Telomeric Regulation of Stem Cell Phenotypes in Stratified Epithelia

Introduction: Oral mucosal regeneration is clinically important in surgical wound healing and periodontal grafting procedures. Epithelialization is an important component of the proliferative phase of wound healing in which oral mucosal stem cells rapidly divide and migrate to cover the wound bed during healing. The capacity of stem cells to self-renew and proliferate is regulated in part by telomeres, which are repetitive DNA-protein structures that cap chromosomes in all cells. Telomere length and stem cell renewal is maintained by several factors. Objectives: We examined the effects of telomere uncapping on oral mucosal stem cells by inhibiting expression of the telomere binding protein Pot1b using mouse genetics. Methods: We used mice with intact Pot1b expression as the control group. We characterized oral mucosal phenotype by histopathology, immunohistochemistry, and immunofluorescence microscopy. We determined oral mucosal stem cell fractions by flow cytometry and programmed cell death activity by TUNEL analysis. Telomere length was determined by quantitative PCR. Cell proliferation was determined by proliferating cell nuclear antigen expression and cell cycle profiling using flow cytometry. Results: Loss of Pot1b expression induced DNA damage response at the telomere characterized by recruitment of 53BP1, activation of ATR and Chk1 kinases, and induction of p53 expression. This increased DNA damage response was associated with telomere shortening, reduction in cell proliferation, and increased programmed cell death in the basal layer of oral mucosa. We localized two distinct stem cell populations in oral mucosa using CD34 or Lgr expression. Both of these cell populations were decreased as a result of Pot1b deficiency. We were able to rescue oral mucosal stem cell depletion in this model by blocking the telomeric DNA damage response. Conclusion: We concluded that telomere uncapping results in activation of the DNA damage response, which limits expansion of oral mucosal stem cells.