Past and Current Research
Epidermal Differentiation, the Cytoskeleton and Blistering Skin Diseases.
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 (Fuchs, 2007; Blanpain and Fuchs, 2009).
Previously, we cloned 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 switch provides a very solid framework for the outer layers of our skin, and is regulated by Notch signaling (Blanpain et al., 2006; Williams et al., 2011).
Keratin filaments are attached through linker proteins (plakins) to specialized integrins that form hemidesmosomes and adhere the epidermis to underlying basement membrane. Plakins also attach keratin filaments to specialized cadherin junctions (desmosomes), promoting cell-cell adhesion. Using gene-knockout technology to remove specific genes, we've been hammering out the relative importance of each of the proteins that participate in integrating the keratin network to cellular junctions. We have learned that severing connections between keratin filaments and cellular junctions has deleterious consequences to the mechanical integrity of epidermal cells (Vasioukhin et al., 2001a and references therein). 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 (Vassar et al., 1991). 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 (Coulombe et al., 1991; reviewed by Fuchs and Cleveland, 1998).
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 (Cheng et al., 1992; Paller et al., 1994). When plakin genes are mutated, complex genetic disorders arise: mutations in the gene encoding 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 (Guo et al., 1995). This complexity occurs because the BPAG1 and plectin genes are expressed not only in epidermis but also in sensory neurons and muscle, respectively. Intriguingly, plakin genes encode multiple isoforms, and through mixing and matching exons, plakin isoforms can link integrins to keratin filaments, actin to neurofilaments, microtubules to neurofilaments or microtubules to actin filaments, to name a few of the functions thus far identified (Yang et al., 1996; 1999; Fuchs and Karakesisoglou, 2001).
We showed that in sensory neurons, BPAG1 isoforms function to stabilize its microtubules necessary to transport neurotransmitter vesicles over long distances ( Yang et al., 1999). 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. The discovery of BPAG1 as an actin-microtubule crosslinking protein also led us to a related protein, ACF7, found not only in mammals, but also flies and worms. Although embryonic lethal when mutated, ACF7 functions in cultured endodermal cells to polarize and stabilize microtubules at a wound front (Kodama et al., 2003). Using conditional targeting, we conditionally ablated ACF7 in the skin stem cells, and showed that when absent, stem cells are not able to efficiently repair wounds in vivo. Increasing evidence suggests that wounding is associated with elevated, localized Wnt signals. We've discovered that GSK3-β, a kinase inhibited by Wnt signaling, controls the phosphosphorylation of ACF7's microtubule binding domain and regulates the polarization process, thereby linking Wnt signaling to stem cell polarization and migration (Wu et al., 2008; 2011).
