Differential regulation of IGF-II-induced IL-8 by extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases in human keratinocytes

In order to study the relationship between insulin like growth factor-II (IGF-II) and interleukin-8 (IL-8) that are upregulated in psoriasis, we monitored IL-8 expression in IGF-II-treated human keratinocytes and explored the signaling pathways of IL-8 expression by IGF-II. IGF-II increased the IL-8 mRNA and protein levels in human keratinocytes. The upregulation of IL-8 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src, PI3-kinase, and ERK, but not by p38. Furthermore, IGF-II remarkably increased the DNA binding activities of NF-kappaB and AP-1, and the IL-8 promoter activity. However, cotransfection with IkappaB mutant blocked the IGF-II-induced IL-8 promoter activity. In addition, cotransfection with dominant negative MEK1 mutant, but not with dominant negative p38 mutant, blocked the IGF-II-induced IL-8 promoter activity. These results suggest that IGF-II is involved in the pathogenesis of psoriasis by inducing IL-8 gene expression through the tyrosine kinase-Src-ERK1/2-AP-1 pathway, and the PI3-kinase and NF-kappaB pathway.     

Farrerol maintains the contractile phenotype of VSMCs via inactivating the extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase signaling

Molecular and Cellular Biochemistry - Farrerol, a dihydroflavone isolated from Rhododendron dauricum L., can inhibit vascular smooth muscle cell (VSMC) proliferation and exert a protective effect...     

Induction of apoptosis in starfish eggs requires spontaneous inactivation of MAPK (extracellular signal-regulated kinase) followed by activation of p38MAPK

Mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase) prevents DNA replication and parthenogenesis in maturing oocytes. After the meiotic cell cycle in starfish eggs, MAPK activity is maintained until fertilization. When eggs are fertilized, inactivation of MAPK occurs, allowing development to proceed. Without fertilization, highly synchronous apoptosis of starfish eggs starts 10 h after germinal vesicle breakdown, which varies according to season and individual animals. For induction of the apoptosis, MAPK should be activated for a definite period, called the MAPK-dependent period, during which eggs develop competence to die, although the exact duration of the period was unclear. In this study, we show that the duration of the MAPK-dependent period was approximately 8 h. Membrane blebbing occurred approximately 2 h after the MAPK-dependent period. Surprisingly, when MAPK was inhibited by U0126 after the MAPK-dependent period, activation of caspase-3 occurred earlier than in the control eggs. Thus, inactivation of MAPK is a prerequisite for apoptosis. Also, even in the absence of the inhibitor, MAPK was inactivated spontaneously when eggs began to bleb, indicating that inactivation of MAPK after the MAPK-dependent period acts upstream of caspase-3. Inactivation of MAPK also resulted in the activation of p38MAPK, which may contribute to apoptotic body formation.     

Eicosanoid activation of extracellular signal-regulated kinase1/2 in human epidermoid carcinoma cells

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase Cgamma1 and activity of protein kinase Calpha (PKCalpha). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nm) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.     

Role of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways in regulating replication of Penicillium marneffei in human macrophages

Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To elucidate the mechanisms involved, we investigated the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) pathways in cytokine expression, phagosome–lysosome fusion and replication of P. marneffei in P. marneffei-infected human macrophages. Analysis of both ERK1/2 and p38 showed rapid phosphorylation in response to P. marneffei. Using specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that ERK1/2 and p38 were essential for P. marneffei-induced tumor necrosis factor-α production, whereas p38, but not that of ERK, was essential for IL-10 production. Furthermore, the presence of PD98059 always decreased phagosomal acidification and maturation and increased intracellular multiplication of P. marneffei, whereas the use of SB203580 always increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that a proper balance of between ERK1/2 and p38 may play an important role in controlling the replication of P. marneffei. Our findings further indicate a novel therapeutic avenue for treating P. marneffei by stimulating ERK1/2 or activating ERK1/2-dependent mechanisms.     

The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils

Phosphorylation of p47 phagocyte oxidase, (p47(phox)), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47(phox) is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47(phox) phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47(phox). However, PD98059 weakly affected PMA-induced p47(phox) phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47(phox) induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47(phox) phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47(phox) phosphorylation either directly or indirectly via other kinases. In PMA-induced p47(phox) phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47(phox) phosphorylation. However, in fMLP-induced p47(phox) phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47(phox) when tested alone, and exerted additive inhibitory effects on p47(phox) phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47(phox). Furthermore, they strongly suggest that p47(phox) is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.     

Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility

We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.     

Visfatin promotes angiogenesis by activation of extracellular signal-regulated kinase 1/2

Adipose tissue is highly vascularized and requires the angiogenic properties for its mass growth. Visfatin has been recently characterized as a novel adipokine, which is preferentially produced by adipose tissue. In this study, we report that visfatin potently stimulates in vivo neovascularization in chick chorioallantoic membrane and mouse Matrigel plug. We also demonstrate that visfatin activates migration, invasion, and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, visfatin evokes activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) in endothelial cells, which is closely linked to angiogenesis. Inhibition of ERK activation markedly decreases visfatin-induced tube formation of HUVECs and visfatin-stimulated endothelial cell sprouting from rat aortic rings. Taken together, these results demonstrate that visfatin promotes angiogenesis via activation of mitogen-activated protein kinase ERK-dependent pathway and suggest that visfatin may play important roles in various pathophysiological angiogenesis including adipose tissue angiogenesis.     

ICAM-1-induced expression of proinflammatory cytokines in astrocytes: involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways

ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer's disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1beta and IL-1alpha protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1alpha and IL-1beta expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.     

Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils

Summary The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca²⁺ ([Ca ) stimulated by PAF and fMLP were due to Ca²⁺ influx and internal Ca²⁺ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca²⁺ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC- isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A₂ (PLA₂) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA₂, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC- isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.     

Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin

We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes for alpha-tropomyosin. Therefore, we evaluated the mechanism by which Ras causes the downregulation of tropomyosin expression. By using RIE-1 cells that harbor inducible expression of activated H-Ras(12V), we determined that the downregulation of tropomyosin expression correlated with the onset of morphological transformation. We found that the reversal of Ras transformation caused by inhibition of extracellular signal-regulated kinase activation corresponded to a restoration of tropomyosin expression. Inhibition of p38 activity in Raf-expressing RIE-1 cells caused both morphological transformation and loss of tropomyosin expression. Thus, a reduction in tropomyosin expression correlated strictly with morphological transformation of RIE-1 cells. However, forced overexpression of tropomyosin in Ras-transformed cells did not reverse morphological or growth transformation, a finding consistent with the possibility that multiple changes in gene expression contribute to Ras transformation. We also determined that tropomyosin expression was low in two human tumor cell lines, DLD-1 and HT1080, that harbor endogenous mutated alleles of ras, but high in transformation-impaired, derivative cell lines in which the mutant ras allele has been genetically deleted. Finally, treatment with azadeoxycytidine restored tropomyosin expression in Ras-transformed RIE-1, HT1080, and DLD-1 cells, suggesting a role for DNA methylation in downregulating tropomyosin expression.     

Rac GTPase activity is essential for lipopolysaccharide signaling to extracellular signal-regulated kinase and p38 MAP kinase activation in rat-2 fibroblasts

Lipopolysaccharide (LPS) has potent proinflammatory properties by acting on many cell types. Recently, mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38 kinase, and c-jun N-terminal kinase (JNK) were shown to be involved in signal transduction in response to LPS. However, the detailed mechanism of LPS-induced signaling in the cell, especially the role of the Rho family GTPases remains largely unknown. In the present study, we investigated the role of Rac1, a member of the Rho family GTPases, in the LPS-induced MAPKs activation in Rat-2 fibroblasts. Our results showed that LPS induced the activation of ERK and p38 MAP kinase in a Rac-dependent manner, suggesting a mediatory role of Rac1 in LPS signaling to MAPKs stimulation. We also observed that LPS caused a time-dependent activation of Rac1. In addition, our results have shown that pretreatment with herbimycin or wortmannin dramatically inhibited Rac1 activation induced by LPS. These suggest that tyrosine kinase(s) and phosphatidylinositol 3-kinase (PI 3-kinase) are possibly acting upstream of Rac1 in the LPS signaling to MAPKs.     

Involvement of protein kinase A and A kinase anchoring protein in the progesterone-initiated human sperm acrosome reaction

The signal transduction pathways involved in the progesterone (P(4))-initiated mammalian sperm acrosome reaction (AR) are not fully understood. To investigate the role of the protein kinase A (PKA) pathway in the P(4)-initiated AR, we probed this pathway by pretreating capacitated human sperm with reagents designed to either inhibit PKA activation or disrupt PKA/A kinase anchoring protein (AKAP) interactions. Preincubation with the stearated (membrane permeable) PKA inhibitor, PKI alpha 5-24 (S-PKI alpha 5-24), significantly inhibited the P(4)-initiated AR at 10 microM as compared to stearated control peptide. In contrast, preincubation with 100 microM nonstearated PKI alpha 5-24 did not significantly inhibit versus solvent control. Preincubation with the PKA inhibitor Rp-8-Br-cAMP at 500 microM and 150 microM significantly inhibited the P(4)-initiated AR versus 8-Br-cAMP and versus solvent. Preincubation with the anchoring inhibitory peptide S-Ht-31 significantly stimulated the P(4)-initiated AR at 10, 3, and 1 microM versus inactive control peptide. The stimulation of the P(4)-initiated AR by 3 microM S-Ht31 was significantly inhibited by the addition of 30 microM S-PKI alpha 5-24 prior to the addition of S-Ht31. Preincubation with S-PKI alpha 5-24 (30 microM) partially inhibited the ionomycin (50 microM)-initiated AR. A role for PKA in the P(4)-initiated AR may exist both upstream and downstream of Ca(2+) entry. Our studies present the first evidence for the participation of PKA in the P(4)-initiated AR and also suggest that AKAPs are involved in the PKA-mediated events.     

Activation of extracellular signal-regulated kinase 1/2 inhibits type I collagen expression by human skin fibroblasts

Treatment with the lipid second messenger, ceramide, activates extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase, and p38 in human skin fibroblasts and induces their collagenase-1 expression (Reunanen, N., Westermarck, J., H?kkinen, L., Holmstr?m, T. H., Elo, I., Eriksson, J. E., and K?h?ri, V.-M. (1998) J. Biol. Chem. 273, 5137-5145). Here we show that C(2)-ceramide inhibits expression of type I and III collagen mRNAs in dermal fibroblasts, suppresses proalpha2(I) collagen promoter activity, and reduces stability of type I collagen mRNAs. The down-regulatory effect of C(2)-ceramide on type I collagen mRNA levels was abrogated by protein kinase C inhibitors H7, staurosporine, and Ro-31-8220 and potently inhibited by a combination of MEK1,2 inhibitor PD98059 and p38 inhibitor SB203580. Activation of ERK1/2 by adenovirus-mediated expression of constitutively active MEK1 resulted in marked down-regulation of type I collagen mRNA levels and production in fibroblasts, whereas activation of p38 by constitutively active MAPK kinase-3b and MAPK kinase-6b slightly up-regulated type I collagen expression. These results identify the ERK1/2 signaling cascade as a potent negative regulatory pathway with respect to type I collagen expression in fibroblasts, suggesting that it mediates inhibition of collagen production in response to mitogenic stimulation and transformation.