Molecular mechanisms mediating the soft tissue attachment to teeth

Stanford University, Stanford CA

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Abstract

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.