PROFILAGGRIN IN EPIDERMAL DIFFERENTIATION AND FUNCTION

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DESCRIPTION: (Adapted from the applicant's abstract) - Profilaggrin is an abundant, highly phosphorylated, calcium-binding protein expressed in differentiating epidermal keratinocytes just prior to formation of cornified cells. Profilaggrin contains an N-terminal calcium-binding domain and multiple filaggrin units. Each region is proposed to have specific, distinct functions in vivo once profilaggrin is cleaved to yield the N-terminal and filaggrin peptides. The goal of this proposal is to understand the function(s) of these domains of profilaggrin by expression in keratinocytes of mutant proteins with a non-functional calcium-binding domain and by targeted disruption in embryonic stem cells to create profilaggrin-deficient mice. New evidence suggests that filaggrin, derived from profilaggrin by specific proteolysis, functions in cultured keratinocytes as a keratin-aggregating protein, leading to collapse of the keratin cytoskeleton, changes in cell shape, and cell cycle arrest. Filaggrin undergoes further proteolysis in vivo to free amino acids which bind water and are thought to maintain normal osmolarity of corneocytes. The hypotheses to be tested are (1) The calcium-binding domain of profilaggrin has a specific function(s) in regulating the calcium-dependent events of epidermal cornification, including profilaggrin expression and proteolytic processing to filaggrin, and (2) filaggrin, through its roles as a keratin-associated protein and as the major source of hygroscopic amino acids, performs critical functions in regulating normal corneocyte maturation and function. To address these questions, the specific aims proposed are to (1) Determine the function of the N-terminal calcium-binding domain of profilaggrin by first abolishing its ability to bind calcium and then expressing it in stable keratinocyte cell lines and in transgenic mice and (2) determine the function(s) of profilaggrin in vivo by targeted disruption in mice. Profilaggrin-deficient mice will allow assessment of the functional significance of profilaggrin in vivo. This work should also provide a firm basis for understanding the biochemical significance of reduced or absent profilaggrin expression in skin diseases such as ichthyosis vulgaris and psoriasis and the failure of profilaggrin processing to filaggrin in the severe scaling disorder Harlequin ichthyosis.