Wound healing is essential for survival. This is a multistep process involving a number of different cell types. Of particular relevance to this project is that wounding activates stem cells in the interfollicular epidermis (IFE) and hair follicles (HF) to proliferate and send their progeny to re-epithelialize the wound and subsequently regenerate the epidermis. Failure to close wounds leads to medical costs estimated in the US at over $25 billion and affecting 6.5 million people. Our previous studies have shown that vitamin D and calcium signaling play important roles in these events. As we show in preliminary data, both the vitamin D receptor (VDR) and calcium sensing receptor (CaSR) are required for the maintenance and activation of the stem cells in the HF and IFE. In the previous funding cycle we showed that lack of the VDR and CaSR are associated with a delay in wound closure. We hypothesized that VDR and CaSR are required both for the maintenance of the stem cell niches and their activation following wounding. This activation stimulates their proliferation and migration to re- epithelialize the wound. Our preliminary data support this hypothesis in that deletion of VDR (VDRKO) results in a reduction in the number of cells in the stem cell niches in both HF and IFE, that proliferation is reduced in the cells at the leading edge of the epithelium after wounding, that expression of axin 2 and CD44 as markers of stem cell activation is reduced, and that the leading edge of the epithelium at the wound is disorganized with reduction in the epithelial junctions (E-cadherin/catenin complexes) that appear to be required for migration of the keratinocytes across the wound as a first step in restoring the epidermis. Moreover, we demonstrated that topical application of a calcimimetic to activate the CaSR or calcitriol to activate the VDR accelerates wound healing, increasing the number and proliferation of the stem cells. Building on these promising preliminary we now propose to determine the mechanisms by which calcium and vitamin D signaling regulate the response of stem cells to wounding and the subsequent ability of their progeny to re-epithelialize the wound as the first step in restoring the epidermis. The hypothesis that we plan to test is: “The VDR and CaSR in keratinocytes are required for the maintenance of the epidermal stem cell niche. Moreover, by regulating intracellular calcium dependent signaling mechanisms they enable the activation, proliferation, and migration of epidermal stem cells and their progeny following wounding to re-epithelialize the wound and subsequently regenerate the epidermis”. To test this hypothesis we propose the following three aims. 1 Determine whether vitamin D and calcium signaling via their receptors play distinct and/or complementary roles in epidermal stem cell function during re-epithelialization. 2. Determine whether VDR and CaSR are essential for the maintenance of the stem cell niches within the epiderm...