Proteasomal Inhibition Enhances Glucocorticoid Receptor Transactivation and Alters Its Subnuclear Trafficking

Abstract

The ubiquitin-proteasome pathway regulates the turnover of many transcription factors, including steroid hormone receptors such as the estrogen receptor and progesterone receptor. For these receptors, proteasome inhibition interferes with steroid-mediated transcription. We show here that proteasome inhibition with MG132 results in increased accumulation of the glucocorticoid receptor (GR), confirming that it is likewise a substrate for the ubiquitin-proteasome degradative pathway. Using the mouse mammary tumor virus (MMTV) promoter integrated into tissue culture cells, we found that proteasome inhibition synergistically increases GR-mediated transactivation. This increased activation was observed in a number of cell lines and on various MMTV templates, either as transiently transfected reporters or stably integrated into chromatin. These observations suggest that the increase in GR-mediated transcription due to proteasome inhibition may occur downstream of the initial chromatin remodeling step. In support of this concept, the increase in transcription did not correlate with an increase in chromatin remodeling, as measured by restriction enzyme hypersensitivity, or transcription factor loading, as exemplified by nuclear factor 1. To investigate the relationship between GR turnover, transcription, and subnuclear trafficking, we examined the effect of proteasome inhibition on the mobility of the GR within the nucleus and association of the GR with the nuclear matrix. Blocking GR turnover reduced the mobility of the GR within the nucleus, and this correlated with increased association of the receptor with the nuclear matrix. As a result of proteasome inhibition, GR mobility within the nucleus was reduced while its association with the nuclear matrix was increased. Thus, while altered nuclear mobility of steroid receptors may be a common feature of proteasome inhibition, GR is unique in its enhanced transactivation activity that results when proteasome function is compromised. Proteasomes may therefore impact steroid receptor action at multiple levels and exert distinct effects on individual receptor types.Glucocorticoids mediate diverse cellular processes such as differentiation, development, and homeostasis through the ligand-activated glucocorticoid receptor (GR) (24). The GR is a versatile transcriptional regulator, and it can activate or repress a large variety of natural promoters in multiple tissues. This versatility is due in part to the numerous mechanisms utilized by the GR to regulate transcription, depending on the cellular and promoter context. These mechanisms include GR modification of chromatin structure through interactions with chromatin remodeling complexes, differential binding of the receptor to both canonical and noncanonical response elements, binding to other regulatory factors such as AP-1 and NF-B, and interaction with coactivators (2,11,46).The activity of the GR is also subject to regulation that occurs at least in part by changes in the half-life of the receptor protein (6). Turnover of t...

The 26S proteasome system degrades the ERM transcription factor and regulates its transcription-enhancing activity

“…It has recently been demonstrated that inhibition of proteosomal degradation enhances the transcriptional response mediated by some nuclear receptors (Blanquart et al, 2002;Deroo et al, 2002;Pollenz, 2002) and the Ets protein E1AF (Takahashi et al, 2005). We show here that blocking protein turnover with the proteasome inhibitor ALLN or MG132 also induces a change in ERM transcriptional activity.…”

Nuclear hormone receptor degradation and gene transcription: An update

“…Hence, it is possible that the UPP may be playing opposite roles with respect to PR and GR. For GR it has been recently demonstrated that proteasome inhibition causes the promoter occupancy of GR to increase and receptor mobility to decrease (55). Although no mechanistic studies have been reported to date for VDR, it appears that a similar mechanism might be operating.…”

“…On the contrary, it appears that ligand may not be acting as a signal in case of VDR and AR, since these receptors are stabilized upon binding to their ligands. Yet, another scenario is with the GR, which undergoes ligand-dependent down regulation but at the same time proteasome inhibitors enhance its transcriptional activity (55). Therefore, the precise role of the proteasome in NHR transcription is quite complex and far from clear.…”

Cellular Processing of the Glucocorticoid Receptor Gene and Protein: New Mechanisms for Generating Tissue-specific Actions of Glucocorticoids

“…3). The receptor is ubiquitinated at a conserved lysine residue located in a PEST degradation motif at the end of the NTD, and this modification targets the receptor for degradation by the proteasome (88,89). Mutation of this lysine residue blocks ligand-dependent down-regulation of GR␣ and enhances its transcriptional activity on reporter genes (89).…”