Differentiation and Development in Normal and Transformed Epidermal Cells
Our long range objective is an understanding of the molecular mechanisms underlying growth, differentiation and development of the epidermis of mammalian skin. In embryonic development, the epidermis begins as a single layered epithelium, which can give rise to neural and ectodermal cells. At the bi-layered epithelial stage, the innermost embryonic basal layer is restricted in its
pluripotency, and can choose only between an epidermal or hair follicle fate. This decision is influenced by whether or not cells come into contact with a specialized mesenchymal component, the dermal papilla cells. In the absence of this stimulus, the epidermal fate is chosen, and cells stratify and differentiate accordingly. Concomitant with commitment to an epidermal cell fate is the expression of keratins, the major structural proteins of the epidermis. The adult epidermis serves an important protective function, being at the interface between the body and the environment. It is composed of about 10 cell layers, the outermost being the skin surface. Only the inner (basal) layer is truly living and undergoes DNA synthesis and cell division. If there are no vacanciesin the basal layer, a daughter cell will move to the first suprabasal layer and, in so doing, undergoes a commitment to terminally differentiate and migrate towards the skin surface. In transit, the cell undergoes a variety of morphological and biochemical changes that are regulated at the transcriptional level. A major change is in keratin gene expression: basal cells express one set of genes encoding keratins 5 and 14, while differentiating epidermal cells express Kl and K10. Both pairs of keratins make 10 nm cytoskeletal filaments which are to the epidermis what globins are to a red blood cell: Kl and K10 constitute up to 85% of the fully differentiated epidermal cell.
We have isolated and characterized a number of keratin genes expressed in human epidermis. Our ultimate goal is to understand what factors control epidermal cell fate determination and how epidermal-specific and differentiation-specific gene expression is orchestrated. To achieve these aims, we have used two model systems. First, we can culture human epidermal cells under conditions where differentiation and stratification can be maintained. We can both transfect these cells with foreign genes, and make nuclear extracts from these cells to examine
keratinocyte-specific transcription factors. Secondly, we use transgenic mice as an in vivo model. These systems have enabled us to identity a number of gene sequences and transcription factors important for
keratinocyte-specific expression in vitro and in vivo.
The keratin promoters have also become invaluable for targeting the expression of foreign genes to the basal and differentiating epidermal layers of transgenic mice. Part of the lab has used these promoters to alter the expression of factors that influence the balance between dividing and differentiating cells. Such factors include growth factors such as TGFa , KGF and
TGFbs, and the retinoic acid receptor (RAR) members of the steroid receptor
superfamily. An analysis of these mice has enabled us to begin to decipher at a molecular level how these factors regulate epidermal differentiation, and how such regulation may go awry in the development of hyperproliferative diseases of the skin, such as psoriasis and skin cancer. In particular, we are interested in how two tyrosine kinase receptors, one for TGFa and one for
KGF, trigger different responses in epidermal cells, and how TGFb's and RARs play a role in epidermal differentiation and cell fate determination.
In addition to examining keratin gene expression, our laboratory studies keratin filament structure. We use molecular genetics to engineer various keratin mutants and examine their behavior in vitro and in transgenic animals. Using this approach, we recently discovered that transgenic mice expressing onemutant keratin gene exhibited the phenotype characteristic of patients with the human blistering skin disease, Epidermolysis Bullosa Simplex
(EBS), while those expressing another mutant keratin gene exhibited the phenotype characteristic another blistering disease, Epidermolytic Hyperkeratosis (EH). In the past two years, we isolated and characterized the genes from patients with these diseases, and showed that they have mutations in the expected
keratingenes. We are now utilizing a combination of molecular genetics, cell biology, embryonic stem cell technology and human genetics to probe further the genetic basis for these and other human genetic skin diseases involving mutations in the genes encoding the structural proteins of the epidermis.
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