Identification of filaggrin in cultured mouse keratinocytes and its regulation by calcium.
Filaggrin is a histidine-rich cationic protein present in cells of the stratum corneum in vivo and derived from a precursor in keratohyalin granules. Biochemical and immunologic methods were used to determine the presence of filaggrin in keratinocytes cultured in vitro and induced to differentiate by increasing the extracellular calcium concentration from 0.07 to 1.2 mM. Indirect immunofluorescence using antibody to rat filaggrin was negative in cells cultured in low-calcium medium but positive in cells switched to high-calcium medium. Large immunofluorescent granules were identified in a perinuclear distribution starting at 6 hours after the shift in calcium concentration, coinciding with the time of appearance of phase-dense cytoplasmic granules. Radiolabeled histidine was preferentially incorporated into proteins of 95, 37, and 27 K. The 37 and 27 K bands were not adsorbed by DE52 cellulose and therefore are cationic. A 27 K cationic, histidine-labeled protein was readily extracted from frozen pellets of cells cultured in high-calcium medium. It comigrates with purified mouse filaggrin (27 K) and reacts with antibody to rat filaggrin on immunoautoradiography. Only trace amounts of this protein could be detected in cells cultured in low-calcium medium. Our observation of filaggrin-immunoreactive granules confirms the previous ultrastructural identification of keratohyalin granules after the shift to high-calcium medium. The results suggest that filaggrin synthesis is stimulated in keratinocytes induced to differentiate by the shift to high extracellular calcium concentration.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects; D000091344:Filaggrin Proteins; D005455:Fluorescent Antibody Technique; D006639:Histidine / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000096:biosynthesis*; D051379:Mice; D008807:Mice, Inbred BALB C
D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D006639:Histidine; D002118:Calcium