The continuing goal of this revised competitive renewal grant proposal is to analyze the molecular mechanisms by which retinoic acid (RA) inhibits 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) gene transcription. We hypothesize that TPA-induced ODC-gene transcription is mediated through the interaction of PKC-modulated trans- acting factor(s) with cis-acting elements in the 5 '-flanking region of the ODC genes and that RA-retinoic acid nuclear receptor(s) (RAR) complex acts as a negative regulator of TPA-induced ODC gene transcription. During the previous award period of this grant application, we isolated an ODC clone which contains about 10 Kb upstream of the ODC gene. A 2.5 Kb of the 5 '-flanking region was sequenced. The sequence data revealed no consensus AP-1 sequences in the 2.5 Kb 5'-flanking region. TPA and RA response was analyzed in several ODC-luciferase (Lu) constructs. TPA induction of luciferase activity (>10-fold) in HeLa cells was reprodicibly observed in -72/+136 ODC Lu. RA treatment inhibited TPA-induced luciferase activity of -72/+136 ODC Lu construct. Another novel observation which supports the above hypothesis is the finding that co-transfection of -72/+136 ODC Lu with PKCalpha or PKCdelta resulted in a dramatic increase in luciferase activity. It is noteworthy that the -72/+136 ODC gene fragment which responds both to TPA and RA neither has putative AP-1 or RA-responsive elements (RARE) sequences as are described in other TPA-inducible (i.e., stromelysine, collagenase) and RA-responsive (i.e., RAR beta, laminin B1) genes. We now propose the following four specific aims to further test the above hypothesis:
Specific Aim 1 : Define the sequences in -72/+136 ODC which respond to TPA and PKC. To achieve the goals of this specific aim, we will: (1) determine by DNase I footprinting analysis which sequences in -72/+136 ODC are recognized by the TPA and PKC-modulated transacting factors; (2) synthesize TPA-responsive (TRE) and PKC responsive (PRE) sequences in - 72/+136 ODC and then test the enhancer ability, such as independence of orientation, distance, and position; (3) define the TRE and PRE by point mutations; (4) determine whether TRE is the same as PRE and define specificity of PRE to PKC isoforms (alpha, beta, gamma, delta, epsilon).
Specific Aim 2 : Determine the nature of PKC and TPA-induced trans-acting factors which may interact with TRE and/or PRE.
Specific Aim 3 : Define RARE in -72/+136 ODC. We will use all the ODC constructs prepared under specific aim 1 to determine RARE in -72/+136 ODC; (2) synthesize and define RARE by site mutagenesis.
Specific Aim 4 : Determine how an RA-RAR complex acts as a negative regulator of TPA-induced ODC gene transcription. We will determine whether RA-RARs inhibits TPA-induced ODC gene transcription by (1) interacting with specific RARE, (2) competing for the TRE, and/or (3) by binding to TPA-induced transcriptional factors (cross-talk).
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