腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响
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摘要
主要嗅觉表皮(main olfactory epithelium,MOE)是哺乳动物感知气味分子的主要嗅觉器官。在MOE组织内,大多数嗅觉神经元通过c AMP信号传导通路感知气味信息。作为嗅觉c AMP信号通路的主要成员之一,腺苷酸环化酶3(adenylyl cyclase 3,ac3)基因敲除小鼠嗅觉探测功能丧失。除c AMP信号传导通路外,MOE内AC3相关因子AC2和AC4,以及肌醇1,4,5-三磷酸(inositol 1,4,5-trisphosphate,IP3)信号通路和Sonic Hedgehog(Shh)信号通路均有表达。然而,敲除ac3是否会对ac2和ac4以及IP3和Shh信号通路成员产生影响,尚不清楚。本文以AC3缺失(AC3-/-)及其野生型小鼠(AC3+/+)MOE为材料,采用实时荧光定量PCR(qRT-PCR)和免疫荧光组织化学方法,发现AC3缺失后,MOE内的ac2和ac4,以及IP3信号通路中的IP3受体ip3r1及钙调蛋白calm1和calm2表达水平均明显降低。Shh信号通路中的受体patched(ptch)与smoothened(smo)、以及核转录因子gli1与gli2的表达也受到了影响。总之,AC3基因缺失不但导致小鼠MOE组织中c AMP信号通路受损,同时AC3相关因子,IP3信号通路和Shh信号通路的传导也受到抑制。本文对于阐明AC3基因敲除小鼠嗅觉丧失的原因及其嗅觉探测机制具有重要启示作用。
The main olfactory epithelium(MOE) is the primary olfactory organ for odorant perception inmammals.Within the MOE,c AMP signal transduction pathway is the main pathway for the detection of odor molecules.As one of the key components of c AMP signaling,adenylyl cyclase 3(AC3) deletion leads to the defects in olfactory detection in mice.In addition to c AMP signaling,the AC3 isoforms,adenylyl cyclase 2(AC2) and adenylyl cyclase 4(AC4) associated with the c AMP signaling pathway,the inositol 1,4,5,-trisphosphate(IP3) and the sonic hedgehog(Shh) signaling pathway components are also expressed in the MOE.However,whether the expression level of AC2,AC4,and the cascade members of IP3 and Shh signaling,will be altered in the MOE after AC3 deletion in mice remains to be determined.In the present study,by real-time fluorescent quantitative PCR(qRT-PCR) and immunofluorescence staining approaches,with MOE from AC3-/-and AC3+/+mice,we demonstrated that the relative expression of ac2 and ac4 was down-regulated in the MOE of AC3-/-mice,compared with AC3+/+controls.In addition,expression level of inositol 1,4,5-trisphosphate receptor 1(ip3r1),calmodulin(CaM) genes calm1,calm2,patched(ptch),smoothened(smo),the nuclear transcription factors gli1 and gli2,components associated with IP3 and Shh signaling pathways,respectively,was also altered in AC3-/-MOE.Therefore,not only the c AMP signaling,but also the IP3 and the Shh signaling pathways were impacted in the MOE of mice with AC3 deletion.This result may help us to understand the underlying molecular mechanisms of olfactory defections in mice with AC3 deletion.
The main olfactory epithelium(MOE) is the primary olfactory organ for odorant perception inmammals.Within the MOE,c AMP signal transduction pathway is the main pathway for the detection of odor molecules.As one of the key components of c AMP signaling,adenylyl cyclase 3(AC3) deletion leads to the defects in olfactory detection in mice.In addition to c AMP signaling,the AC3 isoforms,adenylyl cyclase 2(AC2) and adenylyl cyclase 4(AC4) associated with the c AMP signaling pathway,the inositol 1,4,5,-trisphosphate(IP3) and the sonic hedgehog(Shh) signaling pathway components are also expressed in the MOE.However,whether the expression level of AC2,AC4,and the cascade members of IP3 and Shh signaling,will be altered in the MOE after AC3 deletion in mice remains to be determined.In the present study,by real-time fluorescent quantitative PCR(qRT-PCR) and immunofluorescence staining approaches,with MOE from AC3-/-and AC3+/+mice,we demonstrated that the relative expression of ac2 and ac4 was down-regulated in the MOE of AC3-/-mice,compared with AC3+/+controls.In addition,expression level of inositol 1,4,5-trisphosphate receptor 1(ip3r1),calmodulin(CaM) genes calm1,calm2,patched(ptch),smoothened(smo),the nuclear transcription factors gli1 and gli2,components associated with IP3 and Shh signaling pathways,respectively,was also altered in AC3-/-MOE.Therefore,not only the c AMP signaling,but also the IP3 and the Shh signaling pathways were impacted in the MOE of mice with AC3 deletion.This result may help us to understand the underlying molecular mechanisms of olfactory defections in mice with AC3 deletion.
引文
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[10]李超,杨栋,张梦迪,等.腺苷酸环化酶(AC)基因家族在小鼠骨髓间充质干细胞(BMSCs)中的表达[J].科学技术与工程(Li C,Yang D,Zhang M,et al.The expression of adenylate cyclase(AC)gene family in mouse bone marrow mesenchymal stem cells(BMSCs)[J].Sci Tech Eng)2015,15(16):28-32
[11]Gong Q,Chen H,Farbman AI.Olfactory sensory axon growth and branching is influenced by sonic hedgehog[J].Dev Dyn,2009,238(7):1768-1776
[12]Wang Z,Storm DR.Extraction of DNA from mouse tails[J].Biotechniques,2006,41(4):410-412
[13]Wong ST,Trinh K,Hacker B,et al.Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice[J].Neuron,2000,27(3):487-497
[14]王振山,张喆,翟云鹏,等.盐酸酸化可改善核抗原免疫荧光染色[J].中国生物化学与分子生物学学报(Wang Z,Zhang Z,Zhai Y,et al.Improvement of immunofluorescent staining of nuclear antigens by hydrochloric acid acidification[J].Chin J Biochem Mol Biol),2016,32(2):231-236
[15]Munger SD,Lane AP,Zhong H,et al.Central role of the CNGA4 channel subunit in Ca2+-calmodulin-dependent odor adaptation[J].Science,2001,294(5549):2172-2175
[16]Feinstein PG,Schrader KA,Bakalyar HA,et al.Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain[J].Proc Natl Acad Sci U S A,1991,88(22):10173-10177
[17]Wang Z,Balet Sindreu C,Li V,et al.Pheromone detection in male mice depends on signaling through the type 3 adenylyl cyclase in the main olfactory epithelium[J].J Neurosci,2006,26(28):7375-7379
[18]Wang Z,Storm DR.Maternal behavior is impaired in female mice lacking type 3 adenylyl cyclase[J].Neuropsychopharmacology,2011,36(4):772-781
[19]Livera G,Xie F,Garcia MA,et al.Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function[J].Mol Endocrinol,2005,19(5):1277-1290
[20]Wang Z,Li V,Chan GC,et al.Adult type 3 adenylyl cyclasedeficient mice are obese[J].PLo S One,2009,4(9):e6979
[21]Baker LP,Nielsen MD,Impey S,et al.Regulation and immunohistochemical localization of betagamma-stimulated adenylyl cyclases in mouse hippocampus[J].J Neurosci,1999,19(1):180-192
[22]Choi EJ,Xia Z,Villacres EC,et al.The regulatory diversity of the mammalian adenylyl cyclases[J].Curr Opin Cell Biol,1993,5(2):269-273
[23]Wayman GA,Impey S,Storm DR.Ca2+inhibition of type III adenylyl cyclase in vivo[J].J Biol Chem,1995,270(37):21480-21486
[24]Spehr M,Wetzel CH,Hatt H,et al.3-Phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons[J].Neuron,2002,33(5):731-739
[25]Huque T,Bruch RC.Odorant-and guanine nucleotidestimulated phosphoinositide turnover in olfactory cilia[J].Biochem Biophys Res Commun,1986,137(1):36-42
[26]Boekhoff I,Tareilus E,Strotmann J,et al.Rapid activation of alternative second messenger pathways in olfactory cilia from rats by different odorants[J].EMBO J,1990,9(8):2453-2458
[27]Vogl A,Noe J,Breer H,et al.Cross-talk between olfactory second messenger pathways[J].Eur J Biochem,2000,267(14):4529-4535
[28]Luo J,Chen X,Pan YW,et al.The type 3 adenylyl cyclase is required for the survival and maturation of newly generated granule cells in the olfactory bulb[J].PLo S One,2015,10(3):e0122057
[29]Cai C,Thorne J,Grabel L.Hedgehog serves as a mitogen and survival factor during embryonic stem cell neurogenesis[J].Stem Cells,2008,26(5):1097-1108
[30]Satir P,Pedersen LB,Christensen ST.The primary cilium at a glance[J].J Cell Sci,2010,123(Pt 4):499-503
[2]Belluscio L,Gold G H,Nemes A,et al.Mice deficient in G(olf)are anosmic[J].Neuron,1998,20(1):69-81
[3]Wong ST,Trinh K,Hacker B,et al.Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice[J].Neuron,2000,27(3):487-497
[4]Brunet LJ,Gold GH,Ngai J.General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel[J].Neuron,1996,17(4):681-693
[5]Wang Z,Zhou Y,Luo Y,et al.Gene expression profiles of main olfactory epithelium in adenylyl cyclase 3 knockout mice[J].Int J Mol Sci,2015,16(12):28320-28333
[6]Spehr M,Wetzel CH,Hatt H,et al.3-phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons[J].Neuron,2002,33(5):731-739
[7]Suh BC,Hille B.PIP2 is a necessary cofactor for ion channel function:how and why?[J].Annu Rev Biophys,2008,37:175-195
[8]郝江叶,韩绍芳,罗影涛,等.腺苷酸环化酶III(AC3)在非神经细胞内的定位[J].中国细胞生物学学报(Hao J,Han S,Luo Y,et al.Subcellular localization of adenylate cyclase III in non-neuronal cells[J].Chin J Cell Biol),2015,37(3):378-386
[9]Ho L,Ali S A,Al-Jazrawe M,et al.Primary cilia attenuate hedgehog signalling in neoplastic chondrocytes[J].Oncogene,2013,32(47):5388-5396
[10]李超,杨栋,张梦迪,等.腺苷酸环化酶(AC)基因家族在小鼠骨髓间充质干细胞(BMSCs)中的表达[J].科学技术与工程(Li C,Yang D,Zhang M,et al.The expression of adenylate cyclase(AC)gene family in mouse bone marrow mesenchymal stem cells(BMSCs)[J].Sci Tech Eng)2015,15(16):28-32
[11]Gong Q,Chen H,Farbman AI.Olfactory sensory axon growth and branching is influenced by sonic hedgehog[J].Dev Dyn,2009,238(7):1768-1776
[12]Wang Z,Storm DR.Extraction of DNA from mouse tails[J].Biotechniques,2006,41(4):410-412
[13]Wong ST,Trinh K,Hacker B,et al.Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice[J].Neuron,2000,27(3):487-497
[14]王振山,张喆,翟云鹏,等.盐酸酸化可改善核抗原免疫荧光染色[J].中国生物化学与分子生物学学报(Wang Z,Zhang Z,Zhai Y,et al.Improvement of immunofluorescent staining of nuclear antigens by hydrochloric acid acidification[J].Chin J Biochem Mol Biol),2016,32(2):231-236
[15]Munger SD,Lane AP,Zhong H,et al.Central role of the CNGA4 channel subunit in Ca2+-calmodulin-dependent odor adaptation[J].Science,2001,294(5549):2172-2175
[16]Feinstein PG,Schrader KA,Bakalyar HA,et al.Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain[J].Proc Natl Acad Sci U S A,1991,88(22):10173-10177
[17]Wang Z,Balet Sindreu C,Li V,et al.Pheromone detection in male mice depends on signaling through the type 3 adenylyl cyclase in the main olfactory epithelium[J].J Neurosci,2006,26(28):7375-7379
[18]Wang Z,Storm DR.Maternal behavior is impaired in female mice lacking type 3 adenylyl cyclase[J].Neuropsychopharmacology,2011,36(4):772-781
[19]Livera G,Xie F,Garcia MA,et al.Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function[J].Mol Endocrinol,2005,19(5):1277-1290
[20]Wang Z,Li V,Chan GC,et al.Adult type 3 adenylyl cyclasedeficient mice are obese[J].PLo S One,2009,4(9):e6979
[21]Baker LP,Nielsen MD,Impey S,et al.Regulation and immunohistochemical localization of betagamma-stimulated adenylyl cyclases in mouse hippocampus[J].J Neurosci,1999,19(1):180-192
[22]Choi EJ,Xia Z,Villacres EC,et al.The regulatory diversity of the mammalian adenylyl cyclases[J].Curr Opin Cell Biol,1993,5(2):269-273
[23]Wayman GA,Impey S,Storm DR.Ca2+inhibition of type III adenylyl cyclase in vivo[J].J Biol Chem,1995,270(37):21480-21486
[24]Spehr M,Wetzel CH,Hatt H,et al.3-Phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons[J].Neuron,2002,33(5):731-739
[25]Huque T,Bruch RC.Odorant-and guanine nucleotidestimulated phosphoinositide turnover in olfactory cilia[J].Biochem Biophys Res Commun,1986,137(1):36-42
[26]Boekhoff I,Tareilus E,Strotmann J,et al.Rapid activation of alternative second messenger pathways in olfactory cilia from rats by different odorants[J].EMBO J,1990,9(8):2453-2458
[27]Vogl A,Noe J,Breer H,et al.Cross-talk between olfactory second messenger pathways[J].Eur J Biochem,2000,267(14):4529-4535
[28]Luo J,Chen X,Pan YW,et al.The type 3 adenylyl cyclase is required for the survival and maturation of newly generated granule cells in the olfactory bulb[J].PLo S One,2015,10(3):e0122057
[29]Cai C,Thorne J,Grabel L.Hedgehog serves as a mitogen and survival factor during embryonic stem cell neurogenesis[J].Stem Cells,2008,26(5):1097-1108
[30]Satir P,Pedersen LB,Christensen ST.The primary cilium at a glance[J].J Cell Sci,2010,123(Pt 4):499-503