- 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.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
First Independent Research Support & Transition (FIRST) Awards (R29)
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General Medicine A Subcommittee 2 (GMA)
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Moshell, Alan N
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University of Washington
Schools of Dentistry
United States
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Presland, Richard B; Fleckman, Philip (2005) Tetracycline-regulated gene expression in epidermal keratinocytes. Methods Mol Biol 289:273-86
Presland, Richard B; Coulombe, Pierre A; Eckert, Richard L et al. (2004) Barrier function in transgenic mice overexpressing K16, involucrin, and filaggrin in the suprabasal epidermis. J Invest Dermatol 123:603-6
Presland, Richard B; Jurevic, Richard J (2002) Making sense of the epithelial barrier: what molecular biology and genetics tell us about the functions of oral mucosal and epidermal tissues. J Dent Educ 66:564-74
Pearton, David J; Dale, Beverly A; Presland, Richard B (2002) Functional analysis of the profilaggrin N-terminal peptide: identification of domains that regulate nuclear and cytoplasmic distribution. J Invest Dermatol 119:661-9
Presland, R B; Kuechle, M K; Lewis, S P et al. (2001) Regulated expression of human filaggrin in keratinocytes results in cytoskeletal disruption, loss of cell-cell adhesion, and cell cycle arrest. Exp Cell Res 270:199-213
Presland, R B; Dale, B A (2000) Epithelial structural proteins of the skin and oral cavity: function in health and disease. Crit Rev Oral Biol Med 11:383-408
Presland, R B; Boggess, D; Lewis, S P et al. (2000) Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris. J Invest Dermatol 115:1072-81