RESUMO
Recent studies have demonstrated upregulation of monocyte chemoattractant protein-3 (MCP-3/CCL7) in fibrosis and have suggested that in addition to a major role in regulating leucocyte recruitment this chemokine may also promote extracellular matrix (ECM) overproduction by fibroblasts. In the present study we explore interplay between MCP-3 and transforming growth factor beta (TGFbeta), a potent profibrotic cytokine. We demonstrate that MCP-3 promotes activation of TGFbeta signalling pathways leading to increased type I collagen secretion. In addition we show that MCP-3 gene expression is stimulated by recombinant TGFbeta1, raising the possibility for synergy between these two mediators in the fibrotic microenvironment. Comparison of downstream signalling pathways that regulate collagen gene activation by both cytokines confirms the central role of MAPK pathway activation in mediating the effects of both factors. An additive effect of these two agonists was demonstrated by comparative microarray analysis for key TGFbeta regulated transcripts including PAI-1, OSF2 and IGFBP6. Together, our results confirm cross-talk between MCP-3 and TGFbeta that may be critical in the development of fibrosis.
Assuntos
Quimiocina CCL7/metabolismo , Colágeno/biossíntese , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Quimiocina CCL7/genética , Quimiocina CCL7/farmacologia , Colágeno/genética , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pteridinas/farmacologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To validate the reported association between CC chemokine ligand 2 (CCL2) -2518 G single nucleotide polymorphism and systemic sclerosis (SSc) in a much larger cohort of patients. We also performed subgroup analysis to test the hypothesis that CCL2 variants predispose to specific disease phenotypes. METHODS: Ninety-four Caucasian patients with SSc and 102 matched controls were genotyped by sequence-specific primers-polymerase chain reaction (SSP-PCR) methodology. RESULTS: Six biallelic single-nucleotide polymorphisms (SNP) were investigated (3 in the promoter region, 2 in the exon-coding sequence, and 1 in the 3x untranslated region), in addition to the known functional -2518 (A/G) variant. Six major haplotypes were constructed across all 7 SNP positions. No significant differences in genotype, allele, or haplotype frequency were observed between patients and controls or within disease subgroups. CONCLUSION: Genetic polymorphisms within CCL2 gene are associated with susceptibility neither to SSc nor to specific disease phenotypes.
Assuntos
Quimiocina CCL2/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , Escleroderma Sistêmico/genética , Estudos de Casos e Controles , Mapeamento Cromossômico , Estudos de Coortes , Feminino , Frequência do Gene/genética , Genótipo , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
OBJECTIVE: To investigate expression of the chemokine receptor CCR2 on key cell types involved in the pathogenesis of systemic sclerosis (SSc) and to assess the potential for autocrine activation of SSc dermal fibroblasts via CCL2/CCR2. METHODS: Chemokine receptor expression in skin biopsy tissues and explanted dermal fibroblasts from a well-characterized cohort of SSc patients was examined using immunohistochemistry and flow cytometry techniques. Autocrine regulation of the expression of fibrotic markers in CCR2+ SSc fibroblast cell lines was assessed using specific ligand or receptor antagonists. RESULTS: We identified strong CCR2 expression in skin biopsy samples of early-stage diffuse cutaneous SSc (dcSSc), but not late-stage dcSSc or limited cutaneous SSc. Double labeling confirmed up-regulation of CCL2/CCR2 on myofibroblasts, pericytes, lymphocytes, macrophages, and endothelial cells. Explanted dermal fibroblasts from early dcSSc tissues expressed CCR2 and CXCR2 in 55% and 66% of cell strains, respectively. There was no expression in control fibroblasts. CCR2+ fibroblasts demonstrated a profibrotic phenotype, with overexpression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), and CCL2. Flow cytometric analysis identified a subset of CCR2+ SSc fibroblasts expressing the myofibroblast marker alpha-SMA. In these cultures, specific inhibition of CCL2 or CCR2 attenuated the overexpression of alpha-SMA, but not CTGF or plasminogen activator inhibitor 1. CONCLUSION: Our results show that CCR2 is up-regulated in early dcSSc on cell types known to be activated in the disease, which is consistent with a key role in SSc pathogenesis. CCR2 expression on SSc fibroblasts appears to regulate the expression of CCL2 and alpha-SMA. Our findings suggest potential autocrine regulation of key profibrotic properties via a CCL2/CCR2 loop in early-stage dcSSc.