Mechanism Of Nuclear Phosphatase SCP4 Contributing To Muscle Wasting In Chronic Kidney Disease

Muscle atrophy is a common complication of several diseases.Chronic kidney disease(CKD)and related inflammatory responses stimulate protein-energy wasting(PEW),a complication causing loss of muscle mass.The mechanisms resulting in muscle wasting are complex and primarily result from accelerated protein degradation.However,the mechanism by which proteolysis is regulated during muscle wasting remains unclear.We show that a novel nuclear phosphatase named SCP4/CTDSPL2(the small C-terminal domain phosphatase 2),regulates Fox O 1 and Fox O3 a signaling which influences muscle protein metabolism.CKD stimulates SCP4 expression in a NF-?B-dependent manner,and induces skeletal muscle wasting.Part one Mechanism of Nuclear phosphatase SCP4 Contributing to Protein metabolismObjective:Here we examined how a novel nuclear phosphatase named SCP4/CTDSPL2(the Small C-terminal Phosphatase 4)influences muscle protein metabolism by regulating Fox O transcription factors.Method:For overexpression or knockdown of SCP4,plasmid DNA or si RNA was introduced into cells using an electroporation transfection system.The group of overexpression and control were transfected with GFP-SCP4 and GFP,and then cellswere treated with lactacystin(8uM)and concanamycin A(0.1uM),IGF-1(10ng/ml)and BMP2(100ng/ml)respectively.The groups of knockdown and control were transfected with SCP4-si RNA(si SCP4)and scrambled(si CTL)respectively.We measured protein synthesis and degradation in C2C12 cells using Isotope.The expression of key genes involved in skeletal muscle protein metabolism,function,and disease-related processes in SCP4 forced expression was analyzed using PCR Array,.phosphorylation of multiple proteins were detected using Phospho Explorer antibody microarray after knockdown of SCP4.Western blotting were performed to determine Akt?Fox O1?Fox O3 a and SCP4 protein levels.Real-time PCR was utilized to detect the expression of atrogin-1 ?Mu RF-1 ? MUSA1 ? Myostatin.electroporation was used to generate skeletal muscle-specific deletion of SCP4.Immunofluorescence staining was performed to measure the cross sectional area of TA muscle and the expression of SCP4.Laser scanning confocal microscopy was utilized to detect nuclear translocation of Fox O1 in living cells.Results:1).SCP4 may regulate Fox Os and Smads signaling in skeletal muscle cells.2).Overexpression of SCP4 stimulates proteolysis in C2C12 myoblast.3).Overexpression of SCP4 increases atrogenes expression of Atrogin-1,Mu RF-1,MUSA and Myostatin.4).Overexpression of SCP4 promotes dephosphorylation of Fox O1 and Fox O3 a and blunt them nuclear export.5).Forced expression of SCP4 in muscle results in myofiber atrophy in mice.Conclusion SCP4 enhances the transcriptional activity of Fox O1 and Fox O3 a via regulation of them nuclear export or import,increases the expression of atrogenes,induces proteolysis and muscle wasting.SCP4 retains Fox O1 in the nucleus even p-Akt change.These indicate that SCP4 is a novel regulator for Fox O transcription factors and the cellsignaling of proteolysis.Targeting SCP4 may prevent muscle wasting.Part Two Knockdown of SCP4 prevents muscle wasting induced by Chronic kidney diseaseObjective:In The present study,we investigated the protective effects of knockdown of SCP4 in muscle atrophy induced by CKD and fasting.Methods:C2C12 cell were divided into four groups following electroporation: transfection of scrambled(si CTL),scrambled + serum free(si CTL-SF),SCP4-si RNA(si SCP4),and SCP4-si RNA+ serum-free(si SCP4-SF),the rate of protein degradation was measured by isotope.After establishing CKD mouse model,immunohistochemistry and western-blotting were used to detect the expression of SCP4 protein in skeletal muscle of control and CKD model,human abdominal muscle of healthy and CKD patients.Using electroporation technology to generate skeletal muscle-specific deletion of SCP4 by expression of si SCP4 in sham and CKD.si CTL transfection was used as control,cross sectional area of TA muscle fibers and SCP4 protein was observed by immunofluorescence staining.Western blotting was performed to detects the expression of Akt,Fox O1,Fox O3 a and SCP4 protein level.Real-time PCR was utilized to determine Atrogin-1,Mu RF-1,MUSA1,Myostatin and SCP4 m RNA level.C2C12 cells were treated by CKD-related cytokines IL-6,IFN-?,LPS and combination of them and QNZ(NF-?B inhibitor),then detected SCP4 m RNA and protein levels;Chromatinimmunoprecipitation(Ch IP assay)was used to determine the correlation of NF-?B(p65)and SCP4 promoter region.Results:1).Knockdown of SCP4 blunts proteolysis stimulated by serum-depletion,reduces transcriptional activity of Fox O1/3a,decreases expression of atrogenes of muscle.2).The expression of SCP4 is increased in muscle in Chronic kidney disease mice,SCP4 knockdown prctects CKD-induced loss of muscle mass.3).CKD-related inflammatory factors can stimulate the expression of SCP4 in C2C12 cells.NF-?B signaling might be a major regulator mediating SCP4 expression.Addition of QNZ,an NF-?B inhibitor,completely blocked SCP4 expression stimulated by the cytokine mixture.Conclusion:CKD stimulates SCP4 expression via NF-?B signaling in muscle which increases proteolysis.Knockdown SCP4 provents muscle atrophy induced by CKD and starvation.These results suggest that SCP4 may a new target to prevent muscle wasting in CKD and perhaps other catabolic conditions.