RESUMO
The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFRα to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-ß (gp130/LIFRß) for signaling. Both CLC and CNTFRα are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFRα (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC:CLF-1, as well as the trimeric (CLC:CLF-1:CNTFRα) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC:CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFRα and sorLA, CNTFRα first binds CLC:CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFRα, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC:CLF-1. The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC:CLF-1-dependent downregulation of surface-membrane CNTFRα expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFRα; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFRα.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Citocinas/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de Citocinas/metabolismo , Sítios de Ligação , Western Blotting , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Citocinas/química , Regulação para Baixo , Endocitose , Células HEK293 , Humanos , Imuno-Histoquímica , Proteínas Relacionadas a Receptor de LDL/genética , Ligantes , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/enzimologia , Proteínas de Membrana Transportadoras/genética , Fosforilação , Receptores de Citocinas/química , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Genome-wide association studies have identified a link between genetic variation at the human chromosomal locus 1p13.3 and coronary artery disease. The gene encoding sortilin (SORT1) has been implicated as the causative gene within the locus, as sortilin regulates hepatic lipoprotein metabolism. Here we demonstrated that sortilin also directly affects atherogenesis, independent of its regulatory role in lipoprotein metabolism. In a mouse model of atherosclerosis, deletion of Sort1 did not alter plasma cholesterol levels, but reduced the development of both early and late atherosclerotic lesions. We determined that sortilin is a high-affinity receptor for the proinflammatory cytokines IL-6 and IFN-γ. Moreover, macrophages and Th1 cells (both of which mediate atherosclerotic plaque formation) lacking sortilin had reduced secretion of IL-6 and IFN-γ, but not of other measured cytokines. Transfer of sortilin-deficient BM into irradiated atherosclerotic mice reduced atherosclerosis and systemic markers of inflammation. Together, these data demonstrate that sortilin influences cytokine secretion and that targeting sortilin in immune cells attenuates inflammation and reduces atherosclerosis.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Aterosclerose/imunologia , Interferon gama/imunologia , Interleucina-6/imunologia , Macrófagos/imunologia , Células Th1/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Aloenxertos , Animais , Aterosclerose/genética , Aterosclerose/patologia , Transplante de Medula Óssea , Feminino , Células HEK293 , Humanos , Interferon gama/genética , Interleucina-6/genética , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Células Th1/patologiaRESUMO
Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs.