Sebaceous cell growth and differentiation is regulated mainly by androgen, retinoids, and poorly understood developmental biological factors. Our overall hypothesis is that sebocytes go through specific unidirectional developmental stages, which are each responsive in a specific manner to specific hormonal and stromal signals. The cell types consist of undifferentiated cells and their early-, mid, late, and terminally differentiated progeny. We will begin by characterizing sebocyte stages and substages precisely by fluorescent in situ hybridization (FISH) using molecular markers for mRNA's to co-localize cellular androgen receptor (AR), 5alpha-reductase (5alpha-R), the lipogenic enzymes HMG-CoA reductase (HMGR) and acetyl-CoA carboxylase (ACC), cytokeratin 4 (K4), retinoic acid receptor (RAR), and the retinoid X receptor (RXR). The hypothesis will then be tested that sebocyte stages can be precisely characterized by these molecular characteristics and that sebocytes will go through these early differentiated stages in monolayer culture. Androgen is specifically hypothesized to a) indirectly stimulate undifferentiated preputial cells to proliferate, via an action on preputial stromal cells, and b) directly act on early differentiated sebocytes to inhibit their proliferation, but induce lipid synthesis. We expect that and androgen receptor (AR) and 5alpha-R mRNA will not be expressed in undifferentiated sebocytes, and that they will be expressed in high amounts in preputial fibroblasts. The effects of androgen on lipid synthesis are expected to correspond to increased expression of AR mRNA and subsequent induction by androgen of lipogenic enzyme mRNA's in an early differentiated subpopulation of sebocytes. Retinoic acid (RA) is hypothesized to act directly on an early differentiated subtype of sebocyte to interfere with androgen action by inhibiting both their proliferation and lipogenesis. We will determine whether full differentiation of sebocytes occurs in lifted raft cultures due to the generation of a concentration gradient of RA such that reduced RA levels in supranasal layers permits terminal differentiation of sebocytes. Methodology to be employed is that in use in this laboratory: epithelial cell culture using monolayer and raft technique, histology, histochemistry, Northern analysis, immunocytochemistry, fluorescence microscopy, and RNase protection assay. The latter techniques will be combined in the form of FISH.
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