Abstract
Mutations in the cornified cell envelope protein loricrin have been reported recently in some patients with Vohwinkel syndrome (VS) and progressive symmetric erythrokeratoderma (PSEK). To establish a causative relationship between loricrin mutations and these diseases, we have generated transgenic mice expressing a COOH-terminal truncated form of loricrin that is similar to the protein expressed in VS and PSEK patients. At birth, transgenic mice (ML.VS) exhibited erythrokeratoderma with an epidermal barrier dysfunction. 4 d after birth, high-expressing transgenic animals showed a generalized scaling of the skin, as well as a constricting band encircling the tail and, by day 7, a thickening of the footpads. Histologically, ML. VS transgenic mice also showed retention of nuclei in the stratum corneum, a characteristic feature of VS and PSEK. Immunofluorescence and immunoelectron microscopy showed the mutant loricrin protein in the nucleus and cytoplasm of epidermal keratinocytes, but did not detect the protein in the cornified cell envelope. Transfection experiments indicated that the COOH-terminal domain of the mutant loricrin contains a nuclear localization signal. To determine whether the ML.VS phenotype resulted from dominant-negative interference of the transgene with endogenous loricrin, we mated the ML.VS transgenics with loricrin knockout mice. A severe phenotype was observed in mice that lacked expression of wild-type loricrin. Since loricrin knockout mice are largely asymptomatic (Koch, P.K., P. A. de Viragh, E. Scharer, D. Bundman, M.A. Longley, J. Bickenbach, Y. Kawachi, Y. Suga, Z. Zhou, M. Huber, et al., J. Cell Biol. 151:389-400, this issue), this phenotype may be attributed to expression of the mutant form of loricrin. Thus, deposition of the mutant protein in the nucleus appears to interfere with late stages of epidermal differentiation, resulting in a VS-like phenotype.
Highlights
The formation of the cornified cell envelope (CE)1 in terminally differentiating keratinocytes is generally accepted to be crucial for the barrier function of the skin
On the basis of recent molecular studies, it is clear that Vohwinkel syndrome (VS) associated with deafness is genetically distinct and caused by mutations in connexin 26 (Maestrini et al, 1999), whereas VS associated with ichthyosis is caused by mutations in loricrin (Maestrini et al, 1996; Korge et al, 1997; Armstrong et al, 1998; Takahashi et al, 1999)
To establish a causative relationship between loricrin mutations and the human skin diseases VS and progressive symmetric erythrokeratoderma (PSEK), we introduced a transgene into the germline of mice containing a single nucleotide insertion that mimics frameshift mutations recently identified in VS and PSEK (Maestrini et al, 1996; Ishida-Yamamoto et al, 1997; Korge et al, 1997; Armstrong et al, 1998; Takahashi et al, 1999)
Summary
The formation of the cornified cell envelope (CE) in terminally differentiating keratinocytes is generally accepted to be crucial for the barrier function of the skin Expression of the ML.VS transgene in the loricrin null background (LorϪ/Ϫ) showed a more severe phenotype than ML.VS mice in the Lorϩ/Ϫ background (Fig. 10 a), and as observed previously in the Lorϩ/ϩ background, phenotype severity correlated with transgene dosage as evidenced via ML.VS/LorϪ/Ϫ sibling matings (Fig. 10 b). These mating experiments confirm that the VS mutant protein is not functioning as a classical dominantnegative molecule, i.e., inducing the phenotype by a direct interference with wild-type loricrin. The positive labeling with the LorNAb seen on the cell envelopes of transgenics that express wild-type loricrin (see Fig. 7 a, ϩ/ϩ mice) presumably only detects the presence of wild-type loricrin
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