The three aims of this project are 1) to identify proteins that interact with STAMP, 2) to see if changes in Amax, EC50, and PAA with cofactors such as STAMP can be dissociated, and 3) to search for other activities of STAMP.
Aim 1 is being assessed using our knock out (KO) mice for STAMP, which were employed to prepare immortalized mouse embryo fibroblasts (MEFs) from the KO and wild type mice, and then performing microarray analyses on KO and wild type MEF cells after treatment with steroid or vehicle for 8 hr. High quality data have been obtained for almost 3,000 genes, for which the level of expression changed by ≥1.5 fold after Dex treatment. These studies are on-going, with particular emphasis on the number and nature of those genes that display increased or decreased gene expression in the absence of STAMP, on the ability of STAMP to affect Amax and PAA independently (as we observed for the GILZ gene in PBMCs), and on the identity of cellular pathways that might be especially sensitive to the levels of STAMP.
Aim 2 has been addressed using the ternary system of GR, the coactivator TIF2, and STAMP for two reasons. First, we previously established that TIF2 and STAMP augment the Amax and PAA, and decrease the EC50, of GR-mediated gene expression in a more than additive manner. This cooperative nature of the actions of TIF2 and STAMP amplifies the modulation of the three induction parameters, thereby making it easier to detect productive interactions. Second, the number of permutations of potential biologically active protein surfaces is much higher with three than two factors, thereby increasing the possible combinations that could be involved in selective gene transcription. To answer the question if the three parameters can be independently modified by separate protein domains, we examined the actions of three differently sized fragments of each of three factors (GR, TIF2, and STAMP). The full length proteins are shown to form a ternary complex and to similarly affect the induction properties of transfected and endogenous genes. Twenty five different fragment combinations of the ternary complex were examined for their ability to modulate the Amax, EC50, and PAA of a transiently transfected synthetic reporter gene. Different combinations selectively alter one, two, or all three parameters. It is usually assumed that the modulatory activity of transcriptional cofactors coordinately regulates Amax, EC50, and PAA. Our results clearly demonstrate that Amax, EC50, and PAA can be independently regulated under several conditions by different pathways or molecular interactions. This new mechanistic insight suggests that selected activities of individual transcription factors are viable targets for small molecules, which would have obvious clinical applications for increasing the specificity of steroids during endocrine therapies.
Aim 3 is being pursued by preparing the STAMP KO mice in a homogeneous genetic background. Phenotypic abnormalities in these genetically pure mice would serve as an excellent indicator of processes and proteins that involve interactions with STAMP. These studies are ongoing. In summary, we are using our recently prepared STAMP KO mice to look for associated proteins, as indicated by microarray analyses of immortalized MEF cells and phenotypic defects of KO animals. Detailed studies of the action of the ternary complex of GR/TIF2/STAMP have established that the three parameters of GR-regulated induction (EC50, PAA, and Amax) are not coordinately regulated. Instead, different protein surfaces of the three proteins can selectively modulate each parameter, which indicates that pharmaceutical may exist that can alter the activities of one, two, or all three parameters. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.
|Lee, Geun-Shik; He, Yuanzheng; Dougherty, Edward J et al. (2013) Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility. J Biol Chem 288:15167-80|
|Simons Jr, S Stoney; Chow, Carson C (2012) The road less traveled: new views of steroid receptor action from the path of dose-response curves. Mol Cell Endocrinol 348:373-82|
|Awasthi, Smita; Simons Jr, S Stoney (2012) Separate regions of glucocorticoid receptor, coactivator TIF2, and comodulator STAMP modify different parameters of glucocorticoid-mediated gene induction. Mol Cell Endocrinol 355:121-34|
|He, Yuanzheng; Blackford Jr, John A; Kohn, Elise C et al. (2010) STAMP alters the growth of transformed and ovarian cancer cells. BMC Cancer 10:128|