Our central objective is to explore the mechanisms governing development and differentiation in epidermis and hair of mammalian skin and to understand how these processes go awry in genetic skin diseases. To facilitate these studies, we use cultured keratinocytes from human and mouse skin, one of the few systems where adult stem cells can be maintained and propagated in the laboratory. We also use technologies for targeting foreign genes to epidermis and hairs of transgenic animals, and for removing single epidermal or hair genes from the chromosomes of either mouse embryos (knockout) or skin (conditional inducible knockout). Our global aim is to take a molecular approach to skin biology and to apply our knowledge to human genetics and medical research.

Epidermis is composed of cell layers, the outermost being the skin surface. Only the innermost, basal layer contains living, multiplying cells. When a basal cell ceases to divide and begins its journey to the skin surface, it embarks on a program of terminal differentiation. The cells first protect themselves by producing a mechanically durable, dense intracellular framework of filaments composed of a protein, keratin. Through abundant intercellular junctions, differentiating cells form continuous, interconnected cellular sheets. Later, the cells assemble an indestructible proteinaceous envelope that serves as a scaffold on which lipids are extruded and organized to form the epidermal barrier. Cells reaching the skin surface are sloughed, replaced by inner cells differentiating and moving outward. Every two weeks, the epidermis is nearly brand new.

The epidermal barrier excludes microorganisms and retains body fluids. Without it, we would dehydrate and die. In humans, it is acquired at 8.5 months of prenatal development: prematurely born infants can only survive in incubators. Understanding how the epidermis orchestrates this essential function is a prerequisite to learning how to accelerate barrier formation in premature infants born where medical care is not available, and we have unraveled a number of biochemical and transcriptional changes that orchestrate this process.

We are also interested in understanding how the cells of the epidermis protect themselves, and at the same time maintain their ability to replenish the skin and seal wounds. At the skin's surface, these cells must be extraordinarily resilient. Many years ago, we characterized the genes encoding keratins, proteins that are expressed in epidermis and hairs of mammals but not in fruit flies or other animals possessing protective outer shells or exoskeletons. We showed that basal epidermal cells produce keratins 5 and 14 (K5 and K14), which form filaments that make an internal skeleton (cytoskeleton). In contrast, epidermal cells that stop dividing, detach from the basement membrane, and move outward toward the skin surface, switch to the expression of a new set of keratin genes, producing keratins 1 and 10 (K1 and K10), which form large bundles of keratin filaments. This provides a very solid framework for the outer layers of our skin.

Keratin filaments are attached through linker proteins (plakins) to specialized integrins that adhere the epidermis to underlying basement membrane. Plakins also attach keratin filaments to specialized cadherin junctions (desmosomes), promoting cell-cell adhesion. In order for the cells to move, either in differentiation or in wound healing, a similar adhesive framework must involve the actin cytoskeleton, a network first studied in muscle cells. Epidermal cells that participate in wound healing rely heavily on this actin cytoskeleton.

Using gene-knockout technology to remove specific genes, we have begun to dissect out the relative importance of each of the proteins that participate in forming these two important cellular frameworks. We have learned that severing connections between keratin filaments and cellular junctions has deleterious consequences to the mechanical integrity of epidermal cells. Using transgenic technology to perturb keratin filaments in the basal layer of mouse epidermis, we discovered that similarly, the epidermal cells become fragile, degenerating upon mild rubbing and causing a blister. In humans this condition is known as epidermolysis bullosa simplex (EBS), and it can be caused by mutations in either K5, K14, or epidermal plakin genes.

Our studies on EBS also led us to the genetic basis of epidermolytic hyperkeratosis and a form of epidermal nevi, which arise from gene mutations in K1 or K10. When plakin genes are mutated, complex genetic disorders arise: mutations in the bullous pemphigoid antigen 1 (BPAG1) plakin result in EBS with severe sensory ataxia in mice, and mutations in plectin result in EBS with muscular dystrophy in humans. This complexity occurs because the BPAG1 and plectin genes are expressed not only in epidermis but also in sensory neurons and muscle, respectively. We studied the BPAG1 isoforms in sensory neurons to understand how loss of these plakins leads to degeneration of sensory neurons. Neuronal BPAG1 isoforms integrate all three cytoskeletal networks of the axon, where they function to stabilize its microtubules, the third cytoskeletal framework essential to all cells. Without BPAG1, axonal microtubules are unable to transport neurotransmitter vesicles over long distances. This discovery has important implications for understanding the specialized filament networks of neurons, which like epidermal keratinocytes produce elaborate cytoskeletons. It also contributes to our knowledge of the complexities of human neurodegenerative disorders.

In contrast to the keratin cytoskeleton, the actin cytoskeleton and its connections are essential for cell migration, wound healing, and making a hair follicle. Recently we targeted removal of the b1-integrin gene, a transmembrane protein that links the actin cytoskeleton to the basement membrane that separates the skin epithelium from the underlying dermis. This integrin is required not only for migration but also for the formation of the skin's basement membrane. Since both features are essential to make a hair follicle, the hairs can't form either.

The regulatory regions of highly expressed structural genes of epidermis provide powerful tools for targeting expression of foreign genes in human keratinocytes for use in skin grafting and gene therapy. To this end, we have continued to characterize K5 and K14 promoter and enhancer elements. We have also shown that epidermal keratin promoters can maintain human growth hormone production in transfected keratinocytes and that hormone persists in the bloodstream of mice that receive genetically engineered skin grafts. Thus it should be possible in the future to culture human keratinocytes from a skin biopsy, genetically manipulate them for gene therapy, and then graft them back onto the patient. K14 and K5 promoter and enhancer elements are also valuable for transgenic technology because of their activity in the self-renewing stem cells of the epidermis and hair follicle. We have used these promoters to engineer mice for conditional inducible-knockout technology: upon topical application of an inducing drug, a gene of choice is selectively removed from the patch of epidermis receiving the drug. With this powerful approach, epidermal functions of genes can be analyzed even though their expression is broad. We have already used this technology to begin to uncover insights into mosaic disorders, including skin cancers.

Because K5 and K14 are also expressed in stem cells, we have been able to use their promoters to gain insights into hair growth regulation. The mature follicle consists of a hair shaft of dead epithelial cells, which arise from proliferating matrix cells at the follicle base. The self-renewing, K5/K14 (+) epithelial stem cells are located in the bulge, about a third of the way down the follicle. Postnatally, as matrix cells cease to divide and differentiate, the hair regresses. A specialized pocket of mesenchymal cells at the core of the matrix rises upward with the dying hair, making contact with the bulge and triggering a fresh mesenchymal-epithelial interaction that sets off a spurt of epithelial growth and differentiation.

We have learned that a transcription factor composed of beta-catenin and LEF1 is required for hair follicle morphogenesis in early development and is used again at the start of each new hair cycle. We showed that artificially activating the complex in transgenic mice induces follicle morphogenesis. This finding may be useful for future technologies aimed at controlling hair growth in humans suffering from hirsutism or certain types of balding.

If left uncontrolled, however, excess beta-catenin/LEF signaling leads to tumorigenesis. The mouse tumors guided us to the genetic basis of pilomatricomas, a common human skin tumor, which arises from activating mutations in beta-catenin. In the future, knowledge of how the complex is naturally regulated in the hair follicle will help us to illuminate the mechanism underlying hair development and cycling.

Recently we identified a cousin of LEF1, called TCF3, which is expressed in the stem cells of the bulge. We have discovered that stem cell keratinocytes that receive a signal that stimulates the expression of one type of TCF/LEF1 complex can be coaxed into becoming hair, while those expressing a different TCF/LEF1 complex may become a sebaceous gland or epidermis. This information has provided us with a key clue for understanding how stem cells select specific differentiation pathways in the skin. In the future, this information may help us to assess whether, if given the proper environment, skin stem cells might be coaxed along new pathways, e.g., to produce a neuron.

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