RESUMO
OBJECTIVE: To determine the effect of Huntingtin-associated protein 1 ( Hap1) on fibroblast proliferation. METHODS: Hap1 knockout ( Hap1 -/-) primary fibroblasts were isolated and cultured in vitro. The proliferation of Hap1 -/- fibroblasts was detected by EdU proliferation assay and cell flow assay. Transcriptome sequencing of the wild-type and Hap1 -/- fibroblasts was screened for proliferation-related genes. Real-time quantitative PCR (qPCR) was performed to verify changes in expressions of related genes. Skin repair was examined in Hap1 knockdown mice with skin wounds. The proliferation of fibroblasts during wound repair was detected by PCNA immunohistochemical staining. RESULTS: Hap1 -/- fibroblasts were successfully cultured. Compared with WT, EdU-positive fibroblasts decreased in Hap1 -/-,with less cells entering the S phase. Transcriptome sequencing of primary fibroblasts identified genes of Cdc25C, E2f7, E2f8 and Ccl5. qPCR confirmed that Hap1 knockout increased E2f7 expression. Hap1 +/- mice had larger skin lesions, slower healing and lower positive density of fibroblast proliferation than those of wild type mice. CONCLUSION: Hap1 may positively regulate fibroblast proliferation by inhibiting the expression of cell cycle negative regulator E2f7.Its deletion inhibits fibroblasts entering the S phase, thereby reducing cell proliferation and affecting wound repair.
Assuntos
Proliferação de Células , Fibroblastos/citologia , Proteínas do Tecido Nervoso/genética , Cicatrização , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Pele/patologiaRESUMO
Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aß) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aß production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aß levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aß production in neurons.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Autoantígenos/metabolismo , Biotinilação , Encéfalo/metabolismo , Células Cultivadas , Córtex Cerebral/patologia , Citoplasma/metabolismo , Modelos Animais de Doenças , Endocitose/genética , Chaperona BiP do Retículo Endoplasmático , Ensaio de Imunoadsorção Enzimática , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Choque Térmico/metabolismo , Humanos , Imunoprecipitação , Integrinas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Neurônios/ultraestrutura , Fotodegradação , Presenilina-1/genética , Presenilina-1/metabolismo , Transporte Proteico/genética , Interferência de RNA/fisiologia , Transfecção/métodos , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismoRESUMO
OBJECTIVE: Glucose disposal by insulin-responsive tissues maintains the body glucose homeostasis and insulin resistance leads to a risk of developing type 2 diabetes (T2DM). Insulin stimulates the translocation of glucose transporter isoform 4 (GLUT4) vesicles from intracellular compartments to the plasma membrane to facilitate glucose uptake. However, the underlying mechanisms of GLUT4 vesicle translocation are not well defined. Here we show the role of huntingtin-associated protein 1 (HAP1) in GLUT4 translocation in adipocytes and the pathogenesis of T2DM. RESEARCH DESIGN AND METHODS: The parameters for glucose metabolism including body weight, glucose tolerance and insulin tolerance were assessed in wild-type (WT) and Hap1+/- mice. HAP1 protein expression was verified in adipose tissue. Hap1 mRNA and protein expression was monitored in adipose tissue of high-fat diet (HFD)-induced diabetic mice. Insulin-stimulated GLUT4 vesicle translocation and glucose uptake were detected using immunofluorescence techniques and quantified in primary adipocytes from Hap1-/- mice. The interaction between HAP1 and GLUT4 was assessed by immunofluorescence colocalization and co-immunoprecipitation in HEK293 cells and adipose tissue. The role of sortilin in HAP1 and GLUT4 interaction was approved by co-immunoprecipitation and RNA interference. RESULTS: The expression of Hap1 mRNA and protein was detected in WT mouse adipose tissue and downregulated in adipose tissue of HFD-induced diabetic mice. Hap1+/- mice exhibited increased body weight, pronounced glucose tolerance and significant insulin intolerance compared with the WT mice. HAP1 colocalized with GLUT4 in mouse adipocytes and cotransfected HEK293 cells. Furthermore, the insulin-stimulated GLUT4 vesicle translocation and glucose uptake were defective in Hap1-/- adipocytes. Finally, sortilin mediated the interaction of HAP1 and GLUT4. CONCLUSIONS: Our study showed that HAP1 formed a protein complex with GLUT4 and sortilin, and played a critical role in insulin-stimulated GLUT4 translocation in adipocytes. Its downregulation may contribute to the pathogenesis of diabetes.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Adipócitos , Animais , Diabetes Mellitus Tipo 2/genética , Transportador de Glucose Tipo 4/genética , Células HEK293 , Humanos , Camundongos , Músculo Esquelético , Isoformas de ProteínasRESUMO
OBJECTIVE: To study the mechanism of rosiglitasone to improve glucose-uptake of 3T3-L1 adipocyte with insulin resistance induced by dexamethasone and insulin. METHODS: Insulin resistance was induced to 3T3-L1 adipocyte after chronic treatment of dexamethasone and insulin. The insulin resisted 3T3-L1 adipocyte was treated with 10(-5) mol/L of rosiglitasone for 48 h. The mRNA, protein of glucose transporter GLUT4 and CAP gene were then examined. RESULTS: (1) Rosiglitasone increased the expression of GLUT4 mRNA and protein inhibited by dexamethasone and insulin although it had not reached a normal level. (2) Rosiglitasone increased the mRNA of CAP, which remained unchanged during insulin resistance. CONCLUSION: Rosiglitasone, an insulin sensitizer, might up-regulate GLUT4 and CAP along with the peroxisome proliferators activated receptor gamma (PPAR-gamma), which not only increases the GLUT4, but also activates the CAP-depended signaling pathway; improves the translocating of GLUT4 to the cell membrane; and increases the ability of glucose-uptake of cells.