The Role Of FADD Phosphorylation In Insulin Degrading Enzyme And Renal Fibrosis

In 1995,Fas-associated death domain-containing protein(FADD)was the first to be discovered as an important ligand protein of Fas-induced apoptosis.It is a 23-k Da protein,which contains N-terminal death effector domain(DED)and C-terminus death domain(DD).FADD,a classical apoptotic signaling adaptor,has recently been reported to exhibit a series of non-apoptotic functions,such as inflammation,autophagy,lymphocyte development and proliferation,cell cycle progression,embryonic development,tumorigenesis,glucose metabolism,innate natural immunity,and regeneration and so on.Today,non-apoptotic function of FADD has become a new hotspot.Further studies indicated that the non-apoptotic function of FADD may be correlated with its phosphorylation.The phosphorylation site(serine 194 in human and serine 191 in mouse)of FADD protein is outside of the apoptotic domains.Mice bearing the Asp mutation in the serine 191 of FADD(FADD-D),mimicking constitutive phosphorylation at serine 191,had been used to investigate the physiological functions of FADD phosphorylation.In the present study,the relationship between FADD phosphorylation and glucose metabolism was investigated in FADD-D and control mice,HepG2 cells,HEK293 T cells,and the primary cells extracted from livers.As abnormal glucose metabolism can cause renal dysfunction,the effect of FADD phosphorylation on renal fibrosis was furtherly explored in FADD-D mouse model,FADD-disrupted HEK293 cells,and FADD-deficient MES cells.Because the pancreas is an insulin-producing organ,we extracted the islet cells from the pancreas.The mRNA expression level of insulin was found downregulated in the islets of FADD-D mice.In vitro,it was found that insulin secretion was impaired in FADD-D mice by detecting glucose-stimulated insulin secretion(GSIS)function of islet cells isolated from the pancreas.The results showed that FADD-D mice exhibit reduced serum glucose levels,increased insulin levels,and impaired GSIS function compared to those in control mice.The insulin synthesis was decreased but the accumulation of insulin in serum was increased in FADD-D mice,indicating that the function of insulin degradation was impaired.Because the liver is rich in insulin degrading enzyme(IDE),which is the main organ of insulin metabolism.So if the function of IDE was impaired in the liver,the biological effects and degradation of insulin would be abnormal and cause hyperinsulinemia.Therefore,FADD phosphorylation may affect the expression of IDE and lead to hyperinsulinemia in FADD-D mice.Mice bearing the Asp mutation in serine 191 of FADD were used to investigate the effect of FADD phosphorylation on IDE.Our data showed that the mRNA and protein levels of IDE were significantly downregulated in FADD-D mice compared to control mice by quantitative reverse transcription PCR(RT-q PCR)analysis,Western blotting,and immunohistochemistry staining.The same results were observed in FADD-D primary hepatocytes.The expression and stability of IDE mRNA were performed after FADD knockdown in HepG2 cells.We found that FADD iss able to regulate the expression of IDE at the transcriptional level without affecting the stability of mRNA through qRT-PCR after FADD knockdown in HepG2 cells treated with Atc.D.IDE protein stability was analysed after cyclohexane(CHX)and insulin stimulation in the primary hepatocytes.After the primary hepatocytes were treated with the different time of CHX,IDE expression levels were gradually reduced.After HEK293 T cells were transfected with FADD si RNA / NC fragments,the different concentrations of CHX were treated with cells for 24 h,the same results were observed.After the primary hepatocytes were starved for 24 h,IDE expression levels were also reduced in insulin-stimulated primary hepatocytes of FADD-D mice compared to control mice.FoxO1 is an important transcription factor in glucose and lipid metabolism.The luciferase activity analysis was used to observe the relation between FoxO1 and IDE promotor.We found that transfected FoxO1 and TSS plasmid were able to reduce the expression of IDE at the transcriptional level through promoter luciferase analysis in HEK293 T cells.FoxO1 inhibits the luciferase activity of the IDE promoter as revealed by the results of the luciferase reporter assay.For immunofluorescence assays,nuclear translocation of FoxO1 was observed.SIRT1 was the important regulatory factor in term of glucose and lipid metabolism in the liver.The SIRT1 activator or inhibitor was added into HepG2 cells and the expression of IDE was downregulated or upregulated respectively.The results showed that SIRT1 may regulate the expression of IDE negatively.The expression of SIRT1 was increased in FADD-disrupted HepG2 cells.We speculated that FADD may regulate the expression of SIRT1 negatively.In conclusion,it was found that deficient FADD or its phosphorylation may inhibit IDE expression through FoxO1 and SIRT1,which needs further confirmation.As abnormal glucose metabolism may cause renal function damage,we reported the effect of FADD phosphorylation on the kidney in FADD-D mouse model.Renal fibrosis is a variety of the primary or secondary continuous progression of kidney disease.The normal renal tissue structure is replaced by extracellular matrix(ECM)with renal function irreversible damage of the pathological process.The process of renal fibrosis includes three parts: monocyte / macrophages and other inflammatory cells infiltration,fibrogenic cytokines upregulated,and the increase of matrix protein synthesis and inhibiting matrix degradation.Here,we reported that FADD plays an important role in the development of renal fibrosis.Mice were anatomied to weight the double kidneys after ether anaesthesia.The results showed that the weights were reduced but the relative weights of double kidneys were increased significantly in FADD-D mice.Mice were placed in metabolic cages to collect urine volume / 24 h.And the results showed that urine output was significantly reduced,urinary protein was increased,and hematuria displayed under microscopic by Giemsa staining of urine smears in FADD-D mice.The serum levels of creatinine,urea nitrogen,and uric acid were increased in FADD-D mice by using assay kits.These results indicated that the functions of the glomerular filtration and reabsorption were damaged in FADD-D mice.Proteomics results showed that the glucose metabolism was abnormal,which may cause renal damage.At the same time,it was observed that the capillary was sparse in the surrounding of glomerular and displayed renal vascular damage by two-photon microscope.Renal function damage may be associated with nephritis and renal fibrosis.Neutrophil infiltration in the renal interstitial part,glomerular mesangial cell proliferation,and base-membrane thickness were observed in FADD-D mice by H&E,PAS,and PASM staining.The results of immunofluorescence analysis revealed that macrophage infiltration was significantly enhanced in FADD-D mice.ELISA assay was used to measure the levels of IgA,IgM,IgG,C3,and C1 q in the serum and the kidney,the results of which figured out that IgA was deposed in the kidney of FADD-D mice.The results of immunofluorescence experiment were also found the same profiles in the glomerular.IgA nephropathy is characterized of the recurrent gross or microscopic hematuria,the proliferation of glomerular mesangial cell,and increased matrix.The expression levels of lanminin and collagen Ⅳ were also found to be elevated in FADD-D mice by immunohistochemistry experiments.Renal fibrosis was increased by IgA nephritis in FADD-D mice as evidenced by increased expression levels of Ki67 and PCNA in mice.In addition,the expression levels of α-SMA,Fibronectin,and vimentin were also found to be elevated in FADD-D mice by immunofluorescence experiments.qRT-PCR and western blot analyses indicated that the TLR4 / my D88 / NF-κB signaling pathway was activated in FADD-D mice.The downstream genes of transcription factor NF-κB,such as IFN-γ、IL-6、TGF-β1、COX2、IL-1β,and TNF-αwere also upregulated,as revealed by q PCR analysis.In vitro,the assays indicated that TGF-β1 pathway was also activated and the process of EMT was accelerated in both FADD-disrupted HEK293 cells and FADD-deficient MES cells by western blot.In brief,FADD phosphorylation activated the proteins associated with mTOR and TGF-β / Smad pathways and accelerated the process of EMT.Moreover,the activated degree of TGF-β pathway was enlarged after using TGF-β1 stimulation.Thus,we concluded that FADD phosphorylation could lead to IgA nephritis and eventually result in renal fibrosis.Taken together,our study provides evidence,for the first time,that FADD,especially its phosphorylated form,has an impact on IDE.It was also found that FADD phosphorylation affects nephritis and renal fibrosis.With investigations on IDE and renal function in FADD-D mice,we have expanded non-apoptotic functions of FADD and its phosphorylation,providing a further understanding and insight regarding FADD and its phosphorylation and the development of disease.