Glucocorticoid Repression of AP-1 Is Not Mediated by Competition for Nuclear Coactivators

Glucocorticoid Repression of AP-1 Is Not Mediated by Competition for Nuclear Coactivators Karolien De Bosscher, Wim Vanden Berghe, and Guy Haegeman* Department of Molecular Biology University of Gent-VIB 9000 Gent, Belgium Interleukin-6 (IL-6) is a pleiotropic cytokine that is involved in many autoimmune and inflammatory diseases. Transcriptional control of IL-6 gene expression is exerted by various compounds, among which glucocorticoids are the most potent antiinflammatory and immunosuppressive agents currently in use. Glucocorticoids exert their transrepressive actions by negatively interfering with transcription factors, such as nuclear factor-␬B (NF-␬B) and AP-1. Both factors make use of the coactivator cAMP response element-binding protein (CREB)-binding protein (CBP) to enhance their transcriptional activities, which led to the hypothesis that a mutual antagonism between p65 or cJun and activated glucocorticoid receptor (GR) results from a limited amount of CBP. Recently, we showed that glucocorticoid repression of NF-␬Bdriven gene expression occurs irrespective of the amount of coactivator levels in the cell. In the current study, we extend this observation and demonstrate that also AP-1-targeted gene repression by glucocorticoids is refractory to increased amounts of nuclear coactivators. From results with Gal4 chimeric proteins we conclude that glucocorticoid repression occurs by a promoter-independent mechanism involving a nuclear interplay between activated GR and AP-1, independently of CBP levels in the cell. (Molecular Endocrinology 15: 219–227, 2001) Jun family (c-Jun, junB, and JunD) or among proteins of the Jun and Fos (c-Fos, FosB, Fra1, and Fra2) families, respectively (1); they all belong to the class of the basic zipper (bZIP) family of sequence-specific dimeric DNA-binding proteins (2). The AP-1 binding site is most commonly recognized by c-Jun homodimers or c-Jun/c-Fos heterodimers. AP-1 was originally identified to interact with the control regions of genes, which contain TPA (12-O-tetradecanoyl phorbol 13-acetate)-responsive promoter elements (TRE) and become activated by mitogens, oncoproteins, and UV light. In addition to positive regulatory effects, the AP-1 complex has also been shown to confer negative regulation (3). The transcriptional activity of c-Jun is enhanced by amino-terminal phosphorylation at serine 63 and 73 by Jun amino-terminal kinase (JNK). This inducible phosphorylation step is required to recruit the transcriptional coactivator cAMP response element-binding protein (CREB)binding protein (CBP), which leads to transcriptional enhancement (4, 5). CBP and its homolog p300 are large cointegrator proteins that provide a docking platform for many members from diverging transcription factor families and contain an enzymatic histone acetyltransferase (HAT) activity (6, 7). This HAT activity functions to shift the chromatin structure into a looser configuration, thereby facilitating the access of specific and basal transcription factors and subsequently gene transcription. Other coactivators, belonging to the p160 family, such as steroid coactivator-1 (SRC-1), are suggested to increase the specificity and strength of the interaction of nuclear receptors with members of the CBP family. Interestingly, some of these coactivators, including SRC-1 and its homolog, activator of retinoic acid receptor (ACTR), were recently shown to also contain HAT activities and to associate, similarly as CBP and p300, with another HAT protein, p/CAF (8–10); all together, they give rise to a functional coactivator complex. This additional interaction platform could then potentially provide a link to the core transcriptional machinery (11). Interleukin-6 (IL-6) is a pleiotropic cytokine that is implicated in endocrine and metabolic actions, as well as in immune regulation and aging. IL-6 is thought to INTRODUCTION The transcription factor AP-1 is encoded by protooncogenes and regulates various aspects of cell proliferation and differentiation. AP-1 can be composed of either homo- or heterodimers among members of the 0888-8809/01/$3.00/0 Molecular Endocrinology 15(2): 219–227 Copyright © 2001 by The Endocrine Society Printed in U.S.A. 219 MOL ENDO · 2001 220 play a key role in a number of inflammatory processes, such as rheumatoid arthritis, trauma, and stress, and is also involved in the pathogenesis of osteoporosis, HIV infection, sepsis, and progression of cancer (12, 13). Understanding the regulation of this gene may therefore lead to a controlled and tissue-restricted modulation of its pleiotropic actions. Glucocorticoids not only inhibit proliferation by suppressing AP-1 activity of genes involved in proliferation, such as c-Jun (14), but they can also mediate a strong suppression of AP-1-driven genes involved in inflammation and immune dysregulation, including IL-6. Glucocorticoid action is mediated by binding to the glucocorticoid receptor (GR), which belongs to the family of nuclear hormone receptors. These ligandregulated sequence-specific transcription factors may activate or repress gene expression. Whereas gene activation is generally mediated by binding of homodimeric GR subunits to their cognate DNA elements, experiments with mice expressing a dimerization-defective GR demonstrated that gene repression is mainly conducted by interference of the GR monomer with the activities of other transcription factors, including AP-1 (15). Recently, the negative interference between GR and AP-1 was demonstrated in an in vivo model system of TPA-induced expression of collagenase and stromelysin in skin, and GC repression of these genes was also shown to involve the DNAbinding independent function of GR (16). As CBP can enhance transcriptional activation of AP-1 as well as of nuclear receptors (reviewed in Ref. 17), it was proposed that mutual antagonism between these different signal transduction pathways could be explained by the mutual competition for limiting amounts of CBP within the cell (11). Recent reports also showed that SRC-1 can functionally interact and enhance AP-1-mediated gene expression (18). SRC-1 was originally identified as a coactivator for the nuclear receptor superfamily (19), prompting a role for SRC-1 also in mediating nuclear receptor-dependent gene repression of AP-1-driven genes and vice versa (18). The idea behind a limitation in the amount of coactivator protein such as CBP arose from the observation that a single mutated CBP allele, leading to a heterozygous phenotype, already results in severe developmental defects. This mutation correlates with a disorder called the Rubinstein-Taybi syndrome and includes facial distortions, broadening of thumbs and toes, and mental retardation (20). In a previous study we demonstrated that glucocorticoid repression of various nuclear factor (NF)-␬Bdriven genes occurs independently of coactivator levels in the cell (21). In the present study we demonstrate that glucocorticoids can (...truncated)