1. MAPK signaling and transcriptional control of gene expression

As a strategy to understand the control of keratinocyte differentiation, we study the mechanisms that drive expression of a the involucrin gene. Involucrin is a key structural protein that is expressed in differentiated keratinocytes. We’ve shown that a transcriptional complex which includes jun/fos transcription factor family members (junB, junD, Fra-1) and Sp1, and C/EBP transcription factors binds to unique transcriptional elements (DRR and PRR) in the involucrin promoter to activate gene expression (Fig. 2). We have further shown that the activity and level of this complex is controlled by a multi-protein mitogen-activated protein kinase (MAPK) signalsome-like complex which includes protein kinase c, Ras, MEKK1, MEK3 and p38delta-ERK1/2.  These studies define a pathway of information flow from the cell surface to the nucleus which controls gene expression during differentiation (Efimova and Eckert, J Biol Chem 275:1601-1607, 2000; Efimova et al., Mol Cell Biol 24:8167-8183, 2004; Eckert et al., J Invest Dermatol 123:13-22, 2004).

Ongoing studies 

We are addressing several key questions regarding this regulation, including defining the protein components of the signalsome and transcriptional complexes, understanding how these complexes change when cells are stimulated to differentiate, and how scaffolding proteins influence the flow of information through this signaling cascade.  We are using cell and molecular biological, biochemical, protein purification, proteomic and transgenic approaches in this study.  Our ultimate goal is to understand how these complexes activate involucrin expression in differentiated cells.

2. The TIG3 tumor suppressor protein - keratinocyte survival control
 
We recently identified a novel regulatory protein, TIG3, that is expressed in the upper epidermal layers. TIG3 expression is markedly reduced in diseases of enhanced cell proliferation such as cancer. We believe that this protein regulates cell survival during keratinocyte differentiation.  We have recently demonstrated that TIG3 expression in keratinocytes reduces cell survival by interacting with and activating type 1 transglutaminase (TG1). TG1 is a membrane-localized protein that acts to covalently crosslink intracellular proteins, ultimately causing cell death.  Fig. 3 is a confocal image showing co-localization of full-length TIG3 (TIG31-164) with activated TG1 (Sturniolo et al., J Biol Chem 278:48066-48073, 2003; Sturniolo et al., Oncogene 24:2963-2972, 2005; Jans et al., J. Invest. Dermatol, in press 2007). Also shown, a TIG3 mutant (TIG31-134) which lacks the c-terminal membrane-anchoring domain does not localize with or activate TG1. 

Ongoing studies  

We have shown that TIG3 and TG1 are components of a multiprotein complex that forms in cells.  We are presently addressing several important issues including characterizing the molecular interaction between TIG3 and TG1, identifying other proteins that may be involved in this interaction, monitoring TIG3 and TG1 movement within cells, and elucidating the structure of the TIG3 protein. Methods used include cell and molecular biology, protein structure and proteomics.

3. Epidermal stem cells for cell-based therapy

The keratinocytes that populate the epidermal surface are derived from stem cells located in specific niche areas in the tissue.  These niches harbor a reservoir of cells that could be tapped for therapy applications.  Using the epidermis as a source of cells is an attractive idea because the cells are abundant (the skin is the largest organ of the body) and the epidermis is readily accessible (on the skin surface). The strategy is to convert the epidermal cells into multipotent stem cells and then to "reprogram" them to make other cell types. As part of an ongoing collaboration with Dr. Jackie Bickenbach, Ph.D. (University of Iowa), we are studying how stem cells that are committed to produce keratinocytes can be reprogrammed to produce other cell types (e.g., neuronal cells). If the ability to convert these cells to the multipotent state can be perfected, epidermal cells could provide an abundant and accessible source of cells for therapy.  We have recently shown that these cells can be converted to multipotent status by vector-mediated expression of the embryonic stem cell transcription factor, Oct 4, and that these cells can then be reprogrammed to produce neuronal cells (Grinnell et al., J. Invest. Dermatol 127:372-380, 2007) (Fig. 4).  Oct-4 is required for maintenance of embryonic stem cells. The ability to manipulate cells in this manner has tremendous medical importance for the large scale generation of therapeutically useful cells. Given the number of cells in the skin and the relative ease with which these cells can be harvested, they could provide a potentially huge reservoir of reprogrammable cells for use in cell-based disease therapy.

Ongoing studies
  
A key question is how genes that control stem cell status (such as Oct 4), are regulated in keratinocytes.  We are presently working to identify mechanisms that control expression of several genes involved in this process.  Methods used include cell and molecular biology, cell cloning and cell sorting.

 4. Animal models of disease - scleroderma
  
A major focus of our laboratory is production of genetically engineered transgenic animal (mouse) models that mimic human disease.  The idea is to use these models to understand the disease process and develop therapies.  One such example we are currently pursuing is scleroderma.  Scleroderma, also called systemic sclerosis, is a debilitating disease of tissue thickening and scar formation in the skin and other organs.  The disease is speculated to originate in the skin.  The skin consists of three layers - the epidermis (the outer protective layer), the dermis (the middle connective tissue layer) and the hypodermis (the inner fat layer).  The dermis is regarded as a key tissue in scleroderma, since disease-related damage is observed in this layer, including excess tissue deposition (fibrosis), scarring, inflammation and vascular damage.  In severe forms of this disease, the damage is also observed in the intestine, lung and kidney, and leads to reduced function in these organs.  Unfortunately, the underlying causes of this disease are not known, but are likely to include both environmental and genetic factors.  Although most of the damage is observed in the dermis, it is unlikely that the disease is initiated by dysfunction in this tissue.  Thus, there is an ongoing search for the events that cause scleroderma.

Ongoing studies  

Our recent studies, using a newly developed transgenic animal model, suggest a new way of thinking about this disease.  We propose that the outer layer of the skin, the epidermis, may play a key role in triggering scleroderma - a possibility that has not been considered in the past.  We show that disrupting epidermal function produces major and rapid scleroderma-like changes in the dermis.  This is particularly interesting, since some chemicals that enter the body through the epidermis are thought to promote the disease.  Thus, we have proposed that compromised epidermal function may be a key disease-triggering event.  We believe that initial events in the epidermis trigger the damage in the dermis that ultimately leads to damage in internal organs.  Presentation of our early findings at scientific meetings has stimulated a strong interest in exploring this possibility and we are in the process of testing this hypothesis.  Methods utilized in this study include cell and molecular biology, biochemistry and transgenic mice.

5. Nutrition and cancer prevention

Cancer begins with a normal cell that, due to persistent environmental insult, is transformed, via a series of progressively more insidious steps, into a cancer cell. A major goal of chemopreventive therapy is to alter the normal cell response to the environmental stress agent in order to inhibit disease progression. These agents act via a variety of different mechanisms.  Some chemopreventive agents enhance cell differentiation. (-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive antioxidant, derived from tea, which possesses remarkable cancer preventive properties. Our studies clearly indicate that EGCG markedly increases keratinocyte differentiation. Based on these results, we argue that EGCG acts to prevent cancer development by forcing neoplastic cells to undergo differentiation. This is an important hypothesis, as it indicates that green tea may act to prevent disease before it develops (Balasubramanian et al., J Biol Chem 277:1828-1836, 2002). In addition, our studies show that all chemopreventive agents are not created equal, and that these agents can antagonize each others action (Balasubramanian et al., J Biol Chem 281:36162-36172, 2006; Balasubramanian et al., J Biol Chem 282:6707-6715, 2007).  This implies that the use of these compounds for cancer prevention must be carefully considered.  An immediate goal is to examine how these agents influence epigenetic regulation.

Ongoing studies  

Limited knowledge is available regarding how chemopreventive agents act at the molecular level to prevent tumor cell proliferation and to induce tumor cell survival. We are presently working to identify how these agents act and which kinases are targeted for inactivation or activation. Cell and molecular biology techniques are used in this study.

6. Polycomb genes and epigenetic regulation of survival

The Polycomb Group (PcG) genes are epigenetic suppressors of gene expression that play an important role in development through modification of chromatin. We are interested in these proteins because they enhance normal and cancer stem cell survival. Our studies show that these proteins localize in the nucleus of epidermal keratinocytes. We have also shown that overexpression of one of these proteins, Bmi-1, protects keratinocytes from stress agent-mediated cell death (Fig. 5). This protection is associated with increased levels of cyclin D1 and cyclin-dependent kinases, and reduced activity of apoptotic proteins. Thus, these proteins enhance cell survival by increasing proliferation and reducing apoptosis. We hypothesize that Bmi-1 prevents premature keratinocyte death, thereby assuring proper formation of the stratified epidermis (Lee et al., J Invest Dermatol, in press 2007).

We further show that Bmi-1 levels are elevated in transformed keratinocytes, skin tumors and psoriasis. This is important, since we propose that Bmi-1 may act to promote skin cancer by enhancing survival of preneoplastic keratinocytes (Lee et al., J Invest Dermatol, in press 2007).

PcG gene-dependent gene silencing operates via a mechanism that involves two multiprotein complexes - the eed complex and the Bmi-1 complex. These complexes make sequential modifications to chromatin that result in transcriptional inactivation. The first events are catalyzed by the eed complex. These include specific histone deacetylation followed by methylation of histone H3 at lysine-27 (K27).  The histone H3-methyl-K27 lysine then serves as a binding site for recruitment of the Bmi-1 complex through the specific recognition of methyl-K27. The Bmi-1 complex encodes a histone H2A-K119 ubiquitin E3 ligase which attaches ubiquitin at position K119 of histone H2A.  Ultimately, these sequential events lead to gene silencing.

Ongoing studies  

Biochemical and gene array studies indicate that Bmi-1 overexpression alters function of key proteins that regulate keratinocyte proliferation and apoptosis.  A major immediate goal is to understand the molecular mechanisms whereby this is achieved.  In this context, we are using chromatin immunoprecipitation analysis to examine the impact of Bmi-1 on acetylation, methylation and ubiquitinylation of the Bmi-1-regulated genes.  We are also overexpressing and inhibiting expression of various polycomb genes (using vector-mediated expression and shRNA) to identify their impact on keratinocyte function.

7. S100 protein function in psoriasis and inflammation

S100 proteins are small acidic, calcium binding, EF-hand proteins. S100 proteins have no intrinsic catalytic activity, but in response to increased intracellular calcium, they undergo a conformation change that permits them to bind to and modulate the activity of target proteins.  S100 proteins are expressed in a cell type and tissue-specific manner and have been shown to play an important role in regulating cell differentiation, cell proliferation, and cell shape. Several of these proteins are markedly over-expressed in psoriasis and skin cancer and are thought to be involved in the etiology of these skin diseases, which are associated with increased keratinocyte proliferation. S100 proteins exist as homodimers in cells, and in the presence of increased intracellular calcium they bind to target proteins to alter target protein action and drive end responses (Fig. 6).

We have performed extensive studies on these proteins and have identified several novel functions (Robinson et al., J Biol Chem 273:2721-2728, 1998;  Robinson et al., J Biol Chem 272:12035-12046, 1997; Ruse et al., Biochemistry 40:3167-3173, 2001; Lee and Eckert, J Invest Dermatol 127:945-957, 2007). These work has identified S100 protein target proteins, identified S100 proteins as component of the terminal cell envelope, identified S100A7 as an anti-bacterial defensin-like protein, and mapped intracellular movement. Fig. 7, for example, shows that S100A7 moves to focal adhesion-like structures that contact the cell substratum (white arrow). This finding suggests that it may be involved in regulating cell-substratum interaction. 

Ongoing studies  

We are presently working to identify S100A7 interaction partners that may help us to identify its role in focal adhesion plaques.