Using conventional tissue culture techniques, keratinocytes can be maintained in a basal cell-like proliferative state, or can be induced to differentiate and form cornified envelopes. This type of model has proven useful for understanding many of the molecular events which occur during keratinocyte differentiation and corneocyte formation. However, in many key aspects, the program of differentiation undergone by cultured keratinocytes is different from that seen in vivo. Thus, for example, the biochemical composition of cornified envelopes formed in culture differs markedly from that of the in vivo epidermis. Notably, the marker of late differentiation, loricrin, is largely absent from tissue, culture cornified envelopes. This suggests that, in vitro, the keratinocyte differentiation program is only partially completed, and that results derived from cell culture models should be interpreted cautiously. Unlike cells in vivo, cells grown on tissue culture plastic are nearly completely shielded from mechanical deformation. It is well known that the mechanical environment strongly influences the growth and architecture of many tissues. The epidermis is one of these issues: when skin is subjected to chronic mechanical deformation, one of the resulting responses is the increase in thickness of the stratum corneum, which is structurally the most resistant portion of the epidermis. Possible mechanisms by which stratum corneum thickness can be regulated include synthesis of more envelopes, to over-compensate for increased losses due to mechanical forces, synthesizing better envelopes, i.e. ones which resist mechanical forces better, or creating more cohesion between envelopes, rendering the whole stratum corneum more resistant to desquamation. In the experiments proposed in this Pilot and Feasibility Study application, we will address the hypothesis that keratinocytes grown in can respond to mechanical deformation in a biologically relevant fashion. The goal of each of the aims of this study is to examine one aspect of the possible mechanisms regulating stratum corneum thickness outlined above. We will therefore determine whether specific regimens of mechanical loading influence the proliferation and/or maturation of the keratinocyte by examining cell proliferation and the rate of cornified envelope formation in cultured keratinocytes in response to a series of specific loading regimens. We will next evaluate the expression patterns and levels of the markers of terminal differentiation, involucrin and loricrin to determine if they are affected by the mechanical deformation of cells. We will use immunodetection methods to assay for the presence and relative mounts of these markers in loaded and unloaded keratinocytes. Finally, we will determine whether the expression patterns of desmosomal proteins are affected by mechanical loading of the keratinocyte. We will use immunocytochemistry and confocal microscopy to determine the expression patterns of the desmosomal proteins in mechanically deformed and quiescent keratinocytes.
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