Ethanol modulates the growth of human breast cancer cells in vitro

The role of ethanol or its metabolites on breast neoplasm has not been characterized. We hypothesized that ethanol may alter the growth rate of human breast tumor epithelial cells by modulating putative growth-promoting signaling pathways such as p44/42 mitogen-activated protein kinases (MAPKs). The MCF-7 cell line, considered a suitable model, was used in these studies to investigate the effects of ethanol on [(3)H]thymidine incorporation, cell number, and p44/42 MAPK activities in the presence or absence of a MAPK or extracellular signal-regulated kinase ERK-1, and (MEK1) inhibitor (PD098059). Treatment of MCF-7 cells with a physiologically relevant concentration of ethanol (0.3% or 65 mM) increased p44/42 activities by an average of 400% (P < 0.02), and subsequent cell growth by 200% (P < 0.05) in a MEK1 inhibitor (PD098059)-sensitive fashion, thus suggesting that the Ras/MEK/MAPK signaling pathways are crucial for ethanol-induced MCF-7 cell growth.     

Stimulation of mitogen activated protein kinase by LDL and oxLDL in human U-937 macrophage-like cells

Mitogen activated protein kinase in extracts of U-937 macrophage-like cells was stimulated by LDL and oxLDL. A maximum value (161% of the basal phosphotransferase activity) was obtained after 6 min exposure to oxidized LDL (27 μ/ml) using APRTPGGRR peptide substrate. The activatory effect was more pronounced (LDL 181%, oxLDL 201%) when MAPK of stimulated cells was immunoprecipitated with anti-p42^MAPK antibodies and phosphotransferase activity was assayed in immune complexes. Stimulation produced by oxLDL was inhibited by poly I, fucoidan, dextran sulfate and by the MAPKK inhibitor PD 098059 but not by PMA-mediated depletion of PKC or by pre-treatment with chloroquine or with pertussis toxin. These results suggest a direct mitogenic effect of LDL which, in the case of oxLDL, is dependent on scavenger receptor ligation but not on G-protein mediated or PKC-dependent signal transduction.     

Cancer stem cells in solid tumors: elusive or illusive?

Background

The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.

Results

We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44^MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44^MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44^MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.

Conclusions

These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.     

Hepatocyte growth factor/scatter factor stimulates migration of rat mammary fibroblasts through both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways.

Hepatocyte growth factor/scatter factor (HGF/SF) is considered to be a mesenchymal-derived factor that acts via a dual system receptor, consisting of the MET receptor and proteoglycans present on adjacent epithelial cells. Surprisingly, HGS/SF stimulated the migration of rat mammary (Rama) 27 fibroblasts, although it failed to stimulate their proliferation. HGF/SF stimulated a transient activation of mitogen-activated protein kinases p44 and p42 (p42/44(MAPK)), with a maximum level of dual phosphorylation of p42/44(MAPK) occurring 10-15 min after the addition of the growth factor, which was followed by a rapid decrease to near basal levels after 20 min. Interestingly, a second phase of p42/44(MAPK) dual phosphorylation was observed at later times (3 h to 10 h). PD098059, a specific inhibitor of MEK-1, prevented the dual phosphorylation of p42/44(MAPK) and also the phosphorylation of p90(RSK) (ribosomal subunit S6 kinase), which mirrored the kinetics of p42/44(MAPK) phosphorylation. Moreover, PD098059 prevented the HGF/SF-induced migration of Rama 27 cells. HGF/SF also induced an early increase in the phosphorylation of protein kinase B/Akt. Akt phosphorylation was elevated 15 min after the addition of HGF/SF and then declined to basal levels by 30 min. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), prevented the increase in Akt phosphorylation and abolished HGF/SF-induced migration of fibroblasts. PD098059 also inhibited the stimulation of Akt phosphorylation by HGF/SF and wortmannin similarly inhibited the stimulation of p42/44(MAPK) dual phosphorylation. These results suggest that HGF/SF-induced motility depends on both the transient dual phosphorylation of p42/44(MAPK) and the activation of PtdIns3K in Rama 27 fibroblasts and that these pathways are mutually dependent.     

Signaling through the p38 and p42/44 mitogen-activated families of protein kinases in pancreatic beta-cell proliferation

The present study has focused on the role of the 42- and 44-kDa mitogen-activated protein kinases (p42/44 MAPKs) and the 38-kDa mitogen-activated protein kinase (p38 MAPK) in the proliferation of the pancreatic beta-cell line MIN6. MIN6 beta-cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA. Inhibition of both the p42/44 MAPK pathway using the MEK inhibitor PD098059 (PD) and the p38 MAPK pathway using the p38 inhibitor SB203580 (SB) caused a marked, concentration-dependent reduction in the BrdU immunostaining observed in the presence of 15% FCS when assessed using fluorescence immunocytochemistry. These data provide direct evidence of a role for p42/44 MAPKs in the mitogenic response of MIN6 beta-cells to FCS. Furthermore, these data also suggest a novel role for the p38 MAPK pathway in MIN6 beta-cell proliferation.     

Treatment with IFN‐γ prevents insulin‐dependent PKB,p70S6k phosphorylation and protein synthesis in mouse C2C12 myogenic cells

The purpose of the present study was to examine the potential effect of IFN‐γ (interferon‐γ) on the cellular content and phosphorylation of PKB (protein kinase B), p70^S6k (p70 S6 kinase) and MAPK (mitogen‐activated protein kinase), and on the ability of insulin to stimulate the glucose uptake and protein synthesis in mouse C2C12 myotubes. Insulin (100 nmol/l) stimulated glucose uptake in C2C12 myotubes by 203.4%. Glucose uptake in cells differentiated in the presence of IFN‐γ (10 ng/ml) was increased by 165.8% and was not further significantly modified by the addition of insulin (183.4% of control value). Insulin increased the rate of protein synthesis by 198.8%. The basal rate of protein synthesis was not affected by IFN‐γ; however, this cytokine abolished the insulin effect. Cellular levels of PKB, p70^S6k, p42^MAPK and p44^MAPK were not modified by IFN‐γ. Insulin caused the phosphorylation of PKB and the activation of p70^S6k, but not p42^MAPK and p44^MAPK. In cells differentiated in the presence of IFN‐γ, the insulin‐mediated PKB phosphorylation was significantly diminished, whereas the phosphorylation of p70^S6k was completely prevented. Pretreatment of C2C12 myogenic cells with IFN‐γ led to the marked increase in p42^MAPK phosphorylation. Exposure of C2C12 myoblasts to IFN‐γ impaired MyoD and myogenin expression and decreased the fusion index on the fifth day of differentiation. In conclusion, (i) IFN‐γ present in the extracellular environment during C2C12 myoblast differentiation prevents the stimulatory action of insulin on protein synthesis; (ii) IFN‐γ‐induced insulin resistance of protein synthesis in myogenic cells can be associated with the decreased phosphorylation of PKB and p70^S6k, as well as with the augmented basal phosphorylation of p42^MAPK; (iii) this cytokine effect can be partly explained by alterations in the differentiation process.     

Cyclic AMP inhibits agonist-induced heparin-binding EGF gene expression independently of effects on p42/p44 MAPK activation

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.     

Different signaling responses to anti-proliferative agents in human aortic and venous smooth muscle cells

Proliferation of smooth muscle cells (SMCs) contributes to the stenosis of coronary arteries and vascular grafts. Local delivery of anti-proliferative drugs can prevent vascular stenosis. To understand the cellular responses to anti-proliferative agents, we investigated the signaling events in cultured human aortic SMCs (ASMCs), saphenous venous SMCs (VSMCs), and dermal fibroblasts (DFs) in response to paclitaxel or etoposide. Cellular mitochondrial and proliferative activities were examined with the methylthiazoletetrazolium (MTT) dye reduction and the bromodeoxyuridine (BrdU) incorporation assay, respectively. Cell proliferation was almost completely suppressed by paclitaxel or etoposide, but apoptosis was achieved in only about 50% of cells at the highest drug concentrations, suggesting the presence of compensatory mechanisms to prevent apoptosis. Examination of three important signaling pathways revealed significant differences between ASMCs, VSMCs, and DFs. Treatment with either paclitaxel or etoposide caused a transient phosphorylation/activation of p42 MAPK in ASMCs and DFs, but had no effect on phospho-p42/44 MAPK in VSMCs. High-dose etoposide enhanced p38 MAPK activation in ASMCs, but not in VSMCs. The p38 inhibitor, PD169316, partially inhibited etoposide-induced ASMC apoptosis, but induced apoptosis in VSMCs. The effects of etoposide and paclitaxel on Akt also differed between ASMCs and VSMCs. These observations indicate that ASMCs and VSMCs differ in the response of signaling pathways to anti-proliferative agents. In ASMCs, p42/44 MAPK appears to serve a pro-survival role, whereas p38 MAPK is a pro-apoptotic regulator. In contrast, p38 MAPK is an important pro-survival regulator in VSMCs and p42/44 MAPK appears to play a minor role in responding to anti-proliferative drugs.     

Histone code of genes induced by co-treatment with a glucocorticoid hormone agonist and a p44/42 MAPK inhibitor in human small intestinal Caco-2 cells

Background

Inactivation of glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) is thought to be important in small intestinal maturation and expression of genes related to intestinal differentiation and functions.

Methods

We investigated target genes induced by co-treatment for 48 h with a glucocorticoid hormone agonist, dexamethasone (Dex), and a p44/42 MAPK inhibitor, PD98059 (PD), in a small intestine-like cell line (Caco-2) using microarray analysis. We also investigated whether expression changes of the target genes induced by the co-treatment are associated with histone modifications around these genes.

Results

Co-treatment of Caco-2 cells with Dex and PD enhanced several genes related to intestinal differentiation and functions such as SCNN1A, FXYD3, LCT and LOX. Induction of the SCNN1A gene was associated with increased presence of acetylated histone H3 and H4 and di-methylated histone H3 at lysine (K) 4 around the transcribed region of the gene, and induction of the FXYD3 gene was associated with increased presence of acetylated histones H3 and H4 from the promoter/enhancer to the transcribed region of the gene. Induction of LCT and LOX genes was associated with increased presence of acetylated histone H4 on the promoter/enhancer region of the genes.

Conclusions

Histone acetylation and/or histone H3 K4 methylation around the promoter/enhancer or/and transcribed regions of target genes are associated with induction of the genes by co-treatment with Dex and PD in Caco-2 cells.

General significance

The histone code is specific to each gene with respect to induction by glucocorticoid hormone and inhibition of p44/42 MAPK in Caco-2 cells.     

Regulation of norepinephrine-induced proliferation in cardiac fibroblasts by interleukin-6 and p42/p44 mitogen activated protein kinase

Norepinephrine (NE) is involved in many cardiovascular diseases such as congestive heart failure. We have recently reported that NE had a comitogenic effect in isolated cardiac fibroblasts, and that it activated p42/p44 mitogen activated protein kinase (MAPK). This study was designed to characterize a possible mechanism involved in the proliferative effect of NE. Isolated rat cardiac fibroblasts were exposed to NE (10M) for up to 8 h, and interleukin-6 (IL-6) expression was measured by Ribonuclease Protection Assay and Western blotting. The activity of p42/p44^MAPK was analyzed by Western blotting. Cell number was assessed by use of a Coulter Counter. IL-6/GAPDH mRNA was increased by NE in a time-dependent manner reaching 23 fold stimulation after 1 h compared to untreated samples. Immunoreactivity to IL-6 was not found in controls. After 16h of exposure to NE, IL-6 protein was detected. It further increased up to 48 h. The effect of NE on IL-6 mRNA was abolished by the -adrenoceptor blockers propranolol, metoprolol (₁) and ICI 118.551 (₂), but not by the -adrenoceptor blockers prazosin (₁) and yohimbine (₂). The MAPK-inhibitor PD98059 suppressed the NE-induced MAPK activation in a concentration-dependent fashion after 5 min, attenuated the NE-induced IL-6 expression after 2 h, and suppressed the proliferative effect of NE from 53 to 18% after 48 h. Recombinant IL-6 caused an increase in proliferation by 31% after 48 h. Simultaneous application of the IL-6 antibody reduced the NE-induced proliferation to 34%, and completely prevented the IL-6 induced effect. These results suggest that NE induces proliferation of rat cardiac fibroblasts in part by increasing the expression of IL-6 through regulation of MAPK.     

MKP—1在血管紧张素Ⅱ导致心肌肥大反应中的调控作用

本研究主要从丝裂原活化蛋白激酶磷酸酶－１（ＭＫＰ－１）角度,研究丝裂原活化蛋白激酶（ＭＡＰＫ）信号途径在血管紧张素Ⅱ介导的新生大鼠心肌细胞肥大反应中的作用及调控机制。实验以心肌细胞蛋白合成速率、蛋白含量及细胞表面积作为心肌肥大反应的指标,以凝胶内ＭＢＰ原位磷酸化测定ＭＡＰＫ活性,以免疫印迹法（Ｗｅｓｔｅｒｎ ｂｏｌｔｔｉｎｇ）分别测定ＭＫＰ－１及磷酸化ｐ４４ＭＡＰＫ、ｐ４２ＭＡＰＫ蛋白表达。结果发     

Hypothetical mechanism of sodium pump regulation by estradiol under primary hypertension

Causal relationship between sodium and hypertension has been proposed and various changes in Na⁺,K⁺-ATPase (sodium pump) activity have been described in established primary hypertension. A number of direct vascular effects of estradiol have been reported, including its impact on the regulation of sodium pump activity and vasomotor tone. The effects of estradiol involve the activation of multiple signaling cascades, including phosphatydil inositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44^MAPK). In addition, some of the effects of estradiol have been linked to activity of cytosolic phospholipase A₂ (cPLA₂). One possible cardioprotective mechanism of estradiol involves of the interaction between estradiol and the rennin-angiotensin system (RAS). Elevated circulating and tissue levels of angiotensin II (Ang II) have been implicated in the development of hypertension and heart failure. The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump, in vascular smooth muscle cells (VSMC). The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump activity/expression in VSMC, with particular emphasis on PI3K/cPLA₂/p42/44^MAPK signaling pathways. Our primary hypothesis is that estradiol stimulates sodium pump activity/expression in VSMC via PI3K/cPLA₂/p42/44^MAPK dependent mechanism and, that impaired estradiol-stimulated sodium pump activity/expression in hypertensive rodent models (i.e. SHR), Ang II-mediated vascular impairment of estradiol is related to a decrease ability of estradiol to stimulate the PI3K/cPLA₂/p42/44^MAPK signaling pathways. An important corollary to this hypothesis is that in hypertensive state (i.e. SHR rats) the decreasing in ACE enzyme activity and/or AT1 receptor expression caused by administration of estradiol is accompanying with abrogated ability of Ang II to decrease IRS-1/PI3K association, and consequent PI3K/cPLA₂/p42/44^MAPK activity and associated sodium pump activity/expression.A clear characterization of how Ang II attenuates estradiol signaling may lead to a better understanding of the molecular mechanism(s) underlying pathophysiological conditions such as hypertension and to understanding how certain pathophysiological situations affect sodium pump activity/expression in VSMC.     

Interleukin-1beta induces apoptosis in GL15 glioblastoma-derived human cell line

Interleukin 1-(IL-1) induces apoptosis in a glioblastoma-derived human cell line,exhibiting a poorly differentiated astrocytic phenotype. The apoptoticeffect was demonstrated by analyzing nuclear morphology, in situ DNAfragmentation, and by ELISA detection of cytoplasmatic nucleosomes. Wecorrelated the degree of differentiation of GL15 cells with theapoptotic response: 1) 4',6-diamidino-2-phenylindole staining, combined with glial fibrillary acidic protein (GFAP) immunofluorescence, showed that the cells with apoptotic nuclei expresslow levels of GFAP; and 2) at 13 days of subculture, in amore differentiated state, GL15 cells did not respond with apoptosis toIL-1. In this cell line, nonrandom chromosome changes and theexpression of SV40 early region have been previously shown. Theinvolvement of p42/p44 mitogen-activated protein kinase (MAPK) pathwayin the induction of apoptosis by IL-1 was hypothesized. Previousstudies have shown that SV40 small T antigen partially inhibitsphosphatase 2A, leading to an enhancement of the steady-state activityof p42/p44 MAPK pathway. PD-098059, specific inhibitor of p42/p44 MAPKpathway, counteracts the apoptotic effect of IL-1, whereasSB-203580, specific inhibitor of p38 stress-activated protein kinase(SAPK) pathway, is ineffective. The imbalance between MAPK and SAPKpathways has been proposed as a key factor in determination of cellfate. Our results demonstrate that a further stimulation of p42/p44MAPK pathway can constitute a death signal in tumor cells in whichgenomic damage and MAPK pathway control alterations occur.

     

Treatment with TNF-α and IFN-γ alters the activation of SER/THR protein kinases and the metabolic response to IGF-I in mouse c2c12 myogenic cells

The aim of this study was to compare the effects of TNF-α, IL-1β and IFN-γ on the activation of protein kinase B (PKB), p70^S6k, mitogen-activated protein kinase (MAPK) and p90 ^rsk , and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-α abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-γ increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-α nor IFN-γ influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1β did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-α causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70^S6k, p42^MAPK, p44^MAPK and p90 ^rsk were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42^MAPK (a 2.81-fold increase after 50 min), and the activation of p70^S6k and p90 ^rsk , manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-α or IFN-γ, this IGF-I-mediated PKB and p70^S6k phosphorylation was significantly diminished, and the increase in p42^MAPK and p90 ^rsk phosphorylation was prevented. The basal p42^MAPK phosphorylation in C2C12 cells treated with IFN-γ was high and comparable with the activation of this kinase by IGF-I. Pretreatment of myogenic cells with IL-1β did not modify the IGF-I-stimulated phosphorylation of PKB, p70^S6k, p42^MAPK and p90 ^rsk . In conclusion: i) TNF-α and IFN-γ, but not IL-1β, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-α- and IFN-γ-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70^S6k. iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-γ is PKB independent. iv) The similar effects of TNF-α and IFN-γ on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle.     

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.