Faculty Bibliography

In the area of biology, many laboratories around the world are dissecting and characterizing signal transduction mechanisms and transcription factors responsive to various growth factors and cytokines, in various cell types. However, because of the differences in systems used, it is not clear whether these systems coexist, whether they interact meaningfully, and what their relative roles are. Epidermal keratinocytes are the perfect cell type in which to integrate this knowledge, because in these cells these mechanisms are known to be relevant. Keratinocytes both produce and respond to growth factors and cytokines, especially in pathological conditions and during wound healing, when the physiology of keratinocytes is altered in a way specified by the presence of a subset growth factors and cytokines. In fact, growth factors and cytokines cause the major changes in gene expression and keratinocyte behavior in various cutaneous diseases. In some cases, such as in wound healing, these responses are highly beneficial; in others, such as in psoriasis, they are pathological. It is not clear at present which are operating in which conditions, which are important for the healing process and which are harmful. Growth factors and cytokines affect keratinocytes sometimes simultaneously, at other times individually. In this manuscript we describe the signal transduction pathways responsible for the effects of interferons, the EGF/TGF alpha family and the TNF alpha/IL-1 family of signaling molecules. We also describe the important transcription factors known to be functional in epidermis, with particular emphasis on those factors that are activated by growth factors and cytokines. Finally, we describe what is known about transcriptional regulation of keratin genes, especially those specifically expressed in pathological processes in the epidermis. We expect that the enhanced understanding of the pathways regulating gene expression in keratinocytes will identify the pharmacological targets, the signal transducing proteins and the corresponding transcription factors, used by growth factors and cytokines. This research will led to development of compounds precisely aimed at those targets, allowing us to isolate and inhibit the harmful side effects of growth factors and cytokines. Such compounds should lead to highly specific and therefore more effective treatments of the cutaneous disorders in which these pathways play significant roles

Retinoic acid and thyroid hormone are important regulators of epidermal growth, differentiation, and homeostasis. Retinoic acid is extensively used in the treatment of many epidermal disorders ranging from wrinkles to skin cancers. Retinoic acid and thyroid hormone directly control the transcription of differentiation-specific genes including keratins. Their effect is mediated through nuclear receptors RAR and T3R. We have previously identified the response element in the K14 gene, K14RARE/TRE, to which these receptors bind, and found that it consists of a cluster of five half-sites with variable spacing and orientation. To determine whether this specific structure is found in other keratin genes, we have mapped and analyzed the RARE/TRE elements in three additional epidermal keratin genes: K5, K6, and K17. We used three different approaches to identify these elements: co-transfection of promoter deletion constructs, gel-shift assays, and site-specific mutagenesis. We localized the RARE/TRE elements relatively close to the TATA box in all three promoters. All three RARE/TRE elements have a similar structural organization: they consist of clusters of 3-6 half-sites with variable spacing and orientation. This means that the clustered structure of the RARE/TREs is a common characteristic for keratin genes. RARE and TRE in the K5 promoter are adjacent to each other whereas in the K17 promoter they overlap. All three keratin REs bind specifically both RAR and T3R in gel-shift assays. Interestingly, addition of ligand to the receptor changes the binding pattern ofthe T3R from homodimer to monomer, reflecting the change in regulation from induction to inhibition

Epidermal keratinocytes are subject to a large variety of signals that modulate their differentiation in health and their activation in disease. Hormones and vitamins, which act via nuclear receptors, affect the differentiation process, whereas growth factors and cytokines, which act via cell surface receptors, affect keratinocyte activation and related events. Using expression of keratin genes as markers for keratinocyte phenotype, we examined the interaction between the nuclear receptor and cell surface receptor pathways. We expected to find dominance of one of the pathways. Surprisingly, we found that the two pathways are codominant. Specifically, while EGF induces expression of K6 and K16 keratin genes, retinoic acid suppresses their expression, and when both mediators are present simultaneously, the level of expression is intermediate, a product of both signals. Similar codominant effects were found on other keratin genes using interferon gamma, TGF beta, and thyroid hormone signaling molecules. These codominant effects are specific only for genes that are regulated by both pathways. Our results suggest that a judicious combination of hormones, vitamins, growth factors, and cytokines may be used to target specific expression of appropriate genes in the treatment of human epidermal diseases

Expression of keratin proteins, markers of epidermal differentiation and pathology, is uniquely regulated by the nuclear receptors for retinoic acid (RAR) and thyroid hormone (T3R) and their ligands: it is constitutively activated by unliganded T3R, but it is suppressed by ligand-occupied T3R or RAR. This regulation was studied using gel mobility shift assays with purified receptors and transient transfection assays with vectors expressing various receptor mutants. Regulation of keratin gene expression by RAR and T3R occurs through direct binding of these receptors to receptor response elements of the keratin gene promoters. The DNA binding 'C' domain of these receptors is essential for both ligand-dependent and -independent regulation. However, the NH2-terminal 'A/B' domain of T3R is not required for either mode of regulation of keratin gene expression. Furthermore, v-ErbA, an oncogenic derivative of cT3R, also activates keratin gene expression. In contrast to the previously described mechanism of gene regulation by T3R, heterodimerization with the retinoid X receptor is not essential for activation of keratin gene expression by unliganded T3R. These findings indicate that the mechanism of regulation of keratin genes by RAR and T3R differs significantly from the mechanisms described for other genes modulated by these receptors