| PartⅠTHE MOLECULAR MECHANISM OF JAZF1ONCHOLESTEROL METABOLISMObjective To investigate the effect and molecular metabolism ofJAZF1overexpression on cholesterol metabolism of the mice in vivo or invitro.Methods The mRNA expression of the cholesterol metabolism relatedgenes of JAZF1, HMGCR, LDLR, PPARa, SREBP2, INSIG2, and SCAPwere determined by real-time qPCR. The protein expressions of JAZF1,HMGCR and p-CREB were determined by western blot. The cell model ofJAZF1overexpression was structured by transfection of adenovirus inprimary hepatocytes. The transcriptional activity and binding site ofHMGCR were evaluated by dual-luciferase reporter assay, which wasregulated by JAZF1. Cholesterol and related lipid index were determinedby chemical colorimetric method.Results The mRNA and protein expressions of JAZF1in liver tissuewere increased by51%and60%in C57BL/6J mice treated withAd-JAZF1. The mRNA and protein expressions of JAZF1in liver tissuewere increased by37%and60%in ApoE KO mice treated with Ad-JAZF1, wherase the protein expression of TAK1was decreased by50%. ThemRNA and protein expressions of HMGCR in liver tissue were decreasedby80%and50%in ApoE KO mice treated with Ad-JAZF1. Ad-JAZF1transfection led to a significant increase in mRNA and protein expressionof JAZF1in hepatocytes of mice. Ad-JAZF1treatment of hepatocytes frommice resulted in a27%decrease in intracellular TC. Consistent withdecreased intracellular TC, JAZF1overexpression was also associated witha51%and47%decrease, respectively of HMGCR mRNA and protein inhepatocytes of mice. Luciferase activity were decrease by54%uponAd-JAZF1with pGL3-CRE treatment, whereas JAZF1overexpressionfailed to down-regulate its luciferase activity upon Ad-JAZF1withpGL3-muCRE treatment compared with controls. JAZF1overexpressionled to a26%reduction in the phosphorylation of CREB on Ser133.Conclusion JAZF1overexpression could decrease TAK1, HMGCR,plasma cholesterol, and TC of the liver and hepatocyte, and inhibit thephosphorylation of CREB on Ser133. JAZF1overexpression alsorestrained HMGCR activity and de novo synthesis by CREB pathway. PartⅡTHE MOLECULAR MECHANISM OF KLF14ONATHEROSCLEROSISObjective To investigate the effect and molecular mechanism ofKLF14on lipid metabolism and atherosclerosisMethods pIRES2-EGFP-KLF14， pGenesil-shKLF14andpAd-shKLF14were constructed by gene recombination technology.Overexpression and suppression of KLF14in RAW264.7were constructedby LipofectamineTM2000. RAW264.7-derived foam cell were induced byAc-LDL. The cell viability was detected by MTT. Cholesterol efflux from3H-cholesterol-labeled RAW cells treated with pIRES2-EGFP-KLF14orpGenesil-shKLF14were measured by LSC. The mRNA of KLF14, ABCA1,IL-6, TNFα, and MCP-1were determined by Real-time qPCR. The proteinexpressions of KLF14, ABCA1, p-p38/p38, p-ERK1/2/ERK1/2, andp-JNK/JNK were determined by western blot. The phosphorylation of p38,ERK1/2, and JNK were inhibited by SB203580, PD098059, or SP600125,respectively.24Male ApoE KO mice were randomly divided into high-fatdiet fed+PBS group (HF-A group, n=8), HFD fed+pAd-GFP group(GFP-A group, n=8) and HFD fed+pAd-shKLF14group (shK-A group,n=8). All mice were fed for12weeks. At week8and10of HFD feeding,ApoE KO mice were also treated with pAd-shKLF14, pAd-GFP or sterilesaline by tail vein. We analyzed the lesion area of aortic sinus and aortic lesions en face by oil red O. The serum lipid was detected with enzymaticmethods.Results Both KLF14mRNA and protein expressions weresignificantly increased in HFD of C57BL/6J and apoE KO mice comparedwith SCD of C57BL/6J and apoE KO mice. TC, CE and CE/TC wereincreased about16%,25%and70%accompany with KLF14overexpression in RAW264.7. When Ac-LDL was pretreated, TC, CE andCE/TC were increased about27%,36%and7%for KLF14overexpression.TC, CE and CE/TC were decreased about9%,20%and13%for KLF14supression and decreased about16%,32%and19%for treated withpGenesil-shKLF14plus80μmol/L Ac-LDL. The protein expressions of p38,ERK1/2, but not JNK, were increased significantly by KLF14overexpression in RAW264.7. The mRNA expressions of IL-6, TNFα andMCP-1were increased about68%,120%and238%bypIRES2-EGFP-KLF14transfection. The levels of IL-6, TNFα, and MCP-1were abolished by the inhibition of p38, ERK1/2administration, exceptJNK. However, shKLF14played opposite roles mentioned above. ThemRNA and protein expression of ABCA1and cell cholesterol efflux weresignificantly decreased by KLF14overexpression for about69%,65%and63%. The plaque size and lipid deposition of aortic sinus lesions, the levelsof serum TC were reduced in pAd-shKLF14-treated apoE KO mice.Conclusion The levels of KLF14were increased in the liver and fat tissues of HFD or ApoE KO mice, which was at high risk of atherosclerosis.Cholesterol-loaded cells induction and TC, CE and CE/TC in RAW264.7were inhenced by KLF14overexpression. KLF14overexpressionaggravated inflammatory response and promoted the release ofinflammatory cytokines (IL-6, TNFα, and MCP-1) by p38/ERK1/2pathwayin macrophages. The expression of ABCA1was decreased by KLF14overexpression in macrophages. KLF14supression may inhibitinflammatory response, lower cholesterol, and play important role inatherogenesis. |
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