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.     

PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif

Protein kinases and phosphatases regulate the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by controlling the phosphorylation of specific residues. We report the physical and functional association of ERK1/2 with the PTP-SL and STEP protein tyrosine phosphatases (PTPs). Upon binding, the N-terminal domains of PTP-SL and STEP were phosphorylated by ERK1/2, whereas these PTPs dephosphorylated the regulatory phosphotyrosine residues of ERK1/2 and inactivated them. A sequence of 16 amino acids in PTP-SL was identified as being critical for ERK1/2 binding and termed kinase interaction motif (KIM) (residues 224-239); it was shown to be required for phosphorylation of PTP-SL by ERK1/2 at Thr253. Co-expression of ERK2 with catalytically active PTP-SL in COS-7 cells impaired the EGF-induced activation of ERK2, whereas a PTP-SL mutant, lacking PTP activity, increased the ERK2 response to EGF. This effect was dependent on the presence of the KIM on PTP-SL. Furthermore, ERK1/2 activity was downregulated in 3T3 cells stably expressing PTP-SL. Our findings demonstrate the existence of a conserved ERK1/2 interaction motif within the cytosolic non-catalytic domains of PTP-SL and STEP, which is required for the regulation of ERK1/2 activity and for phosphorylation of the PTPs by these kinases. Our findings suggest that PTP-SL and STEP act as physiological regulators of the ERK1/2 signaling pathway.     

Menadione (vitamin K3) enhances the mitogenic signal of epidermal growth factor via extracellular signal-regulated kinases

The effects of vitamin K3 (VK3) on DNA synthesis, cell proliferation and mitogen-activated protein kinase pathway were investigated in G0-arrested NIH 3T3 fibroblasts. VK3 (5 microM) alone stimulates DNA synthesis by 40% and moderately increases the mitogenic effects of EGF, which is preceded by a rapid phosphorylation of the extracellular signal-regulated kinases (ERKs). At 20 microM, VK3 had an antiproliferative effect. VK3 alone (5 and 50 microM) or in concert with EGF increases the activity of ERK2 (by 2.5 and 5 fold, respectively). Our studies demonstrate that the activation of ERKs by VK3 alone, or VK3 plus EGF can promote either stimulatory or inhibitory effects on the mitogenic signal.     

Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-sevenless 1 (mSos1)

A fragment consisting of residues 584-1071 of the mouse Son-of-sevenless 1 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1. Furthermore, we found that the CDC25H-domain fragment activated the extracellular signal-regulated protein kinases (ERKs), and was mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells. Then, we examined the roles of other mSos1 domains in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments. First, longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions. On the other hand, the preceding Pleckstrin-homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain. By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibited remarkably low ERK activities under serum-starved conditions. This autoinhibitory effect of the DH domain on the CDC25H-domain function was shown to be relieved when cells were stimulated with epidermal growth factor. The DH-domain extension affected neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain. Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli.     

Gene expression of matrix metalloproteinase-1 (interstitial collagenase) and matrix metalloproteinase-3 (stromelysin-1) in basal cell carcinoma by in situ hybridization using chondroitin ABC lyase

We studied the relation between the plasma concentration of brain natriuretic peptide (BNP) and echocardiographic findings to determine the sensitivity of BNP as an indicator of left-ventricular dysfunction in elderly patients with various cardiovascular diseases. The plasma concentration of BNP was positively correlated with left-ventricular end-diastolic and end-systolic dimensions (LVEDD and LVESD, respectively) and inversely correlated with the left-ventricular ejection fraction (LVEF) in patients with prior myocardial infarction, dilated cardiomyopathy, and valvular heart disease. The plasma concentration of BNP decreased significantly in association with an increase in LVEF and decreases in LVEDD and LVESD in patients with congestive heart failure following therapy. These observations indicate that the plasma concentration of BNP is a sensitive marker of impaired left-ventricular function in elderly patients with various cardiovascular diseases and may be useful for evaluating the improvement in left-ventricular function and the efficacy of therapy in patients with congestive heart failure.     

Disruption of fibronectin binding to the alpha 5 beta 1 integrin stimulates the expression of cyclin-dependent kinases and DNA synthesis through activation of extracellular signal-regulated kinase

The alpha 5 alpha 1 integrin, a fibronectin receptor, has been implicated in the control of cell growth and the regulation of gene expression. We report that disruption of ligation between alpha 5 alpha 1 and fibronectin by integrin alpha 5 subunit or fibronectin monoclonal antibodies stimulated DNA synthesis in growth-arrested FET human colon carcinoma cells. This stimulation only occurred when monoclonal antibody was added in the early G1 phase of the cell cycle after release from quiescence by fresh medium. Stimulation of DNA synthesis by alpha 5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed alpha 4 beta 1 integrin (another fibronectin receptor); however, addition of anti-human integrin alpha 4 monoclonal antibody had no effect on DNA synthesis. Treatment with alpha 5 monoclonal antibody led to a marked increase in the expression of CDK4 in G1 phase of the cell cycle and consequently increased the phosphorylation of retinoblastoma protein. alpha 5 monoclonal antibody treatment increased both cyclin A- and cyclin E-associated kinase activity which was accompanied by increased protein levels of CDK2 and cyclin A. Western blotting of immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin A complexes in cells treated with alpha 5 monoclonal antibody. Furthermore, disruption of alpha 5 alpha 1/fibronectin ligation activated mitogen-activated protein kinase p44 and p42 (extracellular signal-regulated kinase 1 and 2). Pretreatment of the cells with a specific inhibitor of MEK-1, PD98059, blocked the alpha 5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented alpha 5 monoclonal antibody-induced DNA synthesis. Since alpha 5 alpha 1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of alpha 5 alpha 1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.     

The effect of retinoic acid on the activation of the human H19 promoter by a 3'' downstream region

The purpose of this study was to identify risk factors in wound dehiscence and to determine which factors might be predictable. Forty patients with abdominal wound dehiscence were compared with 40 control patients standardized by sex and age. Hypoproteinemia, nausea/vomiting, fever, wound infection, abdominal distension, type of suture material, 2 or more abdominal drains, and the surgeon's experience were factors significantly associated with wound dehiscence. Emergency surgery, jaundice, ostomy, total parenteral nutrition, ascites, pulmonary morbidity, co-existence of disease, anemia, leucocytosis, and type of incision were nonsignificant variables. The number of patients with wound dehiscence increased with an increase in the number of risk factors, reaching 100% for patients with 8 risk factors. The risk factors of wound dehiscence can be predicted early and their number can be decreased before and after surgery by an experienced surgeon, leading to a lowered incidence of wound failure.     

Stimulation of the extracellular signal-regulated kinase 1/2 pathway by human beta-3 adrenergic receptor: new pharmacological profile and mechanism of activation

(-)Deprenyl is an irreversible inhibitor of monoamine oxidase B (MAO-B) frequently used as an adjunct therapy in the treatment of Parkinson's Disease. Recent evidence, however, has found that deprenyl's metabolites are associated with an antiapoptotic action within certain neuronal populations. Interestingly, deprenyl's antiapoptotic actions appear not to depend upon the inhibition of MAO-B. Due to a paucity of information surrounding (-)deprenyl's ability to spare neurons in vivo, a series of studies was conducted to further investigate this phenomenon within an apoptotic neuronal death model: kainic acid induced excitotoxicity. Results indicated that (-)deprenyl increased hippocampal neuronal survival compared to saline-matched controls following kainic acid insult. Furthermore, it was discovered that (-)deprenyl treatment could be stopped 14 days following CNS insult by kainate, with evidence of neuronal sparing still present by day 28. In open-field locomotor activity testing of kainate-treated animals, those given subsequent (-)deprenyl treatment showed habituation curves similar to control subjects, while saline-treated animals did not. Given deprenyl's antiapoptotic actions, it is proposed that (-)deprenyl may be beneficial in the treatment of a variety of neurodegenerative diseases where evidence of apoptosis exists, such as Parkinson's and Alzheimer's Disease, by slowing the disease process itself.     

Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor

The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.     

IFN-gamma induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression

The mechanism by which IFN-gamma inhibits tumor cell growth has not been fully understood. Here we report that IFN-gamma up-regulated the expression of Fas and Fas ligand (FasL) on HT29 cells, a human colon adenocarcinoma cell line, and subsequently induced apoptosis of these cells. The kinetics of cell death in IFN-gamma-treated HT29 cells paralleled the increase in the levels of Fas and FasL expression. We further show that IFN-gamma up-regulated the expression of Fas and FasL in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. Correspondingly, IFN-gamma induced cell death in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. IFN-gamma-induced cell death was inhibited by caspase-1 inhibitors. Our results suggest that cell growth inhibition by IFN-gamma is due to apoptosis mediated by Fas and FasL interaction.     

Protein kinase C, but not tyrosine kinases or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes

Angiotensin II (AngII) induces cardiac hypertrophy through activating a variety of protein kinases. In this study, to understand how cardiac hypertrophy develops, we examined AngII-evoked signal transduction pathways leading to the activation of extracellular signal-regulated protein kinases (ERKs), which are reportedly critical for the development of cardiac hypertrophy, in cultured cardiac myocytes isolated from neonatal rats. Inhibition of protein kinase C (PKC) with calphostin C or down-regulation of PKC by pretreatment with a phorbol ester for 24 h abolished AngII-induced activation of Raf-1 and ERKs, and addition of a phorbol ester conversely induced a marked increase in the activities of Raf-1 and ERKs. Pretreatment with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, genistein and tyrphostin, did not attenuate AngII-induced activation of ERKs. In contrast, genistein strongly blocked insulin-induced ERK activation in cardiac myocytes. Although pretreatment with manumycin, a Ras farnesyltransferase inhibitor, or overexpression of a dominant-negative mutant of Ras inhibited insulin-induced ERK activation, neither affected AngII-induced activation of ERKs. Overexpression of a dominant-negative mutant of Raf-1 completely suppressed ERK2 activation by AngII, endothelin-1, and insulin. These results suggest that PKC and Raf-1, but not tyrosine kinases or Ras, are critical for AngII-induced activation of ERKs in cardiac myocytes.     

Induction of Epstein-Barr virus early antigens by corticosteroids: inhibition by TPA and retinoic acid

Corticosteroids can induce the synthesis of EBV antigens in the Burkitt lymphoma line Daudi. As early as 12 h after application of the drug, an increase of EA-positive cells can be seen, the maximum induction being reached after 2 days. Nanogram amounts per ml of hormone are sufficient for measurable effects. Early antigen induction by corticosteroids does not require replication of viral DNA. Induction by corticosteroid differs from induction by other systems in two major respects: (1) it does not cooperate with other inducers, and (2) it is specifically inhibited by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Induction by corticosteroids, however, shares at least one retinoic acid-sensitive step with induction by chemicals such as TPA, 5-iodo-2-deoxyuridine (IdUrd), n-butyric acid (n-BA) or inducing serum factor. This study defines three qualitatively different effects of TPA in Daudi cells: an inhibitory effect on EBV induction by corticosteroids and two differential types of synergistic effects with serum factor or n-BA, respectively. In this particular cell line, TPA exhibits no inducing capacity when applied alone.     

Extracellular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain: regional distribution and regulation by chronic morphine

Quantitative blot immunolabeling techniques were used to determine the concentrations of ERK1 (M(r) 44 kDa) and ERK2 (M(r) 42 kDa), the two major extracellular signal-regulated protein kinases, in different regions of rat brain. The aggregate ERK concentrations (ERK1 and ERK2) were relatively high in each of the brain regions studied, ranging from approximately 0.35 ng/microgram protein in cerebellum to approximately 1.2 ng/microgram protein in nucleus accumbens. However, differences in the regional distributions of ERK1 and ERK2 resulted in ratios of their relative abundance that differed by close to 10-fold among the regions studied. The ratios of ERK1 protein to ERK2 protein varied along a rostral-caudal gradient from a low of 0.16 in frontal cortex to a high of 1.5 in pons/medulla. In hypotonic homogenates from regions at either extreme of the gradient, ERK1 and ERK2 were both found to be predominantly (> 80%) soluble. In subcellular fractions prepared from sucrose homogenates of frontal cortex and pons/medulla, both ERK1 and ERK2 were enriched in the synaptosomal and cytosolic fractions, whereas ERK2 was also enriched in the microsomal fraction. By contrast, in subfractions containing purified nuclei, levels of ERK1 and ERK2 were about one-third of those seen in homogenates and, in subfractions enriched in mitochondria, both ERK1 and ERK2 were barely detectable. The catalytic activity of the ERKs paralleled their protein levels in all of the brain regions except the hippocampus, in which the activity and phosphotyrosine content were disproportionately high. As a possible explanation for this apparent disparity, the regional distribution of ERK kinase (MEK), which phosphorylates and activates the ERKs, was also investigated. The levels of immunoreactivity of the M(r) 45 kDa ERK kinase band differed by about threefold among the brain regions, with the highest levels being present in nucleus accumbens, hippocampus, substantia nigra, and caudate/putamen. Therefore, a higher concentration of ERK kinase immunoreactivity did not appear to account for the disproportionate levels of ERK activity and phosphotyrosine content in the hippocampus. Potential regulation of ERK and ERK kinase levels was also investigated in rats subjected to chronic morphine treatment. ERK1 and ERK2 levels were increased selectively in locus coeruleus and caudate/putamen after chronic morphine treatment, whereas ERK kinase immunoreactivity remained unchanged in all of the brain regions analyzed. In summary, the regional differences in ERK and ERK kinase expression and the region-specific regulation of ERK expression suggest that ERK-related signaling may play an important role in CNS function and its adaptive responses.     

Retinoic acid induced mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase-dependent MAP kinase activation needed to elicit HL-60 cell differentiation and growth arrest

Retinoic acid (RA) activated the extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein kinase (MAPK) of HL-60 human myeloblastic leukemia cells before causing myeloid differentiation and cell cycle arrest associated with hypophosphorylation of the retinoblastoma (RB) tumor suppressor protein. ERK2 activation by mitogen-activated protein/ERK kinase (MEK) was necessary for RA-induced differentiation in studies using PD98059 to block MEK phosphorylation. G0 growth arrest and RB tumor suppressor protein hypophosphorylation (which is typically associated with induced differentiation and G0 arrest), two putatively RB-regulated processes, also depended on ERK2 activation by MEK. Activation of ERK2 by RA occurred within hours and persisted until the onset of RB hypophosphorylation, differentiation, and arrest. ERK2 activation was probably needed early, because delaying the addition of PD98059 relative to that of RA restored most of the RA-induced cellular response. In contrast to RA (which activates RA receptors (RARs) and retinoid X receptors in HL-60 cells with its metabolite retinoids), a retinoid that selectively binds RAR-gamma, which is not expressed in HL-60 cells, was relatively ineffective in causing ERK2 activation. This is consistent with the need for a nuclear retinoid receptor function in RA-induced ERK2 activation. RA reduced the amount of unphosphorylated RAR-alpha, whose activation is necessary for RA-induced differentiation and arrest. This shifted the ratio of phosphorylated:unphosphorylated RAR-alpha to predominantly the phosphorylated form. Unlike other steroid thyroid hormone receptors susceptible to phosphorylation and activation by MAPKs, RAR-alpha was not phosphorylated by the activated ERK2 MAPK. The results thus show that RA augments MEK-dependent ERK2 activation that is needed for subsequent RB hypophosphorylation, cell differentiation, and G0 arrest. The process seems to be nuclear receptor dependent and an early seminal component of RA signaling causing differentiation and growth arrest.     

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.