Dental Enamel Structure Is Altered by Expression of Dominant Negative RhoA in Ameloblasts

Li, Yong; Pugach, Megan K.; Kuehl, Melissa A.; Peng, Li; Bouchard, Jessica R.; Hwang, Soon Young; Gibson, Carolyn W.

doi:10.1159/000324559

Cited by 12 publications

(21 citation statements)

References 36 publications

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“…Previous in vivo studies done with RhoA DN transgenic mice that express RhoA with a dominant negative N19 mutation to decrease RhoA activity in ameloblasts [Qiu et al, 1995] concluded that interference with RhoA activity leads to surface defects in enamel [Li et al, 2011a]. Because the defects seen in RhoA DN transgenic mice were somewhat similar to those seen in cases of human enamel fluorosis, in the present study ALCs were treated with NaF to determine if the cultured cells respond through cell signaling pathways and how treatment affects the cross-talk between Wnt and Rho in ameloblast cells, leading to alterations in pathway component expression or activity.…”

Section: Discussionmentioning

confidence: 99%

“…The RhoA DN (dominant negative RhoA) plasmid includes amelogenin regulatory sequences and partially blocks endogenous RhoA in ameloblasts by the N19 dominant negative mutation [Qiu et al, 1995;Li et al, 2011b]. Four micrograms of RhoA DN plasmid was transfected to the RhoA pathway blocking groups using 10 μl of Lipofectamine 2000 in serum-free Opti-MEM (Invitrogen).…”

Section: Treatment With Rhoa Dn Plasmid and Rock Inhibitormentioning

confidence: 99%

“…In severe cases, the enamel pitting and porous regions increase in size, and discoloration of the enamel surface is seen [Bronckers et al, 2009]. When a transgenic mouse model expressing a dominant negative RhoA (RhoA DN ) in secretory ameloblasts was generated, some similarities to enamel fluorotic defects in the cutting edge of incisors as well as cusps in the molar were observed [Li et al, 2011a]. Moreover, three independent transgenic mouse strains showed that fluoride-induced enamel alterations have similar changes in cell signaling mechanisms.…”

Section: Introductionmentioning

confidence: 99%

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Wnt-RhoA Signaling Pathways in Fluoride-Treated Ameloblast-Lineage Cells

Shusterman¹,

Gibson²,

Li³

et al. 2014

Cells Tissues Organs

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This study examined the effect of sodium fluoride (NaF) on the Wnt and RhoA signaling pathways in murine ameloblast-lineage cells (ALCs) to better understand the developmental mechanisms of dental fluorosis. Wnt and Rho pathway activities were investigated when ALCs were treated with 1.5 mM NaF, dickkopf-related protein-1 (Dkk-1), secreted frizzled related-protein-2 (sFRP-2), β-catenin siRNA dominant negative RhoA (RhoA^DN) plasmid and Y-27632. Wnt pathway activity was investigated via RT-PCR, Western blot and Topflash luciferase assay. The activity of the RhoA pathway was analyzed via Rho pull-down assay and immunoprecipitation. The differentiation of ALCs was analyzed by alkaline phosphatase assay. Western blot and Topflash luciferase assay results verified that both the Wnt and Rho pathways were upregulated by 1.5 mM NaF. Wnt was discovered to be located upstream from the Rho pathway, as confirmed by treatment with Wnt pathway cell receptor inhibitors Dkk-1 and sFRP-2, leading to a decrease in RhoA and ROCK activity. Inhibition of the Rho pathway with RhoA^DN plasmid and Y-27632 caused upregulation of Wnt pathway activity which could be further increased by 1.5 mM NaF. The increased Wnt pathway activity was found to negatively regulate ALC differentiation. These data suggest that fluoride could induce the cross-talk between Wnt and RhoA signaling pathways, and these responses are predicted to contribute to the development of enamel fluorosis.

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Section: Discussionmentioning

confidence: 99%

Section: Treatment With Rhoa Dn Plasmid and Rock Inhibitormentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Wnt-RhoA Signaling Pathways in Fluoride-Treated Ameloblast-Lineage Cells

Shusterman¹,

Gibson²,

Li³

et al. 2014

Cells Tissues Organs

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“…The transgenic mice in which dominant-negative T19N RhoA was expressed under the control of the amelogenin regulatory sequences showed enamel hypoplasia and surface defects in the molar cusps [17], [18]. In the Tg molar, amelogenin, E-cadherin, and Ki67 (proliferation marker) were reduced, and both canonical and noncanonical Wnt signaling pathways were activated [17], [19].…”

Section: Implication Of Rho Gtpases Signaling In Ameloblastsmentioning

confidence: 99%

“…Further, they are involved in osteoclastogenesis as well as in hematopoiesis and hemopathies [12], [13]. Recently, evidence has emerged showing the involvement of Rho signaling in tooth development [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. In this article, we summarize some interesting recent findings that provide molecular insights into how signaling by Rho GTPases results in tooth development, focusing on ameloblast differentiation in particular.…”

Section: Introductionmentioning

confidence: 99%

Rho GTPases in ameloblast differentiation

Otsu

Harada

2016

Japanese Dental Science Review

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SummaryDuring tooth development, ameloblasts differentiate from inner enamel epithelial cells to enamel-forming cells by modulating the signal pathways mediating epithelial–mesenchymal interaction and a cell-autonomous gene network. The differentiation process of epithelial cells is characterized by marked changes in their morphology and polarity, accompanied by dynamic cytoskeletal reorganization and changes in cell–cell and cell–matrix adhesion over time. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete enamel-specific proteins. After deposition of the full thickness of enamel matrix, ameloblasts become smaller and regulate enamel maturation. Recent significant advances in the fields of molecular biology and genetics have improved our understanding of the regulatory mechanism of the ameloblast cell life cycle, mediated by the Rho family of small GTPases. They act as intracellular molecular switch that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. In our review, we summarize studies that provide current evidence for Rho GTPases and their involvement in ameloblast differentiation. In addition to the Rho GTPases themselves, their downstream effectors and upstream regulators have also been implicated in ameloblast differentiation.

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The Semaphorin 4D-RhoA-Akt Signal Cascade Regulates Enamel Matrix Secretion in Coordination With Cell Polarization During Ameloblast Differentiation

Otsu

Ida‐Yonemochi

Fujiwara

et al. 2016

Journal of Bone and Mineral Research

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During tooth development, oral epithelial cells differentiate into ameloblasts in order to form the most mineralized tissue in the vertebrate body: enamel. During this process, ameloblasts directionally secrete enamel matrix proteins and morphologically change from low columnar cells to polarized tall columnar cells, both of which are essential for the proper formation of enamel. In this study, we elucidated the molecular mechanism that integrates ameloblast function and morphology. Immunohistochemistry revealed that the restricted expression of semaphorin 4D (Sema4D) and RhoA activation status are closely associated with ameloblast differentiation in mouse incisors. In addition, in vitro gain-of-function and loss-of-function experiments demonstrated that Sema4D acts upstream of RhoA to regulate cell polarity and amelogenin expression via the Plexin B1/Leukemia-associated RhoGEF (LARG) complex during ameloblast differentiation. Experiments in transgenic mice demonstrated that expression of a dominant-negative form of RhoA in dental epithelium hindered ameloblast differentiation and subsequent enamel formation, as well as perturbing the establishment of polarized cell morphology and vectorial amelogenin expression. Finally, we showed that spatially restricted Akt mediates between Sema4D-RhoA signaling and these downstream cellular events. Collectively, our results reveal a novel signaling network, the Sema4D-RhoA-Akt signal cascade, that coordinates cellular function and morphology and highlights the importance of specific spatiotemporally restricted components of a signaling pathway in the regulation of ameloblast differentiation. © 2016 American Society for Bone and Mineral Research.

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Dental Enamel Structure Is Altered by Expression of Dominant Negative RhoA in Ameloblasts

Cited by 12 publications

References 36 publications

Wnt-RhoA Signaling Pathways in Fluoride-Treated Ameloblast-Lineage Cells

Wnt-RhoA Signaling Pathways in Fluoride-Treated Ameloblast-Lineage Cells

Rho GTPases in ameloblast differentiation

The Semaphorin 4D-RhoA-Akt Signal Cascade Regulates Enamel Matrix Secretion in Coordination With Cell Polarization During Ameloblast Differentiation

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