Thyroid Hormone Regulates KiSS1 Gene Expression Through PI3K/Akt-mTOR Signaling Pathway And Specificity Protein-1

Objective: Thyroid hormones are essential for the normal development and differ entiation of human cells.Thyroid function is closely related to reproduction,Thy roid dysfunctions often cause reproductive health problems.The integrity and fun ction of the HPG axis(hypothalamic-pituitary-gonadal axis)is the key to maintai ning puberty development and normal reproductive function.KiSS1 / KiSS1 R sys tem is the gatekeepers for the onset of puberty,and is the key to maintain HPG function.The Thyroid Hormone Receptor were shown expressed in the GnRH Neurons of Hypothalamus and its upstream Kisspeptin Neurons.There is evidenc e that the leptin-mTOR-Kisspeptin pathway plays an important role for the regul ation of reproductive metabolism.Our previous studies have found that adiponect in inhibits the expression of KiSS1 gene through AMPK and the translocation of Sp1 protein in the hypothalamic GT1-7 neurons.Therefore,Our goal was to in vestigate whether T3 affects the expression of KiSS1 gene through mTOR regula ting the translocation of Sp1 protein.METHODS: 1.The first part of this study focused on whether T3 regulates Ki SS1 gene expression by TR(thyroid hormone receptor)at the level of KiSS1 ge ne promoter.Western blot was used to detect whether the TR?1 localized in the cytoplasm of GT1-7 cells.The TR?was silenced with siRNA,Western blot was used to analyze whether the regulation of Kisspeptin protein expression is throug h TR?-dependent pathways.After GT1-7 neuron cells were treated with 200ng/ml T3 for different times or siRNA of TR?,the KiSS1 gene promoter activity in t he various treatment groups was determined using the luciferase assay system(Pr omega).2.The second part of this study mainly discussed whether PI3K(phosphatidylinositol 3-kinase)/ Akt-mTOR(the mammalian target of rapamycin)signali ng pathway is involved in T3-induced KiSS1 Gene Expression.We treated GT1-7 cells with Wortmannin and LY294002,the PI3 K / Akt inhibitor and Rapamyci n,the mTOR inhibitor followed by T3,Real-time PCR,Western blot and lucifer ase assay system were used to determine Whether the PI3 K / Akt-mTOR signali ng pathway is involved in T3-induced KiSS1 Gene Expression.We showed Co-I P(co-immunoprecipitation)of TR?and the p85? subunit of PI3 K.The PI3 K activi ty was then measured by ELISA.We further studied cell signaling protein activa tion by Western blot analysis of phosphorylated Akt and mTOR.3.The third part of this study mainly discussed whether PI3 K / Akt-mTOR signaling pathway is mediated by Sp1 in T3-induced KiSS1 Gene Expression.To investigate which r egion of the human KiSS1 promoter was responsible for the stimulatory effect o f T3,truncated deletions of the KiSS1 promoter were performed.The region re sponsible for the T3-regulated increase in promoter activity was submitted to JA SPAR(http://jaspar.genereg.net/cgi-bin/jaspar_db.pl)and analyzed.To analyze the contribution of Sp1 to the activity of the KiSS1 promoter,GT1-7 cells were cot ransfected with promoter reporters and different amounts of pcDNA-Sp1 construct or treated with Mithramycin A,a specific inhibitor of Sp1 protein.We fraction ated cells into nuclear and cytosolic extracts followed by immunoblotting to exa mined the effect of PI3K/Akt-mTOR pathway modulators on Sp1 protein nuclear and cytosolic abundance.We also investigated their effect on three phosphorylate d modifications of Sp1 known to possibly affect its nuclear abundance.We also e xamined the effect of T3 and PI3 K / Akt-mTOR signaling on Sp1 subcellular tr anslocation and phosphorylation in GT1-7 cells;Finally,we performed performed EMSA(electrophoretic mobility shift assay)and ChIP(Chromatin immunoprecip itation assay)to determined whether Sp1 associate with the two putative Sp1 site s in the KiSS1 gene promoter and the effect of T3 and PI3 K / Akt-mTOR sign aling on the binding of Sp1 to KiSS1 gene promoter.Result: 1.We showed that cytoplasmic localization of TR?1 was found in GT1-7cells.TR?-siRNA1259 inhibited the T3-induced up-regulation of Kisspeptin protein expression.The promoter activity of human KiSS1 gene were significantly increased in a time-dependent,that of 48 h group was most obvious,and TR?siR NA1259 played an inhibitory effect.2.Wortmannin,LY294002 and Rapamycin re sults in a decrease of T3-induced KiSS1 Gene expreion.Co-IP studies showed a direct interaction of cytosol-localized TR?1 and the p85?subunit of PI3 K in a lig and-independent manner.T3 treatment rapidly increased PI3 K activity,which result ed in increased phosphorylation of downstream kinases Akt and mTOR.3.Deletio n analysis of KiSS1 promoter indicates that the T3-regulated increase in promote r activity depends on the Sp1 sites of the proximal promoter-191 bp ~-100 bp r egion.Overexpression of Sp1 enhanced the activity of KiSS1 gene promoter.The i nduction of KiSS1 gene promoter activation by Sp1 and T3 was inhibited by Mi thramycin A.T3 increased the nuclear proportion of Sp1,whereas LY294002 and rapamycin decreased it.The nuclear Sp1 abundance was positively correlated with total phospho-serine Sp1.Using both EMSA and ChIP analysis,we confirmed th at Sp1 directly binds to proximal-191 bp ~-100 bp region of the human KiSS1 gene promoter which contains two Sp1 binding site,whereas T3 enhanced the bi nding,while rapamycin and LY294002 had the opposite effect.Conclusion: 1.we show that T3 regulated KiSS1 mRNA and Kisspeptin protein l evels through TR? by modulating KiSS1 gene promoter activity in GT1-7 cells.2.PI3 K / Akt-mTOR Signaling pathway was involved in T3-induced KiSS1 Gene Expression.3.T3-dependent KiSS1 Gene Expression was mediated by PI3 K / Akt-mTOR signaling pathway through the interaction of Sp1 protein with the GC-rich motifs of KiSS1 gene promoter.4.Together,our study provides a molecular mechanism for how T3 regulates KiSS gene expression,which is important for revealing the relationship between thyroid function and reproduction.