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Principal Investigator: Jill A Helms
Organization: STANFORD UNIVERSITY
Fiscal Year: 2023
Award: $706,137
Funding agency: National Institute of Dental and Craniofacial Research
Project abstract
Maintaining the junctional epithelium (JE) is of primary importance if preservation of the cementum, periodontal ligament
(PDL), and alveolar bone is to be achieved. New insights into JE barrier functions came with our discovery of a Wnt-
responsive stem cell niche in the JE. In this proposal, our goal is to characterize the cellular players, niche signals, and
regulatory mechanisms that control and maintain the JE stem cell niche in health, and after damage or disease.
AIM 1 experiments will first test whether Wnt/β-catenin signaling is necessary for JE maintenance. Pathway inhibition will
be achieved in Axin2LacZ/+ mice using adenovirus expressing the soluble Wnt inhibitor Dkk1; controls will receive adenovirus
encoding the Fc portion of immunoglobulin G. At defined intervals, quantitative analyses will assess endogenous Wnt/β-
catenin signaling via Xgal staining; JE hemidesmosomal gene and protein distribution via quantitative
immunohistochemistry (qIHC); JE and GE cell cycle kinetics by EdU/BrdU labeling; and inflammatory cell infiltration in
connective tissues underlying the JE and GE by FACS. Second, whether Wnt/β-catenin signaling is necessary for JE
regeneration will be determined by subjecting Wnt lineage tracer e.g., Axin2CreERT2/+;R26RmTmG/+ mice to partial gingivectomy
followed by Ad-Dkk1/Ad-Fc delivery. Lineage-tracing and quantitative analyses will establish a relationship between Wnt-
responsive cell progeny, cell cycle kinetics, hemidesmosomal gene and protein distribution, and regeneration of JE barrier
functions.
AIM 2 experiments will evaluate the ability of a stabilized formulation of WNT protein to regenerate a functional JE. In one
injury-repair model the JE will be surgically excised; in a second model, JE breakdown will be triggered via a ligature-induced
periodontitis; both will be carried out in Axin2LacZ/+ and Axin2CreERT2/+;R26RmTmG/+ mice. Delivery of the WNT therapeutic will
be followed at multiple timepoints by quantitative analyses to assess re-establishment of JE barrier functions.
AIM 3 experiments will characterize Wnt-responsive JE stem cells and their progeny. Wnt-responsive stem cell pools from
adjacent gingival epithelium (GE) will serve as control. Axin2CreERT2/+;R26RmTmG/+ mice will be exposed to tamoxifen, followed
by harvest of JE and GE tissues at defined timepoints. GFP+ cells will be sorted by flow cytometry. Gene expression profiling
of GFP+ cells will focus stem cell and differentiation markers. Fluorescent in situ hybridization will confirm gene expression
patterns using RNA probe libraries corresponding to stem cell markers, components of Wnt/β-catenin, Notch, and Bone
Morphogenetic Protein (BMP) pathways. Collectively, this proposal promises to provide important new insights into the
requirement for Wnt/β-catenin signaling in maintaining the JE stem cell niche; regulating JE and GE cell proliferation and
differentiation; and influencing hemidesmosomal-mediated attachment to the tooth surface. In addition, it should resolve
the current debate over the molecular identities of JE v. GE stem cell pools and their differentiation potential. The proposed
work also has the potential to identify an innovative therapeutic strategy for rebuilding a damaged JE and thus open new
avenues for the restoration of the soft tissue attachment following periodontal diseases.
Terms: <7S Gamma Globulin><Abscission><Address><Adenoviridae><Adenoviruses><Alveolodental Ligament><Alveolodental Membrane><Anatomic Sites><Anatomic structures><Anatomy><Autoregulation><BUdR><Beta Cadherin-Associated Protein><Beta-1 Catenin><Biochemical><Body Tissues><Bone Morphogenetic Protein Gene><Bone Morphogenetic Proteins><BrdU><Bromodeoxyuridine><Bromouracil Deoxyriboside><Broxuridine><Buccal Cavity><Buccal Cavity Head and Neck><CUL-2><Cavitas Oris><Cell Body><Cell Communication and Signaling><Cell Cycle><Cell Cycle Kinetics><Cell Differentiation><Cell Differentiation process><Cell Division Cycle><Cell Kinetics><Cell Signaling><Cells><Cementum><Chronic Periodontitis><Clinical><Connective Tissue><Data><Dental Cementum><Differentation Markers><Differentiation Antigens><Differentiation Markers><Disease><Disorder><Embryo><Embryonic><Epithelial Attachment><Epithelial Cell Junction><Epithelial Cell Proliferation><Epithelial Cells><Epithelium><Excision><Exposure to><Expression Signature><Extirpation><FISH Technic><FISH Technique><FISH analysis><FISH assay><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Fluorescence In Situ Hybridization><Fluorescent in Situ Hybridization><Formulation><Gene Expression Monitoring><Gene Expression Pattern Analysis><Gene Expression Profile><Gene Expression Profiling><Genes><Gingiva><Gingival><Gingivectomy><Goals><Harvest><Health><Hemidesmosomes><Heterogeneity><Histology><Homeostasis><Human><IgG><Immunoglobulin G><Immunohistochemistry><Immunohistochemistry Cell/Tissue><Immunohistochemistry Staining Method><Infiltration><Inflammatory><Injury><Intracellular Communication and Signaling><Investigators><Jaw><Junctional Epithelium><Knowledge><Label><Libraries><Ligature><Maintenance><Marker Antigens><Mediating><Mice><Mice Mammals><Modeling><Modern Man><Molecular><Mouth><Murine><Mus><Natural regeneration><Operative Procedures><Operative Surgical Procedures><Oral><Oral cavity><PRO2286><Parodontosis><Pathway interactions><Periodontal Diseases><Periodontal Ligament><Periodontal Membrane><Periodontitis><Physiological Homeostasis><Placebos><Population><Preventative strategy><Prevention><Prevention strategy><Preventive strategy><Progenitor Cells><Proliferating><RNA Gene Probes><RNA Probes><Recombinants><Regeneration><Regenerative capacity><Removal><Reporter><Research Personnel><Researchers><Role><Series><Sham Treatment><Signal Transduction><Signal Transduction Systems><Signaling><Site><Skeleton><Sorting><Source><Staining method><Stains><Surgical><Surgical Interventions><Surgical Procedure><Surgical Removal><Tamoxifen><Testing><Therapeutic><Tissues><Tooth><Tooth structure><Tracer><Transcript Expression Analyses><Transcript Expression Analysis><WNT Signaling Pathway><WNT signaling><Wnt proteins><Wnt-3A protein><Work><Wound Repair><alveolar bone><alveolar supporting bone><analyze gene expression><beta catenin><biological signal transduction><bone morphogenic protein><epithelial progenitor cell><epithelial stem cell><epithelium regeneration><experiment><experimental research><experimental study><experiments><flow cytophotometry><gene expression analysis><gene expression assay><gene expression pattern><gene expression signature><inhibitor><injuries><injury and repair><innovate><innovation><innovative><insight><lipid based nanoparticle><lipid nanoparticle><maxilla alveolar process><native protein drug><new drug target><new druggable target><new pharmacotherapy target><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutic target><new therapy approaches><new therapy target><new treatment approach><new treatment strategy><notch><notch protein><notch receptors><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapeutic target><novel therapy approach><novel therapy target><pathway><periodontal disorder><periodontium disease><periodontium disorder><pharmaceutical protein><preservation><protein distribution><protein drug agent><protein-based drug><regenerate><regenerate epithelium><regeneration ability><regeneration capacity><repair model><resection><restoration><self-renew><self-renewal><sham therapy><skeletons><social role><socket wall><soft tissue><stem cell biomarkers><stem cell markers><stem cell niche><stem cell regeneration><stem cell self renewal><stem cells><surgery><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><teeth><therapeutic protein><tooth surface><transcriptional profile><transcriptional profiling><transcriptional signature><wound healing><wound resolution><β-catenin>