Cyclic strain stimulates monocyte chemotactic protein-1 mRNA expression in smooth muscle cells

Hemodynamic forces are important determinants for the formation of atherosclerotic plaques. The recruitment of circulating monocytes into the arterial wall is an important step during atherogenesis. Monocyte chemotactic protein-1 (MCP-1) has been shown to be a key factor for monocyte transmigration. This study examined the effects of cyclic strain on MCP-1 mRNA expression levels of cultured rat aortic smooth muscle cells. The MCP-1 mRNA levels of aortic smooth muscle cells first increased as the duration of cyclic strain increased, reaching the maximum at 6-12 h, maintained at high levels throughout the 48-h strain period. To explore signaling pathways mediating cyclic strain-stimulated MCP-1 mRNA expression, we examined the involvement of tyrosine kinase and protein kinase C (PKC). Tyrosine kinase inhibitors, genistein and tyrphostin 51, at 50 microM blocked cyclic strain-stimulated MCP-1 mRNA expression. Preincubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA), 2 microM, for 24 h to downregulate PKC did not decrease cyclic strain-induced MCP-1 mRNA expression. A 6-h incubation with 0. 1 microM PMA to activate PKC, which stimulated MCP-1 expression when applied alone, abolished the stimulatory effects of cyclic strain. A specific PKC inhibitor, calphostin C (0.1 microM), diminished cyclic strain-stimulated MCP-1 mRNA expression. Angiotensin II at 10 or 1,000 nM induced a moderate upregulation of MCP-1 mRNA, and no synergistic effects were observed between angiotensin II and cyclic strain. These results indicate that cyclic strain stimulates MCP-1 mRNA expression in smooth muscle cells through signaling pathway(s) mediated by tyrosine kinase activation.     

Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.     

The mitogen-activated protein kinase p38 is necesssary for interleukin 1beta-induced monocyte chemoattractant protein 1 expression by human mesangial cells

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1beta (IL-1beta)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1beta-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1beta treatment. p38 kinase immunoprecipitated from IL-1beta-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1beta-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-kappaB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1beta induction of NF-kappaB was measured. SB 203580 (up to 25 microM) had no effect on IL-1beta-induced NF-kappaB nuclear translocation or DNA binding activity. Our previous work showed that IL-1beta also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1beta-induced MCP-1 mRNA or protein levels, or on IL-1beta activation of NF-kappaB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1beta, but is not involved at the level of cytoplasmic activation of NF-kappaB. In contrast, ERK2 does not mediate IL-1beta induced MCP-1 gene expression.     

Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

Role of 12/15-lipoxygenase in the expression of MCP-1 in mouse macrophages

Monocyte chemoattractant protein (MCP)-1 plays a key role in atherosclerosis and inflammation associated with visceral adiposity by inducing mononuclear cell migration. Evidence shows that mouse peritoneal macrophages (MPM) express a 12-lipoxygenase (12/15-LO) that has been clearly linked to accelerated atherosclerosis in mouse models and increased monocyte endothelial interactions in both rodent and human cells. However, the role of 12/15-LO products in regulating MCP-1 expression in macrophages has not been clarified. In this study, we tested the role of 12/15-LO products using MPM and the mouse macrophage cell line, J774A.1 cells. We found that 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] increased MCP-1 mRNA and protein expression in J774A.1 cells and MPM. In contrast, 12(R)-HETE, a lipid not derived from 12/15-LO, did not affect MCP-1 expression. 15(S)-HETE also increased MCP-1 mRNA expression, but the effect was less compared with 12(S)-HETE. MCP-1 mRNA expression was upregulated in a macrophage cell line stably overexpressing 12/15-LO (Plox-86 cells) and in MPM isolated from a 12/15-LO transgenic mouse. In addition, the expression of MCP-1 was downregulated in MPM isolated from 12/15-LO knockout mice. 12(S)-HETE-induced MCP-1 mRNA expression was attenuated by specific inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein kinase (p38). 12(S)-HETE also directly activated NADPH oxidase activity. Two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium chloride, blocked 12(S)-HETE-induced MCP-1 mRNA. Apocynin attenuated 12(S)-HETE-induced MCP-1 protein secretion. These data show that 12(S)-HETE increases MCP-1 expression by inducing PKC, p38, and NADPH oxidase activity. These results suggest a potentially important mechanism linking 12/15-LO activation to MCP-1 expression that induces inflammatory cell infiltration.     

Endothelin-1 stimulates c-fos mRNA expression in C6 glioma cells via MAP kinase pathway.

Exposure of C6 glioma cells to endothelin-1 (ET-1) caused dose-dependent (10(-11) M to 10(-7) M) increments in intracellular calcium concentration ([Ca2+]i) and c-fos mRNA expression (4.5-fold) that were abolished by the endothelinA receptor antagonist, BQ610, and by inhibition of phospholipase C with U73122. ET-1 stimulated c-fos mRNA expression was also inhibited by protein kinase C inhibition (chelerythrine) and by the MAP kinase kinase inhibitor PD98059, but not by inhibitors of tyrosine kinases, protein kinase A type I or II, calmodulin kinase II, or calcium channel blockade. C6 cells treated with ET-1 demonstrated a significant increase in MAP kinase activity as evidenced by Western blotting. These results indicate a mechanism of long-term signaling by ET-1 involving an ET(A) receptor-mediated, phospholipase C(beta)-linked pathway that is dependent on protein kinase C and MAP kinase activation.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

PI 3 kinase-dependent Akt kinase and PKCepsilon independently regulate interferon-gamma-induced STAT1alpha serine phosphorylation to induce monocyte chemotactic protein-1 expression

Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1.     

Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-kappaB in THP-1 macrophages

Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05-0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-kappaB due to increased phosphorylation of the inhibitory protein (IkappaB-alpha) as well as reduced expression of IkappaB-alpha mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-kappaB activation.     

Astragaloside IV attenuates glycated albumin-induced epithelial-to-mesenchymal transition by inhibiting oxidative stress in renal proximal tubular cells

In diabetic kidney disease (DKD), epithelial-to-mesenchymal transition (EMT) is a classic pathological process in tubular damage. Oxidative stress is considered to play an important role in DKD. Astragaloside IV (A-IV), one of the main active ingredients of Astragalus membranaceus, exhibits a wide range of biological activities. However, the effect of A-IV on regulating EMT in tubular cells is unclear. This study aims to determine whether A-IV could attenuate glycated albumin (GA)-induced EMT in the NRK-52E cell line by inhibiting oxidative stress. GA and A-IV-induced cytotoxicity were assayed by CCK-8. The intercellular reactive oxygen species (ROS) level was detected by H₂DCFDA. The activity of NADPH oxidase was assayed by adding exogenous NADPH oxidase, and the superoxide dismutase (SOD) units were observed by NBT. We used a microscope to examine the morphology of the NRK-52E cell line. We conducted a wound healing assay to measure cell mobility. To determine mRNA and protein expressions of α-SMA and E-cadherin, we used real-time polymerase chain reaction (real-time PCR), immunofluorescence, and western blot analysis. A-IV significantly attenuated GA-induced amplification of ROS, lowered the increased level of NADPH oxidase activity, and elevated the decreased level of SOD units. The GA-induced NRK-52E cell line showed increased expression of α-SMA and decreased expression of E-cadherin in mRNA and protein levels, whereas A-IV alleviated the expression of α-SMA and increased the expression of E-cadherin. Our data demonstrate that GA could induce NRK-52E cell line EMT through oxidative stress. This effect could be attenuated by A-IV via regulation of the impaired redox balance.     

Geldanamycin induces cell cycle arrest in K562 erythroleukemic cells

Geldanamycin (GA), a benzoquinone ansamycin, is one of the specific inhibitors of 90-kDa heat shock protein and induces growth inhibition and apoptosis in certain cancer cell lines. We have investigated the mechanism of GA-induced growth inhibition in K562 erythroleukemic cells. DNA flow-cytometric analysis indicated that GA-induced growth arrest was associated with G2/M phase arrest of the cell cycle. GA treatment down-regulated the expression of cyclin B1 and inhibited phosphorylation of Cdc2 protein, both key regulatory proteins at the G2/M boundary. GA also markedly inhibited the Cdc2 kinase activity, which may be in part a result of up-regulation of p27KIP1 by GA. The present results suggest a novel mechanism that p27KIP1 could be involved in the regulation of G2 to M phase transition.     

Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta1 integrin expression at the mRNA and protein levels; 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60c-src; and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line.     

Isoproterenol stimulates monocyte chemoattractant protein-1 expression and secretion in 3T3-L1 adipocytes

Recently, monocyte chemoattractant protein (MCP)-1 has been characterized as a novel adipocytokine upregulated in obesity and insulin resistance which impairs insulin signaling in muscle and fat in vitro. Growing evidence, on the other hand, suggests that increased activity of the sympathetic nervous system is an integral part in the development of insulin resistance. In the current study, the impact of the beta-adrenergic agonist isoproterenol on MCP-1 mRNA synthesis and secretion was determined in 3T3-L1 adipocytes. Interestingly, isoproterenol increased MCP-1 secretion 3-fold. Furthermore, 10 microM isoproterenol acutely induced MCP-1 mRNA by up to 5.3-fold in a time-dependent fashion with significant stimulation seen at concentrations as low as 0.3 microM effector. Studies using pharmacological inhibitors suggested that basal and isoproterenol-induced MCP-1 expressions are mediated via beta-adrenergic receptors and protein kinase A. Moreover, acute activation of adenylyl cyclase by forskolin was sufficient to mimic the effects of isoproterenol. Taken together, our results demonstrate that isoproterenol induces MCP-1 expression and secretion via a classical GS-protein-coupled pathway and support the notion that MCP-1 might be an interesting novel candidate linking obesity and insulin resistance.