The Journal of Steroid Biochemistry and Molecular Biology
Conditional modulation of glucocorticoid receptor activities by CREB-binding protein (CBP) and p300
Introduction
Steroid hormones, including glucocorticoids, exert their effects on their target cells through nuclear receptors, ligand-specific and -dependent transcription factors. Binding of glucocorticoids to their receptors causes them to dissociate from a hetero-oligomer of heat shock proteins and to translocate into the nucleus, where they bind as homodimers and/or monomers, respectively, to specific DNA enhancer elements or other transcription factors, such as AP-1, NFκB and SP1 [1]. The hormone responsive element-bound receptors interact with several newly described nuclear proteins, the coactivators, which help transduce the hormonal signal to the transcription initiation complex [2]. Some coactivators have intrinsic histone acetyltransferase activity, through which they have been proposed to loosen chromatin structure and to facilitate the binding of transcription machinery components to DNA [3], [4]. One of these coactivator molecules, cAMP-responsive element binding protein (CREB)-binding protein (CBP) was originally described as a coactivator of CREB [5], [6]. CBP and the closely related protein p300 [7], [8], may in fact serve as macromolecular docking ‘platforms’ for transcription factors from several signal transduction cascades, including in addition to CREB, nuclear receptors, such as the glucocorticoid, progesterone and estrogen receptors, AP-1, NFκB, p53, Ras-dependent growth factor and several STATs [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. Because of their central position in many signal transduction cascades, CBP/p300 have been also called co-integrators [21]. Nuclear receptor-specific coactivators were described recently; these include steroid receptor coactivator-1 (SRC-1) [22], glucocorticoid receptor interacting polypeptide-1 (GRIP-1) [23], p300/CBP/co-integrator-associated protein (p/CIP) [21], and ACTR [24], all of which also possess histone acetyltransferase activity [4], [24]. Both CBP/p300 and SRC-1/GRIP-1/p/CIP/ACTR contain one or more copies of the signature motif sequence LXXLL, which is necessary for interaction with the nuclear hormone receptors [25].
By modulating nuclear receptor signal pathways, coactivators may participate in defining the sensitivity of a cell to steroid hormones either in a tissue-specific or generalized fashion. Changes in the sensitivity of tissues to steroid hormones may be important in both physiologic and pathologic conditions. In the case of glucocorticoids, tissue sensitivity change may participate in the maintenance of resting and stress-related homeostasis [26]. A change of the host tissue sensitivity to glucocorticoids, expressed either as hypersensitivity or resistance, has been encountered or inferred in several common pathologic conditions, such as, respectively, visceral obesity or hypertension, and glucocorticoid resistant asthma or other autoimmune diseases [27].
Since CBP is thought to be a crucial interaction point between nuclear receptors, such as the glucocorticoid receptor, and other signal transduction pathways, we investigated the role of this protein in altering cell sensitivity to steroid hormones on well characterized glucocorticoid-responsive reporter plasmids in two human cell lines. We report here that CBP may function not only as a coactivator of nuclear receptors but also as a corepressor, depending on its absolute or relevant to other coactivators concentrations and the cell type employed.
Section snippets
Plasmids
The mouse CBP expression vector pRcRSV–CBP–HA–RK, the CREB expression vector pRcRSV–CREB341, the constitutive active form of protein kinase A (PKA) expression vector RSV–PKA and the cAMP-responsive somatostatin promoter-driven CAT reporter plasmid p(−71)SRIF–CAT were kind gifts from Dr R.H. Goodman (Oregon Health Science University, Portland, Oregon) [5]. The deletion mutant of mouse CBP expression vector pRcRSV–CBP(1–1098) was constructed by digesting pRcRSV–CBP–HA–RK with XbaI. This treatment
Results
We examined the consequence of overexpressing CBP on the activity of the MMTV promoter in HeLa and A204 cells. As shown in Fig. 1A and B, CBP strongly suppressed dexamethasone-stimulated MMTV promoter activity in a dose-dependent fashion in both cell lines (HeLa cells, 70%, A204 cells, 90%, respectively). For comparison, we employed the Simian virus 40, Rous sarcoma virus (RSV) and Cytomegalovirus (CMV) promoters which do not contain GREs. CBP had no or minimal effects on these promoters in
Discussion
CBP suppressed the activity of the glucocorticoid-responsive MMTV promoter in HeLa and A204 cells in a dose-dependent manner. On the other hand, p300 and SRC-1a enhanced GR-activated MMTV promoter activity in the same system in which CBP showed functional suppressive activity. Although CBP and p300 are highly homologous, our results showed differentiation between these two proteins, suggesting that these molecules are not functionally interchangeable, at least in the ‘overexpressed’ condition.
Acknowledgements
We thank Drs R.H. Goodman, D.M. Livingston, J.N. Miner, B.W. O’Malley, R.M. Evans, G. Hager, P. Chambon, S. Simons Jr, J. Segars, A.J. Levine, B. Vogelstein, S.J. Tapscott for plasmids, and K. Alexander and A. Verbalis for technical assistance.
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