bromochloroacetic-acid and monodansylcadaverine

We previously demonstrated that the aspartate protease cathepsin D is activated by ceramide derived from acid sphingomyelinase. Increased expression of cathepsin D in the skin has been reported in wound healing, psoriasis and skin tumors. We explored specific functions of cathepsin D during epidermal differentiation. Protein expression and enzymatic activity of cathepsin D increased in differentiated keratinocytes in both stratified organotypic cultures and in mouse skin during epidermal barrier repair. Treatment of cultured keratinocytes with exogenous cathepsin D increased the activity of transglutaminase 1, known to cross-link the cornified envelope proteins involucrin and loricrin during epidermal differentiation. Inhibition of cathepsin D by pepstatin A suppressed the activity of transglutaminase 1. Cathepsin D-deficient mice revealed reduced transglutaminase 1 activity and reduced protein levels of the cornified envelope proteins involucrin and loricrin. Also, amount and distribution of cornified envelope proteins involucrin, loricrin, filaggrin, and of the keratins K1 and K5 were significantly altered in cathepsin D-deficient mice. Stratum corneum morphology in cathepsin D-deficient mice was impaired, with increased numbers of corneocyte layers and faint staining of the cornified envelope only, which is similar to the human skin disease lamellar ichthyosis. Our findings suggest a functional link between cathepsin D activation, transglutaminase 1 activity and protein expression of cornified envelope proteins during epidermal differentiation.

Topics: Animals; Cadaverine; Cathepsin D; Cell Differentiation; Cells, Cultured; Enzyme Activation; Filaggrin Proteins; Gene Deletion; Intermediate Filament Proteins; Keratinocytes; Keratins; Membrane Proteins; Mice; Pepstatins; Protein Precursors; Protein Processing, Post-Translational; Skin; Transglutaminases; Wound Healing

Topics: Animals; Cadaverine; Culture Techniques; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Epidermal Cells; Epidermis; Filaggrin Proteins; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Keratins; Proteins; Skin

Cultured bovine keratinocytes contain two major classes of transglutaminase substrate proteins. These may be separated on Sephadex G-75 into a 30,000-to-50,000-dalton fraction and a fraction which is heavier than 75,000 daltons. By SDS-electrophoresis, the lighter fraction consists of two components of molecular weights 9,000 and 18,000 daltons, while the heavier fraction consists of a doublet of about 100,000 daltons. Bovine epidermis contains substrate proteins which apparently correspond to the two components of the lighter class. The 2 classes of substrate proteins may be analogous to two substrate proteins isolated from cultured human keratinocytes which are thought to be precursors of cornified envelope. The major transglutaminase substrate present in both newborn rat epidermis and cultured keratinocytes has a native molecular weight of greater than 75,000 daltons but an SDS-dissociated molecular weight of 22,000 daltons. The sera of 5 of 6 rabbits react immunologically with a protein present in cultured human keratinocytes which is a poor or minor substrate of transglutaminase. The cross-linking of keratins may play a role in epidermal differentiation since keratins from more differentiated areas of epidermis may contain a higher level of interchain isopeptide bonds. Keratin may be cross-linked itself or to stratum corneum basic protein, in vitro, to form both soluble and insoluble aggregates. No adducts between keratin and purified cornified envelope were formed when both were treated with active epidermal transglutaminase.

Topics: Animals; Animals, Newborn; Aspartate Aminotransferases; Cadaverine; Cell Differentiation; Epidermal Cells; Epidermis; Humans; In Vitro Techniques; Keratins; Molecular Weight; Rats