RESUMEN
BACKGROUND: Recent studies have suggested that caveolin-1 (cav-1) plays an important role in the regulation of transforming growth factor (TGF)-ß1 signalling and participates in the pathogenesis of tissue fibrosis. However, its effects on dermal fibrosis keloids are unknown. OBJECTIVES: To investigate the effect of cav-1 in the pathogenesis of tissue fibrosis by keloid fibroblasts. METHODS: Keloid fibroblasts were cultured and exposed to different concentrations of cav-1 cell-permeable peptides (cav-1p) in the presence of TGF-ß1. Keloid fibroblast phenotypes and protein production were analysed by real-time reverse transcriptase-polymerase chain reaction, Western blot, and multiplex enzyme-linked immunosorbent assay techniques. The effect of cav-1p on cell viability was evaluated by MTT assay. RESULTS: Cav-1 was markedly decreased in the keloid-derived fibroblasts. Moreover, cav-1p significantly reduced TGF-ß receptor type I levels and Smad2/3 phosphorylation in response to added TGF-ß1. Additionally, TGF-ß1 decreased cav-1 expression in human skin fibroblasts. Cav-1 was able to suppress TGF-ß1-induced extracellular matrix production in cultured keloid fibroblasts through regulation of the mitogen-activated protein kinase pathway. CONCLUSIONS: Cav-1 appears to participate in the pathogenesis of tissue fibrosis in keloid. Restoration of cav-1 function by treatment with a cell-permeable peptide corresponding to the cav-1 scaffolding domain may be a novel therapeutic approach in keloid.
Asunto(s)
Caveolina 1/farmacología , Queloide/metabolismo , Western Blotting , Caveolina 1/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Humanos , Fenotipo , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND: Vitamin D and its metabolites play an important role in calcium homeostasis, bone remodelling, hormone secretion, cell proliferation and differentiation. Recent studies also suggest a beneficial role of vitamin D in slowing the progression of tissue fibrosis. However, their effects on dermal fibrosis and keloids are unknown. Objectives To investigate the effect of 1,25-dihydroxyvitamin D3 (1,25D) in the pathogenesis of tissue fibrosis by keloid fibroblasts (KFs). METHODS: KFs were cultured and exposed to different concentrations of 1,25D in the presence or absence of transforming growth factor (TGF)-ß1. KF phenotypes and protein production were analysed by real-time reverse transcriptase-polymerase chain reaction, Western blot, immunofluorescence and multiplex enzyme-linked immunosorbent assay techniques. Collagen synthesis was evaluated by measuring (3) H-proline incorporation. The effect of 1,25D on cell proliferation and viability was evaluated by Formazan assay, proliferating cell nuclear antigen expression and the colorimetric conversion of 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. RESULTS: We confirmed the presence of vitamin D receptors (VDRs) in cultured keloid fibroblasts. Fibroblasts transfected with a vitamin D response element reporter construct and exposed to the active vitamin D metabolite 1,25D showed increased promoter activity indicating VDR functionality in these cells. Incubation of KFs with 1,25D suppressed TGF-ß1-induced collagen type I, fibronectin and α-smooth muscle actin expression. 1,25D also modulated plasminogen activator inhibitor-1 and matrix metalloproteinase-9 expression induced by TGF-ß1. Interestingly, 1,25D induced hepatocyte growth factor mRNA expression and protein secretion in keloid fibroblasts. CONCLUSIONS: This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for keloid and related fibrotic disorders.
Asunto(s)
Dihidroxicolecalciferoles/farmacología , Fibroblastos/efectos de los fármacos , Queloide/tratamiento farmacológico , Vitaminas/farmacología , Adolescente , Adulto , Western Blotting , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Colágeno/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Técnicas de Inmunoadsorción , Queloide/metabolismo , Queloide/patología , Masculino , Fenotipo , Receptores de Calcitriol/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/farmacología , Adulto JovenRESUMEN
An assay has been developed to quantitatively measure the tension and elasticity of the cytoskeleton in living plant cells. The cell optical displacement assay (CODA) uses a focused laser beam to optically trap and displace transvacuolar and cortical strands through a defined distance within the cell. Results from these experiments provide evidence for the classification of at least two rheologically distinct cytoskeletal assemblies, cortical and transvacuolar, that differ in their tension and response to both signaling molecules and reagents that perturb the cytoskeleton. It is further demonstrated that the tension of the transvacuolar strands can be significantly decreased by the addition of either linoleic acid, 1,2 dioctanoyl-sn-glycerol, or 1,3 dioctanoylglycerol. These decreases in tension could also be induced by lowering the cytoplasmic pH. In contrast, addition of Ca2+, Mg2+, or the ionophore A23187 to the cells caused a considerable increase in the tension of the transvacuolar strands. The data provides evidence that: (a) linoleic acid may be a signaling molecule in plant cells; (b) diacylglycerol functions as a signaling molecule through a protein kinase C-independent pathway mediated by PLA2; and (c) Ca2+ and pH have regulatory roles for controlling cytoskeleton tension and organization.