RESUMEN
A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner.
Asunto(s)
Proteína ADAM17/genética , Citoesqueleto de Actina/metabolismo , Anfirregulina/genética , Proteínas Cullin/genética , Fibroblastos/metabolismo , Proteína ADAM17/antagonistas & inhibidores , Proteína ADAM17/metabolismo , ADP Ribosa Transferasas/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Anfirregulina/metabolismo , Animales , Toxinas Botulínicas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/antagonistas & inhibidores , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/metabolismo , Citocalasina D/antagonistas & inhibidores , Citocalasina D/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Tiazolidinas/antagonistas & inhibidores , Tiazolidinas/farmacología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
AIMS/INTRODUCTION: Aldose reductase inhibitors (ARIs) are a useful therapy for diabetic neuropathy. Nerve damage is associated with delayed wound healing of skin ulcers in diabetic patients. Therefore, we hypothesized that ARI supplementation would improve diabetic wound healing. MATERIALS AND METHODS: Control and streptozotocin-induced diabetic mice were fed either control diet or diet containing the ARI Epalrestat (40 mg/kg). After 12 weeks, we created skin wounds on the backs of the mice. Wound healing was determined by measuring the reduction in wound area. RESULTS: The wound gap of the diabetic group was significantly larger 9 days after creating the wounds when compared to the other groups (p<0.01). Interestingly, wound healing in the diabetic mice fed Epalrestat was comparable to the non-diabetic mice. To clarify the mechanism(s) behind this improved wound healing, mRNA expression of growth factors reported to be involved in wound healing were examined. Among the growth factors investigated, only the expression of nerve growth factor (NGF) was -significantly decreased (54.0%) in the healing lesions of diabetic mice. Similarly, NGF protein expression was decreased in diabetic mice and recovered in Epalrestat treated diabetic mice. Inhibition of NGF via 2 separate inhibitors (K252a and BSO) reduced the ability of Epalrestat to improve wound healing in diabetic mice. CONCLUSIONS: These findings suggest that Epalrestat is a potential therapy for improving diabetic wound healing and the mechanism involves upregulation of NGF.