclear factor-kappaB (NF-kappaB) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-kappaB activation pathway similar to that of IL-1beta and TNF alpha in cultured rat VSMCs. Neutral SMase, although less potently than IL-1beta and TNF alpha, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1beta, and TNF alpha activated NF-kappaB, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-kappaB activation stimulated by TNF alpha, but not by IL-1beta. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-kappaB activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1beta and TNF alpha, but not neutral SMase, caused a transient decrease in IkappaB-alpha protein levels, whereas IkappaB-beta protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated IkappaB-alpha degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-kappaB less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-kappaB activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated IkappaB-alpha degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).     

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.     

Ceramide accumulation during oxidant renal tubular injury: mechanisms and potential consequences

Ceramide is an important signaling molecule that is typically generated via sphingomyelinase (SMase)-mediated sphingomyelin (SM) hydrolysis. Although diverse forms of renal injury elicit ceramide accumulation, the molecular determinants of this change and its contribution to tissue damage are poorly defined. The present study uses iron (Fe/hydroxyquinoline)-mediated injury of cultured human proximal tubular (HK-2) cells to gain additional insights into these issues. A 4-h Fe exposure doubled ceramide levels in the absence of cell death. This was independent of de novo synthesis, since ceramide synthase inhibition (with fumonisin B1) had no effect. Oxidant stress directly suppressed, rather than stimulated, SMase activity by: (1) decreasing SMase levels; (2) depleting SMase-stimulating glutathione; and (3) increasing SM resistance to SMase attack. Fe suppressed cell sphingosine levels (3 to 4 times ceramide/sphingosine ratio increments), suggesting a possible ceramidase block. Fe did not directly affect HK-2 ceramidase levels. However, arachidonic acid (C20:4) accumulation, a consequence of oxidant-induced phospholipase A2 (PLA2) activation, markedly suppressed ceramidase and stimulated SMase activity. Exogenous C20:4, as well as PLA2 (in doses simulating Fe-induced deacylation) recapitulated Fe's ceramide-generating effect. Because C20:4 is directly cytotoxic, it was hypothesized that ceramide might offset some of C20:4's adverse effects. Supporting this possibility were the following: (1) C20:4 exacerbated Fe toxicity; (2) this was abrogated by ceramide treatment; and (3) ceramide blunted Fe-mediated cell death. Conclusions: (1) ceramide accumulation during acute cell injury can be an adaptive response to PLA2 activation/C20:4 generation; (2) C20:4-induced ceramidase inhibition, coupled with SMase stimulation, may trigger this result; and (3) these ceramide increments may exert a "biostat" function, helping to offset C20:4/PLA2- and "catalytic" iron-mediated tubular cell death.     

p38 Kinase is a negative regulator of angiotensin II signal transduction in vascular smooth muscle cells: effects on Na+/H+ exchange and ERK1/2

Activation of the Na+/H+ exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II-mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na+/H+ exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na+/H+ exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na+/H+ exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na+/H+ exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II-stimulated Na+/H+ exchange. First, angiotensin II activation of ERK1/2 was increased 1. 5- to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II-stimulated MAP kinases to phosphorylate a glutathione S-transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II-mediated regulation of the Na+/H+ exchanger in VSMC.     

Negative regulation of interleukin-1beta-activated neutral sphingomyelinase by protein kinase C in rat mesangial cells

Endogenous ceramide is produced by the action of acidic or neutral sphingomyelinases (SMase) in response to stimuli such as proinflammatory cytokines or other inducers of stress. Interleukin-1beta (IL-1beta) is known to stimulate ceramide formation in rat renal mesangial cells; however, the respective subtype of SMase and its regulation have not been investigated. We found that IL-1beta induced an increase in endogenous ceramide levels via the action of a neutral SMase but not an acidic SMase in rat mesangial cells. Cytokine-induced activation of neutral SMase was inhibited by stimulation of protein kinase C (PKC) by the phorbol ester TPA which caused a reduction of ceramide back to control levels. This inhibitory effect of TPA was reversed by the specific PKC-inhibitor Ro-318220. Long-term incubation (24 h) of mesangial cells with TPA, which downregulates PKC-alpha, -delta, and -epsilon isoenzymes, resulted in a recovery of IL-1beta-stimulated neutral SMase activity as well as ceramide formation. These data implicate an important modulatory function of PKC in ceramide production in IL-1beta-activated mesangial cells.     

Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC8) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC8 or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-espilon to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C2-Cer also inhibited the activation of phospholipase D activity induced by either diC8 (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.     

Cerebral changes in hepatic encephalopathy

Two subgroups of mitogen-activated protein kinases, c-jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), are thought to be involved in cultured cardiac myocyte hypertrophy and gene expression. To examine the in vivo activation of these kinases, we measured cardiac JNK and ERK activities in conscious rats subjected to acute or chronic angiotensin II (Ang II) infusion, by using in-gel kinase methods. About 50 mm Hg rise in blood pressure by Ang II (1000 ng . kg-1 . min-1) infusion caused larger activation of left ventricular JNK than ERK, via the AT1 receptor. In spite of short duration (about 30 minutes) of maximal blood pressure elevation by Ang II, JNK sustained the peak value (more than 5-fold increase) from 15 minutes up to at least 3 hours. Similar activation of JNK was seen in the right ventricle. Thus, cardiac JNK activation by Ang II seems to be in part mediated by its direct action via the AT1 receptor. The dose-response relationships for Ang II-induced rises in blood pressure and cardiac JNK and ERK activation indicated that cardiac JNK or ERK was not activated by a mild increase in blood pressure and that cardiac JNK was activated by Ang II-mediated hypertension in a more sensitive manner than ERK. Cardiac hypertrophy, induced by chronic Ang II infusion, was preceded by JNK activation without ERK activation. Furthermore, gel mobility shift analysis showed that cardiac JNK activation was followed by increased activator protein-1 DNA binding activity due to c-Fos and c-Jun. These results provided the first evidence for the preferential activation of cardiac JNK in Ang II-induced hypertension and suggested that JNK might play some role in Ang II-induced cardiac hypertrophic response in vivo. However, further study is needed to elucidate the role of JNK in cardiac hypertrophy in vivo.     

Purification of a newly identified alkaline sphingomyelinase in human bile and effects of bile salts and phosphatidylcholine on enzyme activity

The hydrolysis of sphingomyelin (SM) generates important signals regulating cell proliferation and apoptosis. Acid and neutral sphingomyelinases (SMase) have been identified and their biological effects intensively studied. We recently found in human bile a novel alkaline SMase that may have important roles in hepatobiliary diseases. In this work, we purified the enzyme and studied the factors influencing enzyme activity. Purification steps included Sephadex G25, diethylaminoethyl (DEAE)-Sepharose, Sephacryl S-200, and sphingosylphosphorylcholine (SPC) affinity chromatographies. A single protein of 92 kd was obtained with the specific enzyme activity increased 1,154-fold. The enzyme specifically hydrolyzed SM to ceramide, had a weaker activity against phosphatidylcholine (PC), and no activity against either phosphatidylethanolamine (PE) or p-nitrophenyl phosphate. Its optimum pH was 9.0 and its Vmax and Km were 45 micromol/h/mg and 2.5 x 10(-5) mol/L, respectively. The enzyme activity was dependent on concentration and structure of bile salts. Both trihydroxy and dihydroxy bile salts at concentrations up to their critical micellar concentrations activated the alkaline SMase, trihydroxy bile salts being more potent than dihydroxy ones. The side chain of trihydroxy bile salts was also important. Taurocholate (TC) was most effective in activating SMase, followed by glycocholate (GC), and cholate. 3-((3-cholamidopropyl)dimethylammonio)-propanesulfonate (CHAPS) alone had no effect on SMase activity but inhibited TC-induced activation of SMase. PC competitively inhibited bile alkaline SMase activity, with the 50% inhibition occurring at a PC/SM ratio of approximately 28. In conclusion, we purified a novel alkaline SMase from human bile and found that its activity is dependent on the levels of two major biliary components: PC and bile salts.     

Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.     

Early activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in response to binding of simian immunodeficiency virus to Jurkat T cells expressing CCR5 receptor

We have shown that the binding of simian immunodeficiency virus (SIV) to Jurkat T cells expressing CD4 receptor strongly induces mitogen-activated protein (MAP) kinase kinase (MEK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and only weakly induces p38 MAP kinase and c-Jun N-terminal kinase (JNK). Similarly, T-tropic NL4-3 virus, which uses both CD4 and CXCR4 receptors for entry, stimulated in these cells the MEK/ERK MAP kinase (MAPK) pathway in a CD4 receptor-dependent manner (Popik and Pitha, 1998). In contrast, both macrophage-tropic SIVmac316 and T cell-tropic SIVmac239, which in addition to CD4 require CCR5 coreceptor for entry, significantly enhanced early MEK/ERK, p38 MAPK, and JNK signaling in Jurkat cells expressing constitutively or transiently the CCR5 receptor. Together, this study provides the evidence that viruses using CXCR4 or CCR5 receptors for entry may differentially use signaling properties of their specific coreceptors to stimulate MAP kinase cascades. In addition, although SIVmac239 and SIVmac316 use different structural domains of the CCR5 receptor for entry, both viruses stimulate early phosphorylation of MEK, ERK, p38, and JNK independently of their tropism and replication.     

Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stress-activated protein kinases, in in vitro propagated endothelial cells

We investigated the involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways in human endothelial cells in response to shear stress and alterations of these kinases in in vitro-propagated endothelial cells (ECs). Potent activation (10-fold) of extracellular signal-regulated kinase (ERK2), a member of the MAPK family, occurred within 10 min of shear stress (5 dynes/cm2), whereupon rapid inactivation ensued. Shear stress also induced activation of stress-activated protein kinase (SAPK) or c-Jun NH2-terminal protein kinase (JNK) in ECs. Suramin pretreatment completely inhibited shear stress stimulation of ERK2, but not SAPK/JNK, highlighting a role for growth factor receptors in ERK activation. Translocation of ERK2 from the cytoplasm to the nucleus was observed in shear-stressed endothelial cells. In addition, we compared activities of MAPKs in shear-stressed cells derived from passages 4 and 10 (older). The magnitude of ERK2 activation was significantly lower in aged ECs compared to those of passage 4, while SAPK/JNK was not altered in the in vitro aged ECs. A similar level of ERK2 activation was found in both young and older cells stimulated with phorbol-12-myristate-13-acetate (PMA), indicating an age-related alteration of the plasma membrane. Taken together, these findings suggest that MAP kinase activation may be crucial for the expression of many genes in ECs stimulated by shear stress, and that an alteration in MAPK activities could contribute to the age-related decline in proliferative capacity.     

TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase

Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes. It has recently been reported that ceramide activates stress-activated protein kinase (SAPK, also known as c-Jun NH2-terminal kinase JNK), a subfamily member of mitogen-activated protein kinase superfamily molecules and that the ceramide/SAPK/JNK signaling pathway is required for stress-induced apoptosis. However, the molecular mechanism by which ceramide induces SAPK/JNK activation is unknown. Here we show that TAK1, a member of the mitogen-activated protein kinase kinase kinase family, is activated by treatment of cells with agents and stresses that induce an increase in ceramide. Ceramide itself stimulated the kinase activity of TAK1. Expression of a constitutively active form of TAK1 resulted in activation of SAPK/JNK and SEK1/MKK4, a direct activator of SAPK/JNK. Furthermore, expression of a kinase-negative form of TAK1 interfered with the activation of SAPK/JNK induced by ceramide. These results indicate that TAK1 may function as a mediator of ceramide signaling to SAPK/JNK activation.     

Activation of signal transduction kinases by tamoxifen

PURPOSE: To study the signal transduction mechanisms of tamoxifen via the activation of MAPKs, JNK and ERK in order to understand its regulation of gene expression. METHODS: The effects of tamoxifen (TAM) on the activation of serine/threonine mitogen-activated protein kinase (MAPK, p42/ERK2) and the stress-activated protein kinases (p46 SAPK or c-Jun N-terminal kinase, JNK1) were evaluated using a human cervical epitheloid carcinoma HeLa cell line. RESULTS: TAM activated both JNK1 and ERK2 activities in a time- and dose-dependent manner in HeLa cells. The activation of JNK1 was enhanced when the cells were pretreated with prooxidant H2O2. CONCLUSIONS: These studies show that TAM activates the signal transduction kinases, JNK1 and ERK2, which may play important roles in the regulation of gene expression by TAM.     

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.     

Activation of mitogen-activated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury

Smooth muscle cell proliferation is a key event in neointimal formation after balloon angioplasty. The molecular signals that mediate this process have yet to be identified. Mitogen-activated protein (MAP) kinases are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate whether the signal transduction pathways of MAP kinases were involved in the development of restenosis in the injured arteries. Rat carotid arteries were isolated at various time points after balloon injury, and activities of MAP kinases, including extracellular signal-regulated kinases (ERK), and stress activated protein kinases (SAPK)/c-Jun N-terminal protein kinases (JNK), were determined in protein extracts of the vasculature using protein kinase assay and Western blot analysis. After balloon angioplasty, ERK2 and JNK1 activities in the vessel wall increased rapidly, reached a high level in 5 minutes and maintained for 1 hour. A sustained increase in ERK2 kinase activity was observed over the next 7 days in the arterial wall and 14 days in neointima after injury. In contrast, opposite and uninjured arteries did not show significant changes in these kinase activities. Concomitantly, Western blot analysis confirmed that the ERK2 kinase in the injured vessels was indeed activated or phosphorylated, showing a slowly migrating species of a 42-kDa protein containing phosphorylated tyrosine. Kinase activation is followed by an increase in c-fos and c-jun gene expression and enhanced activator protein 1 (AP-1) DNA-binding activity. Thus, balloon injury rapidly activates the MAP kinases in rat carotid arteries. These kinase activations may be crucial in mediating smooth muscle cell proliferation in response to vascular angioplasty.     

Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals

T cells from elderly humans often display impaired IL-2 production, but the mechanisms are unknown. Because the activities of extracellular signal-regulated kinases (ERK) and c-Jun NH2-terminal kinases (JNK) are important for IL-2 production, the current study evaluated if aberrancies in the expression and activation of ERK2 or JNK might underlie decreased IL-2 production by human T cells during aging. The present results show that diminished ERK2 and JNK catalytic activities were commonly detected in T cells from elderly humans stimulated with anti-CD3 mAb OKT3 plus PMA. These reductions did not represent temporal shifts in activation or altered expression of ERK2 or JNK. In addition, the reductions of ERK2 activation in stimulated T cells from elderly individuals were accompanied by decreased Raf-1 kinase activation and could be observed without coexisting impairments in JNK activation. Stimulation of ERK2 activation in elderly T cells correlated with IL-2 production and decreased ERK2 activation was consistently associated with reduced IL-2 production. Although the age-related decreases in JNK activation were accompanied by reduced IL-2 production, substantial impairments of JNK activation were observed with diminished ERK2 activation. Moreover, anti-CD3/PMA-stimulated T cells from elderly individuals that displayed normal JNK activation and impaired ERK2 activation continued to demonstrate reduced IL-2 production. These findings show that impairments in the activation of ERK2 and JNK can accompany decreased IL-2 production by T cells from elderly humans and further suggest that aberrancies in TCR/CD3-dependent activation of the Raf-1/MEK/ERK2 cascade may be rate-limiting for the full induction of IL-2.