Paradoxical activation of Raf by a novel Raf inhibitor.

BACKGROUND: Raf is a proto-oncogene that is activated in response to growth factors or phorbol esters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade. RESULTS: The compound ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro. Paradoxically, exposure of cells to ZM 336372 induces > 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKK1 or p42 MAPK/ERK2. The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras and is not prevented by inhibition of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase. ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or p42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only other protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38. Although ZM 336372 is structurally unrelated to SB 203580, a potent inhibitor of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SB 203580. CONCLUSIONS: Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. These observations imply that some agonists reported to trigger the cellular activation of c-Raf might actually be inhibitors of this enzyme, and that compounds which inhibit the kinase activity of Raf might not be useful as anticancer drugs. The binding sites for ZM 336372 and SB 203580 on Raf and SAPK2/p38 are likely to overlap.     

Rottlerin enhances IL-1β-induced COX-2 expression through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells

Cyclooxygenase-2 (COX-2) is an important enzyme in inflammation. In this study, we investigated the underlying molecular mechanism of the synergistic effect of rottlerin on interleukin1β (IL-1β)-induced COX-2 expression in MDA-MB-231 human breast cancer cell line. Treatment with rottlerin enhanced IL-1β-induced COX-2 expression at both the protein and mRNA levels. Combined treatment with rottlerin and IL-1β significantly induced COX-2 expression, at least in part, through the enhancement of COX-2 mRNA stability. In addition, rottlerin and IL-1β treatment drove sustained activation of p38 Mitogen-activated protein kinase (MAPK), which is involved in induced COX-2 expression. Also, a pharmacological inhibitor of p38 MAPK (SB 203580) and transient transfection with inactive p38 MAPK inhibited rottlerin and IL-1β-induced COX-2 upregulation. However, suppression of protein kinase C δ (PKC δ) expression by siRNA or overexpression of dominant-negative PKC δ (DN-PKC-δ) did not abrogate the rottlerin plus IL-1β-induced COX-2 expression. Furthermore, rottlerin also enhanced tumor necrosis factor-α (TNF-α), phorbol myristate acetate (PMA), and lipopolysaccharide (LPS)-induced COX-2 expression. Taken together, our results suggest that rottlerin causes IL-1β-induced COX-2 upregulation through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells.     

Interleukin-1beta stimulates matrix metalloproteinase-2 expression via a prostaglandin E2-dependent mechanism in human chondrocytes

IL-1beta is known promote cyclooxygenase-2 (COX- 2) and matrix metalloproteinase-2 (MMP-2) expression. This study focuses on the characterization of the signaling cascade associated with IL-1beta-induced matrix metalloproteinase-2 (MMP-2) regulation in human chondrocytes. The decrease in collagen levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that IL-1beta promotes the proteolytic process leading to MMP-2 activation. IL-1beta-related MMP-2 expression was found to be dependent on prostaglandin E2 (PGE2) production. In addition, the induction of COX-2 and MMP-2 was inhibited by the pretreatment of chondrocytes with a SB203580 or Ro 31-8220, indicating the involvement of protein kinase C (PKC) or p38 mitogen-activated protein kinase (MAPK). However, there is no cross-talk between PKC and p38 MAPK in the IL-1beta-induced MMP-2 activation. Taken together, these results demonstrated that IL-1beta induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.     

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ialpha. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.     

Role of p38 mitogen-activated protein kinase and caspases in UV-B-induced apoptosis of murine peritoneal macrophages

The mechanisms of ultraviolet-B (UV-B)-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in murine peritoneal macrophages. Exposure of murine peritoneal macrophages to UV-B irradiation induced rapid apoptosis concurrent with DNA fragmentation and activation of caspase-3 but did not activate caspase-1. UV-B irradiation (100 mJ/cm2) also induced expression of phospho-p38 and -c-Jun N-terminal kinase (JNK) MAPK; however, no significant expression of phospho-p42/44 was observed 120 min after exposure. Pretreatment of macrophages with a p38 MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190), and a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-CHO, suppressed UV-B irradiation-induced apoptosis as observed by DNA laddering and DNA fragmentation estimation quantitatively. Pretreatment with caspase-1 inhibitor, N-acetyl-Tyr-Val-Ala-Asp-CHO, had no effect. UV-B-induced caspase-3 activation resulted in the cleavage of poly-(ADP-ribose) polymerase (PARP), which was inhibited by the caspase-3 inhibitor. SB202190 pretreatment also prevented activation of caspase-3 and the cleavage of PARP. However, the caspase-3 and -1 inhibitors did not affect UV-B-induced expression of phospho-p38 and -JNK. These results suggest that activation of p38 MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UV-B irradiation.     

Catechins inhibit angiotensin II-induced vascular smooth muscle cell proliferation via mitogen-activated protein kinase pathway

Catechins, components of green tea, reduce the incidence of cardiovascular diseases such as atherosclerosis. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMC), resulting in atherosclerosis. The acting mechanisms of the catechins remain to be defined in the proliferation of VSMC induced by Ang II. Here we report that catechin, epicatechin (EC), epicatechingallate (ECG) or epigallocatechingallate (EGCG) significantly inhibits the Ang II-induced [3H]thymidine incorporation into the primary cultured rat aortic VSMC. Ang II increases the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2), c-jun-N-terminal kinase 1/2 (JNK 1/2), or p38 mitogen-activated protein kinases (MAPKs) and mRNA expression of c-jun and c-fos. The EGCG pretreatment inhibits the Ang II-induced phosphorylation of ERK 1/2, JNK 1/2, or p38 MAPK, and the expression of c-jun or c-fos mRNA. U0126, a MEK inhibitor, SP600125, a JNK inhibitor, or SB203580, a p38 inhibitor, attenuates the Ang II-induced [3H]thymidine incorporation into the VSMC. In conclusion, catechins inhibit the Ang II-stimulated VSMC proliferation via the inhibition of the Ang II-stimulated activation of MAPK and activator protein-1 signaling pathways. The antiproliferative effect of catechins may be associated with the reduced risk of cardiovascular diseases by the intake of green tea. Catechins may be useful in the development of prevention and therapeutics of vascular diseases.     

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.     

Prunus yedoensis Matsum. stimulates glucose uptake in L6 rat skeletal muscle cells by activating AMP-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways

Prunus yedoensis Matsum. is used as a medicinal plant to alleviate symptoms of diabetes; however, the molecular mechanism underlying its antihyperglycaemic activity is unknown. In this study, we investigated the antihyperglycaemic effects of P. yedoensis and its molecular mechanism. Prunus yedoensis leaf extract (PLE) increased the glucose uptake of phosphorylatinginsulin receptor substrate (IRS)-1, 3'-phosphoinositide-dependent kinase (PDK)-1 and Akt PLE, and also increased the phosphorylation of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK). PLE-stimulated glucose uptake was blocked by an AMPK inhibitor (Compound C) and a p38 MAPK inhibitor (SB203580). Inhibition of AMPK activity reduced p38 MAPK phosphorylation, whereas the inhibition of p38 MAPK activity did not affect AMPK phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 and Compound C reduced PLE-stimulated glucose uptake. Our results demonstrate that PLE stimulated glucose uptake by activating both insulin signalling and AMPK-p38 MAPK pathways. PLE shows potential as a natural antihyperglycaemic agent.     

Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution

Background: Specific inhibitors of protein kinases have great therapeutic potential, but the molecular basis underlying their specificity is only poorly understood. We have investigated the drug SB 203580 which belongs to a class of pyridinyl imidazoles that inhibits the stress-activated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38β2 but not other mitogen-activated protein kinase family members. Like inhibitors of other protein kinases, SB 203580 binds in the ATP-binding pocket of SAPK2a/p38. Results: The SAP kinases SAPK1γ/JNK1, SAPK3 and SAPK4 are not inhibited by SB 203580, because they have methionine in the position equivalent to Thr106 in the ATP-binding region of SAPK2a/p38 and SAPK2b/p38β2. Using site-directed mutagenesis of five SAP kinases and the type I and type II TGFβ receptors, we have established that for a protein kinase to be inhibited by SB 203580, the sidechain of this residue must be no larger than that of threonine. Sensitivity to inhibition by SB 203580 is greatly enhanced when the sidechain is even smaller, as in serine, alanine or glycine. Thus, the type I TGFβ receptor, which has serine at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38β2, is inhibited by SIB 203580. Conclusions: These findings explain how drugs that target the ATP-binding site can inhibit protein kinases specifically, and show that the presence of threonine or a smaller amino acid at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38β2 is diagnostic of whether a protein kinase is sensitive to the pyridinyl imidazole class of inhibitor.     

Regulation of hepatocyte growth factor-mediated urokinase plasminogen activator secretion by MEK/ERK activation in human stomach cancer cell lines

The regulatory mechanisms for the proliferation and the particular invasive phenotypes of stomach cancers are not still fully understood. Up-regulations of hepatocytes growth factor (HGF), its receptor (c-Met), and urokinase-type plasminogen activator (uPA) are correlated with the development and metastasis of cancers. In order to investigate roles of HGF/c-Met signaling in tumor progression and metastasis in stomach cancers, we determined effects of a specific MEK1 inhibitor (PD098059) and a p38 kinase inhibitor (SB203580) on HGF-mediated cell proliferation and uPA expression in stomach cancer cell lines (NUGC-3 and MKN-28). HGF treatment induced the phosphorylations of ERK and p38 kinase in time- and dose- dependent manners. Pre-treatment with PD098059 reduced HGF-mediated cell proliferation and uPA secretion. In contrast, SB203580 pre-treatment enhanced cell proliferation and uPA secretion due to induction of ERK phosphorylation. Stable expression of dominant negative-MEK1 in NUGC-3 cells showed a decrease in HGF-mediated uPA secretion. These results suggest that interaction of a MEK/ERK and a p38 kinase might play an important role in proliferation and invasiveness of stomach cancer cells.     

p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.     

PKCalpha induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKCa isoform. When PKCalpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKCalpha kinase active mutant (PKCalpha- CAT) in the undifferentiated keratinocyte, but not PKCbeta-CAT, also increased differentiation marker expressions. On the other hand, PKCalpha dominant negative mutant (PKCbeta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKCbeta-KR did not, suggesting that PKCalpha is responsible for keratinocyte differentiation. When downstream pathway of PKCalpha in Ca2+ -mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+ -mediated differentiation, and that only ERK1/2 pathway is specific for PKCalpha-mediated differentiation in mouse keratinocytes.     

The p38 mitogen-activated protein kinase is involved in stress-induced phospholipase D activation in vascular smooth muscle cells

Oxidative stress has been implicated in mediation of vascular disorders. Earlier study showed that the exposure of vascular smooth muscle cells (VSMC) to pervanadate (hydrogen peroxide plus orthovanadate) resulted in the accumulation of [3H]phosphatidylbutanol. In this study, the effect of pervanadate on the activation of p38 mitogen-activated protein kinase (p38 MAPK) was studied in the VSMC. Pervanadate treatment activated p38 MAPK in a dose-and time-dependent manner. Interestingly, specific inhibition of p38 MAPK with SB203580 attenuated pervanadate-induced PLD activation. This correlates with the finding that expression of dominant negative mutants of MKK3/6 inhibited the PLD activation. SB203580 pretreatment also inhibited other cellular stressors (i.e. high osmolarity and UV light)-induced PLD activation. The possible correlationship of p38 MAPK activation with PKC was examined since PKC is reported to be involved in the pervanadate-induced PLD activation. Calphostin C, a PKC inhibitor, suppressed pervanadate-induced p38 MAPK and PLD activation in a dose-dependent manner. These results suggest that PKC-p38 MAPK may represent an upstream pathway of PLD in the signal transduction of cellular stress.     

Thromboxane A2 modulates migration, proliferation, and differentiation of adipose tissue-derived mesenchymal stem cells

Prostanoid metabolites are key mediators in inflammatory responses, and accumulating evidence suggests that mesenchymal stem cells (MSCs) can be recruited to injured or inflamed tissues. In the present study, we investigated whether prostanoid metabolites can regulate migration, proliferation, and differentiation potentials of MSCs. We demonstrated herein that the stable thromboxane A₂ (TxA₂) mimetic U46619 strongly stimulated migration and proliferation of human adipose tissue-derived MSCs (hADSCs). Furthermore, U46619 treatment increased expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, in hADSCs, suggesting differentiation of hADSCs into smooth muscle-like cells. U46619 activated ERK and p38 MAPK, and pretreatment of the cells with the MEK inhibitor U0126 or the p38 MAPK inhibitor SB202190 abrogated the U46619-induced migration, proliferation, and α-SMA expression. These results suggest that TxA₂ plays a key role in the migration, proliferation, and differentiation of hADSCs into smooth muscle-like cells through signaling mechanisms involving ERK and p38 MAPK.     

Involvement of MAPK pathway in hypoxia-induced up-regulation of urokinase plasminogen activator receptor in a human prostatic cancer cell line, PC3MLN4

Clinical studies have shown that tumor hypoxia is associated with invasive growth and metastasis and may be an important prognostic factor adversely influencing survival in patients with tumors. To investigate the mechanisms involved in hypoxia-induced invasive growth and metastasis, hypoxia-mediated urokinase plasmalogen activator receptor (uPAR) expression, cellular invasiveness, and mitogen activated protein kinase (MAPK) activation were measured in a prostate cancer cell line, PC3MLN4. The levels of uPAR expression and cellular invasiveness were increased in hypoxic cells. Hypoxia-induced cellular invasiveness was blocked by an anti-uPAR monoclonal antibody. Phosphorylations of ERK and p38 kinases were also more extensive in hypoxic cells than in normoxic cells. Hypoxia-induced uPAR up-regulation was inhibited by pre-treatments with a specific inhibitor of MEK, PD98059 and a specific inhibitor of p38 MAP kinase, SB203580. Cell growth also increased in hypoxic cells. From these results, hypoxia increased tumor cell invasion by up-regulating uPAR expression, which might be mediated through ERK and p38 kinase signaling pathways in PC3MLN4 prostate cancer cell line.     

Oligonol inhibits UVB-induced COX-2 expression in HR-1 hairless mouse skin--AP-1 and C/EBP as potential upstream targets

Oxidative stress and inflammatory tissue damage are two major events frequently implicated in carcinogenesis. Numerous polyphenolic compounds derived from plants possess antioxidant and anti-inflammatory activities and are hence effective in preventing cancer. Oligonol is a polyphenol formulation enriched with catechin-type oligomers. As an initial approach to assess the chemopreventive potential of oligonol, we have determined its effects on inflammatory as well as oxidative damage in mouse skin irradiated with UVB. Topical application of oligonol onto the dorsal skin of male HR-1 hairless mice 30 min prior to UVB exposure diminished epidermal hyperplasia and formation of 4-hydroxynonenal, a biochemical hallmark of lipid peroxidation. Topical application of oligonol also significantly inhibited UVB-induced cyclooxygenase (COX-2) expression in mouse skin. Oligonol diminished the DNA binding of activator protein-1 (AP-1) and CCAAT/enhancer binding protein (C/EBP), and the expression of C/EBPdelta in mouse skin exposed to UVB. Our study also revealed that oligonol attenuated UVB-induced catalytic activity as well as expression of p38 mitogen-activated protein (MAP) kinase. Moreover, UVB-induced phosphorylation of another upstream kinase Akt was attenuated by oligonol. Taken together, oligonol showed antioxidative and anti-inflammatory effects in UVB-irradiated mouse skin by inhibiting COX-2 expression via blockade of the activation of AP-1 and C/EBP, and upstream kinases including p38 MAP kinase and Akt.