EXPRESSION, STRUCTURE AND FUNCTION OF FILAGGRIN

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Profilaggrin is a major structural protein expressed in the granular layer of the epidermis. It is a polyprotein precursor consisting 10, 11 or 12 filaggrin repeats which are adjoined by a short linker sequences. During terminal differentiation, this precursor is cleaved into individual filaggrin molecules, which based on in vitro experiments, are thought to be involved in the aggregation and specific alignment of the keratin intermediate filaments in the fully differentiated, dead, cornified cell layers of the epidermis. In earlier studies, we have explored the regulation of expression of the profilaggrin gene and have characterized its proximal promoter. An AP1 site and its cognate binding c-fos and c-jun proteins confer keratinocyte-specific expression to the gene, in concert with neighboring Sp1, ets-like, and NF-KB elements. We have discovered a novel ets transcription protein that seems to function in the regulation of expression of several late differentiation genes in the epidermis, including profilaggrin. Moreover, studies by Morasso and collaborators have identified a homeodomain transcription factor, known as Distal-less 3 (Dlx3), which plays a central role in activating the expression of profilaggrin (and other structural proteins of the epidermis) that are necessary for the formation of the cornified layer. During epidermal development, the expression of Dlx3 is restricted to the differentiated (suprabasal) cells, predominantly the granular cells, of the mouse stratified epidermis. Gain-of-function experiments by ectopically driving the expression of Dlx3 in the basal cells of transgenic mice using the basal-specific keratin 5 promoter, resulted in a severely abnormal epidermal phenotype leading to perinatal lethality because of the inability to form a functional cornified layer, that constitutes the water-barrier of the skin and prevents dehydration. Importantly, the basal cells in the transgenic epidermis ceased to proliferate and expressed late differentiation epidermal markers such as loricrin and profilaggrin. Consistent with these findings, we identified a binding site for Dlx3 in the proximal promoter of the profilaggrin gene. The importance of Dlx3 in the patterning and development of structures derived from epithelial-mesenchymal interactions during embryogenesis (i.e. tooth, hair) is corroborated by the effects of mutations in the DLX3 gene in patients with the autosomal dominant Tricho-Dento-Osseous (TDO) syndrome. We are presently developing a transgenic model for the TDO syndrome, which should allow us to obtain a better understanding of Dlx3 function and the molecular basis for the pathologies associated with TDO. The major focus of this project has now changed to an understanding the molecular mechanisms that regulate the early stages of epidermal differentiation, using the effects of the Dlx3 system on expression of the profilaggrin gene. Epidermis has been used as an excellent model for studying the process of cellular differentiation because the cells form a stratified structure during development, with each strata being easily identified by morphology and specific markers. The aims of the current studies are to: determine the role of Dlx3 in the regulation of late differentiation structural genes using profilaggrin as the paradigm; determine the signaling pathway through which Dlx3 acts and controls transcription; and analyze functional role of Dlx3 through mouse model of TDO syndrome. Use of gene targeting to study Dlx3 function In a parallel approach to understand the functional role of Dlx3, a targeted deletion of the gene was performed and the analysis of the phenotype has been completed. Targeted deletion of the DLX3 gene results in embryonic developmental arrest around day 9.5-10, associated with a gross failure of the placenta to undergo proper morphogenesis. It was not possible to assess the effects of Dlx3 loss of function on epidermal differentiation, since embryonic death occurs significantly earlier than the onset of epidermal stratification (E15.5). Of the large number of targeted mutagenesis of genes that lead to early death in embryogenesis, several of those due to defects in placenta development have been rescued by tetraploid aggregation experiments (i.e. Mash-2, Ets-2). Rescue of the trophoblast defect in the Dlx3 -/- embryos by tetraploid aggregation experiments has been initiated to determine if in this context, the null animals are viable or present a phenotype in the specific structures were Dlx3 is expressed throughout embryonic development, with special interest in the stratified epidermis. Generating mouse models for TDO Recently, Hart and collaborators reported that the genetic abnormality in the hereditary disease Tricho-Dento-Osseous syndrome (TDO) was a four nucleotide deletion in the human DLX3 gene, immediately downstream from the homeodomain coding region. This syndrome is inherited as an autosomal dominant trait, and includes abnormalities in the teeth, hair and facial bones. Mice heterozygous for the Dlx3 disruption (?knockout? project) do not show any such abnormalities, suggesting that the dominance of the TDO syndrome is due to formation of nonfunctional heteromeric complexes involving the truncated Dlx3 protein, as opposed to haploinsufficiency. We have generated transgenic mice to serve as a model for the TDO syndrome. The exact mutation present in the reported human families was introduced in the mouse DLX3 gene and driven under the regulatory control of a Dlx3 promoter region that has been previously characterized. Analyses of these mice will help to understand the structure of Dlx3, the effect of the mutation on the protein:protein interactions, and on the DNA binding and/or activation of transcription capability of Dlx3 that results in defects in several of the structures were Dlx3 is known to be expressed. Regulation of Dlx3 expression in differentiating keratinocytes In order to determine the signaling pathway by which Dlx3 is activated in the epidermis, we have studied mouse keratinocytes cultivated in vitro.