Keratinocyte transglutaminase (TGK) is a membrane protein responsible for cross-linked envelope formation during terminal differentiation in epidermal cells. In cultured keratinocytes, involucrin is its major substrate. Ordinarily neither protein is present in basal cells; expression occurs in maturing cells committed to terminal differentiation. The first part of the proposal exploits this lab's recent success in identifying response elements in the proximal promoter that are important for expression of the TGK gene. The DNA sequence requirements for protein binding to these elements will be determined by saturation binding mobility shift assay (to obtain estimates of kd), mutation analysis and methylation interference. Molecular weight estimates of binding protein will be obtained by UV crosslinking and/or Southwestern blotting. Since these identified sites all resemble recognition sites for AP2, mobility supershift experiments with AP2 antibodies will test for the presence of AP2 or an immunochemically related protein in the DNA/protein complexes. cDNA as encoding TGK transcription factors will be cloned either by screening an expression library with binding site probes or after partial sequence analysis of the purified protein and PCR amplification. The second part of the proposal investigates the regulation of keratinocyte transcription by arsenate, an agent shown by this lab to increase substantially cellular phosphotyrosine levels, to suppress involucrin (and to a lesser extent TGK) expression and to decrease greatly AP2 binding and AP1 transactivating activities. First, elements of the involucrin promoter required for arsenate sensitivity will be determined by transient transfections and arsenate-induced alterations in protein binding will be sought using footprinting. Second, molecular mechanisms will be investigated for suppression oa AP2 DNA binding (including alternative splicing, postranslational modification and protein/protein interactions) AP1 transactivating activity (Jun and Fos family members, phosphorylation status). Comparison of TGK and involucrin promoter elements that confer transcriptional activity may provide insight into aspects of common and distinct regulation. Of particular interest is the frequent occurrence of AP2-like elements that contribute to transcription of TGK, involucrin and other keratinocyte genes.
| Reznikova, Tatiana V; Phillips, Marjorie A; Rice, Robert H (2009) Arsenite suppresses Notch1 signaling in human keratinocytes. J Invest Dermatol 129:155-61 |
| Ngo, Mai A; Sinitsyna, Nadezda N; Qin, Qin et al. (2007) Oxygen-dependent differentiation of human keratinocytes. J Invest Dermatol 127:354-61 |
| Patterson, Timothy J; Rice, Robert H (2007) Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential. Toxicol Appl Pharmacol 221:119-28 |
| Lee, Young Jin; Rice, Robert H; Lee, Young Moo (2006) Proteome analysis of human hair shaft: from protein identification to posttranslational modification. Mol Cell Proteomics 5:789-800 |
| Rea, Miguel A; Zhou, Lei; Qin, Qin et al. (2006) Spontaneous immortalization of human epidermal cells with naturally elevated telomerase. J Invest Dermatol 126:2507-15 |
| Rice, Robert H; Crumrine, Debra; Uchida, Yoshikazu et al. (2005) Structural changes in epidermal scale and appendages as indicators of defective TGM1 activity. Arch Dermatol Res 297:127-33 |
| Patterson, Timothy J; Reznikova, Tatiana V; Phillips, Marjorie A et al. (2005) Arsenite maintains germinative state in cultured human epidermal cells. Toxicol Appl Pharmacol 207:69-77 |
| Lee, Chan; Lee, Young Moo; Rice, Robert H (2005) Human epidermal cell protein responses to arsenite treatment in culture. Chem Biol Interact 155:43-54 |
| Phillips, Marjorie A; Jessen, Bart A; Lu, Ying et al. (2004) A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes. BMC Dermatol 4:2 |
| Rea, Miguel A; Gregg, Jeff P; Qin, Qin et al. (2003) Global alteration of gene expression in human keratinocytes by inorganic arsenic. Carcinogenesis 24:747-56 |
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