[unreadable] Our overall goal is to test the hypothesis that hyaluronan (hyaluronic acid, HA), an abundant matrix component in the intercellular space between keratinocytes of the skin, has a crucial role in epidermal homeostasis and participates in a mechanism to coordinate keratinocyte proliferation and differentiation during epidermal formation and wound-healing. Our preliminary data indicate that HA is not simply a passive occupant of the extracellular space, but rather has an active, instructive role in regulating the epidermal differentiation program. Using an in vitro model specifically tailored for these studies (a rat keratinocyte line that stratifies in raft culture), we will examine epidermal HA metabolism and its functional role in regulating epidermal differentiation. Aim 1 will quantify the production of HA, expression of the major HA synthase enzymes, and expression of hyaluronidases produced by the keratinocytes during differentiation. HA will be experimentally removed from the intercellular space using Streptomyces hyaluronidase, to create a "functional HA-knockout," in which effects on differentiation and proliferation can be assessed. Aim 2 will more-carefully explore the synthetic side of HA metabolism, using a tet-inducible (tetON) gene-switch system to overexpress HAS2 in the keratinocytes at different times during epidermal differentiation. Aim 3 will take a similar approach on the catabolic side, by inducibly overexpressing HYAL2, the major hyaluronidase expressed and regulated in the REK cells. HYAL2 will be overexpressed, and the consequences of increased HA degradation determined. In related experiments, CD44 (the major hyaluronan-receptor on the keratinocyte plasma membrane, and implicated in the uptake and degradation of HA) will be targeted. A dominant-negative form of CD44 that lacks the cytoplasmic anchoring tail will be inducibly overexpressed to interfere with native CD44 binding and function. Aim 4 will apply information gained from the prior Aims to experiments in hairless mouse skin in vivo. Using two well-known models of epidermal hyperplasia, namely (i) application of topical retinoids, and ii) barrier disruption of the stratum corneum, we will determine whether HA has a role in responses to epidermal-specific irritation and injury. [unreadable] [unreadable]