The major purpose of this project is to understand the molecular mechanisms that regulate the early stages of epidermal differentiation. Epidermis has been used as an excellent model for studying the process of cellular differentiation because the cells form a stratified structure during development, and each strata is easily identified by morphology and expression of specific markers. Studies by Morasso and collaborators have focused on a homeodomain transcription factor, known as Distal-less 3 (Dlx3), that plays a central role in activating the expression of structural proteins that are necessary for the formation of the cornified layer. During epidermal development, the expression of Dlx3 is restricted to the differentiated (suprabasal) cells, predominantly the granular cells of the mouse stratified epidermis. Transgenic temporal and spatial mis-expression of Dlx3 in the pre-differentiated basal layer caused a highly abnormal skin phenotype, characterized by cessation of proliferation and premature differentiation of the basal cells judged by the upregulation of expression of late differentiation markers such as loricrin and filaggrin. The importance of Dlx3 in the patterning and development of ectodermal structures derived from epithelial-mesenchymal interactions during embryogenesis (i.e. tooth, hair) is corroborated by the effects of DLX3 mutations in patients with the autosomal dominant Tricho-Dento-Osseous (TDO) syndrome. The objectives of this project are to study the processes that contribute to epidermal cell fate and differentiation by: 1) characterizing the pathway(s) that regulate transcriptional activity during epidermal differentiation; 2) exploring the function, interacting factors and target genes of the homeodomain protein Dlx3 as a crucial transcriptional regulator of terminal differentiation in the skin. To elucidate the functional role of Dlx3, a targeted deletion of the gene was performed and the analysis of the phenotype. Targeted deletion of Dlx3 results in embryonic developmental arrest around day 9.5-10, associated with a gross failure of the placenta to undergo proper morphogenesis. It was not possible to assess the effects of Dlx3 loss of function on epidermal differentiation, since embryonic death occurs significantly earlier than the onset of epidermal stratification (E15.5). These results have led us to commence a project for conditional targeting of Dlx3 to be able to specifically assess the role of this homeodomain transcription factor in epidermal stratification. We have studied the regulatory control of the Dlx3 gene and the biochemical characteristic of the protein by analyzing the different functional domains of Dlx3 and the overall role of these regions in the transcriptional function of Dlx3 in epidermal differentiation. To determine the factors that induce Dlx3 gene expression, we have cloned the 1.2-kb and 2.4 kb proximal region of the mouse gene and analyzed its cis-regulatory elements and potential trans-acting factors. The regulatory aspects are centered in the calcium inducublity of the gene and are being characterized using transgenic mice with the beta-galactosidase reporter gene. This activationof Dlx3 is mediated through a Protein Kinase C-dependent (PKC) pathway. We investigated whether PKC can modulate the activity of murine Dlx3 protein. Using in vitro kinase assays, we show that PKC enzymes phosphorylate the Dlx3 protein. Using keratinocytes nuclear extracts for the kinase reaction, we determined that Dlx3 protein is phosphorylated, and the phosphorylation is inhibited by the PKC-specific inhibitor GF109203X, suggesting that Dlx3 is phosphorylated by PKC in vivo. Of the PKC isoforms present in the epidermis, we tested a, d, e and z. Dlx3 is primarily phosphorylated by PKCa. By deletion and mutational analysis, we show that the serine residue S138, located in the homeodomain of Dlx3 protein, was specifically phosphorylated by PKC. The phosphorylation of purified Dlx3 proteins by PKC partially inhibited formation of complexes between Dlx3 protein and DNA. These results suggest that Dlx3 protein can be directly phosphorylated by PKC and this affects the DNA binding activity of Dlx3.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2001
Total Cost
Indirect Cost
Name
Arthritis, Musculoskeletal, Skin Dis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Hwang, Joonsung; Kalinin, Alexandr; Hwang, Meeyul et al. (2007) Role of Scarf and its binding target proteins in epidermal calcium homeostasis. J Biol Chem 282:18645-53
Radoja, Nadezda; Guerrini, Luisa; Lo Iacono, Nadia et al. (2007) Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias. Development 134:13-8
Hassan, Mohammad Q; Tare, Rahul S; Lee, Suk Hee et al. (2006) BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network. J Biol Chem 281:40515-26
Mukhopadhyay, Mahua; Gorivodsky, Marat; Shtrom, Svetlana et al. (2006) Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium. Development 133:2149-54
Berghorn, K A; Clark, P A; Encarnacion, B et al. (2005) Developmental expression of the homeobox protein Distal-less 3 and its relationship to progesterone production in mouse placenta. J Endocrinol 186:315-23
Depew, Michael J; Simpson, Carol A; Morasso, Maria et al. (2005) Reassessing the Dlx code: the genetic regulation of branchial arch skeletal pattern and development. J Anat 207:501-61
Morasso, Maria I; Radoja, Nadezda (2005) Dlx genes, p63, and ectodermal dysplasias. Birth Defects Res C Embryo Today 75:163-71
Hwang, M; Kalinin, A; Morasso, M I (2005) The temporal and spatial expression of the novel Ca++-binding proteins, Scarf and Scarf2, during development and epidermal differentiation. Gene Expr Patterns 5:801-8
Morasso, Maria I; Tomic-Canic, Marjana (2005) Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing. Biol Cell 97:173-83
Hassan, Mohammad Q; Javed, Amjad; Morasso, Maria I et al. (2004) Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol 24:9248-61

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