HDAC inhibition prevents NF-κB activation by suppressing proteasome activity: Down-regulation of proteasome subunit expression stabilizes IκBα
Introduction
Butyrate (BA) is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fiber within the gastrointestinal tract. It is actively absorbed by intestinal epithelial cells and is the major luminal source of energy for colonocytes [1], [2]. In addition to being a fuel source, butyrate has a number of other profound biological effects [3], [4], [5], [6]. For instance, butyrate can readily induce apoptosis of transformed cell lines [7], [8]. The ability for butyrate to induce apoptosis of cancer cells may contribute to the cancer preventive effects ascribed to dietary fiber [9], [10], [11]. Short chain fatty acids, particularly butyrate, can also suppress intestinal inflammation. Clinical trials have shown that topical butyrate applications alleviate symptoms in patients with mild and moderate ulcerative colitis [12]. Butyrate is also effective at treating other inflammatory conditions of the distal gastrointestinal tract [6], [13], [14]. In association, deficiencies in luminal butyrate production have been linked to colonic inflammation [9]. Such results suggest that butyrate may play an important role in regulating intestinal inflammation, as well as suppressing cellular transformation.
Biochemically, butyrate is a histone deacetylase (HDAC) inhibitor [15]. HDAC inhibitors in general have been noted for their ability to induce cell cycle arrest, differentiation and apoptosis of a wide spectrum of transformed cells. It has been proposed that HDAC inhibitors serve to normalize HDAC activity in transformed cells, which often express elevated levels of certain HDAC proteins [16], [17], [18], [19]. These findings have prompted clinical trials to assess the cancer therapeutic activity of HDAC inhibitors (such as SAHA and LAQ824) [20], [21], [22]. Gene expression profiles of cells treated with HDAC inhibitors have revealed that roughly 2% of the expressed genes are altered following HDAC inhibition [23]. Interestingly, within this population of genes, roughly half increase in expression, while the other half decreases. These selective changes in gene expression likely result from the enhanced acetylation of gene-regulatory transcription factors (e.g., GATA, p53, Sp1 and Sp3), in addition to the enhanced acetylation of histone proteins [24], [25], [26], [27].
Recently, HDAC inhibitors have been reported to modulate the activity of the transcription factor NF-κB in a number of different cell types including colon cancer cell lines and macrophages isolated from the lamia propria of the colon [3], [28], [29], [30], [31]. NF-κB is a central mediator of the immune and inflammatory response and has been implicated in promoting tumorogenesis by protecting cancer cells from apoptosis [32]. Upon activation, NF-κB rapidly enhances the expression of proinflammatory genes such as cytokines and cell adhesion molecules, as well as genes involved in promoting proliferation, angiogenesis and cell survival [33], [34], [35]. The ability of butyrate and other HDAC inhibitors to modulate NF-κB activity coincides with its proposed cancer suppressing and anti-inflammatory activities.
NF-κB is regulated through the binding of inhibitory molecules collectively referred to as the IκB proteins [36], [37]. IκB family members include IκBα, IκBɛ, IκBγ, IκBζ, Bcl-3, p105, p100, splicing variants IκBβ1 and IκBβ2 [37], [38], [39], [40], [41], [42]. Perhaps the most important and well-characterized inhibitor of the IκB family is IκBα. It is the most abundant inhibitor overall, and is responsible for the rapid activation of NF-κB [43], [44], [45], [46], [47]. During NF-κB activation, IκBα is phophorylated by the IκB kinase (IKK) complex and subsequently ubiquitinated by the multisubunit E3 ubiquitin-ligation enzyme, SCFβTrCP[48], [49]. Ubiquitinated IκBα is then rapidly degraded by the proteasome, releasing NF-κB to influence target gene expression [50], [51], [52]. It has been reported that butyrate's ability to suppress NF-κB activity depends in part on its ability to suppress cellular proteasome activity [30], [53].
Here, we provide evidence that HDAC inhibitors butyrate and trichostatin A (TSA) suppress proteasome activity by down-regulating the expression of select proteasome subunits. This ultimately prevents the ubiquitin-mediated, proteasome-dependent degradation of IκBα limiting NF-κB activation. Furthermore, we find that HDAC inhibitors selectively interfere with the proteasome-dependent activation of NF-κB, while having little effect on the proteasome-independent pathway. These findings further classify the cellular activities of a promising new class of anti-inflammatory/anti-neoplastic agents.
Section snippets
Cell culture and treatments
All cell lines were purchased from American Type Culture Collection (Manassas, VA). Caco-2 cells were propagated in minimal essential media containing 2 mM l-glutamine and Earle's salts (E-MEM) supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, streptomycin (50 mg/ml) and penicillin (50 U/ml). HT-29 and SW480 cells were propagated in McCoy's 5A medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and antibiotics. All medium
TNF-α-induced degradation of IκBα is proteasome-dependent in Caco-2 cells
Recent studies have indicated that not all activators of NF-κB induce the degradation of IκBα via the ubiquitin-proteasome system [56], [57], [58], [59]. In fact, it has been reported that the proteasome does not play a predominant role in the IL-1β-induced activation of NF-κB in colonic epithelial cells [60]. Therefore, we sought to determine the involvement of the proteasome in the induced degradation of endogenous IκBα by proinflammatory cytokines TNF-α and IL-1β in the Caco-2 colonic
Discussion
Numerous lines of evidence indicate that NF-κB promotes tumorigenesis [33], [35]. The sustained activation of NF-κB suppresses apoptosis by increasing the expression of cellular survival signals. In fact, the increased activity of NF-κB has been found in many types of solid tumors and transformed cell lines, including those isolated from the colonic epithelium [74], [75]. NF-κB also plays a pivitol role in the inflammatory response by inducing the expression of numerous inflammatory-related
Acknowledgement
This work was supported in part by an award from the National Cancer Institute to C.G. (R29CA79656)
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