Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by cartilage destruction and extracellular matrix degradation in multiple joints (1). The pathogenesis of RA is not clearly understood; however, tumor necrosis factor α (TNFα) is involved in its development, a conclusion which is supported by successful treatments with anti-TNFα reagents (2). The production of TNFα was found to be increased in rheumatoid synovium, followed by the induction of other proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and IL-8, as well as matrix metalloproteinases involved in cartilage and bone destruction in RA (3–5). These cytokines are involved in synovial cell activation and proliferation, leading to generation of pannus (6).
Synovial tissue consists of heterogeneous immune and non–immune cell populations, including fibroblast-like synoviocytes, macrophage-like synoviocytes (MLS), lymphocytes, dendritic cells, and endothelial cells (7). Among these populations, MLS originating from bone marrow–derived monocytes are largely responsible, upon activation, for the production of TNFα protein (6). Monocytes can give rise to osteoclasts involved in rheumatoid bone destruction (8). The mechanisms of MLS activation have been only partially understood. Macrophages express TNFα via activation of Toll-like receptor 4–NF-κB signaling, and this type of activation may be mimicked experimentally by administration of lipopolysaccharide (LPS) (9, 10).
TNFα-mediated chronic inflammation has also been studied in inflammatory bowel diseases and in animal models of such diseases. In those studies, physiologic roles for short-chain fatty acids have been identified (11, 12). Short-chain fatty acids are a natural product of colonic anaerobic fermentation of dietary fiber by luminal microflora. These are the preferred sources of energy for the normal colonic epithelial cell, and they can modulate a variety of fundamental cellular processes to induce cell-cycle arrest, differentiation, and apoptosis in transformed cells (13, 14). These molecules, especially butyrate, have potent antiinflammatory effects and can modulate TNFα expression in colonic epithelial cells and in monocytes (15). It has also been shown that administration of butyrate suppresses experimental enteritis induced in mice by dextran sulfate sodium (16). Experiments in colonocytes revealed that butyrate down-regulates TNFα expression by modulating NF-κB–DNA binding activity (15), although the precise mechanism is not fully understood. In addition, butyrate is known to function as a histone deacetylase (HDA) inhibitor in cells, and it can induce alteration of the chromatin structure (17, 18), although the effect of this activity on TNFα down-regulation is not yet understood. We investigated whether butyrate could suppress TNFα production in activated synovial cells and macrophages, in order to evaluate short-chain fatty acids as a potential investigational new treatment for chronic inflammation in RA.
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- MATERIALS AND METHODS
In this report, we have shown that butyrate strongly down-regulated the production of TNFα in primary synoviocytes, peripheral monocytes, and murine RAW264.7 macrophages at the mRNA level. Our results also indicated that butyrate induced an ARE-binding protein, TIS11B, that could facilitate TNFα mRNA degradation. The collective data led us to believe that the down-regulation of TNFα expression by butyrate was mediated through the TIS11B protein that bound to a specific ARE and facilitated mRNA degradation.
TNFα is a key cytokine in the pathogenesis of RA. Therefore, inhibition of the action of TNFα may improve the clinical course of patients with RA. Biologic agents designed to interfere with the action of TNFα have been approved for the treatment of RA, and they provide dramatic efficacy for the affected patients. However, the disadvantages of biologic agents, such as their high cost, their instability, the difficulty of producing them, and the immune reaction of the host toward them, remain to be addressed. Therefore, new chemical agents that down-regulate TNFα production have been researched extensively. Our current findings may provide a framework for the therapeutic down-regulation of TNFα production by butyrate or its analogs.
Butyrate facilitated the degradation of luciferase transcripts when the TNFα 3′-UTR cDNA sequence was included in the reporter construct. This effect appeared to be dependent on an ARE, since a mutation in the ARE in the reporter plasmids resulted in the loss of the butyrate-induced changes in the transcript turnover. Thus, butyrate could down-regulate TNFα mRNA expression by facilitating TNFα mRNA degradation through a mechanism that was dependent on the specific ARE.
AREs are several repetitive sequences of AUUUA or UUAUUUAUU present in the 3′-UTRs of various mRNA. The AREs are clustered into several categories and have been identified in a panel of genes other than TNFα (27–30). These genes largely encode proteins that regulate cellular growth and the body's response to exogenous agents such as microbes and inflammatory and environmental stimuli. The mRNA containing AREs are commonly short lived, rapidly expressed in response to stimuli, and rapidly degraded once their critical role in gene regulation ceases.
To date, several ARE-binding proteins have been reported that can stabilize or destabilize the target mRNA by binding competitively to their AREs. TTP (also known as TIS11, Nup475, and G0S24) is one of these ARE-binding proteins, and it has been characterized as a critical factor that controls TNFα mRNA turnover (31). Interestingly, genetically manipulated mice that lack TTP showed an abundance of endogenous TNFα production, an RA-like arthritis, and autoimmunity, presumably mediated by an excess of TNFα (32–34). TTP-related proteins, designated TIS11B and TIS11D, are structurally similar to TTP in their zinc-finger motifs, and they share functions with TTP, although their unique functions have yet to be characterized (35). These TTP-related proteins are rapidly expressed in response to 12-O-tetradecanoylphorbol-13-acetate and other diverse stimuli in various types of eukaryotic cells (23, 36). The expression of these proteins differs depending on the circumstances, hence they may have their own roles in controlling mRNA turnover under different circumstances (37). Among these proteins, TTP is induced by TNFα, suggesting that TTP may function as a feedback regulator of TNFα gene expression (31, 38). Notably, a TIS11B homolog expressed rapidly in response to butyrate has also been discovered in human cells and characterized as butyrate response factor 1 (BRF1) (39).
Consistently, butyrate rapidly induced the expression of TIS11B in RAW264.7 cells (Figure 4A). In addition, the induction of TNFα mRNA by LPS stimulation was strongly inhibited when TIS11B was overexpressed in these cells (Figure 4B). Stoecklin et al identified BRF1 as a regulator of ARE-dependent mRNA decay, and also showed that BRF1 can bind directly to ARE and promote the degradation of ARE-containing mRNA (40). Thus, we postulated a model whereby butyrate induced the expression of TIS11B/BRF1, followed by the binding of this ARE-binding protein to the ARE, thus facilitating TNFα mRNA degradation. Our cDNA microarray (GeneNavigator cDNA Array System; Toyobo) revealed that butyrate suppressed expression of mRNA containing AREs in their 3′-UTRs (e.g., mRNA for IL-1β, IL-15, and granulocyte–macrophage colony-stimulating factor) (data not shown). This result further supported the relevance of our hypothesis.
TIS11B has a unique character that facilitates TNFα mRNA degradation; therefore, analyzing the regulation of this molecule would provide a novel approach to the control of TNFα production. TNFα expression is activated mainly by the transcription factor NF-κB and by the MAP kinase (MAPK) pathways (the ERK, JNK, and p38 MAPK pathways). Recent studies have shown that the p38 MAPK pathway in particular plays an important role in posttranscriptional regulation that leads to mRNA stabilization (41). The p38 MAPK pathway also strongly induces and activates TTP, which down-regulates TNFα (42–45). Those studies suggested that the p38 MAPK pathway may play a crucial role in regulating the expression of TNFα (involving a TTP-dependent mechanism); however, the precise mechanism is not completely understood, and less is known about the relationship between TIS11B and the p38 MAPK pathway. Since butyrate induced TIS11B expression and has been shown to affect the p38 MAPK pathway (46), it may be that butyrate influences TIS11B expression through the p38 MAPK pathway. Analysis of the relationship between TIS11B and the p38 MAPK pathway would be important for understanding the effect of butyrate.
The effects of butyrate on TNFα gene expression, other than those involving TIS11B- and ARE-dependent mechanisms, also need to be addressed. In previous studies, it was shown that butyrate can inhibit the binding of NF-κB to DNA (12, 15, 47). In contrast, in the reporter gene assays, butyrate enhanced the transcriptional activity driven by NF-κB sites and the TNFα promoter in a dose-dependent manner (Figures 3A and B). This phenomenon may be explained in part by the HDA-inhibitory effects of butyrate. Butyrate strongly inhibits HDA activity in cells, and it can cause hyperacetylation of nucleotides and thereby nonspecifically enhance transcriptional activity (18, 48, 49). As in the case of genomic DNA, transfected plasmid DNA has been shown to be assembled with histones to form a “minichromosome” that is sensitive to histone hyperacetylation (50). Thus, butyrate can function as a nonspecific transcriptional enhancer for transfected plasmid DNA; hence, cotransfection with an internal control was not informative in the current experiments.
Analysis of the regulation of TNFα by butyrate provides information for a novel approach to the treatment of patients with RA. Further investigation of the regulation of TIS11B expression, including study of its gene promoter, promises to pave the way for therapeutic approaches that address ARE-dependent cytokine gene regulation.