ABSTRACT
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-RhoADN transgenic mice and wild type (WT) mice were observed by scanning electronic microscopy at 20 kV, and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by ImageJ (Rasband, 1997-2009). The enamel organs from mandibular first molars from postnatal-4-day (P4) EGFP-RhoADN mice 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-RhoADN transgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay.@*RESULTS@#The EGFP-RhoADN transgenic 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-RhoADN transgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoADN transgenic 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-RhoADN transgenic 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-RhoADN transgenic mice, the enamel formation was disrupted and the adherens junctions of EGFP-RhoADN transgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.
Subject(s)
Animals , Humans , Mice , Adherens Junctions , Ameloblasts , Amelogenesis , Antigens, CD , Cadherins/metabolism , Dental Enamel/metabolism , Enamel Organ , Mice, Transgenic , Molar , Signal Transduction , alpha Catenin , beta Catenin , rhoA GTP-Binding Protein/physiologyABSTRACT
<p><b>BACKGROUND</b>Intermediate filament (IF) proteins have been thought to play a role in nuclear centration, organelle movement and maintenance of cell shape. dl-praeruptorin A (Pd-Ia), a novel Ca2+-influx blocker and K+-channel opener isolated from Chinese traditional herbal medicine Qian-Hu, has been demonstrated to inhibit expression of apoptosis related proteins and reduce the level of proinflammatory factors in ischemia/reperfusion myocardiocytes. Morphologically, whether Pd-Ia effects myocardiocyte IFs remains unclear. The purpose of this study was, for the first time, to evaluate the in vivo effects of Pd-Ia on IF desmin and vimentin content in order to further explore its cardioprotection against ischemia and elucidate its mechanism of action.</p><p><b>METHODS</b>Rats underwent a 30 minutes coronary occlusion followed by 120 minutes reperfusion. Assessment of desmin and vimentin content of myocardiocytes was performed by immunohistochemistry, Western blot, Hematoxylin-Eosin staining and densitometry.</p><p><b>RESULTS</b>Pretreatment with i.v. infusion of Pd-Ia prior to ischemia significantly increased desmin and vimentin content and alleviated defects caused by the ischemia/reperfusion insult, e.g. with Pd-Ia at a dose of 0.5 or 1.0 mg/kg, integrated density values of desmin were increased from 61 478 +/- 10 074 to 177 408 +/- 10 395 and 195 784 +/- 20 057, and vimentin from 59 189 +/- 19 853 to 164 781 +/- 19 543 and 185 696 +/- 20 957 (P < 0.01, vs placebo), respectively. The recovery of desmin seemed to be stronger and appeared earlier than that of vimentin.</p><p><b>CONCLUSION</b>Pd-Ia protectively increased IF desmin and vimentin content in ischemia/reperfusion myocardiocytes, which might be partially responsible for its cardioprotection against ischemia.</p>