ANTISENSE PROFILAGGRIN RNA DELAYS AND DECREASES PROFILAGGRIN EXPRESSION AND ALTERS IN-VITRO DIFFERENTIATION OF RAT EPIDERMAL-KERATINOCYTES

Ichthyosis vulgaris is an epidermal disorder in which profilaggrin exp ression is decreased or absent. To determine whether the ichthyosis vu lgaris phenotype could be mimicked by eliminating profilaggrin express ion, a rat epidermal cell line was transfected with a plasmid that dir ects the constitutive expression of an RNA that is antisense to normal profilaggrin mRNA. Non-transfected and neomycin-resistant cells not c ontaining antisense plasmid that were grown in the neomycin analogue G 418 served as controls. Immunoblot and immunofluorescence analysis sho wed that profilaggrin protein expression and processing to filaggrin w ere delayed by 3 to 4 d and decreased in transfected cells. Profilaggr in mRNA was detected in both control and transfected cells only after the cells reached confluence, whereas antisense RNA was detected in tr ansfectants at all times, even prior to confluence. Ultrastructural ex amination revealed that keratohyalin granules were decreased in number , globular, and heterogeneous in appearance in transfected cells in co ntrast to angular structures seen in control cells. Unexpectedly, stra tification was impaired, intermediate filaments were noticeably reduce d, and cornified cell envelope formation was delayed in transfectants. Unlike ichthyosis vulgaris keratinocytes, where keratin expression is unaffected, appearance of K1 and K10 was delayed and K1/K10 synthesis was delayed and decreased in transfected cells. The precipitous drop in S-35-merhionine incorporation into cytoskeletal protein seen at con fluence in control cells was delayed by 3 d in transfected cells. We c onclude that, rather than producing the ichthyosis vulgaris phenotype, antisense profilaggrin RNA has more broad-reaching effects on in vitr o differentiation of rat epidermal keratinocytes.