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Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβ plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.
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Objective:To observe the expression of tumor necrosis factor receptor-associated death domain (TARDD), nuclear transcription factor-κB inhibiting protein α(IκBα)IκB kinase-α (IKKα) and nuclear transcription factor (NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant (CFA) rats after treatment with Xiao Chaihutang (XCHT). Method:In animal experiments, SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table, the rats were randomly divided into the normal group, the model group, the low-dose XCHT group, the medium-dose XCHT group, the high-dose XCHT group, and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5.94 g·kg-1),medium-dose XCHT group(11.88 g·kg-1),high-dose XCHT group(23.76 g·kg-1),Tripterygium glucosides group(0.006 3 g·kg-1) were given corresponding drugs by gavage for three times a day, 2 mL/time. The histopathology of rat ankle joint was observed, and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot. Result:With the increase of the dosage of XCHT, the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats, compared with the model group, the statistical results of the low-dose XCHT group showed decreased protein expressions (PPPα, IκB α, NF-κB p65 in the NF-κB signaling pathway were significantly increased (PPα, IκBα, NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower (PPConclusion:This study shows that as the dose of Xiao Chaihutang increases, it could effectively improve synovitis, and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB, thereby preventing inflammation and suppressing bone erosion.
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Objective To investigate the expression and significance of Maspin and IKKα in nasosinusoidal mucosa of rats with fungal rhinosinusitis (FRS).Methods A total of 40 SD rats were used to establish the FRS model, and randomly divided into nasal obstruction group, FRS group, immunosuppressive group and invasive FRS group, 10 rats in each group.Another 10 normal rats were used as control group.Mice in the control group were fed with normal diet.In the nasal obstruction group, the mice had only hemostatic cotton stuffed in the nasal cavity and injection of 0.9% NaCl in the abdominal and nasal cavities.In the FRS group, the mice were injected Aspergillus fumigatus spore suspension into the nasal cavity and 0.9% NaCl i.p.The mice of the immunosuppressive group were given cyclophosphamide i.p.and 0.9% NaCl injection into the nasal cavity.The invasive FRS group was injected with cyclophosphamide i.p.and Aspergillus fumigatus spore suspension into the nasal cavity.The serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA).The expression of Maspin and IKKα in nasosinusoidal mucosa was detected by immunohistochemical staining.The expression of Maspin mRNA and IKKα mRNA in the nasosinusoidal mucosa was detected by fluorescence quantitative PCR.Results The serum levels of IL-6 and TNF-α in different groups were significantly different (P 0.05).Theresult of immunohistochemical staining showed that the protein expression of Maspin in the FRS group and invasive FRS group was significantly lower than that in the control group, nasal obstruction group and immunosuppressive group, while the expression of IKKα protein was significantly higher than that of control group, nasal obstruction group and immunosuppressive group (P< 0.05).The protein expression of Maspin in the invasive FRS group was significantly lower than that in the FRS group, by contrast, the expression of IKKα protein was significantly higher (P< 0.05).The PCRresult revealed that the expression levels of Maspin and IKKα mRNA were (0.217 ± 0.013) and (0.193 ± 0.012), significantly lower than that in the control, obstruction and immunosuppressive groups [(0.309 ± 0.021), (0.302 ± 0.017), and (0.293 ± 0.02)] (P< 0.05), while the expressions level of IKKα mRNA were significantly higher [(0.319 ± 0.043), (0.384 ± 0.048) vs (0.169 ± 0.015), (0.171 ± 0.018), and (0.175 ± 0.019)] (P< 0.05).Conclusions Down-regulation of Maspin expression after IKKα activation is the main cause of the onset of FRS, which may also be one of the mechanisms of invasive FRS.
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Objective To explore the signal transduction mechanism of inhibitor kappa B kinase α( IKKα) , one of the catalytic subunits of IKK complex , for regulating p53 transactivation in the cellular ultraviolet radiation ( UVB) repsonse. Methods The transactivation of p53 was determined by dual-luciferase reporter gene analysis system while the expression and activation of IKKα, IKKβ, p53 and p38K was detected by Western blotting assay .Results UVB exposure induced activation and transactivation of p 53 in the wild type mouse fibroblasts ,but the effect was blocked by IKKa deficiency and recovered by reconstitution of IKKαexpression.Under the same conditions , IKKαregulated p38K activation, while inhibi-ting p38K activation down-regulated p53 transactivation under UVB exposure .Conclusion IKKαregulates UVB-induced phosphorylation and activation of p 53 in a p38K-dependent manner .
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Objective To reveal the role of inhibitor of nuclear factor kappa B kinase alpha (IKKα) in renal inflammation after renal ischemia-reperfusion (IR) injury and its potential associated mechanism.Methods Ischemia-reperfusion injury models were induced in a total of 24 healthy C57BL/6 male mice.Renal function and histological changes were estimated.The expression and site of IKKα,p52,RelB,IL-10 and IL-18 were determined by immunohistochemistry and Western blotting.After the short hairpin RNA(shRNA)targeting IKKα was injected into renal parenchyma,renal function and protein expressions of IKKα,p52,RelB,IL-10,IL-18 were detected.Results Compared with sham-operated group[Scr(7.30±0.13) μmol/L,BUN (8.39± 0.30) mmol/L],levels of Scr [(29.80± 2.10)μmol/L,(27.00±3.40) μmol/L,(23.00±3.70) μmol/L] and BUN [(9.47±3.50) mmol/L,(11.68 ±4.30)mmol/L,(13.12±2.10) mmol/L] were higher on day 1,3,7 and the injury of kidney was serious in IR injury group.Immunohistochemical expression of both IL-18 and IL-10 were increased.Markedly increased IKKα,p52 and RelB protein expression were noted in experiments from day 1 to day 7 during kidney recovery period,with a peak on day 3 and then decreasing toward baseline after day 7.Compared with IR injury group,low-expression of IKKα by injection of shRNA up-regulated the expression of IL-18 and down-regulated the expression of IKKα,p52,RelB and IL-10.Conclusions The NF-κB pathway is activated and IKKα expression is up-regulated during the kidney ischemiareperfusion injury,low-expression of IKKα may block inflammation resolution via down-regulation of alternative NF-κB pathway family members of both p52 and RelB.
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Objective To investigate the mRNA expression of IKB kinase (IKK-α) and interferon-α (IFN-α) in the peripheral blood leukocytes of patients with systemic lupus erythematosus (SLE), and to explore the role of IKK-α in the production of IFN-α in SLE patients. Methods SYBR green dye I based real-time quantitative PCR was used to detect the mRNA expression levels of IKK-α and IFN-α in the peripheral blood leucocytes of SLE patients and healthy controls. Serum levels of IFN-α were measured with ELISA method. Results IKK-α mRNA expression levels in SLE patients were significantly higher than those of normal controls (P<0.05). IKK-α mRNA expression levels in SLE patients with active disease were significantly higher than patients with stable disease (P<0.01). IFN-α mRNA expression level in SLE patients was significantly lower than that of the normal controls (P<0.01). IFN-α mRNA expression levels in SLE patients with active disease were significantly higher than patients with stable disease (P<0.01). Serum levels of IFN-α in SLE patients with active disease was significantly higher than that of the normal controls and patients with stable disease (P<0.05). The anti-dsDNA antibody correlated positively, and complement C3 correlated negatively with serum concentration of IFN-α. IKK-α mRNA expression levels in SLE patients correlated positively with serum concentration of IFN-α. Conclusion IKK-α correlates positively with serum concentration of IFN-α. The IFN-α level is significantly correlated with disease activity, This suggests that IKK-α may play an important role in the pathogenesis of SLE.
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Objective To study the effect of p50 on IKKα at IL-1β promoter in LPS tolerant cells and to reveal the mechanism of the inhibition of IL-1β mRNA by pS0. Methods THP-1 human promono-cyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype was used. Chromatin immu-noprecipitation assay(CHIP) and real-time PCR were applied to quantify the binding of p50 and IKKα to IL-1βpromoter. IL-1β mRNA transcription was studied after knocking-down of p50 and/or IKKα. Results With LPS stimulation, p50 binding did not reduce but somewhat increased at IL-1β promoter in tolerant THP-1 cells. Knocking-down of p50 increased the transcription of IL-1β mRNA, which revealed the inhibi-tory effect of p50 in tolerant cells. In contrast, the accumulation of IKKα to IL-1β promoter decreased with LPS stimulation in tolerant cells; However, IKKα binding increased after p50 gene knock-down. In the meantime, IL-1β mRNA transcription increased; At last, IL-1β mRNA decreased again after double-knoc-king down of p50 and IKKα. Conclusion p50 is an inhibitory protein at IL-1β promoter in tolerant THP-1 cells. The unresponsiveness of IL-1β mRNA transcription to LPS at least partly results from the inhibition of IKKα binding to IL-1β promoter by p50.