Effect Of LRP16Gene On Leptin Of3T3-L1Adipocytes And The Molecular Mechanism

Objective:Leukemia-related protein16(LRP16), localized on chromosome11q12.1, is anovel gene cloned from human lymphocyte cells by our group in the year1999. Thatstudy found that estrogen down-regulated mRNA expression of LRP16by binding tothe LRP16promoter, which was either the estrogen-responsive element half-site (at–246to–242bp) or the GC-rich Sp1binding site (at–236to–227bp). We also foundthat LRP16was a co-activator of ERα. Recently, there is strong evidence from largerandomized controlled trials that E2-based hormone therapy attenuates weight gain inpostmenopausal women. Data from clinical trials have revealed menopausal hormonetherapy (MHT) with combined estrogen-progestins can reduce weight gain and elicitadipose tissue redistribution in postmenopausal women. Studies have also that theestrogen receptors alpha (ERα) and ERβ are present in white adipocytes, and estrogenscan regulate leptin expression and secretion in these cells. This indicates that LRP16gene, as a downstream targeted gene of estrogen receptors alpha (ERα), may regulatethe leptin gene expression and protein secretion in white adipocytes. In this study, weutilized an over-expression strategy to regulate the LRP16gene's expression andthereby demonstrated effect of LRP16gene on leptin of3T3-L1adipocytes and themolecular mechanism.Methods:1) Construction the3T3-L1cell with over expression of LRP16The cell were seeded in6cm culture dish with density of80%,24hours later,pcDNA-3.1-16and pcDNA-3.1were transfected into these cells using superfecttransfection according to the introduction. Another24hours later, the DMEMcontaining G418(800μg/ml) was used to screening for resistance to3weeks. Thenthe cells was cultured with the DMEM containing G418(400μg/ml) to construct cell lines with LRP16over-expression and the control cells.2) Effect of LRP16gene on expression of leptin in3T3-L1adipocytea) The protein expression of leptin and LRP16were measured in3T3-L1cellsduring differentiation (0d,4d,8d) using Western blot.b) Measurement of ERа protein expression in mature3T3-L1cells which wereserum-starved for12h and then, incubated with or without chemical reagents atvarious concentrations for24h (PPT: ERа selective agonist and MPP ERаselective antagonist) using Western blot.c) Measurement of leptin mRNA in both3T3-L1-16and3T3-L1-3.1cells whichwere serum-starved for12h and then, incubated with or without PPT or MPP at10-510-7and10-9mol/L for24h using Real-time Quantitative PCR.d) Measurement of leptin protein in both3T3-L1-16and3T3-L1-3.1cells whichwere incubated with PPT or MPP for24h or48h at10-510-7and10-9mol/Lusing ELSIA.e) Measurement of leptin protein in both3T3-L1-16and3T3-L1-3.1cells whichwere incubated with PPT or MPP for24h at t10-5mol/L using Western blot.3) Effect of LRP16gene on JAK/STAT signaling in3T3-L1adipocytesa) Measurement of JAK2mRNA STAT3mRNA and SOCSmRNA in both3T3-L1-16and3T3-L1-3.1using Real-time Quantitative PCR. Measurementof pJAK2tyr (1007/1008)and pSTAT3Tyr (705)protein expression using western blot.b) Measurement of p-JAK2tyr (1007/1008)and p-STAT3Tyr (705)protein expression inboth3T3-L1-16and3T3-L1-3.1cells which were incubated with MPP for24hat10-5mol/L using western blot.4) Effect of LRP16gene on transcriptional activity of leptin genea) The structural expression vector of leptin(-762bp～+29bp) was constructed andconnected with pGL3-basic vector (p(-762)lep-luc)b) The structural expression vectors, including p(-762)lep-luc and ERa,pcDNA-3.1,pcDNA-3.1-16and the internal control vector pRL-TK weretransiently co-transfected into MCF-7cells COS-7cells and Hela cells, then relative luciferase activity of p(-762)lep-luc was detected using Dual-luciferaseReporter Gene Assay system. Effect of ERa on p(-762)lep-luc relative luciferaseactivity was measured after incubating with ERa antagonist.Results:1) Construction the3T3-L1cell lines with over-expression of LRP16Lipidosome transfection was used to construct the cell lines with over-expression ofLRP16. The stable transfected cells were selected with G418. Western blotindicated that LRP16protein in3T3-L1-16was2.37fold of that of3T3-L1-3.1cells(p<0.05).2) Effect of LRP16gene on expression of leptin in3T3-L1adipocytesa) The protein of leptin and LRP16in3T3-L1cells increased significantly duringdifferentiation.b) The ERa expression in3T3-L1cells increased significantly after treatment withhigh concentration (10-5mol/L) of PPT (P<0.01). By contrast, the ERaexpression decreased in cells treated with high concentration ofMPP(10-5mol/L), compared to controls (p<0.01) and the cells treated with lowconcentrations(10-7mol/L and10-9mol/L).c) The leptin mRNA was significantly increased in3T3-L1-16than3T3-L1-3.1.The leptinmRNA was not significantly increased in both3T3-L1-16and3T3-L1-3.1which were incubated with PPT than in groups without PPT.By contrast, the leptinmRNA was decreased in both3T3-L1-16and3T3-L1-3.1which were incubated with MPP than that in groups without MPP. MPPtreatment for24h led to decline of leptin mRNA expression in both3T3-L1-16and3T3-L1-3.1in a dose-dependent manner.d) The stimulatory action of PPT on leptin protein expression in both3T3-L1-16and3T3-L1-3.1was dose dependant and maximal effect was achieved at10-5mmol/L for PPT in both3T3-L1-16and3T3-L1-3.1.The inhibitory actionof the MPP on leptin protein expression in both3T3-L1-16and3T3-L1-3.1wasdose dependant and maximal effect was achieved for MPP at10-5mmol/L in both3T3-L1-16and3T3-L1-3.1e) The leptin protein expression increased significantly in both3T3-L1-16and3T3-L1-3.1with PPT at10-5mmol/L, decreased significantly with MPP at10-5mmol/L using western blot3) Effect of LRP16gene on JAK/STAT signaling in3T3-L1adipocytesa) The expression of JAK2mRNA,STAT3m RNA and SOCS3mRNA were about1.46fold (P<0.01)1.25fold(P<0.01) and1.49fold(P<0.01) of3T3-L1-3.1byQ-PCR; The protein expression of p-JAK2(tyr1007/1008),p-STAT3of3T3-L1-16were about1.97fold (P<0.01) and1.18fold(P<0.05) of3T3-L1-3.1usingwestern blot.b) The expression of JAK2mRNA,STAT3mRNA and SOCSmRNA of3T3-L1-16with MPP (10-5mmol/L) were31.4%(P<0.01),57.1%(P<0.01)and91.5%of3T3-L1-16without MPP. The protein expression of p-JAK2(tyr1007/1008) andp-STAT3of3T3-L1-16with MPP were about48.2%(P<0.01) and53.4%(P<0.01) of3T3-L1-16without MPP.4) Effect of LRP16gene on transcriptional activity of leptina) pGL3-lep(-762)was successfully constructed which the ob gene promoter(-762bp to+29bp). The transactivation of leptin promoter (-762bp to+29bp) was enhanced by LRP16in a dose-dependent manner in ERα-positiveMCF-7cells in the presence of E2, but not in ERα-negative cos-7cells.b) LRP16enhanced the transcriptional activity of leptin in a dose dependentmanner, the relative luciferase activity of p(-762)lep-luc accounted for2.63fold (P<0.05) of the control group at pcDNA-3.1-160.5μg in MCF-7. Thereporter activitives of p(-762)lep-luc were enhanced by LRP16in COS-7and Hela cells after being co-transfected with ERα, and the relativeluciferase activity of p(-762)lep-luc accounted for2.6foldand2.7fold ofthe control group at pcDNA-3.1-160.5μg. MPP decreased thetranscriptional activity of leptin. the luciferase activity of leptin accountedfor41.3%(P<0.01) of the control group at pcDNA-3.1-160.5μg. Conclusions:1) We successfully constructed LRP16over-expression and control cell lines, and hadprovided a stable experimental cell model for the follow-up study.2) The LRP16gene stimulated leptin expression and secretion in3T3-L1adipocytes.The effect was inhibited by ERa antagonist.3) The LRP16gene stimulated pJAK2tyr (1007/1008)and pSTAT3protein expression in3T3-L1adipocytes. The effect was inhibited by ERa antagonist at10-5mol/L4) The LRP16gene stimulated transcriptional activation of leptin gene which waspartly via ERa.In a word, the LRP16gene plays a key role in the transcriptional regulation ofleptin, and stimulated the JAK/STAT signaling. The stimulation of LRP16on leptinpartly was partly via ERa.