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Principal Investigator: Jill A Helms
Organization: STANFORD UNIVERSITY
Fiscal Year: 2023
Award: $38,268
Funding agency: National Institute of Dental and Craniofacial Research
Summary of Parent R01
Maintaining the junc-onal epithelium (JE) is essen-al to preserve cementum, periodontal ligament (PDL), and alveolar bone.
New insights into JE barrier func-ons came with our discovery of a Wntresponsive 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 aGer damage or disease. AIM 1 experiments will test whether Wnt/β-catenin signaling is
necessary for JE maintenance. Pathway inhibi-on will be achieved in Axin2LacZ/+ mice using adenovirus expressing the soluble
Wnt inhibitor Dkk1; controls will receive adenovirus encoding the Fc por-on of immunoglobulin G. Quan-ta-ve analyses
will assess endogenous Wnt/βcatenin signaling via Xgal staining; JE hemidesmosomal gene and protein distribu-on via
quan-ta-ve immunohistochemistry (qIHC); JE and GE cell cycle kine-cs by EdU/BrdU labeling; and inflammatory cell
infiltra-on in connec-ve -ssues underlying the JE and GE by FACS. Second, whether Wnt/β-catenin signaling is necessary for
JE regenera-on will be determined by subjec-ng Wnt lineage tracer e.g., Axin2CreERT2/+;R26RmTmG/+ mice to par-al
gingivectomy followed by Ad-Dkk1/Ad-Fc delivery. Lineage-tracing and quan-ta-ve analyses will establish a rela-onship
between Wntresponsive cell progeny, cell cycle kine-cs, hemidesmosomal gene and protein distribu-on, and regenera-on
of JE barrier func-ons. AIM 2 experiments will evaluate the ability of a stabilized formula-on of WNT protein to regenerate
a func-onal 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 periodon--s; both will be carried out in Axin2LacZ/+ and Axin2CreERT2/+;R26RmTmG/+ mice. Delivery of the
WNT therapeu-c will be followed to assess re-establishment of JE barrier func-ons. 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 -ssues at
defined -mepoints. GFP+ cells will be sorted by flow cytometry. Gene expression profiling of GFP+ cells will focus stem cell
and differen-a-on markers. Fluorescent in situ hybridiza-on will confirm gene expression pacerns using RNA probe libraries
corresponding to stem cell markers, components of Wnt/β-catenin, Notch, and Bone Morphogene-c Protein (BMP)
pathways. Collec-vely, this proposal has the poten-al to iden-fy an innova-ve therapeu-c strategy for rebuilding a damaged
JE and thus open new avenues for the restora-on of the soG -ssue acachment following periodontal diseases.
Proposed Supplement
The JE acts as an essen-al barrier against bacterial infec-on, which is crucial in preven-ng periodontal diseases. In this
proposed diversity supplement, we will look to elucidate the role of Wnt/β-catenin signaling in maintaining the barrier and
acachment func-ons of the JE. Extended Aim 2 experiments will use a transgenic mouse model with a defect in Wnt
signaling, e.g., OCN-Cre;Wlsfl/fl, to evaluate if the JE and Wntresponsive stem cell niche become disrupted. Alveolar bone
morphology will be assessed by µCT tomography; inflammatory cell response and JE hemidesmosomal proteins via qIHC;
and bone remodeling ac-vity by TRAP/ALP assays. This proposal has the poten-al to advance Wnt therapeu-c treatments
for periodontal diseases, which can then be made more uniformly accessible to disadvantaged communi-es.
Terms: <7S Gamma Globulin><Abscission><Address><Adenoviridae><Adenoviruses><Alveolodental Ligament><Alveolodental Membrane><Anatomic Sites><Anatomic structures><Anatomy><Assay><Autoregulation><BUdR><Beta Cadherin-Associated Protein><Beta-1 Catenin><Bioassay><Biochemical><Biologic Assays><Biological Assay><Body Tissues><Bone Morphogenetic Protein Gene><Bone Morphogenetic Proteins><Bone remodeling><BrdU><Bromodeoxyuridine><Bromouracil Deoxyriboside><Broxuridine><Buccal Cavity><Buccal Cavity Head and Neck><C protein><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><Defect><Dental Cementum><Differentation Markers><Differentiation Antigens><Differentiation Markers><Disadvantaged><Disease><Disorder><Embryo><Embryonic><Epithelial Attachment><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><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><In Situ><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><Morphology><Mouth><Murine><Mus><Natural regeneration><Operative Procedures><Operative Surgical Procedures><Oral><Oral cavity><PRO2286><Parents><Parodontosis><Pathway interactions><Periodontal Diseases><Periodontal Ligament><Periodontal Membrane><Periodontitis><Physiological Homeostasis><Placebos><Population><Preventative strategy><Prevention strategy><Preventive strategy><Progenitor Cells><Proliferating><Property><Proteins><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><Tissues><Tooth><Tooth structure><Tracer><Transcript Expression Analyses><Transcript Expression Analysis><Transgenic Mice><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><bone morphogenic protein><bone remodelling><citrate carrier><citrate periplasmic carrier protein><citrate transporter><citrate-binding transport 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><genetic approach><genetic strategy><inhibitor><injuries><injury and repair><insight><lipid based nanoparticle><lipid nanoparticle><maxilla alveolar process><mouse model><murine model><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><parent><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><response><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><tomography><tooth surface><transcriptional profile><transcriptional profiling><transcriptional signature><tricarboxylate carrier><tricarboxylate transporter><tricarboxylate-binding C protein><wound healing><wound resolution><β-catenin>