RESUMO
Stabilin-1 is specifically expressed in alternatively activated macrophages. These macrophages participate in anti-inflammatory and healing processes, and display a high phagocytic capacity. In this study, we provide evidence that stabilin-1 is a membrane receptor that performs a crucial function in the clearance of cell corpses. Stabilin-1 is expressed on the cell surface of alternatively activated macrophages and is recruited to the sites of recognition and engulfment of apoptotic bodies, as well as to early phagosomes. Blocking stabilin-1 in macrophages results in defective engulfment of aged red blood cells. Ectopic expression of stabilin-1 induces the binding and engulfment of aged cells in mouse fibroblast L cells. The binding and phagocytosis are dependent on phosphatidylserine (PS), which is well known as an engulfing ligand. Furthermore, using PS-coated beads, we demonstrate that PS directly interacts with stabilin-1 and is sufficient for stabilin-1-mediated phagocytosis. EGF-like domain repeat in stabilin-1 is responsible for PS recognition and binding. Thus, our results demonstrate that stabilin-1, found on alternatively activated macrophages, is a phagocytic receptor mediating the clearance of apoptotic cells in a PS-dependent manner. Therefore, this protein might play an important role in the maintenance of tissue homeostasis and prevention of autoimmunity.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/metabolismo , Fagocitose , Fosfatidilserinas/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular Neuronais/química , Linhagem Celular , Senescência Celular/efeitos dos fármacos , Dexametasona/farmacologia , Fator de Crescimento Epidérmico/química , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Humanos , Interleucina-4/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Fagocitose/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Receptores de Retorno de Linfócitos/químicaRESUMO
The clearance of apoptotic cells is critical during cellular homeostasis as well as inflammation resolution. Recently, we found that stabilin-1 is a phagocytic receptor that is involved in the clearance of apoptotic cells. However, the downstream signaling pathway of stabilin-1-mediated phagocytosis remains to be investigated. Here we identify that GULP is able to specifically interact with the NPxF/Y motif of stabilin-1 cytoplasmic region. The PTB domain of GULP is necessary for interaction with stabilin-1. GULP is enriched around PS-coated beads for phagocytosis and co-localized with stabilin-1. Downregulation of endogenous GULP expression decreased stabilin-1-mediated phagocytosis. Thus, these results indicate that GULP functions as an adaptor protein for stabilin-1-mediated phagocytosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Fagocitose , Receptores de Retorno de Linfócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células CHO , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Retorno de Linfócitos/genéticaRESUMO
betaig-h3 is an extracellular matrix (ECM) protein induced by TGF-beta, and it has motifs interacting with the alpha3beta1, alphavbeta5, and alphavbeta3 integrins. Our previous study shows the role of betaig-h3 in osteoblast differentiation and its involvement in melorheostosis, a rare bone disease. Here we demonstrate that betaig-h3 expression is down-regulated during the early stage of differentiation of the murine preosteoblastic cell line, KS483. The recombinant betaig-h3 and its FAS1 domain significantly inhibited in vitro osteoblast differentiation as evaluated by matrix mineralization/bone nodule formation. Furthermore, inhibition of expression of osteoblast differentiation marker genes [such as type I collagen, alkaline phosphatase, and osteocalcin (OC)] was accompanied by suppression of osteoblast-specific transcription factors, Cbfa1/Runx2 and osterix. Flow cytometric analyses, cell adhesion, and inhibition assays disclosed alphavbeta3 and alphavbeta5 as the principal integrins mediating the adhesion of osteoblastic cells to betaig-h3. The disruption of interactions between betaig-h3 and osteoblasts by a function-blocking antibody specific for alphavbeta3 but not for alphavbeta5 abolished the inhibitory effect of betaig-h3 on osteoblast differentiation. We suggest that these interacting integrins may play an important role in betaig-h3-mediated inhibition of osteoblast differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Inibidores do Crescimento/fisiologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Sequência de Aminoácidos , Animais , Matriz Óssea/metabolismo , Matriz Óssea/fisiologia , Calcificação Fisiológica/fisiologia , Adesão Celular/fisiologia , Linhagem Celular , Células Cultivadas , Regulação para Baixo/fisiologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genéticaRESUMO
The prompt clearance of cells undergoing apoptosis is critical during embryonic development and normal tissue turnover, as well as during inflammation and autoimmune responses. We recently demonstrated that stabilin-2 is a phosphatidylserine receptor that mediates the clearance of apoptotic cells, thereby releasing the anti-inflammatory cytokine, transforming growth factor-beta. However, the downstream signaling components of stabilin-2-mediated phagocytosis are not known. Here, we provide evidence that the adaptor protein, GULP, physically and functionally interacts with the stabilin-2 cytoplasmic tail. Using fluorescent resonance energy transfer analysis and biochemical approaches, we show that GULP directly binds to the cytoplasmic tail of stabilin-2. Knockdown of endogenous GULP expression significantly decreased stabilin-2-mediated phagocytosis. Conversely, overexpression of GULP caused an increase in aged cell engulfment. The phosphotyrosine binding (PTB) domain of GULP was sufficient for the interaction with stabilin-2; therefore, transduction of TAT fusion PTB domain acts as a dominant negative, resulting in impaired engulfment of aged red blood cells in stabilin-2 expressing cells. In addition, the PTB domain of GULP was able to specifically interact with the NPXY motif of the stabilin-2 cytoplasmic tail. Taken together, these results indicate that GULP is a likely downstream molecule in the stabilin-2-mediated signaling pathway and plays an important role in stabilin-2-mediated phagocytosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Animais , Apoptose , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Eritrócitos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Biológicos , Ligação Proteica , Transdução de SinaisRESUMO
Fibronectin (FN) plays a role in various biological processes such as fibrosis and tumor metastasis. In this study, we investigated the regulation of FN gene expression by NF-kappaB transcription factor. Transient expression of NF-kappaB p65 increased FN promoter activity in rat hepatocytes. Deletion analysis of FN promoter localized the NF-kappaB-responsive region at the position between -1214 and -1126. Mutations in a putative NF-kappaB element (5(')-GAGAATTTCC-3(')) at -1180 blocked most of the p65-induced promoter activity. Electromobility shift assays showed that the expression of p65 induced the binding of the p65/p65 homodimer to the NF-kappaB site at -1180. Stably p65-expressing cells showed increase of promoter activity, FN protein, and its mRNA levels over control cells. Furthermore, treatment of cells with interleukin-1beta, a NF-kappaB-stimulating cytokine, also increased promoter activity, FN production, and mRNA levels. These results show that NF-kappaB activates FN gene expression by binding to the responsive element at -1180 as the p65/p65 homodimer in rat hepatocytes.