RESUMEN
The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1highCD11bhighVLA-3high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities.
Asunto(s)
Inflamación/inmunología , Integrina alfa3beta1/metabolismo , Pulmón/inmunología , Insuficiencia Multiorgánica/inmunología , Neutrófilos/inmunología , Peritonitis/inmunología , Proteína C/metabolismo , Animales , Terapia Biológica , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Activación Neutrófila , Unión Proteica , Proteína C/genética , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: Chinese medicine comprised of all natural herbs is widespread used in the treatment of diabetic nephropathy (DN). Podocyte contributes to the integrity of glomerular filtration barrier whose injury plays an important role in the initiation and progression of DN. Our study aimed to investigate the effect of Qiwei granules on podocyte lesion in diabetic KK-A(y) mice kidney and its underlying mechanism. METHODS: Twelve-week-old male KK-A(y) mice were randomly divided in vehicle group and Qiwei granules group, while C57BL/6J mice were used as normal control. The mice were gavage with 1.37 g/kg/day Qiwei granules or water for 10 weeks. We measured water, food intake and body weight (BW) and fasting blood glucose (FBG) every 2 weeks, and urine protein every 4 weeks. At the end of the experiment, all surviving mice were sacrificed. The kidney weight and serum renal parameters were measured, and the renal morphology was observed. To search the underlying mechanism, we examined the podocyte positive marker, slit diaphragm protein expression and some involved cell signal pathway. RESULTS: Qiwei granules treatment significantly improved the metabolic parameters, alleviated the urinary protein, and protected renal function in KK-A(y) mice. In addition, the glomerular injuries and podocyte lesions were mitigated with Qiwei granules treatment. Furthermore, Qiwei granules increased expression of nephrin, CD2AP, and integrin alpha3beta1 in the podocytes of KK-A(y) mice. Qiwei granules improved the phosphoration of Akt and inhibited cleaved caspase-3 protein expression. CONCLUSION: These finding suggest that Qiwei granules protects the podocyte from the development of DN via improving slit diaphragm (SD) molecules expression and likely activating Akt signaling pathway in KK-A(y) mice.
Asunto(s)
Nefropatías Diabéticas/tratamiento farmacológico , Medicamentos Herbarios Chinos/uso terapéutico , Riñón/efectos de los fármacos , Magnoliopsida , Fitoterapia , Podocitos/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Biomarcadores/metabolismo , Peso Corporal , Caspasa 3/metabolismo , Proteínas del Citoesqueleto/metabolismo , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Medicamentos Herbarios Chinos/farmacología , Integrina alfa3beta1/metabolismo , Riñón/metabolismo , Riñón/patología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Podocitos/patología , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Cordycepin, also known as 3-deoxyadenosine, is an analogue of adenosine extracted from the traditional Chinese medicine "Dong Chong Xia Cao". Cordycepin is an active small molecular weight compound and is implicated in modulating multiple physiological functions including immune activation, anti-aging and anti-tumor effects. Several studies have indicated that cordycepin suppresses tumor progression. However, the signaling pathways involved in cordycepin regulating cancer cell motility, invasiveness and epithelial-mesenchymal transition (EMT) remain unclear. In this study, we found that cordycepin inhibits hepatocellular carcinoma (HCC) cell proliferation and migration/invasion. Treatment of cordycepin results in the increasing expression of epithelial marker, Ecadherin while no significant effect was found on N-cadherin α-catenin and ß-catenin. Furthermore, although the expression of focal adhesion kinase (FAK) was slightly reduced, the level of phosphorylated FAK was significantly reduced by the treatment of cordycepin. In addition, cordycepin significantly suppresses the expression of integrin α3, integrin α6 and integrin ß1 which are crucial interacting partners of FAK in regulating the focal adhesion complex. These results suggest cordycepin may contribute to EMT, antimigration/ invasion and growth inhibitory effects of HCC by suppressing E-cadherin and integrin/FAK signaling. Thus, cordycepin is a potential therapeutic or supplementary agent for preventing HCC tumor progression.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma Hepatocelular/metabolismo , Desoxiadenosinas/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrina alfa3beta1/metabolismo , Integrina alfa6beta1/metabolismo , Neoplasias Hepáticas/metabolismo , Cadherinas/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Integrina alfa3beta1/genética , Integrina alfa6beta1/genética , Neoplasias Hepáticas/patología , Transducción de SeñalRESUMEN
We show that human osteosarcoma cells (Saos-2) have downregulation of alpha3beta1-integrin compared to normal bone cells; this was further described in human osteosarcomas and in a primary murine sarcoma. The alpha3 gene was silenced in Saos-2 cells causing a low expression of alpha3beta1-integrin and reduction in collagen attachment with increasing migratory capacity. Chromatin immunoprecipitation assay performed on alpha3 promoter established that Myc and Yin Yang protein (YY1) cooperate in tandem to downregulate the alpha3 gene. This silencing mechanism involves the binding of Myc and YY1 to DNA and formation of complexes among Myc/Max, YY1, CREB-binding protein and deacetylation activity. The promoter containing deletions of E-boxes or YY1 cassettes failed to downregulate the transcription of a reporter gene as well as the inhibition of deacetylation activity. Overexpression of both Myc and YY1 was necessary to determine the alpha3-integrin promoter downregulation in normal osteoblasts. This downregulation of alpha3beta1-integrin can contribute to the acquisition of a more aggressive phenotype. YY1 regulated negatively the Myc activity through a direct interaction with the Myc/Max and deacetylase complexes. This represents a novel silencing mechanism with broad implications in the transcription machinery of tumours.
Asunto(s)
Silenciador del Gen , Integrina alfa3/genética , Integrina alfa3beta1/metabolismo , Osteosarcoma/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Transcripción YY1/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Western Blotting , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Inmunoprecipitación de Cromatina , Colágeno/metabolismo , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Laminina/metabolismo , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Osteoblastos/citología , Osteoblastos/metabolismo , Osteosarcoma/genética , Osteosarcoma/patología , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Factor de Transcripción YY1/antagonistas & inhibidores , Factor de Transcripción YY1/genéticaRESUMEN
Beta ig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-beta1. Whereas beta ig-h3 is known to mediate keratinocyte adhesion and migration, its effects on keratinocyte differentiation remain unclear. In the present study, it was demonstrated that expression of both beta ig-h3 and TGF-beta1 was enhanced during keratinocyte differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in beta-h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of beta ig-h3 by transfection with antisense beta ig-h3 cDNA constructs effectively inhibited keratinocyte differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was an approximately 2-fold increase in mitotic capacity of the cells. Conversely, overexpression of beta ig-h3, either by transfection with beta ig-h3 expression plasmids or by exposure to recombinant beta ig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant beta ig-h3 also promoted keratinocyte adhesion through interaction with integrin alpha3beta1. Changes in beta ig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant beta ig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced beta ig-h3, induced by enhanced TGF-beta during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin alpha3beta1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that beta ig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation.