This proposal has been prepared as a supplement to our grant RO1 AR050023 of the same name. The basis for this project is the knowledge that the transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator and corepressor complexes, which bind to the VDR in a ligand (1,25(OH)2D3) dependent (coactivators) or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the vitamin D receptor interacting protein (DRIP) complex (also known as Mediator or Thyroid Receptor Activating Protein {TRAP} complex) and the steroid receptor coactivator (SRC) complexes, which like DRIP have multiple names. Of the three SRC proteins critical for formation of SRC complexes, only SRC 2 and 3 have been found in keratinocytes, and of these two SRC3 plays the dominant role in the processes we have examined. The DRIP complex spans the distance from vitamin D response elements (VDRE) in the promoters of vitamin D regulated genes to the transcription start site, linking the VDR to the RNA polymerase machinery. The SRC complexes contain histone acetyl transferase (HAT) activity and are thought to open up the gene to enable the transcriptional machinery to bind to the gene being regulated. These coactivator complexes play different roles in regulating keratinocyte proliferation and differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating keratinocyte, whereas SRC2 and 3 and their associated proteins are the major coactivators binding to VDR in the differentiated keratinocyte. Furthermore, DRIP205, the protein in the DRIP complex that binds directly to VDR, is abundantly expressed in proliferating keratinocytes in vitro and in vivo, whereas SRC3 is expressed primarily in differentiated keratinocytes and in the upper (differentiated) layers of the epidermis. Moreover, we have found a specific role for DRIP205 in the regulation of wnt/2-catenin signaling pathways regulating keratinocyte proliferation, whereas SRC3 regulates the ability of 1,25(OH)2D3 to induce more differentiated functions such as lipid synthesis and processing required for permeability barrier formation and the innate immune response triggered by disruption of the barrier. These findings provide a basis by which we can understand how one receptor (VDR) and one ligand (1,25(OH)2D3) can regulate a large number of genes in a sequential and differentiation specific fashion. Furthermore, these observations indicate the potential for discovering small molecules that by selectively modulating the interactions between the coactivators and VDR can manifest specificity in regulating VDR function not achievable by ligands such as 1,25(OH)2D3 and its analogs. We have successfully addressed all aims in the original proposal and verified our hypothesis that the VDR coactivators DRIP and SRC are sequentially utilized during vitamin D regulated keratinocyte differentiation. The preliminary data section in this competitive revision application will demonstrate that fact. During the coming year we want to move beyond the aims of the current grant to test the following hypothesis. The two main coactivator complexes, DRIP and SRC, differentially modulate the ability of VDR and its ligand 1,25(OH)2D3 to inhibit keratinocyte proliferation, while promoting their differentiation leading to a protective epidermal barrier. We are developing mice in which DRIP205 and SRC3 are selectively deleted in keratinocytes and using high throughput screening to identify molecules that will differentially regulate VDR binding to DRIP205 and SRC3 to test this hypothesis. We propose 2 aims.
Aim 1. Determine the selective roles of DRIP205 and SRC3 in VDR regulation of epidermal proliferation, differentiation, and protective barrier formation in mouse models in which DRIP205 and SRC3 are selectively deleted in keratinocytes.
Aim 2. Develop the means to selectively inhibit DRIP205 and SRC3 modulation of VDR function using high throughput screening to identify molecules selective for disruption of the binding of VDR to DRIP205 and SRC3 and use these drugs to selectively modulate the ability of VDR/1,25(OH)2D3 to induce (suppress) vitamin D target genes and functions differentially regulated by DRIP205 and SRC3. We anticipate that progress in these two aims over the coming year will put us in excellent position for a successful restoration of full funding for this project.
The mechanisms by which 1,25(OH)2D3 and its receptor VDR regulate keratinocyte differentiation and function remain unclear. We have discovered that different coactivators, DRIP and SRC in particular, regulate the transcriptional activity of VDR differently for different genes, and thus selectively affect different functions in the keratinocyte. By developing mice lacking either DRIP205 or SRC3, we will further explore these differences in an effort to more fully understand VDR transcriptional activity in the skin and develop means of selectively regulating these processes therapeutically.
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