RhoA对小鼠成釉细胞黏附连接的影响
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摘要
目的:引进在成釉细胞中表达RhoA显性抑制基因的EGFP-RhoADominant Negative(EGFP-RhoADN)转基因鼠模型,研究RhoA通路对成釉细胞黏附连接复合体的影响,探索EGFP-RhoADN转基因鼠牙釉质发育缺陷的机制,为牙胚发育及牙齿再生的研究提供实验依据。方法:所有实验均以EGFP-RhoADN转基因鼠为实验组,以同时期野生型小鼠为对照组。使用扫描电子显微镜观察1月龄的两组小鼠下颌第一磨牙牙釉质外形,每组样本量为20,记录牙釉质厚度。采用RT-PCR、Western blot方法检测并比较出生后4 d(postnatal-4-day,P4)的两组小鼠下颌第一磨牙上皮层细胞间黏附连接复合体成分基因和蛋白表达的改变。结果:1月龄的EGFP-RhoADN转基因鼠下颌第一磨牙牙釉质厚度比同时期野生型小鼠牙釉质厚度下降[(84.60±0.20)μm vs.(106.24±0.24)μm,P<0.05],出生后4 d的EGFP-RhoADN转基因鼠成釉细胞层上皮钙黏附蛋白(epithelium-cadherin,E-cadherin)、α-上皮连环蛋白(α-E-catenin)、pan-钙黏附蛋白(pan-cadherin)表达水平较同时期野生型小鼠下调,β-连环蛋白(β-catenin)表达水平较同时期野生型小鼠上调,E-cadherin基因表达量较同时期野生型小鼠下调(0.93±0.01 vs.1.00±0.02,P<0.05),β-catenin基因表达量较同时期野生型小鼠上调(1.23±0.03 vs.1.00±0.05,P<0.05)。结论:EGFP-RhoADN转基因鼠的下颌第一磨牙牙釉质发育出现缺陷,牙釉质厚度下降,这一现象可能是由于RhoA通路抑制引起成釉细胞黏附连接的改变,从而影响了牙釉质正常发育。
Objective: To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoADominant Negative( EGFP-RhoADN) transgenic mice model,from the aspect of adherens junctions,and to provide a theory basis for mechanism of enamel development defects.Methods: The enamel thickness of mandibular first molars of EGFP-RhoADNtransgenic mice and wild type( WT) mice were observed by scanning electronic microscopy at 20 k V,and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by Image J( Rasband,1997-2009). The enamel organs from mandibular first molars from postnatal-4-day( P4) EGFP-RhoADNmice and wild type mice were isolated,and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoADNtransgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay. Results: The EGFP-RhoADNtransgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group( n = 20),and enamel thickness was( 84. 60 ± 0. 20) μm vs.( 106. 24 ± 0. 24) μm,P < 0. 05. The protein expressions of E-cadherin,α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoADN transgenic mice molars were down-regulated,and the protein level of β-catenin in ameloblasts layer of P4 EGFP-RhoADNtransgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoADNtransgenic mice molars was down-regulated versus that of WT mice,and the gene expression of E-cadherin was 0. 93 ± 0. 01 vs. 1. 00 ± 0. 02,P < 0. 05. The mRNA level of β-catenin in ameloblasts layer of P4 EGFP-RhoADNtransgenic mice molars was up-regulated versus that of WT mice,and the gene expression of β-catenin was 1. 23 ± 0. 03 vs. 1. 00 ± 0. 05,P < 0. 05. Conclusion:In the mandibular first molars of EGFP-RhoADNtransgenic mice,the enamel formation was disrupted and the adherens junctions of EGFP-RhoADNtransgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.
Objective: To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoADominant Negative( EGFP-RhoADN) transgenic mice model,from the aspect of adherens junctions,and to provide a theory basis for mechanism of enamel development defects.Methods: The enamel thickness of mandibular first molars of EGFP-RhoADNtransgenic mice and wild type( WT) mice were observed by scanning electronic microscopy at 20 k V,and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by Image J( Rasband,1997-2009). The enamel organs from mandibular first molars from postnatal-4-day( P4) EGFP-RhoADNmice and wild type mice were isolated,and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoADNtransgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay. Results: The EGFP-RhoADNtransgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group( n = 20),and enamel thickness was( 84. 60 ± 0. 20) μm vs.( 106. 24 ± 0. 24) μm,P < 0. 05. The protein expressions of E-cadherin,α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoADN transgenic mice molars were down-regulated,and the protein level of β-catenin in ameloblasts layer of P4 EGFP-RhoADNtransgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoADNtransgenic mice molars was down-regulated versus that of WT mice,and the gene expression of E-cadherin was 0. 93 ± 0. 01 vs. 1. 00 ± 0. 02,P < 0. 05. The mRNA level of β-catenin in ameloblasts layer of P4 EGFP-RhoADNtransgenic mice molars was up-regulated versus that of WT mice,and the gene expression of β-catenin was 1. 23 ± 0. 03 vs. 1. 00 ± 0. 05,P < 0. 05. Conclusion:In the mandibular first molars of EGFP-RhoADNtransgenic mice,the enamel formation was disrupted and the adherens junctions of EGFP-RhoADNtransgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.
引文
[1]Hu C,Fukae M,Simmer J,et al.Cloning and characterization of porcine enamelin mRNAs[J].J Dent Res,1997,76(11):1720-1729.
[2]Hu C,Fukae M,Simmer J,et al.Sheathlin:cloning,c DNA/polypeptide sequences,and immunolocalization of porcine enamel sheath proteins[J].J Dent Res,1997,76(2):648-657.
[3]Smith C.Ameloblasts:secretory and resorptive functions[J].J Dent Res,1979,58(Spec Issue B):695-707.
[4]Schmitz A,Govek E,B9ttner B,et al.Rho GTPases:signaling,migration,and invasion[J].Exp Cell Res,2000,261(1):1-12.
[5]Etiennemanneville S,Hall A.Rho GTPases in cell biology[J].Nature,2002,420(6916):629.
[6]Leung T,Chen X,Manser E,et al.The p160 Rho A-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton[J].Mol Cell Biol,1996,16(10):5313-5327.
[7]Bartlett J,Dobeck J,Skobe Z,et al.Targeted p120-Catenin ablation disrupts dental enamel development[J].PLo S One,2010,5(9):1-11.
[8]Watanabe T,Sato K,Kaibuchi K.Cadherin-mediated intercellular adhesion and signaling cascades involving small GTPases[J].Cold Spring Harb Perspect Biol,2009,1(3):a3-20.
[9]Hall A.Rho GTPases and the actin cytoskeleton[J].Science,1998,279(5350):509-514.
[10]Braga VM,Machesky LM,Hall A,et al.The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts[J].J Cell Biol,1997,137(6):1421-1431.
[11]Takaishi K,Sasaki T,Kotani H,et al.Regulation of cell-cell adhesion by Rac and Rho small G proteins in MDCK cells[J].J Cell Biol,1997,139(4):1047-1059.
[12]Otsu K,Ida-Yonemochi H,Fujiwara N,et al.The semaphorin4D-Rho A-Akt signal cascade regulates enamel matrix secretion in coordination with cell polarization during ameloblast differentiation[J].J Bone Miner Res,2016,31(11):1943-1954.
[13]Li Y,Decker S,Yuan ZA,et al.Effects of sodium fluoride on the actin cytoskeleton of murine ameloblasts[J].Arch Oral Biol,2005,50(8):681-688.
[14]Li Y,Pugach MK,Kuehl MA,et al.Dental enamel structure is altered by expression of dominant negative Rho A in ameloblasts[J].Cells Tissues Organs,2011,194(2/3/4):227-231.
[15]Biz MT,Marques MR,Crema VO,et al.GTPases Rho A and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts[J].Cell Tissue Res,2010,340(3):459-470.
[16]Baum B,Georgiou M.Dynamics of adherens junctions in epithelial establishment,maintenance,and remodeling[J].J Cell Biol,2011,192(6):907-917.
[17]Sahai E,Marshall CJ.ROCK and Dia have opposing effects on adherens junctions downstream of Rho[J].Nat Cell Biol,2002,4(6):408-415.
[18]Shewan A M,Maddugoda M,Kraemer A,et al.Myosin 2 is a key Rho kinase target necessary for the local concentration of E-cadherin at cell-cell contacts[J].Mol Biol Cell,2005,16(10):4531.
[19]Martin AC,Kaschube M,Wieschaus EF.Pulsed contractions of an actin-myosin network drive apical constriction[J].Nature,2009,457(7228):495-499.
[20]Katayama K,Melendez J,Yoshida Y,et al.Loss of Rho A in neural progenitor cells causes the disruption of adherens junctions and hyperproliferation[J].Proc Natl Acad Sci USA,2011,108(18):7607-7612.
[21]Terling C,Heymann R,Rozell B,et al.Dynamic expression of Ecadherin in ameloblasts and cementoblasts in mice[J].Eur J Oral Sci,1998,106(S1):137-142.
[22]Sorkin BC,Wang MY,Dobeck JM,et al.The cadherin-catenin complex is expressed alternately with the adenomatous polyposis coli protein during rat incisor amelogenesis.[J].J Histochem Cytochem,2000,48(3):397-406.
[2]Hu C,Fukae M,Simmer J,et al.Sheathlin:cloning,c DNA/polypeptide sequences,and immunolocalization of porcine enamel sheath proteins[J].J Dent Res,1997,76(2):648-657.
[3]Smith C.Ameloblasts:secretory and resorptive functions[J].J Dent Res,1979,58(Spec Issue B):695-707.
[4]Schmitz A,Govek E,B9ttner B,et al.Rho GTPases:signaling,migration,and invasion[J].Exp Cell Res,2000,261(1):1-12.
[5]Etiennemanneville S,Hall A.Rho GTPases in cell biology[J].Nature,2002,420(6916):629.
[6]Leung T,Chen X,Manser E,et al.The p160 Rho A-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton[J].Mol Cell Biol,1996,16(10):5313-5327.
[7]Bartlett J,Dobeck J,Skobe Z,et al.Targeted p120-Catenin ablation disrupts dental enamel development[J].PLo S One,2010,5(9):1-11.
[8]Watanabe T,Sato K,Kaibuchi K.Cadherin-mediated intercellular adhesion and signaling cascades involving small GTPases[J].Cold Spring Harb Perspect Biol,2009,1(3):a3-20.
[9]Hall A.Rho GTPases and the actin cytoskeleton[J].Science,1998,279(5350):509-514.
[10]Braga VM,Machesky LM,Hall A,et al.The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts[J].J Cell Biol,1997,137(6):1421-1431.
[11]Takaishi K,Sasaki T,Kotani H,et al.Regulation of cell-cell adhesion by Rac and Rho small G proteins in MDCK cells[J].J Cell Biol,1997,139(4):1047-1059.
[12]Otsu K,Ida-Yonemochi H,Fujiwara N,et al.The semaphorin4D-Rho A-Akt signal cascade regulates enamel matrix secretion in coordination with cell polarization during ameloblast differentiation[J].J Bone Miner Res,2016,31(11):1943-1954.
[13]Li Y,Decker S,Yuan ZA,et al.Effects of sodium fluoride on the actin cytoskeleton of murine ameloblasts[J].Arch Oral Biol,2005,50(8):681-688.
[14]Li Y,Pugach MK,Kuehl MA,et al.Dental enamel structure is altered by expression of dominant negative Rho A in ameloblasts[J].Cells Tissues Organs,2011,194(2/3/4):227-231.
[15]Biz MT,Marques MR,Crema VO,et al.GTPases Rho A and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts[J].Cell Tissue Res,2010,340(3):459-470.
[16]Baum B,Georgiou M.Dynamics of adherens junctions in epithelial establishment,maintenance,and remodeling[J].J Cell Biol,2011,192(6):907-917.
[17]Sahai E,Marshall CJ.ROCK and Dia have opposing effects on adherens junctions downstream of Rho[J].Nat Cell Biol,2002,4(6):408-415.
[18]Shewan A M,Maddugoda M,Kraemer A,et al.Myosin 2 is a key Rho kinase target necessary for the local concentration of E-cadherin at cell-cell contacts[J].Mol Biol Cell,2005,16(10):4531.
[19]Martin AC,Kaschube M,Wieschaus EF.Pulsed contractions of an actin-myosin network drive apical constriction[J].Nature,2009,457(7228):495-499.
[20]Katayama K,Melendez J,Yoshida Y,et al.Loss of Rho A in neural progenitor cells causes the disruption of adherens junctions and hyperproliferation[J].Proc Natl Acad Sci USA,2011,108(18):7607-7612.
[21]Terling C,Heymann R,Rozell B,et al.Dynamic expression of Ecadherin in ameloblasts and cementoblasts in mice[J].Eur J Oral Sci,1998,106(S1):137-142.
[22]Sorkin BC,Wang MY,Dobeck JM,et al.The cadherin-catenin complex is expressed alternately with the adenomatous polyposis coli protein during rat incisor amelogenesis.[J].J Histochem Cytochem,2000,48(3):397-406.