Cornification of epidermal cells involves two major events: processing of profilaggrin to the keratin filament aggregating protein filaggrin and formation of cornified envelopes. Profilaggrin processing occurs in two stages; the first stage produces intermediates which are then processed to filaggrin in the second stage. Preliminary experiments indicate that the second stage is initiated by increased cytoplasmic Ca2+, which activates calpain. Calpain appears to then activate the protease that initates second stage processing: thus a cytoplasmic protease cascade is proposed. These studies will test this model and also test whether the initiating Ca2+ influx occurs through a voltage-sensitive plasma membrane calcium channel. Increased [Ca2+]i is known to be involved in cornified envelope formation, but the role of Ca2+ in profilaggrin processing has been previously unrecognized; the possible coupling of these two events by Ca2+ will be analyzed. Finally,activation of the Ca2+ channel and alteration of profilaggrin processing in response to irritation of skin will be examined.
Specific aims are to 1) examine the regulation of [Ca2+}i in cultured keratinocytes and epidermis: 2) test whether formation of cornified envelopes and profilaggrin processing are linked by increased [Ca2+]i in cultured keratinocytes; 3) identify the second stage protease that processes intermediates to filaggrin; 4) correlate changes in profilaggrin processing with Ca2+ channel activity in irritated skin. [Ca2+}i will be measured using fluorescent indicator dyes and ion-capture cytochemistry. Quantification of SDS/mercaptoethanol-resistant cell envelopes will provide a measure of cornification. Profilaggrin processing will be assessed by Western blotting using specific antibodies. Processing proteases will be distinguished from other proteases in epidermis by: 1) structural analysis of the new amino- and carboxyl-termini formed by digestion of intermediates; and 2) comparing the specificity of inhibitors acting in vitro and in intact cultured cells. Morphological studies will involve electron and light microscopy. These biochemical studies will lay the groundwork for understanding the regulation of profilaggrin processing and the coordination of events occurring during cornification. The experimental accessibility of these events makes this an ideal system to examine the interplay of internal and external Ca2+ stores in regulating [Ca2+]i. Finally, defining the role of calpain in this system would provide a paradigm for the Ca2+ regulation of proteolysis in cells and exemplify the first known cytoplasmic zymogen cascade.
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