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.     

Natriuretic peptide gene expression after beta-adrenergic stimulation in adult mouse cardiac myocytes

The role of A- and B-type natriuretic peptides (ANP and BNP) in cardiac pathophysiology are of increasing interest. Isolated neonatal mouse cardiac myocytes express increased levels of ANP mRNA in the absence of growth factors in culture. Expression of ANP and BNP mRNA has not been studied in isolated adult mouse cardiac myocytes (AMCM). We examined expression of ANP and BNP mRNA in isolated AMCM with and without stimulation with beta-adrenergic receptor agonists and antagonists. AMCM were isolated and maintained in culture for 24-48 h with and without stimulation with the beta-adrenergic receptor agonist isoproterenol (Iso), the beta1-antagonist CGP20712A (CGP), or the beta2-antagonist ICI-118,551 (ICI). Northern blot analysis was performed using probes for mouse ANP and BNP mRNA. TUNEL assay was performed after beta-adrenergic receptor stimulation of AMCM. BNP mRNA expression was increased fivefold (P < 0.001) after 48 h in culture without adrenergic stimulation. BNP mRNA expression was reduced (P < 0.0001) after stimulation with Iso while ANP expression remained similar to unstimulated cells. CGP prevented the Iso reduction in BNP mRNA. Iso stimulation at doses that reduced BNP mRNA expression increased TUNEL positive nuclei, an effect blocked by the beta1-antagonist CGP. In conclusion, we have demonstrated differential gene expression of ANP and BNP in AMCM in culture. Expression of BNP mRNA increases in AMCM in culture and beta1-adrenergic receptor stimulation attenuates increased BNP gene expression and results in apoptosis.     

IL-18 induces monocyte chemotactic protein-1 production in macrophages through the phosphatidylinositol 3-kinase/Akt and MEK/ERK1/2 pathways

Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-gamma or TNF-alpha. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IkappaB kinase/NF-kappaB pathway, evidenced by no degradation of IkappaBalpha and no translocation of NF-kappaB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.     

PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 muM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.     

Adipophilin affects the expression of TNF-α, MCP-1, and IL-6 in THP-1 macrophages

Macrophages accumulated in the arterial intima play an important role in the development of atherosclerosis by producing a large number of proinflammatory cytokines which accelerate the disease. Recent studies show that adipophilin might be involved in inflammatory processes in macrophages. In this study, we observe the effect of adipophilin on proinflammatory cytokine expression and secretion in THP-1 macrophages. SiRNA and adipophilin gene overexpression mediated by an pEGFP-C3 vector were used to observe the effect of adipophilin on proinflammatory cytokines in THP-1 macrophages in vitro. Realtime PCR and enzyme-linked immunosorbent assay (ELISA) were applied to detect the production of tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). It was found that acetylated low-density lipoprotein (AcLDL), pioglitazone [a peroxisome proliferator-activated receptor γ (PPARγ) agonist] increased adipophilin expression in macrophages, while glucose had no such affect. It was also shown that adipophilin augments TNF-α, MCP-1, and IL-6 expression in AcLDL induced macrophages. Our results suggest that adipophilin augment inflammation in macrophages, which might be one role of adipophilin in atherosclerosis.     

Neurohormonal regulation of myocardial cell apoptosis during the development of heart failure

Adult cardiac myocytes are terminally differentiated cells that are no longer able to divide. Accumulating data support the idea that apoptosis in these cells is involved in the transition from cardiac compensation to decompensated heart failure. Since a number of neurohormonal factors are activated in this state, these factors may be involved in the positive and negative regulation of apoptosis in cardiac myocytes. beta1-Adrenergic receptor and angiotensin type 1 receptor pathways, nitric oxide and natriuretic peptides are involved in the induction of apoptosis in these cells, while alpha1- and beta2-adrenergic receptor and endothelin-1 type A receptor pathways and gp130-related cytokines are antiapoptotic. The myocardial protection of the latter is mediated, at least in part, through mitogen-activated protein kinase-dependent pathways, compatible with the findings in other cell types. In contrast, signaling pathways leading to apoptosis in cardiac myocytes are distinct from those in other cell types. The cAMP/PKA pathway induces apoptosis in cardiac myocytes and blocks apoptosis in other cell types. The p300 protein, a coactivator of p53, mediates apoptosis in fibroblasts but appears to play a protective role in differentiated cardiac myocytes. The inhibition of myocardial cell apoptosis in heart failure may be achieved by directly blocking apoptosis signaling pathways or by modulating neurohormonal factors involved in their regulation. These may provide novel therapeutic strategies in some forms of heart failure.     

Coxsackievirus group B type 3 infection upregulates expression of monocyte chemoattractant protein 1 in cardiac myocytes, which leads to enhanced migration of mononuclear cells in viral myocarditis

Coxsackievirus group B type 3 (CVB3) is an important cause of viral myocarditis. The infiltration of mononuclear cells into the myocardial tissue is one of the key events in viral myocarditis. Immediately after CVB3 infects the heart, the expression of chemokine(s) by infected myocardial cells may be the first trigger for inflammatory infiltration and immune response. However, it is unknown whether CVB3 can induce the chemokine expression in cardiac myocytes. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemokine that stimulates the migration of mononuclear cells. The objective of the present study was to investigate the effect of CVB3 infection on MCP-1 expression in murine cardiac myocytes and the role of MCP-1 in migration of mononuclear cells in viral myocarditis. Our results showed that the expression of MCP-1 was significantly increased in cardiac myocytes after wild-type CVB3 infection in a time- and dose-dependent manner, which resulted in enhanced migration of mononuclear cells in mice with viral myocarditis. The migration of mononuclear cells was partially abolished by antibodies specific for MCP-1 in vivo and in vitro. Administration of anti-MCP-1 antibody prevented infiltration of mononuclear cells bearing the MCP-1 receptor CCR2 in mice with viral myocarditis. Infection by UV-irradiated CVB3 induced rapid and transient expression of MCP-1 in cardiac myocytes. In conclusion, our results indicate that CVB3 infection stimulates the expression of MCP-1 in myocardial cells, which subsequently leads to migration of mononuclear cells in viral myocarditis.     

Calcium-independent phospholipase A(2) is required for human monocyte chemotaxis to monocyte chemoattractant protein 1.

Monocyte chemoattractant protein 1 (MCP-1) has an important influence on monocyte migration into sites of inflammation. Our understanding of the signal transduction pathways involved in the response of monocytes to MCP-1 is quite limited yet potentially significant for understanding and manipulating the inflammatory response. Prior studies have demonstrated a crucial regulatory role for cytosolic phospholipase A(2) (cPLA(2)) in monocyte chemotaxis to MCP-1. In these studies we investigated the role for another PLA(2), calcium-independent PLA(2) (iPLA(2)) in comparison to cPLA(2). Pharmacological inhibitors of PLA(2) were found to substantially inhibit chemotaxis. Using antisense oligodeoxyribonucleotide treatment we found that iPLA(2) expression is required for monocyte migration to MCP-1. Complete blocking of the chemotactic response was observed with inhibition of either iPLA(2) or cPLA(2) expression by their respective antisense oligodeoxyribonucleotide. In reconstitution experiments, lysophosphatidic acid completely restored MCP-1-stimulated migration in iPLA(2)-deficient monocytes, whereas lysophosphatidic acid was without effect in restoring migration in cPLA(2)-deficient monocytes. To the contrary, arachidonic acid fully restored migration of cPLA(2)-deficient monocytes while having no effect on the iPLA(2)-deficient monocytes. Additional studies revealed that neither enzyme appears to be upstream of the other indicating that iPLA(2) and cPLA(2) represent parallel regulatory pathways. These data demonstrate novel and distinct roles for these two phospholipases in this critical step in inflammation.     

Cyr61 induces the expression of monocyte chemoattractant protein-1 via the integrin ανβ3, FAK,PI3K/Akt,and NF-κB pathways in retinal vascular endothelial cells

Diabetes causes a number of metabolic and physiological abnormalities in the retina. Many of the molecular and physiological abnormalities that develop during diabetic retinopathy are due to inflammation. Monocyte chemoattractant protein-1 (MCP-1) is an important factor involved in diabetic retinopathy. In a previous study, we found that cysteine-rich 61 (Cyr61), an important angiogenic factor, also plays an important role in diabetic retinopathy. In addition to the direct effects of Cyr61, we observed that Cyr61 can induce the expression of MCP-1. However, the mechanism through which this occurs is not completely understood in chorioretinal vascular endothelial cells. We therefore investigated the effects of Cyr61 on MCP-1 expression in this cell type. Cyr61 stimulated the expression of MCP-1 at the mRNA, protein, and secreted protein levels in a dose-dependent and time-dependent manner. Both total MCP-1 levels and secreted MCP-1 levels were attenuated during the response to Cyr61 stimulation by pretreatment with integrin ανβ3-blocking antibodies, a FAK inhibitor (PF573228), a PI3K inhibitor (LY294002), and an Akt inhibitor (A6730). Electrophoretic mobility shift assays revealed that the above inhibitors suppressed the activation of NF-κB. Additionally, deletion of the NF-κB-binding element in the MCP-1 gene promoter led to a decrease in expression in luciferase reporter assays. These results show that the induction of MCP-1 by Cyr61 is mediated through the activation of the integrin ανβ3, FAK, PI3K/Akt, and IKK/NF-κB pathways in chorioretinal vascular endothelial cells.     

Thrombin induces MCP-1 expression through Rho-kinase and subsequent p38MAPK/NF-κB signaling pathway activation in vascular endothelial cells

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.     

Hypertrophic Gene Expression Induced by Chronic Stretch of Excised Mouse Heart Muscle

Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O₂/ 5% CO₂, and 34°C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.