Effect And Mechanism Of Col XV On Adipocyte Differentiation And Adipose Metabolism

Collagen XV(ColXV)is a member of the collagens family belonged to extracellular matrix contents.It is distributed on the surface of cell base membraneand plays an important role in animal physiology and pathology.ColXVbelongs to the category of MULTIPLEXINs/endostatin-producing collagens together with collagen type XVIII(ColXVIII).MULTIPLEXINs have similar structures and they are involved in angiogenesis,inflammation,tumor formation,and adipose metabolism in a similar manner.Studies have reported that Col XVIII promotes adipocyte differentiation,increases visceral fat lipid deposition,increases tissue remodeling,and maintains energy homeostasis.Col XV is upregulated in differentiated adipocytes and is involved in obesity-related diseases,tissue regeneration and energy stabilization.However,the effectand its molecular mechanism of Col XV on adipocyte differentiation remain to be studied.In order to clarify the molecular mechanism of Col XV on adipocyte differentiation,wetransfected Col XV overexpression vector and Col XV interferencevector in adipocytes,and obtained the following results:1.Col XV promoted adipocyte differentiation and inhibitedlipolysis.Col XV expressions in abdomen fat,gonadal fat were higher than that in brown fat,liver,muscle and heart.Especially,the expressions of Col XV in subcutaneous and visceralwhite adipose tissues were significantly higher than that in brown adiposetissue(P<0.01).The expression of Col XV in inguinal whitetissue,gonadal white tissueand adiposetissue was significantly increased by HFD(P<0.01).In adipocyte,the mRNA expressions of Col XV was significantly increased with the up-regulation of C/EBP?,PPAR? and FABP4,and the expression of CREB was significantly decreased(P<0.01).Oil red O staining and Bodipy staining showed that Col XV promoted the differentiation and accelerated the accumulation of lipid droplets in adipocytes.The expressions of C/EBP?,PPAR? and FABP4 were significantly up-regulated by Col XV overexpression at 0d,4d and 8d(P<0.05).In addition,overexpression of Col XV promoted the expressions of ACC? and FASN,and inhibited the expressions of HSL,LPL and ATGL(P<0.05).The expressions of these genes were opposite in Col XV interference group.Overexpression of Col XV reduced cAMP level and p-PKAThr197 in adipocytes,Forskolin reversed these inhibitory effects,but H89 enhanced this inhibition.Interference of Col XV activated cAMP/PKA signaling pathway.The data indicated that Col XV promoted adipocyte differentiation and inhibited lipolysis by inhibiting cAMP/PKA signaling pathway.2.Col XV regulated adipocyte differentiation and lipid metabolism through CREB negative transcription of its promoter.It was well established of luciferase reporter vector with a gradually decreasing length on the Col XV promoter and CRE site mutation luciferase reporter vector.It revealed that CREB bound to the-354 bp to-334 bp of the Col XV promoter and inhibited Col XV transcription activity by luciferase activity analysis.Oilred O staining showed that overexpression of Col XV promoted lipid accumulationin adipocyte.Instead,interfering Col XV reduced lipid production,ablation of lipid droplets after CREB agonist Forskolin treatment.Overexpression of Col XV promoted the expressions of C/EBP?,PPAR? and FABP4 genes,interfering Col XV inhibited the expression of these genes,whereas Forskolin treatment further reduced the expressions of the adipogenic genes.Overexpression of Col XV promoted the expression of FASN and ACC? and blunted HSL expression.Simultaneously,Forskolin reverses the effects of Col XV on lipid metabolism.Interfering Col XV reduced the expression of FASN and ACC?,increased HSL expression,and Forskolin aggravated the effectsof Col XV interference on lipid metabolism.It is indicated that CREB negatively transcriptional regulated adipocyte differentiation and lipid metabolism through binding the promoter of Col XV.3.Col XV regulated adipocyte differentiation and adipose metabolism by DNA methylation.Compared with undifferentiated adipocytes,DNA methylation level of Col XV declined,but the expression of Col XV rised in mature adipocytes.The mRNA expressions of methyltransferasesDnmt1,Dnmt3 a,Dnmt3b and CpG binding protein Mbd3 significantly reduced during adipocyte differentiation.In undifferentiated adipocytesand differentiated adipocytes,Col XV overexpression inhibited the expressionof Dnmt1,methyltransferase inhibitors 5-azacytidine(5-Aza-dC)aggravated the decrease,but had no effects onDnmt3 a,Dnmt3b and Mbd3.5-Aza-dC inhibited the DNA methylation level of Col XV and promoted its mRNA expression.The expressions of C/EBP? and PPAR? significantly increased in Col XV overexpressed group,and 5-Aza-dC treatment reversed the effectsat 0 d.However,the increased expressions of C/EBP? and PPAR? werefurther promoted when DNA methylation of ColXV down-reglated at4 d.The HSLmRNA expressionwas significantly decreased in Col XV overexpression,and the inhibitory effect was exacerbated in low methylation of both at the 0 d and 4 d after adipocyte differentiation.Oil red O staining showed consistent results that Col XV overexpression promoted lipid deposition in differentiated adipocytes,Col XV interference inhibited lipid deposition,and lipid droplets formation was acceleratedwhen Col XV methylation was inhibited.In summary,our datasuggested that Col XV promoted adipocyte differentiation and inhibitedlipolysis bydown-regulating DNA methylation.In conclusion,the study showed that Col XV promoted adipocyte differentiation and inhibited lipolysis by inhibiting cAMP/PKA signaling pathways,and confirmed that Col XV enhanced adipocyte differentiation and blunted lipolysis through CREB negatively regulatingits transcription and DNA methylation inhibition.