Monocyte Chemoattractant Protein-1 Promoter Activity Research Articles

Androgen-androgen receptor system improves chronic inflammatory conditions by suppressing monocyte chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation

Age-related decreases in sex hormones are closely related to chronic inflammation in obesity and metabolic diseases. Particularly, the molecular basis of androgen activity in regulating inflammation and controlling metabolism remains largely unknown. Obese adipocytes secrete monocyte chemoattractant protein-1 (MCP-1), a key chemokine that promotes the infiltration of monocytes/macrophages into adipose tissue, thereby leading to metabolic disorders. Here, we studied the role of androgen-androgen receptor (AR) action in regulating MCP-1 expression in adipose tissue. We observed the induction of Mcp-1 expression in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. Additionally, Mcp-1 expression was upregulated by culturing in conditioned medium derived from inflammatory macrophages (M1-Mφ) containing tumor necrosis factor-alpha (TNF-α). We found that sex hormones downregulated TNF-α-induced Mcp-1 and interleukin (Il)-6 expression in 3T3-L1 adipocytes. Furthermore, luciferase-reporter analysis indicated that MCP-1 promoter activity was predominantly suppressed by dihydrotestosterone (DHT)-AR interactions through functional canonical nuclear factor-kappa B (NF-κB) sites, whereas non-canonical NF-κB site containing important flanking sequences exhibited minor contributions to DHT-AR transcriptional repression. These findings suggested that androgen-AR suppressed obesity-induced chronic inflammation in adipose tissue.

Abstract 139: Id3 Regulation of High-Fat-Diet--Induced Visceral Adipose Tissue Macrophages

BACKGROUND: Global deletion of the helix-loop-helix (HLH) transcription factor Inhibitor of Differentiation 3 (Id3), attenuates HFD-induced obesity. Specifically, Id3 -/- mice have less visceral adipose tissue (VAT); an effect that may be linked to VAT inflammation. Adipose tissue macrophages (ATMs) play a key role in HFD-induced obesity. Monocyte chemoattractant protein-1 (MCP-1) is a key cytokine regulating VAT macrophage accumulation, yet the role of Id3 in regulating MCP-1 expression and ATM accumulation is unknown. RESULTS: Id3 -/- mice and C57Bl/6 litter-mate controls were fed 4 weeks of HFD. The epididymal adipose tissue (VAT) and sera were analyzed for MCP-1 expression. Consistent with published findings, results demonstrated that HFD induced MCP-1 mRNA (analyzed via qPCR) and protein expression (analyzed via ELISA) in VAT. Notably, there was marked attenuation of HFD-induced MCP-1 expression in Id3 -/- mice. Epididymal adipose tissue from both WT and knockout mice were cultured with and without IL-1β. MCP-1 protein levels were the same in both sets of mice, when fat from chow-fed mice was cultured in media alone. However, IL-1β induced MCP-1 protein expression in VAT, an effect significantly attenuated in mice null for Id3. Flow cytometry analysis revealed that at early time-points (4 weeks HFD), WT mice have a significant increase in M1 macrophage numbers in epididymal VAT in response to HFD. However, Id3 null mice had an attenuation of this response to HFD. In contrast, HFD induced a statistically significant increase in M2 macrophages in both WT and Id3 -/- mice. RAW 264.7 cells were stimulated with 100ng/ml of LPS for 0, 0.5, 3, 6 and 24 hours. LPS treatment led to a significant and rapid induction of Id3 mRNA expression, which preceded upregulation of MCP-1 mRNA and protein levels. NIH 3T3 cells were co-transfected with a 1-kb MCP-1 luciferase promoter construct, as well as an expression plasmid encoding E12, a member of the bHLH E-protein family of transcription factors. Increasing concentrations of E12 led to repression of MCP-1 promoter activity, seen as a decrease in RLU/s compared to transfection with the promoter alone.. CONCLUSION: The loss of Id3 decreases local and systemic MCP-1 expression, and decreases M1 macrophage numbers in visceral adipose tissue. Id3’s partner E12 represses MCP-1 promoter activity. Based on the Id3-E-protein paradigm, it is likely that Id3 would restore MCP-1 promoter activity. These data suggest that Id3 promotes HFD-induced MCP-1 expression and macrophage-mediated visceral adipose tissue expansion. Further studies are needed to confirm Id3 regulation of MCP-1 specifically in macrophages.

SP600125, an inhibitor of c-Jun NH2-terminal kinase, blocks expression of angiotensin II-induced monocyte chemoattractant protein-1 in human mesangial cells

We investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, in the expression of angiotensin II (Ang II)-induced monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-1 (TGF-1), and in the production of fibronectin (FN), by human mesangial cells (HMCs). JNK activation in cultured human mesangial cells was determined by Western blotting with an antibody against the phosphorylated Ser63 residue of c-Jun. Binding of the activator protein (AP-1) to the MCP-1 AP-1 motif was detected via the electrophoretic mobility shift assay (EMSA). The transient luciferase reporter was used to examine MCP-1 promoter activity; an RNase protection assay and ELISA were used respectively to detect the expression of MCP-1 mRNA and production of MCP-1, TGF-beta and FN. Anthra (1,9-cd) pyrazol-6(2H)-one (SP600125), a pharmacological inhibitor of JNK, almost completely abolished Ang II-induced Ser63 phosphorylation of c-Jun at concentrations of 5-20 micromol/L: JNK activity was reduced by 75% with 10 micromol/L SP600125, and by 90% with 20 micromol/L. Ang II increased AP-1 binding to the MCP-1 AP-1 motif in a time-dependent manner, as detected by EMSA, while SP600125 effectively blocked this increased AP-1 binding in a concentration-dependent manner. Treatment with 100 nmol/L Ang II led to a steady increase in MCP-1 mRNA expression, and to an enhanced production of MCP-1, TGF-beta and FN. These effects were blocked by SP60025 in a dose-dependent manner. SP600125 also reduced MCP-1 mRNA stability: the halflife of MCP-1 mRNA was approximately 5 hours in cells treated with Ang II only, but was reduced to 2 hours when treated with a combination of Ang II and SP600125. These results show that the JNK/AP-1 pathway is involved in the expression of MCP-1 and TGF-beta, and in extracellular matrix production. JNK is an important therapeutic target for glomerulonephritis and glomerulosclerosis.

C-Jun N-terminal kinases inhibitor suppresses the TNF-α induced MCP-1 expression in human umbilical vein endothelial cells

Monocyte chemoattractant protein-1 (MCP-1) is a 76-amino-acid chemokine that is considered to be an important chemotactic factor for monocytes. MCP-1 is expressed in the macrophage-rich areas of atherosclerotic lesions. A recent report indicated that MCP-1 expression in human umbilical vein endothelial cells (HUVECs) is induced by the stimulation of tumor necrosis factor (TNF)-alpha via the c-Jun N-terminal kinases (JNK) pathway. In this study, we examined the effects of JNK inhibitor (JNKI-1), on MCP-1 expression. The results of this study indicated that the expression of MCP-1 mRNA and protein were stimulated in the presence of TNF-alpha. TNF-alpha stimulated the phosphrylation of JNK, however, JNKI-1 inhibited the TNF-alpha stimulated MCP-1 secretion and gene expression. As expected, JNKI-1 blocked the stimulatory effect of TNF-alpha on the MCP-1 promoter activity. In conclusion, JNKI-1 partially inhibits the TNF-alpha-induced MCP-1 expression in HUVECs, and therefore JNKI-1 may be of therapeutic value in the treatment of diseases such as atherosclerosis.

Transcription Factor Activating Protein-2β: A Positive Regulator of Monocyte Chemoattractant Protein-1 Gene Expression

We previously reported an association between the activating protein (AP)-2beta transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and inducing insulin resistance in 3T3-L1 adipocytes. In this study, we found that overexpression of AP-2beta in 3T3-L1 adipocytes accelerated the promoter activity of monocyte chemoattractant protein-1 (MCP-1) and subsequently increased both mRNA and protein expression and protein secretion. Furthermore, knockdown of endogenous AP-2beta by RNA interference reduced the mRNA and the protein expression of MCP-1. EMSAs and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to MCP-1 promoter regions, in vitro and in vivo. Additionally, site-directed mutagenesis of the AP-2 binding site located at -137 to -129 relative to the transcription start site markedly diminished MCP-1 promoter activity, whereas other putative AP-2 binding sites did not. Our results clearly show that AP-2beta directly enhanced MCP-1 secretion by binding to its promoter. Thus, we propose that AP-2beta positively regulates MCP-1 expression; subsequently contributes to the infiltration of macrophages to adipose tissue; and leads to insulin resistance, type 2 diabetes, and cardiovascular diseases.

Monocyte Chemoattractant Protein-1 Expression Is Enhanced by Granulocyte-Macrophage Colony-stimulating Factor via Jak2-Stat5 Signaling and Inhibited by Atorvastatin in Human Monocytic U937 Cells

The proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in inflammatory and atherosclerotic lesions. GM-CSF is known to enhance monocytic expression of monocyte chemoattractant protein-1 (MCP-1). However, the molecular mechanism(s) by which GM-CSF up-regulates the MCP-1 expression remains to be clarified. Thus, in this study, we examined our hypothesis that GM-CSF up-regulates the MCP-1 expression via Jak2-Stat5 signaling pathway. In human monocytic cell line U937, GM-CSF increased MCP-1 expression in protein and mRNA levels. Furthermore, analysis of the GM-CSF promoter element revealed that the STAT5 (signal transducer and activator of transcription-5) transcription factor binding site, located between -152 and -144 upstream of the transcription start site, as well as Janus kinase-2-mediated Stat5 activation were necessary for the GM-CSF-induced transcriptional up-regulation of the MCP-1 gene. This GM-CSF-induced MCP-1 expression, measured as both protein and mRNA levels, was down-regulated by atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. However, this decrease in MCP-1 expression was not at the transcriptional level of MCP-1 gene but rather at the level of the stability of MCP-1 mRNA. These results indicate that GM-CSF regulates MCP-1 expression via Janus kinase-2-Stat5 pathway and by a novel regulatory mechanism of statins to reduce inflammatory reactions by down-regulating the expression of monocytic MCP-1, which promotes atherogenesis.

D-Psicose inhibits the expression of MCP-1 induced by high-glucose stimulation in HUVECs

Monocyte chemoattractant protein-1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. MCP-1 is found in macrophage-rich areas of atherosclerotic lesions. Recent report indicates that MCP-1 is induced by glucose-stimulation, raising the important link between diabetes mellitus and atherosclerosis. One of the rare sugars, d-psicose ( d-ribo-2-hexulose) is present in small quantities in commercial carbohydrate complexes, however the physiological functions of d-psicose have not been evaluated. In this study, we examined the effects of d-psicose on MCP-1 expression in human umbilical vein endothelial cells (HUVECs). Results showed that MCP-1 mRNA and protein were stimulated following exposure to 22.4 mM glucose. Transcriptional activity of MCP-1 promoter paralleled endogenous expression of the gene and this activity was dependent on the dose of d-glucose. d-Psicose inhibited these effects. Next we used inhibitors of selected signal transduction pathways to show that high-glucose (HG) stimulated MCP-1 promoter activity was sensitive to p38-Mitogen-Activated Protein Kinase (p38-MAPK) pathway inhibitor. As expected, a dominant-negative p38-MAPK abolished the stimulatory effect of HG on the promoter activity. To incubate the cells with HG and d-psicose reduced the activation of p38-MAPK. Together, these results indicate that the d-psicose suppression of HG induced MCP-1 expression is mediated in part by inhibition of the p38-MAPK pathway and raise the possibility that d-psicose may be of therapeutic value in the treatment of diseases such as atherosclerosis.

Role of redox factor-1 in hyperhomocysteinemia-accelerated atherosclerosis

Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. We have previously shown that homocysteine can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes in vitro. In the present study, we investigated whether redox factor-1 (Ref-1) is involved in HHcy-accelerated atherosclerosis. We used a mild HHcy animal model, aortic roots and peritoneal macrophages were isolated for immunohistochemistry and Western blotting, from apoE −/− and C57BL/6J mice fed a high Hcy diet (1.8 g/L) for 4 or 12 weeks. Four-week HHcy apoE −/− mice showed more plaques and significantly increased immunostaining of Ref-1 and MCP-1 in foam cells, and HHcy mice showed enhanced Ref-1 expression in peritoneal macrophages. To explore the mediating mechanism, incubation with Hcy (100 μM) increased Ref-1 protein level and translocation in human monocytes in vitro. In addition, Hcy-induced NADPH oxidase activity mediated the upregulation of Ref-1. Furthermore, overexpressed Ref-1 upregulated NF-κB and MCP-1 promoter activity, and antisense Ref-1 reduced Hcy-induced NF-κB DNA-binding activity and MCP-1 secretion. These data indicate that Hcy-induced ROS upregulate the expression and translocation of Ref-1 via NADPH oxidase, and then Ref-1 increases NF-κB activity and MCP-1 secretion in human monocytes/macrophages, which may accelerate the development of atherosclerosis.

Bidirectional regulation of monocyte chemoattractant protein-1 gene at distinct sites of its promoter by nitric oxide in vascular smooth muscle cells

We have previously reported that chronic activation of phosphatidylinositol 3-kinase (PI3-kinase) by the overexpression of membrane-targeted p110CAAX induced proinflammatory gene expression in rat vascular smooth muscle cells (VSMCs) through the induction of CCAAT/enhancer binding protein-beta (C/EBP-beta) and C/EBP-delta. To examine the anti-inflammatory effect of nitric oxide (NO) on proinflammatory gene expression, we have investigated the effects of sodium nitroprusside (SNP) on the monocyte chemoattractant protein-1 (MCP-1) gene expression in VSMCs under chronic activation of PI3-kinase. At low concentrations (0.05 mM) of SNP, but not at high concentrations (0.5-1.0 mM), MCP-1 mRNA and protein expression as well as its transcriptional activity were significantly reduced. We found that SNP induced C/EBP homologous protein (CHOP) expression, which inhibited C/EBP binding activity and reduced the C/EBP activity induced by chronic activation of PI3-kinase in a dose-dependent manner up to 1.0 mM. Consistently, the increase in CHOP expression significantly reduced the MCP-1 promoter activity induced by PI3-kinase. However, the overexpression of CHOP alone upregulated MCP-1 promoter activity in a dose-dependent manner up to high concentrations. Deletion analysis of MCP-1 promoter and electrophoretic mobility shift assay identified the CHOP-response element (CHOP-RE) at the region between -190 and -179 bp of MCP-1 promoter. By using CHOP-RE as a decoy, we significantly suppressed the increase in promoter activity of MCP-1 induced by either CHOP or SNP. Thus CHOP induced by an NO donor has bidirectional effects on MCP-1 gene expression: it decreases gene expression by inhibition of C/EBPs, and it increases the gene expression through CHOP-RE.

Myocyte enhancer factor 2 mediates vascular inflammation via the p38-dependent pathway.

Although it has been established that myocyte enhancer factor 2 (MEF2) plays pivotal roles in the development of the cardiovascular system as well as skeletal muscle cells, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To investigate the role of MEF2 in vascular inflammation and that of p38 in the activation of MEF2, we infected cultured rat vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a dominant-negative mutant of MEF2A (MEF2ASA) or mitogen-activated protein kinase kinase 6 (MEK6AA), and examined their effects on the expression of monocyte chemoattractant protein-1 (MCP-1), which is known to play important roles in vascular inflammation. We also examined the role of MEF2 in vivo using a rat model of transluminal wire-induced injury of the femoral artery. Angiotensin II (Ang II)-induced expression of MCP-1 mRNA was significantly inhibited by infection with adenoviruses encoding MEF2ASA (AdMEF2ASA) or MEK6AA. Ang II-induced increase of MCP-1 promoter activity was also significantly suppressed by overexpression of MEF2ASA or MEK6AA. Ang II stimulated the transactivating function of MEF2A and this activation was inhibited by overexpression of MEK6AA. Infection with AdMEF2ASA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. AdMEF2ASA infection also inhibited macrophages infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that MEF2 activation via the p38-dependent pathway mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophages infiltration.

Calcineurin promotes the expression of monocyte chemoattractant protein-1 in vascular myocytes and mediates vascular inflammation.

Although the role of the calcineurin-dependent pathway in the development of cardiac hypertrophy has been intensively studied, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To help elucidate the role of calcineurin in vascular inflammation, we infected cultured vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a constitutively active mutant of calcineurin, and examined its effect on the expression of monocyte chemoattractant protein-1 (MCP-1). We also examined the role of calcineurin in vivo using a transluminal wire injury model of the rat femoral artery. Forced activation of calcineurin significantly increased the expression of MCP-1 both at the transcriptional and protein levels. Angiotensin II (Ang II) also significantly stimulated MCP-1 expression, and this increase was significantly inhibited by cyclosporin A (CyA). Constitutive activation of calcineurin stabilized MCP-1 mRNA without enhancing MCP-1 promoter activity. In accordance with the results, Ang II-induced increase of MCP-1 promoter activity was not suppressed by CyA. Ang II stabilized MCP-1 mRNA, and this effect of Ang II was diminished by CyA. CyA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. CyA also inhibited macrophage infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that calcineurin mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophage infiltration.

Molecular mechanism and role of endothelial monocyte chemoattractant protein-1 induction by vascular endothelial growth factor.

We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular permeability and the underlying molecular mechanism of VEGF-induced endothelial MCP-1 expression in vitro and in vivo. We used an anti-MCP-1 neutralizing antibody for specific inhibition of MCP-1. VEGF increased tubule formation in the angiogenesis assay and vascular permeability in the Miles assay, and these effects were markedly inhibited by anti-MCP-1 antibody. Using a luciferase MCP-1 promoter-gene assay, we found that the activator protein-1 (AP-1) binding site of the MCP-1 promoter region contributes to the increase in MCP-1 promoter activity by VEGF. To specifically inhibit AP-1, we used recombinant adenovirus containing a dominant-negative c-Jun (Ad-DN-c-Jun). Ad-DN-c-Jun inhibited VEGF-induced endothelial MCP-1 mRNA expression and promoter activity in vitro. In vivo gene transfer of DN-c-Jun into rat carotid artery, with the hemagglutinating virus of the Japan liposome method, significantly blocked VEGF-induced MCP-1 and macrophage/monocyte (ED1) expression in endothelium. These results reveal that endothelial MCP-1 induced by VEGF seems to participate in angiogenesis, vascular leakage, or arteriosclerosis. AP-1 plays a critical role in the molecular mechanism underlying induction of MCP-1 by VEGF.

17Beta-estradiol inhibits MCP-1 production in human keratinocytes.

A chemokine, monocyte chemoattractant protein 1 (MCP-1), attracts macrophages. The production of MCP-1 is enhanced in keratinocytes of psoriatic lesions, which may contribute to macrophage infiltration into the lesions. It is known that estrogen regulates the course of psoriasis. We examined in vitro effects of 17beta-estradiol (E2) on MCP-1 production by human keratinocytes. E2 inhibited constitutive and 12-O-tetradecanoylphorbol-13-acetate-induced MCP-1 secretion, mRNA expression, and promoter activity in keratinocytes, and these effects of E2 were counteracted by estrogen receptor antagonist ICI 182 780. GC-rich Sp1 element and activator protein 1 (AP-1) element on MCP-1 promoter were required for constitutive and 12-O-tetradecanoylphorbol-13-acetate-induced transcription, respectively, and involved in transrepression by E2. E2 inhibited constitutive Sp1 and 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 transcriptional activities whereas it did not inhibit DNA binding of Sp1 or AP-1 c-Fos/c-Jun. E2 inhibited Sp1 and AP-1 transcriptional activities and MCP-1 promoter activity in estrogen receptor beta (ERbeta) transfected SKBR3 cells. Deletion of the A/B region or mutation of activation function 2 in ERbeta abrogated E2-dependent transcriptional inhibition by ERbeta whereas mutation of DNA-binding domain retained the inhibitory effects. Transfection of ERbeta enhanced the inhibitory effects of E2 on Sp1 and AP-1 transcriptional activities and MCP-1 promoter activities in nontransfected keratinocytes. Coimmunoprecipitation studies showed an E2-dependent association of ERbeta with Sp1 or AP-1 in ERbeta-transfected keratinocytes. These results suggest that E2-bound ERbeta may inhibit MCP-1 gene expression by inhibiting Sp1 and AP-1 transcriptional activities in keratinocytes. A/B region and intact activation function 2 of ERbeta may be responsible for the effects of E2.

17β-Estradiol Inhibits MCP-1 Production in Human Keratinocytes

A chemokine, monocyte chemoattractant protein 1 (MCP-1), attracts macrophages. The production of MCP-1 is enhanced in keratinocytes of psoriatic lesions, which may contribute to macrophage infiltration into the lesions. It is known that estrogen regulates the course of psoriasis. We examined in vitro effects of 17β-estradiol (E2) on MCP-1 production by human keratinocytes. E2 inhibited constitutive and 12-O-tetradecanoylphorbol-13-acetate-induced MCP-1 secretion, mRNA expression, and promoter activity in keratinocytes, and these effects of E2 were counteracted by estrogen receptor antagonist ICI 182 780. GC-rich Spl element and activator protein 1 (AP-1) element on MCP-1 promoter were required for constitutive and 12-O-tetradecanoylphorbol-13-acetate-induced transcription, respectively, and involved in transrepression by E2. E2 inhibited constitutive Spl and 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 transcriptional activities whereas it did not inhibit DNA binding of Sp1 or AP-1 c-Fos/c-Jun. E2 inhibited Sp1 and AP-1 transcriptional activities and MCP-1 promoter activity in estrogen receptor P (ERβ) transfected SKBR3 cells. Deletion of the A/B region or mutation of activation function 2 in ERβ abrogated E2-dependent transcriptional inhibition by ERβ whereas mutation of DNA-binding domain retained the inhibitory effects. Transfection of ERβ enhanced the inhibitory effects of E2 on Sp1 and AP-1 transcriptional activities and MCP-1 promoter activities in nontransfected keratinocytes. Coimmunoprecipitation studies showed an E2-dependent association of ERβ with Sp1 or AP-1 in ERβ-transfected keratinocytes. These results suggest that E2-bound ERβ may inhibit MCP-1 gene expression by inhibiting Sp1 and AP-1 transcriptional activities in keratinocytes. A/B region and intact activation function 2 of ERβ may be responsible for the effects of E2.

Molecular regulation of monocyte chemoattractant protein-1 expression in pancreatic beta-cells.

Pancreatic beta-cells are selectively destroyed during the course of type 1 diabetes. In the early stages of the disease, inflammatory infiltrates of mononuclear cells, containing predominantly monocytes and T-cells, are present in the islets (insulitis). Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), play a key role in the recruitment and activation of these immunocytes. We have previously described cytokine-induced MCP-1 gene expression in human and rat pancreatic islets. In the present study, the transcriptional regulation by cytokines of the rat MCP-1 gene in fluorescence-activated cell sorting-purified rat beta-cells, insulin-producing INS-1E cells, and RINm5F cells was investigated. Transient transfections with luciferase-reporter constructs identified an interleukin (IL)-1beta-responsive enhancer region between -2,180 bp and -2,478 bp. Mutation of either of the two nuclear factor (NF)-kappaB sites present in this region abrogated IL-1beta-induced MCP-1 promoter activity. Binding of NF-kappaB to the two sites was shown in vitro by gel shift assays, while supershift assays revealed the presence of p65/p50 heterodimers and p65 homodimers. In vivo binding of NF-kappaB was confirmed by chromatin immunoprecipitation assay. Blocking of NF-kappaB activation in cytokine-exposed primary beta-cells by an adenovirus overexpressing a nondegradable form of IkappaBalpha or by pyrrolidine dithiocarbamate decreased IL-1beta-induced MCP-1 mRNA expression. We conclude that NF-kappaB plays an important role for MCP-1 expression in beta-cells. This transcription factor may be an interesting target for ex vivo gene therapy before islet transplantation.

Insulin Activates CCAAT/Enhancer Binding Proteins and Proinflammatory Gene Expression through the Phosphatidylinositol 3-Kinase Pathway in Vascular Smooth Muscle Cells

Phosphatidylinositol 3-kinase (PI3K) is a key molecule mediating signals of insulin in vascular smooth muscle cells (VSMCs). To examine the effect of chronic activation of PI3K on the gene expression of VSMCs, membrane-targeted p110CAAX, a catalytic subunit of PI3K, was overexpressed in rat VSMCs by adenovirus-mediated gene transfer. Similar to insulin's effects, cells overexpressing p110CAAX exhibited a 10- to 15-fold increase in monocyte chemoattractant protein-1 (MCP-1) mRNA expression as compared with the control cells. Electrophoretic mobility shift assay analysis showed that the overexpression of p110CAAX activated neither the NF-kappaB binding nor the activator protein (AP-1) binding activities. We found that two CCAAT/enhancer binding protein (C/EBP) binding sites located between 2.6 and 3.6 kb upstream of the MCP-1 gene were responsible for the induction by p110CAAX. The overexpression of C/EBP-beta and C/EBP-delta but not C/EBP-alpha caused 6- to 8-fold induction of MCP-1 promoter activity. Consistently, the overexpression of p110CAAX as well as insulin induced mRNA expression and nuclear expression of C/EBP-beta and C/EBP-delta in VSMCs. These results clearly indicate that the activation of PI3K induced proinflammatory gene expression through activating C/EBP-beta and C/EBP-delta but not NF-kappaB, which may explain the proinflammatory effect of insulin in the insulin-resistant state.