AC3缺失对小鼠MOE组织内相关因子及信号通路的影响
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摘要
腺苷酸环化酶3(Adenylate cyclase 3,AC3)是膜整合蛋白,可将ATP变成cAMP,使cAMP信号通路受损,进而导致嗅觉丧失。主要嗅觉表皮(main olfactory epithelium,MOE)组织是感知和传导嗅觉信号的重要器官,主要通过环磷酸腺苷cAMP、肌醇1,4,5-三磷酸(Inositol 1,4,5-trisphosphate,IP3)、刺猬(Hedgehog,Hh)等3个信号通路进行嗅觉信号转导。MOE组织中有AC2、AC3、AC4等表达,为探讨当AC3缺失时,AC2和AC4是否可以发挥AC3作用介导嗅觉信号转导;AC3缺失是否通过引起IP3和Hh信号通路的改变而介导cAMP信号通路,本研究拟选取AC3缺失的小鼠和野生型小鼠各4只,以小鼠的MOE组织为实验材料,利用实时荧光定量PCR(q PCR)技术检测小鼠MOE组织内的AC2、AC4;检测IP3信号通路中的ip3r1、calm1、calm2;检测Hh信号通路中的patched、smoothened、gli1和gli2等基因的表达情况。结果显示,AC3缺失的小鼠中AC3几乎无表达;和野生型小鼠相比,AC3缺失的小鼠MOE组织中AC2、AC4表达量均显著下降(p<0.05),分别下调1倍和4倍;AC3缺失的小鼠IP3信号通路中的三磷酸肌醇Ⅰ型受体(type 1 inositol1,4,5-trisphosphate receptor,ip3r1)、calm1、calm2表达量均显著下降(p<0.05),分别下调0.3倍、10倍及4倍;AC3缺失的小鼠Hh信号通路中patched、smoothened、gli1和gli2的表达量均显著下降(p<0.05),分别下调0.4倍、10倍、10倍、4倍。因此,AC3缺失会导致cAMP信号通路的调节受损,进而影响AC3相关因子AC2和AC4的表达,同时抑制IP3和Hh信号通路的传导,在探究AC3基因敲出小鼠嗅觉丧失方面具有一定的指导意义。
Adenylate cyclase 3(AC3) is a membrane-bound protein that converts ATP into cAMP, which damages the cAMP signaling pathway, leading to loss in sense of smell. Main olfactory epithelium(MOE) is the important organ for perceiving and conducting olfactory signals. It is mainly composed of cyclic adenosine monophosphate(cAMP), inositol 1,4,5-trisphosphate(IP3), Hedgehog(Hedgehog, Hh) and other three signaling pathways for olfactory signal transduction. There are expression of AC2, AC3 and AC4 in the MOE tissues. We investigated whether AC2 and AC4 could exert AC3-mediated olfactory signal transduction when AC3 was absent. 4 AC3-deficient mice and 4 wild-type mice were used to induce cAMP signal pathways by changing the signal transduction pathways of IP3 and Hh. Real-time fluorescence quantitative PCR was used to detect AC2 and AC4 in MOE tissues of those mouse, and ip3 r1, calm1, calm2 in IP3 signal pathway, as well as the expression of patched, smoothened, gli1 and gli2 genes in the Hh signaling pathway. The results showed that AC3-deficientmice had almost no AC3 expression. Compared with wild-type mice, the expression of AC2 and AC4 in MOE tissues with AC3 significantly decreased(p<0.05), down 1 and 4 times respectively. The expression of type 1 inositol 1,4,5-trisphosphate receptor(ip3 r1), calm1 and calm2 significantly decreased in IP3 signaling pathway of AC3-deficient mouse(p<0.05), down 0.3, 10, and 4 times respectively. The expression of tpatched,smoothened, gli1, and gli2 significantly decreased in Hh signaling pathway of AC3-deficient mouse(p<0.05), down0.4, 10, 10 and 4 times, respectively. Therefore, the deletion of AC3 might lead to the impairment of the regulation of cAMP signaling pathway, which might affect the expression of AC3 and AC2, and inhibit the conduction of IP3 and Hh signaling pathways. It would provide a guide to the effect of AC3 on olfactory loss in mice.
Adenylate cyclase 3(AC3) is a membrane-bound protein that converts ATP into cAMP, which damages the cAMP signaling pathway, leading to loss in sense of smell. Main olfactory epithelium(MOE) is the important organ for perceiving and conducting olfactory signals. It is mainly composed of cyclic adenosine monophosphate(cAMP), inositol 1,4,5-trisphosphate(IP3), Hedgehog(Hedgehog, Hh) and other three signaling pathways for olfactory signal transduction. There are expression of AC2, AC3 and AC4 in the MOE tissues. We investigated whether AC2 and AC4 could exert AC3-mediated olfactory signal transduction when AC3 was absent. 4 AC3-deficient mice and 4 wild-type mice were used to induce cAMP signal pathways by changing the signal transduction pathways of IP3 and Hh. Real-time fluorescence quantitative PCR was used to detect AC2 and AC4 in MOE tissues of those mouse, and ip3 r1, calm1, calm2 in IP3 signal pathway, as well as the expression of patched, smoothened, gli1 and gli2 genes in the Hh signaling pathway. The results showed that AC3-deficientmice had almost no AC3 expression. Compared with wild-type mice, the expression of AC2 and AC4 in MOE tissues with AC3 significantly decreased(p<0.05), down 1 and 4 times respectively. The expression of type 1 inositol 1,4,5-trisphosphate receptor(ip3 r1), calm1 and calm2 significantly decreased in IP3 signaling pathway of AC3-deficient mouse(p<0.05), down 0.3, 10, and 4 times respectively. The expression of tpatched,smoothened, gli1, and gli2 significantly decreased in Hh signaling pathway of AC3-deficient mouse(p<0.05), down0.4, 10, 10 and 4 times, respectively. Therefore, the deletion of AC3 might lead to the impairment of the regulation of cAMP signaling pathway, which might affect the expression of AC3 and AC2, and inhibit the conduction of IP3 and Hh signaling pathways. It would provide a guide to the effect of AC3 on olfactory loss in mice.
引文
Boto T.,Louis T.,Jindachomthong K.,Jalink K.,and Tomchik SM.,2014,Dopaminergic modulation of c AMP drives nonlinear plasticity across the Drosophila mushroom body lobes,Curr.Biol.,24(8):822-831
Cao Z.L.,Hao J.Y.,Zhou Y.F.,Zhang Z.,Ni Z.H.,Hu Y.X.,Liu W.L.,Li Y.C.,Storm D.R.,Ma R.Z.,and Wang Z.S.,2014Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3by using suppression subtractive hybridization approach Yichuan(Hereditas),36(6):574-583(曹振龙,郝江叶,周艳芬,张喆,倪志华,胡远想,刘伟丽,李永超,Storm D.R.,马润林,王振山,2014,利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因,遗传,36(6)574-583)
Chen X.,Xia Z.,and Storm D.R.,2012,Stimulation of electro-olfactogram responses in the main olfactory epithelia by airflow depends on the type 3 adenylyl cyclase,J.Neurosci.32(45):15769-15778
Feng S.P.,2016,Research advance on mi RNA q PCR methods and its application,Shengwu Jishu Tongbao(Biotechnology Bulletin),32(2):59-69(冯世鹏,2016,mi RNA q PCR检测方法研究进展及其应用,生物技术通报,32(2):59-69)
Gao X.D.,Cui N.,An L.F.,Lang J.Q.,and Zhu D.D.,2014,Etiologies,treatment and prognosis of olfactory dysfunction Linchuang Erbi Yanhou Toujing Waike Zazhi(Journal of Clinical Otorhinolaryngology),28(20):1623-1627(高旭栋崔娜,安立峰,郎金琦,朱冬冬,2014,嗅觉障碍常见病因、治疗及预后,临床耳鼻咽喉头颈外科杂志,28(20)1623-1627)
Juhász T.,Szentléleky E.,Somogyi C.S.,Takács R.,Dobrosi N.Engler M.,Tamás A.,Regl觟di D.,and Zákány R.,2015,Pituitary adenylate cyclase activating polypeptide(PACAP)pathway is induced by mechanical load and reduces the activity of hedgehog signaling in chondrogenic micromass cell cultures,Int.J.Mol.Sci.,16(8):17344-17367
Luo J.,Chen X.,Pan Y.W.,Lu S.,Xia Z.,and Storm D.R.,2015,The type 3 adenylyl cyclase is required for the survival and maturation of newly generated granule cells in the olfactory bulb,PLo S One,10(3):e0122057
Mariman E.C.,Vink R.G.,Roumans N.J.,Bouwman F.G.,Stumpel C.T.,Aller E.E.,van Baak M.A.,and Wang P.,2016,The cilium:a cellular antenna with an influence on obesity risk,Br.J.Nutr.,116(4):576-592
Rattazzi L.,Cariboni A.,Poojara R.,Shoenfeld Y.,and D'Acquisto F.,2015,Impaired sense of smell and altered olfactory system in RAG-1(-/-)immunodeficient mice,Front Neurosci.,9:318
Vuolo L.,Herrera A.,Torroba B.,Menendez A.,nd Pons S.,a2015,Ciliary adenylyl cyclases control the hedgehog pathway,J.Cell Sci.,128(15):2928-2937
Wang Z.,Phan T.,and Storm D.R.,2011,The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory:role of c AMP signaling in primary cilia,J.Neurosci.,31(15):5557-5561
Wang Z.,Zhou Y.,Luo Y.,Zhang J.,Zhai Y.,Yang D.,Zhang Z.,Li Y.,Storm D.R.,and Ma R.Z.,2016,Gene expression profiles of main olfactory epithelium in adenylyl cyclase 3knockout mice,Int.J.Mol.Sci.,16(12):28320-28333
Wang Z.S.,Zhang Z.,Zhai Y.P.,Liu M.C.,Zhang Y.H.,and Zhou Y.F.,2016,Improvement of immunofluorescent staining of nuclear antigens by HCl acidification,Zhongguo Shengwu Huaxue Yu Fenzishengwu Xuebao(Chinese Journal of Biochemistry and Molecular Biology),32(2):231-236(王振山,张喆,翟云鹏,刘明琛,张燕花,周艳芬,2016,盐酸酸化可改善核抗原免疫荧光染色,中国生物化学与分子生物学报,32(2):231-236)
Zeng X.P.,Chen H.X.,Fu Q.L.,Xu R.,Weng W.P.,Shi J.B.,and Xu G.,2014,Relationship between olfactory function and the number of mature olfactory neurons in olfactory epithelium of the adult mouse,Zhongguo Linchuang Jiepouxue Zazhi(Chinese Journal of Clinical Anatomy),32(2):192-195(曾宪平,陈合新,付清玲,徐睿,文卫平,史剑波,许庚,2014,成年小鼠嗅觉功能与嗅上皮中成熟嗅觉神经元数量的实验研究,中国临床解剖学杂志,32(2):192-195)
Cao Z.L.,Hao J.Y.,Zhou Y.F.,Zhang Z.,Ni Z.H.,Hu Y.X.,Liu W.L.,Li Y.C.,Storm D.R.,Ma R.Z.,and Wang Z.S.,2014Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3by using suppression subtractive hybridization approach Yichuan(Hereditas),36(6):574-583(曹振龙,郝江叶,周艳芬,张喆,倪志华,胡远想,刘伟丽,李永超,Storm D.R.,马润林,王振山,2014,利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因,遗传,36(6)574-583)
Chen X.,Xia Z.,and Storm D.R.,2012,Stimulation of electro-olfactogram responses in the main olfactory epithelia by airflow depends on the type 3 adenylyl cyclase,J.Neurosci.32(45):15769-15778
Feng S.P.,2016,Research advance on mi RNA q PCR methods and its application,Shengwu Jishu Tongbao(Biotechnology Bulletin),32(2):59-69(冯世鹏,2016,mi RNA q PCR检测方法研究进展及其应用,生物技术通报,32(2):59-69)
Gao X.D.,Cui N.,An L.F.,Lang J.Q.,and Zhu D.D.,2014,Etiologies,treatment and prognosis of olfactory dysfunction Linchuang Erbi Yanhou Toujing Waike Zazhi(Journal of Clinical Otorhinolaryngology),28(20):1623-1627(高旭栋崔娜,安立峰,郎金琦,朱冬冬,2014,嗅觉障碍常见病因、治疗及预后,临床耳鼻咽喉头颈外科杂志,28(20)1623-1627)
Juhász T.,Szentléleky E.,Somogyi C.S.,Takács R.,Dobrosi N.Engler M.,Tamás A.,Regl觟di D.,and Zákány R.,2015,Pituitary adenylate cyclase activating polypeptide(PACAP)pathway is induced by mechanical load and reduces the activity of hedgehog signaling in chondrogenic micromass cell cultures,Int.J.Mol.Sci.,16(8):17344-17367
Luo J.,Chen X.,Pan Y.W.,Lu S.,Xia Z.,and Storm D.R.,2015,The type 3 adenylyl cyclase is required for the survival and maturation of newly generated granule cells in the olfactory bulb,PLo S One,10(3):e0122057
Mariman E.C.,Vink R.G.,Roumans N.J.,Bouwman F.G.,Stumpel C.T.,Aller E.E.,van Baak M.A.,and Wang P.,2016,The cilium:a cellular antenna with an influence on obesity risk,Br.J.Nutr.,116(4):576-592
Rattazzi L.,Cariboni A.,Poojara R.,Shoenfeld Y.,and D'Acquisto F.,2015,Impaired sense of smell and altered olfactory system in RAG-1(-/-)immunodeficient mice,Front Neurosci.,9:318
Vuolo L.,Herrera A.,Torroba B.,Menendez A.,nd Pons S.,a2015,Ciliary adenylyl cyclases control the hedgehog pathway,J.Cell Sci.,128(15):2928-2937
Wang Z.,Phan T.,and Storm D.R.,2011,The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory:role of c AMP signaling in primary cilia,J.Neurosci.,31(15):5557-5561
Wang Z.,Zhou Y.,Luo Y.,Zhang J.,Zhai Y.,Yang D.,Zhang Z.,Li Y.,Storm D.R.,and Ma R.Z.,2016,Gene expression profiles of main olfactory epithelium in adenylyl cyclase 3knockout mice,Int.J.Mol.Sci.,16(12):28320-28333
Wang Z.S.,Zhang Z.,Zhai Y.P.,Liu M.C.,Zhang Y.H.,and Zhou Y.F.,2016,Improvement of immunofluorescent staining of nuclear antigens by HCl acidification,Zhongguo Shengwu Huaxue Yu Fenzishengwu Xuebao(Chinese Journal of Biochemistry and Molecular Biology),32(2):231-236(王振山,张喆,翟云鹏,刘明琛,张燕花,周艳芬,2016,盐酸酸化可改善核抗原免疫荧光染色,中国生物化学与分子生物学报,32(2):231-236)
Zeng X.P.,Chen H.X.,Fu Q.L.,Xu R.,Weng W.P.,Shi J.B.,and Xu G.,2014,Relationship between olfactory function and the number of mature olfactory neurons in olfactory epithelium of the adult mouse,Zhongguo Linchuang Jiepouxue Zazhi(Chinese Journal of Clinical Anatomy),32(2):192-195(曾宪平,陈合新,付清玲,徐睿,文卫平,史剑波,许庚,2014,成年小鼠嗅觉功能与嗅上皮中成熟嗅觉神经元数量的实验研究,中国临床解剖学杂志,32(2):192-195)