The Role And Mechanism Of DNA Polymerase Gamma In The Calcification Of Vascular Smooth Muscle Cells

Background:Mitochondria are the most important organelles in cells.It can integrate metabolic products to produce ATP and guarantee the energy required for normal operation of cells.Meanwhile,mitochondria also affect a variety of cellular functions and run through the whole process of cell fate.Therefore,the stability of mitochondrial function is the premise of normal cellular homeostasis,any mitochondrial damage or dysfunction could lead to serious outcomes such as cellular senescence and calcification.Mitochondrial DNA polymerase γ(Pol γ)is the only multi-functional enzyme combined with polymerase and exonuclease in mitochondria,which is responsible for the replication,proof and correction of mitochondrial DNA(mtDNA),to guarantee the expression of respiratory chain complex related protein subunits and maintain mitochondrial function.Pol γ can stabilize the mitochondrial function by affecting the biological behaviors of mitochondria and regulating the biogenesis and quality control of mitochondria.POLGD257A/D257A mice are characteristic as defective mitochondria caused by point mutation of POLG,D257A mutation leads to the deficiency of 3’-5’ exonuclease activity,resulting in a significant increment of the number of mutant or defective mtDNA and bring a series of serious phenotypic changes,such as premature aging,spinal deformity and severe dilated cardiomyopathy.Vascular calcification(VC)is the ectopic deposition of insoluble calcium and phosphorus salts on the vascular wall.It is common in the intima or middle layer of the artery.VC is recognized as a degenerative pathological phenomenon related to aging.Vascular smooth muscle cells(VSMCs)calcification is commonly seen in the end-stage of most chronic diseases such as hypertension,diabetes and chronic kidney disease.Moreover,the appearance of VC usually indicates adverse clinical outcomes.It has been found that mitochondrial dysfunction and alterations of cellular metabolism could often be observed in calcified vessels.However,the research on VC in POLGD257A/D257A mice has not been fully carried out,it is still unknown whether Pol γ participate in the process of calcification and the role of Pol γ in VC is still not clear.Meanwhile,whether there will be functional differences between this specific mutant Pol γ and normal Pol γ under calcification stimulation condition remains to be studied.P53 is the most important transcription factor in cells and participates in a variety of cellular biological processes,one of them is to regulate mitochondria by a variety approaches.Some studies believed that p53 could regulate the redox homeostasis and metabolism of mitochondria and suggested that p53 could control the quality of mitochondria by regulating the biogenesis of mitochondria and affecting the autophagy and apoptosis of mitochondria.In addition,it has been reported that p53 could promote the level of aerobic metabolism by transcriptional regulation of different mitochondrial related genes and affect the function of mitochondria indirectly.In terms of response to injury,some research suggested that p53 would interact with pol y to repair damaged mtDNA.However,it is still unclear whether p53 would also interact with pol y to participate in the process of calcification and the maintenance of mitochondrial function under calcification stimulation,and whether mutant pol y could affect the biological behavior and function of p53 remains to reveal.Objective:The present study based on the POLGD257A/D257A mitochondrial defective mouse model and conducted the related research around the relationship among pol y,P53 and calcification.We aim to identify the causal relationship between mitochondrial function and calcification,reveal the role of Pol y in maintaining mitochondrial function and VSMCs calcification,and clarify the potential mechanism of Pol y and p53 in the regulating mitochondrial function and VSMCs calcification by comparing the different status of pol y.Methods:In the first part,we induced VC in POLGWT/WT mice and POOLGD257A/D257A mice using Vitamin D3(Vit D3)and compared the degree of aortic VC between these two mice.Meanwhile,Pol y overexpressed and knockdown VSMCs were constructed and treatment with l0mmol beta-glycerophosphate(β-GP)for in vitro calcification.Alterations of mitochondrial function and calcification degree under different Pol y quantity were detected.On this basis,we designed Pol y point mutant plasmids,and compared the function alterations of stabilizing mitochondria and inhibiting calcification before and after Pol y mutation by overexpressing different types of Pol y in VSMCs.Besides,we performed WT-Pol y and Mutant-Pol y back-complementation expression in ShPol y VSMCs to compare their different roles in rescuing mitochondrial functions and to reveal observe the effect recovered mitochondrial function of in inhibiting calcification.In the aspect of mitochondrial function detection,we detected the mitochondrial membrane potential alterations,ATP production and ND-1 mRNA level respectively to reflect the changes in mitochondrial structure,function and mitochondria quantity.In terms of calcification,Alizarin red staining and quantitative detection of calcium ions deposition were conducted in this study to reflect the formation of calcified nodules and calcium salt deposition level,and the calcification degree of vascular tissues or VSMCs was comprehensively evaluated by detecting the expression level of core proteins in the calcification process.In the second part of the experiment,we constructed p53 knockdown VSMCs and examined the changes in cellular mitochondrial function under different p53 levels and calcification conditions.Pol γ plasmids were overexpressed in p53 knockout VSMCs,and Pol γ plasmids were overexpressed in p53 knockout VSMCs under 10mmolβ-GP calcification conditions.By comparing the mitochondrial functions of these two systems to reveal the roles of Pol γ and p53 in maintaining mitochondrial stability and inhibiting cellular calcification.Meanwhile,the mitochondria of VSMCs were isolated under the condition of calcification induced by β-GP,and the changes in the expression of p53 in different subcellular structures as well as the effect of calcification on the changes in the location of p53 were examined.Finally,co-immunoprecipitation(CO-IP)experiments were performed in Vit D3-induced vascular tissues of POLGWT/WT mice and POLGD257A/D257A mice to clarify the potential mechanism of Pol γ co-operate with p53 to maintain the mitochondrial homeostasis and regulate the calcification process.And we also conducted in vitro semi-exogenous overexpression to confirm and complement the in vivo binding alterations.Results:In the first part of the functional research we observed that POLGD257A/D257A mice presented more severe VC degree than POLGWT/WT mice under Vit D3 induced calcification conditions.The results of in vitro experiments showed that the overexpression of Pol γ could upregulate various mitochondrial functional indices,and the VSMCs in the related group showed an attenuated calcification states.Meanwhile,Pol γ deficiency could lead to a significant decrease in mitochondrial membrane potential,ATP content and mtDNA copy number under β-GP-stimulated conditions,and correspondingly the calcification degree was significantly increased.And furthermore,this mitochondrial functional impairment and aggravated calcification could be rescued by overexpression of Pol γ.However,the Mutant-Pol γ overexpression alone could not improve mitochondrial function and alleviate the extent of calcification.Moreover,unlike WT-Pol γ,we didn’t observe the overexpression Mutant-Pol γ based on ShPol γ VSMCs bring the upregulated effects of mitochondrial function and reduction of calcification under β-GP conditions,the cells still showed impairment of mitochondrial function and severe calcification.In the second part of the mechanism experiments,we observed that Pol γ overexpression significantly upregulated mitochondrial functional indices.Besides,calcification stimulation promoted p53 phosphorylation at the Ser392 site and translocated into mitochondria.WT Pol γ could interact with p53 and form a complex and this binding was significantly enhanced by calcification stimulation.However,the binding of Mutant Pol γand p53 has already been maintained at a high level under physiological conditions,and the exogenous calcification stimulation failed to further enhance the binding level of Mutant Pol γ-p53 complex and even this reduce interaction.Conclusion:Mutant Pol γ induced mitochondrial function impaired could promote the development of calcification in VSMCs under the exogenous stimuli.Pol y maintains mitochondrial stability and rescues mitochondrial function against exogenous calcification.However,Pol γ would fail to maintain mitochondrial stability in response to damaging stimuli after the D257A point mutation,and could not rescue mitochondrial dysfunction caused by defective Pol γ,and no longer had a role in inhibiting calcification in VSMCs,which suggesting that the integrity of Pol γ nucleic acid exonuclease function is essential in maintaining stable mitochondrial function and inhibiting calcification.Meanwhile,Polγ is the execution molecule which directly decide mitochondrial function,and p53 could downstream regulate and enhance Pol γ in mtDNA correction and stabilization which indirectly participating in the process of stabilizing mitochondrial function.And the mitochondria impaired under calcification conditions could promote the translocation of p53 into mitochondria to assist in maintaining mitochondrial function,and phosphorylated Ser392 position of p53 plays an important role in the translocation process of p53.In addition,the ability of Pol γ to form a complex with p53 before and after the D257A mutation in response to calcification-induced mtDNA damage to initiate repair mechanisms is different,and Mutant Pol γ-p53 has a reduced ability to respond to damage stimuli and cannot be further activated to maintain mtDNA stability and inhibit calcification in the presence of pathological damage.