Hyperphosphatemia Exacerbated Atherosclerosis By Aberrant Activation Of α-Mannosidase Promoting A Complex-Type Conversion Of SCAP N-Glycans

BackgroundAtherosclerotic cardiovascular disease(ASCVD)is the leading cause of mortality in patients with chronic kidney diseases(CKD).Hyperphosphatemia is a common complication of CKD,especially ESRD.A large number of epidemiological and clinical studies have shown that the increase of blood phosphorus level is closely related to dyslipidemia and the risk of atherosclerosis(AS).However,the mechanism is unclear.ObjectiveOn the basis of previous animal experiments and epidemiological studies,the present study is aimed to reveal the effect of hyperphosphatemia on cholesterol homeostasis in vascular smooth muscle cells(VSMCs),and to further explore the molecular mechanism of hyperphosphatemia accelerating the abnormal synthesis and accumulation of cholesterol in VSMCs,leading to atheroma plaque foam cell formation,by altering the glycans of the cholesterol sensor sterol regulatory elementbinding protein cleavage activating protein(SCAP).Methods1.Primary human aortic VSMCs were used as the cell model,which was treated by various concentrations of phosphate(Pi 1.0-3.0 mmol/L),the specific blocker for sodium phosphorus cotransporter(NPC)phosphonoformic acid(PFA),or small inference RNA(siRNA)of SCAP and sterol regulatory element binding protein 2(SREBP2).By neutral lipid oil red O staining,enzyme quantitative cholesterol determination,real-time PCR,Western blotting and laser confocal methods,the effects of high phosphorus environment(Pi 3.0mmol/L)on cholesterol level,mRNA expression,protein level and function of the key molecules in cholesterol metabolism,such as 3-hydroxy-3-methyl-glutaryl coenzyme A reductase(HMGCR),Low density lipoprotein receptor(LDLR),SREBP2 and SCAP in VSMCs were detected.2.By means of protein half-life detection,N-glycosylation induction and inhibition test,enzyme digestion identification of SCAP N-glycans and laser confocal methods,the key molecular mechanism of abnormal transactivation of SREBP2 by SCAP caused by hyperphosphatemia was explored.3.The function of α-mannosidase(α-MAN)Ⅱ,the key enzyme for complex-type conversion of N-glycans,in promoting intracellular cholesterol biosynthesis mediated by SCAP,was detected by enzymaticreaction colorimetry,real-time PCR and Western blotting.The contributions of α-MAN 2A1 and 2A2 in enhancing conversion of SCAP N-glycans from high mannose-type to complex-type were further illustrated by shRNA transfection.Results1.Under the high phosphorus environment,neutral lipids accumulation was significantly increased in VSMCs.The levels of total cholesterol(TC)and cholesterol ester(CE)are dramatically increased.High phosphorus environment did not affect the mRNA and protein levels of liver X receptor(LXR α),adenosine triphosphate binding cassette transporter 1(ABCA1),but significantly increased the gene transcription of SREBP2,HMGCR,LDLR,elevated the protein level of NH2-terminal fragment of SREBP2(SREBP2-N),HMGCR and SCAP.In high phosphorus environment,the fluorescence staining of SCAP was significantly enhanced and its co localization with the Golgi was increased.Excess phosphate up regulated HMGCR and SCAP protein levels in a dose-dependent manner(Pi 1.0-5.0 mmol/L).After treatment with 3.0mmol/L of phosphate,SCAP protein was significantly increased in 4 hours and HMGCR was significantly increased in 16 hours.PFA,a phosphorus channel blocker,can block the gene and protein expression changes of above molecules,and counteract the accumulation of cholesterol in VSMCs in high phosphorus environment.The gene silencing of SCAP or SREBP2 can significantly reduce the expression of HMGCR gene and the protein level of SREBP2-N in VSMCs,and overrode the effect of high phosphorus environment on cholesterol accumulation in VSMCs.2.Under physiological conditions(Pi 1.0 mmol/L),8 hours after the inhibition of protein synthesis by cycloheximide(CHX)in VSMCs,the protein level of SCAP decreased significantly;24 hours after CHX treatment,the protein level of SCAP decreased to 22% of 0 hours(P <0.01).However,under high phosphorus environment,24 hours after CHX treatment,the protein level of SCAP decreased significantly slower,and maintained 48.9% of that at 0 h(P < 0.01).GlcNAc(20mmol/L),the intermidate product facilitating N-glycosylation,significantly increased the expression of SREBP2 and HMGCR,while N-glycosylation inhibitor tunicamycin(Tuni,1μg/mL)counteract the up-regulation of the above moleculars expression in high phosphorus environment.Under physiological conditions,most of the N-glycans of SCAP protein are high-mannose type which is sensitive to Endo.H,while under high phosphorus environment,most of them are complex-type glycans resistant to Endo.H.After the conversion of SCAP N-glycans to complex-type,the degradation of SCAP protein was significantly delayed,which promoted the transactivation of SREBP2 and expression of its downstream molecular.GlcNAc enhanced the fluorescence intensity of SCAP in VSMCs and its co localization with the Golgi,which was similar to that in high phosphorusenvironment.Tuni weakened the fluorescence intensity of SCAP and its co localization with the Golgi in VSMCs,and counteracted the influence of high phosphorus environment on the fluorescence intensity of SCAP and its co localization with the Golgi.3.In high phosphorus environment,the activity of α-MAN Ⅱ in VSMCs increased significantly,which could be counteracted by Tuni or PFA.The activity of α-MANⅡ increased with the raising of phosphorus concentration.Silencing α-MAN 2A1 or α-MAN 2A2 gene can block the enhancement of α-MAN Ⅱ activity by excess phosphate in VSMCs,restoring the degradation rate of SCAP protein,weaken the co localization of SCAP protein with the Golgi,and reduce the mRNA expression of SREBP2 and HMGCR.Conclusions1.Under the high phosphorus condition,excess cholesterol was significantly accumulated in VSMCs,which was specifically mediated by SCAP-SREBP2 signaling pathway.At the posttranscriptional level,the protein content of SCAP increased significantly,enhanced its function of transferring SREBP2 from ER to the Golgi,increased SREBP2-N translocation into the nucleus,and upregulated HMGCR gene transcription.The latter,as the rate limiting enzyme of endogenous cholesterol synthesis in eukaryotic cells,significantly enhanced cholesterol synthesis in cells,and eventually led to abnormal cholesterol accumulation.2.Promoting or inhibiting N-glycosylation can regulate the expression of HMGCR and SREBP2.Under high phosphorus environment,the conversion of SCAP N-glycans from high mannose-type to complex-type increased significantly,which resulted in slower degradation of SCAP protein and prolonged half-life in VSMCs.The latter increased the activation of recycling transport of SREBP2 to the Golgi,and ultimately up regulated HMGCR gene transcription and endogenous cholesterol synthesis.3.Hyperphosphatemia promoted the complex-type conversion of SCAP N-glycans by increasing the activity of α-MANⅡ in VSMCs.The normal degradation of SCAP in VSMCs can be restored by blocking the uptake of phosphorus into cells from the external environment of VSMCs or inhibiting the conversion of SCAP N-glycans from high mannose-type to complex-type,maintaining the regulatory function of SCAP on SREBP2 transactivation and cholesterol homeostasis.