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Inhibition by sodium butyrate of enzyme induction by glucocorticoids and dibutyryl cyclic AMP
Abstract
We have found that butyrate selectively inhibits hormonal induction of a few specific proteins and messenger RNAs in hepatoma cells. The fatty acid salt reversibly abolishes induction of tyrosine aminotransferase by dexamethasone and dibutyryl cyclic AMP in HTC cells by inhibiting the production of tyrosine aminotransferase messenger RNA. Half-maximal inhibition of enzyme induction occurred in 0.9 mM butyrate. This effect is highly specific, since 4 h after the addition of butyrate to induced HTC cells, the relative abundance of only five messenger RNA species out of several hundred observable on two-dimensional gels of translational products is changed. Upon removal of the butyrate from cell cultures pretreated with dexamethasone, tyrosine aminotransferase activity begins to increase more rapidly than if dexamethasone is added to control cultures, indicating that part of the induction process occurs in the presence of butyrate. A dose-dependent reduction of fast histone acetylation by butyrate was demonstrated by treating cells with butyrate followed by a short pulse with [3H]acetate and chase in a high concentration of butyrate. The butyrate concentration test range over which rapid histone acetylation is inhibited is similar to that which inhibits enzyme induction to the same extent. In contrast, the slow form of histone acetylation is unaffected in the concentration range examined. The induction of tyrosine aminotransferase by dexamethasone is delayed in hypoacetylated cells. This lag is consistent with the time required to initiate the recovery of the fast form of histone acetylation after its transient disappearance (Covault, J., Perry, M., and Chalkley, R. (1982) J. Biol. Chem. 257, 13433-13440). We conclude that sodium butyrate interferes with the ability of dexamethasone and dibutyryl cyclic AMP to increase production of several specific species of messenger RNA in hepatoma cells. This effect correlates well with its ability to reduce rapid acetylation of histones in HTC cells; we discuss potential roles of rapid histone acetylation in modulating hormonal stimulation of transcription.
... Sodium butyrate (NaB) modulates the expression of specific proteins in a variety of cell types (reviewed in 1). In some cases butyrate causes a change in the differentiated state (2); in other cases this agent affects the synthesis of a limited number of proteins (3,4). Butyrate can also inhibit the steroid-hormone induced transcription of certain genes (5). ...
... butyrate induction of MT RNA is apparently a broadly distributed phenomenon. The number of mRNAs whose levels are altered by butyrate in rat hepatoma cells is reported to be quite restricted (4). We similarly find the mRNA for both the actively transcribed actin gene and the liver-specific, dex-inducible TAT gene unchanged by butyrate. ...
... the steroid hormone induction of certain genes(4,5). probe was in excess was demonstrated by incubating increasing amnounts of probe with Cd-induced RNA and analyzing the amount of the protected bands (data not shown). ...
Sodium butyrate selectively induces accumulation of metallothionein-I (MT-I) RNA in H4IIE rat hepatoma cells. The induction
1s rapid; significant elevation in cytoplasnric MT-I RNA can be observed within three hours after exposure to 5 mM butyrate.
Maximal levels of MT-I RNA are obtained after eight hours. Butyrate stimulates MT RNA accumulation in the absence of de novo protein synthesis, indicating that MT induction by butyrate is not a distal step in a cascade of gene activation events.
Butyrate blocks the induction of tyrosine amino transferase by dexamethasone. in contrast, butyrate and dexamethasone induced
MT RNA elevations are additive. Butyrate induced MT-I RNA transcripts initiate at the correct start site. Measurements of
the transcriptional activity of the MT-I gene indicate that butyrate stimulates MT-I transcription. The rapid, direct nature
of the induction of MT-I by butyrate, combined with the extensive characterization of the metallothionein gene, provide an
excellent system in which to study the effects of butyrate on a small, well-defined, responsive region of chromatin.
... Thus, in accordance with general models of the role of KATs and KDACs in transcription, KATs are generally considered to facilitate steroid receptor-activated gene expression whereas KDACs repress or prevent it. However, very early studies of the HDACi, sodium butyrate, showed that it impaired steroid-mediated activation of several genes (37)(38)(39)(40), first raising the possibility that KDACs play a more complicated and versatile role in steroid receptor signaling. More recent studies have supported these original findings and provided some insight into the mechanism by which this may occur. ...
... The activation of several genes by glucocorticoids was shown in the early 1980s to be impaired by cotreatment with the class I-selective HDACi, sodium butyrate (37,38). However, because KDACs and KATs had not yet been identified, the mechanism remained undefined. ...
Lysine deacetylases have been known to regulate nuclear receptor function for many years. In the unliganded state, nuclear receptors that form heterodimers with retinoid X receptors, such as the retinoic acid and thyroid hormone receptors, associate with deacetylases to repress target genes. In the case of steroid receptors, binding of an antagonist ligand was initially reported to induce association of deacetylases to prevent activation of target genes. Since then, deacetylases have been shown to have diverse functions in steroid receptor signaling, from regulating interactions with molecular chaperones to facilitating their ability to activate transcription. The purpose of this review is to summarize recent studies on the role of deacetylases in steroid receptor signaling, which show deacetylases to be highly versatile regulators of steroid receptor function.
... HTC cells were (like H4IIE cells) derived from rat liver. They possess the capacity to respond to glucocorticoids, as evidenced by the induction of tyrosine aminotransferase (32). However, these cells have lost the capacity to synthesize PEPCK mRNA, even though the PEPCK gene sequence is still present and, as far as can be determined by restriction mapping, is intact (data not shown). ...
... The line represents DNA, and the circles represent nucleosomal structures. Nuclease-HS sites are indicated by V's and are designated as sites A to E. The lower portion is an enlargement of site C, which is the promoterhormone-responsive region of the gene(2,29,32). The brackets above the sequence show the in vitro footprints obtained with rat liver extracts (Wong and Granner, unpublished data). ...
We used indirect end labeling to identify a series of five hypersensitive (HS) sites in the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells. These sites were found at -4800 base pairs (bp) (site A), at -1300 bp (site B), over a broad domain between -400 and -30 bp (site C), at +4650 bp (site D), and at +6200 bp (site E). Sites A to D were detected only in cells capable of expressing the PEPCK gene, whereas site E was present in all of the cells examined thus far. The HS sites were present in H4IIE cells even when transcriptional activity was reduced to a minimum by treatment with insulin. Stimulation of transcription by a cyclic AMP analog to a 40-fold increase over the insulin-repressed level did not affect the main features of the HS sites. Furthermore, increased transcription did not disrupt the nucleosomal arrangement of the coding region of the gene, nor did it affect the immediate 5' region (site C), which is always nucleosome-free. In HTC cells, a rat hepatoma line that is hormonally responsive but unable to synthesize PEPCK mRNA, the four expression-specific HS sites were totally absent. Our experimental results also showed that, although there is a general correlation between lack of DNA methylation and transcriptional competence of the PEPCK gene, the role, if any, of methylation in the regulation of PEPCK gene activity is likely to be exerted at very specific sites.
... This raises the intriguing possibility that, like protein phosphorylation, histone acetylation may be a multifaceted post-translational modification with respect to gene expression (7). For example, studies have shown the inhibitory effects of histone hyperacetylation induced by deacetylase inhibitors on steroid inducible genes such as ovalbumin (15), tyrosine aminotransferase (16), prolactin receptor (17), interleukin-2 (18), mouse mammary tumor virus (MMTV) (19,20) and more recently, vitamin-D regulation of the osteocalcin gene (21). 4 The progesterone (PR), glucocorticoid (GR), androgen (AR) and mineralocorticoid (MR) receptors are members of the steroid hormone receptors (SHRs), a class of receptors that belong to a large nuclear hormone receptor superfamily of hormone-activated transcriptional regulators (22). ...
... We demonstrate that for MMTV, exposure of cells to levels of TSA that results in global histone hyperacetylation inhibits PR-mediated transcription. These observations are consistent with previous studies where inhibition of deacetylase activity is associated with gene inactivation (15)(16)(17)(18)21). In the case of the nuclear receptors, it has been proposed that histone acetylation may be viewed as a molecular switch between the inactive and active forms of the receptor, suggesting that action of both acetylases and deacetylases is important in the regulation of many genes. ...
Post-translational modifications of histones play an important role in modulating gene transcription within chromatin. We used the mouse mammary tumor virus (MMTV) promoter, which adopts an ordered nucleosomal structure, to investigate the impact of a specific inhibitor of histone deacetylase, trichostatin A (TSA), on progesterone receptor-activated transcription. TSA induced global histone hyperacetylation, and this effect occurred independently of the presence of hormone. Interestingly, chromatin immunoprecipitation analysis revealed no significant change in the level of acetylated histones associated with the MMTV promoter following high TSA treatment. In human breast cancer cells, in which the MMTV promoter adopts a constitutively "open" chromatin structure, treatment with TSA converted the MMTV promoter into a closed structure. Addition of hormone did not overcome this TSA-induced closure of the promoter chromatin. Furthermore, TSA treatment resulted in the eviction of the transcription factor nuclear factor-1 from the promoter and reduced progesterone receptor-induced transcription. Kinetic experiments revealed that a loss of chromatin-remodeling proteins was coincident with the decrease in MMTV transcriptional activity and the imposition of repressed chromatin architecture at the promoter. These results demonstrate that deacetylase inhibitor treatment at levels that induce global histone acetylation may leave specific regulatory regions relatively unaffected and that this treatment may lead to transcriptional inhibition by mechanisms that modify chromatin-remodeling proteins rather than by influencing histone acetylation of the local promoter chromatin structure.
... HDACi prevented activation of transiently transfected, episomal, or chromosomal MMTV promoters by glucocorticoids (Mulholland et al., 2003;Kinyamu and Archer, 2004). Although sodium butyrate inhibited glucocorticoid induction of the tyrosine aminotransferase gene in rat HTC cells (Plesko et al., 1983), it enhanced glucocorticoid induction of alkaline phosphatase in HeLa S3 cells (Littlefield and Cidlowski, 1984). Finally, trichostatin A induced estrogen-dependent transcription in MCF-7 cells (Ruh et al., 1999) and in stably transfected HepG2 cells (Mao and Shapiro, 2000). ...
... The strong dose-dependent stimulatory effects of HDACi on GRE5-TATA-CAT and endogenous TAT gene expression differ markedly from previously reported results demonstrating down-regulation of the stimulatory effect of glucocorticoids on the MMTV promoter in various cell types (Mulholland et al., 2003;Kinyamu and Archer, 2004) or on the TAT gene in rat hepatoma cells (Plesko et al., 1983). Our results, in contrast, are compatible with earlier observations that sodium butyrate enhances dexamethasone responsiveness of the alkaline phosphatase gene in HeLa S3 cells (Littlefield and Cidlowski, 1984). ...
Histone deacetylase inhibitors (HDACi), which have emerged as a new class of anticancer agents, act by modulating expression of genes controlling apoptosis or cell proliferation. Here, we compared the effect of HDACi on transcriptional activation by estrogen or glucocorticoid receptors (ER and GR, respectively), two members of the steroid receptor family with cell growth regulatory properties. Like other transcription factors, steroid receptors modulate histone acetylation on target promoters. Using episomal reporter vectors containing minimal promoters to avoid promoter-specific effects, we observed that long-term (24-h) incubation with HDACi strongly stimulated GR-dependent but markedly repressed ER-dependent signaling in ER+/GR+ human endometrial carcinoma Ishikawa cells. These effects were reproduced on endogenous target genes and required incubation periods with HDACi substantially longer than necessary to increase global histone acetylation. Repression of estrogen signaling was due to direct inhibition of transcription from multiple ERalpha promoters and correlated with decreased histone acetylation of these promoters. In contrast, the strong HDACi stimulation of GR-dependent gene regulation was not accounted for by increased GR expression, but it was mimicked by overexpression of the histone acetyltransferase complex component transcriptional intermediary factor 2. Together, our results demonstrate striking and opposite effects of HDACi on ER and GR signaling that involve regulatory events independent of histone hyperacetylation on receptor target promoters.
... The data described in this report are consistent with the model that ISG transcription depends on HDAC activity to facilitate the transition from committed initiation to processive elongation through, at least in part, the targeted recruitment of P-TEFb to ISG promoter-proximal regions mediated by the Brd4 protein, and that sequestration of Brd4 by acetylated chromatin impairs ISG expression (Fig. 6 F). Previous reports have substantiated a requirement of HDAC activity for IFN-stimulated transcription (Génin et al., 2003;Nusinzon and Horvath, 2003;Chang et al., 2004;Sakamoto et al., 2004), and this observation has been extended to additional inducible expression systems, such as in response to IFN-γ (Zupkovitz et al., 2006), glucocorticoids (Tichonicky et al., 1981;Plesko et al., 1983;Bresnick et al., 1990;Mulholland et al., 2003;Kadiyala et al., 2013) or in STAT5-dependent transcription (Rascle et al., 2003;Xu et al., 2003). Broad requirement for HDAC activity during acute induction of gene expression from stimulus-dependent promoters suggests a common positive role for HDAC enzymes for this subset of genes, in addition to its well-appreciated repressive role of maintaining histone deacetylation on silent chromatin. ...
In contrast to the common role of histone deacetylases (HDACs) for gene repression, HDAC activity provides a required positive function for IFN-stimulated gene (ISG) expression. Here, we show that HDAC1/2 as components of the Sin3A complex are required for ISG transcriptional elongation but not for recruitment of RNA polymerase or transcriptional initiation. Transcriptional arrest by HDAC inhibition coincides with failure to recruit the epigenetic reader Brd4 and elongation factor P-TEFb due to sequestration of Brd4 on hyperacetylated chromatin. Brd4 availability is regulated by an equilibrium cycle between opposed acetyltransferase and deacetylase activities that maintains a steady-state pool of free Brd4 available for recruitment to inducible promoters. An ISG expression signature is a hallmark of interferonopathies and other autoimmune diseases. Combined inhibition of HDAC1/2 and Brd4 resolved the aberrant ISG expression detected in cells derived from patients with two inherited interferonopathies, ISG15 and USP18 deficiencies, defining a novel therapeutic approach to ISG-associated autoimmune diseases.
... I suggested the use of synchronized HTC cells as a means of studying this process in precisely defined segments of the cell replication cycle (13). We also studied the role of histone acetylation in the regulation of TAT gene expression a few years before this became a topic of broad interest (14). ...
If you don't know where you're going, you might wind up someplace else—Yogi Berra
My professional journey, once I accepted the fact that I would not play first base for the Chicago Cubs, was not accomplished in a straight line. I entered medical school intending to become a family physician but soon became interested in research. I took an internal medicine residency at the University of California, San Francisco (UCSF), then a long research fellowship in the nascent field of molecular biology at the National Institutes of Health (NIH), and followed that with training in clinical endocrinology at the University of Wisconsin. I spent the next fifteen years dividing my time between the clinic and laboratory at the University of Iowa. Finally, I decided to concentrate on research and spent the next twenty-three years at Vanderbilt University. For the past eleven years, I have been at the University of Iowa, where I helped establish a new diabetes research center. My research journey also had several different directions. Upon reflection, I guess I ended up “someplace else” by choice, and it certainly has been an interesting trip. It is one I unreservedly recommend.
... Sodium butyrate, an inhibitor of HDACs (histone deacetylases), has been used as a tool to induce histone hyperacetylation to study the effects of hyperacetylated histone on chromatin structure and function. In mammalian cells, however, butyrate does not cause an increase in histone hyperacetylation, and in fact just the opposite is observed (Covault et al., 1982;Plesko et al., 1983). Butyrate blocks MyoD-mediated chromatin reorganization and transcription initiation, but cannot block transcription when added after chromatin remodeling had occurred (Gerber et al., 1997;Johnston et al., 1992). ...
MyoD and myogenin (Myog) recognize sets of distinct but overlapping target genes and play different roles in skeletal muscle differentiation. MyoD is sufficient for near-full expression of early targets, while Myog can only partially enhance expression of MyoD-initiated late muscle genes. However, the way in which Myog enhances the expression of MyoD-initiated late muscle genes remains unclear. Here, we examine the effects of Myog on chromatin remodeling at late muscle gene promoters and their activation within chromatin environment. Chromatin immunoprecipitation (ChIP) assay showed that Myog selectively bound to the regulatory sequences of late muscle genes. Overexpression of Myog was found to overcome sodium butyrateinhibited chromatin at late muscle genes in differentiating C2C12 myoblasts, shifting the transcriptional activation of these genes to an earlier time period. Furthermore, overexpression of Myog led to increased hyperacetylation of core histone H4 in differentiating C2C12 myoblasts but not NIH3T3 fibroblasts, and hyperacetylated H4 was associated directly with the late muscle genes in differentiating C2C12, indicating that Myog can induce chromatin remodeling in the presence of MyoD. In addition, co-immunoprecipitation (CoIP) revealed that Myog was associated with the nuclear protein Brd4 in differentiating C2C12 myoblasts. Together, these results suggest that Myog enhances the expression of MyoD-initiated late muscle genes through MyoD-dependent ability of Myog to induce chromatin remodeling, in which Myog-Brd4 interaction may be involved.
... This interaction inhibits CBP-associated histone acetyltransferase activity resulting in the active recruitment of histone deacetylase (Ito et al., 2000;Matthews et al., 2004;Tsaprouni et al., 2002;Espada and Esteller, 2007;Ito et al., 2006) and dephosphorylation complexes (Parra et al., 2007;Kim et al., 2008). Deacetylation and dephosphorylation lead to chromatin modification, resulting in immune dysregulation (Kagoshima et al., 2001;Mishra et al., 2001;Plesko et al., 1983). Epigenetic patterns have been shown to regulate IFN gamma production (Avni et al., 2002;Muegge et al., 2003;Mishra et al., 2001;Parra et al., 2007;Kim et al., 2008) as well as molecules that contribute to NKCA (e.g. ...
The molecular basis for psychosocial-distress mediated immune-dysregulation is not well understood. The purpose of this study was to determine whether peripheral blood mononuclear cell (PBMC) epigenetic pattern associates with this form of immune dysregulation. Women newly diagnosed with early stage breast cancer were enrolled into the study and psychosocial, immunological and epigenetic assessments were made at diagnosis and four months later, after completion of cancer treatment. At diagnosis women reported increased perceived stress, anxiety, and mood disturbance and the PBMC of these women exhibited reduced natural killer cell activity and reduced production of interferon gamma, which contrasted with results, obtained after completion of treatment. At the epigenetic level, a PBMC subset derived from women at diagnosis exhibited a distinct epigenetic pattern, with reduced nuclear acetylation of histone residues H4-K8 and H4-K12, as well as reduced phosphorylation of H3-S10, when compared to similar cells derived after the completion of treatment. Natural killer cell activity and interferon-gamma production were associated with nuclear acetylation and phosphorylation status of these histone residues. These findings demonstrate associations among nuclear epigenetic pattern and the immune dysregulation that accompanies psychosocial distress.
... perforin, granzymes, and NK cell Ig-like receptors) (Araki et al., 2008;Lu et al., 2003;Santourlidis et al., 2002;Chan et al., 2005) and to control IFN gamma production (Hatton et al., 2006;Chang and Aune, 2005;Yano et al., 2003;Jones and Chen, 2006). The effects of glucocorticoids on chromatin structure and on immune response genes have been shown to be recovered by HDAC inhibitors, like Trichostatin A (TSA) (Kagoshima et al., 2001;Mishra et al., 2001;Plesko et al., 1983). ...
It is well-established that psychological distress reduces natural killer cell activity (NKCA) and dysregulates cytokine balance. This may be mediated by stress-induced release of glucocorticoids, which have broad effects on the immune system, including the suppression of NKCA and alteration of cytokine production. The purpose of this study was to evaluate epigenetic mechanisms that may underlie the effect of glucocorticoids on NK cells, using the human NK cell line, NK92. Treatment of NK92 cells with the synthetic glucocorticoid, dexamethasone, at a concentration of 10⁻⁷M, produced a significant reduction in NKCA. Glucocorticoid inhibition was a consequence of not only a reduced capacity of the NK cells to bind to tumor targets but also a reduced production of granule constituents (perforin and granzyme B) with no detectable effect on granule exocytosis. Glucocorticoids also reduced the constitutive and the stimulated production of the cytokines, IL-6, TNF alpha and IFN gamma, and reduced the surface expression of LFA-1. Glucocorticoid treatment also reduced global histone acetylation, the acetylation of histone 4 lysine position 8, and the accessibility of the proximal promoters of perforin, interferon gamma and granzyme B. Histone acetylation was recovered by treatment of the NK cells with a histone deacetylase inhibitor, which also restored NKCA and IFN gamma production. These results demonstrate glucocorticoids to dysregulate NK cell function at least in part through an epigenetic mechanism, which reduces promoter accessibility through modification of histone acetylation status. This epigenetic modification decreases the expression of effector proteins necessary to the full functional activity of NK cells.
... HDAC inhibitors induce specific changes in gene expression that influence a variety of other processes, including growth arrest, differentiation, cytotoxicity, and induction of apoptosis (17). It has been reported that sodium butyrate, an HDAC inhibitor, interferes with GR-activated transcription (20). However, the roles of HDACs in GR-mediated mechanisms are still obscure. ...
The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as "sLZIP" contains a deleted putative transmembrane domain (amino acids 229-245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.
... Also dexamethasone has been shown to affect glycosyltransferase activities (51) and to interfere with the action of butyrate and TPA (16, 32, 44), but it was unable to induce synthesis of Shiga toxin-binding sites in MDCK cells. Butyric acid has been found to modulate the expression of a number of genes (13, 14, 19, 25, 31, 32, 34). This could be because of its ability to cause changes in phosphorylation of nuclear proteins (4, 11), changes in DNA g I I LU ~_Figure 9. Transport rate of Shiga toxin from the apical and basolateral side of MDCK cells to the Golgi apparatus , t25I-labeled Shiga toxin was added to the basolateral or the apical side of butyric acid-treated MDCK cells, and the fraction of labeled toxin in the Golgi apparatus (peak I,Fig. ...
The glycolipid-binding cytotoxin produced by Shigella dysenteriae 1, Shiga toxin, binds to MDCK cells (strain 1) only after treatment with short-chain fatty acids like butyric acid or with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. The induced binding sites were found to be functional with respect to endocytosis and translocation of toxin to the cytosol. Glycolipids that bind Shiga toxin appeared at both the apical and the basolateral surface of polarized MDCK cells grown on filters, and Shiga toxin was found to be endocytosed from both sides of the cells. This was demonstrated by EM of cells incubated with Shiga-HRP and by subcellular fractionation of cells incubated with 125I-labeled Shiga toxin. The data indicated that toxin molecules are endocytosed from coated pits, and that some internalized Shiga toxin is transported to the Golgi apparatus. Fractionation of polarized cells incubated with 125I-Shiga toxin showed that the transport of toxin to the Golgi apparatus was equally efficient from both poles of the cells. After 1-h incubation at 37 degrees C approximately 10% of the internalized toxin was found in the Golgi fractions. The results thus suggest that glycolipids can be efficiently transported to the Golgi apparatus from both sides of polarized MDCK cell monolayers.
... Histone deacetylation is promptly inhibited about 40-fold and hyperacetylation of the histones ensues (1)(2)(3). The ability to induce tyrosine aminotransferase is also rapidly inhibited (4,5). Over the following 24 h the rate of histone acetylation decreases and overall RNA synthesis declines by about 50%, though the complexity of the products is largely unchanged (6,7). ...
We have obtained a number of variant HTC cells which are capable of vigorous replication in the presence of 6 mM sodium butyrate. These cells show characteristic changes in histone acetylation. H2A/H2B are no longer modified and the turnover of histones H3/H4 acetate is about 4-fold greater than in control HTC cells at the same butyrate concentration. Histone deposition continues successfully even though histones H3/H4 become hyperacetylated upon association with the chromatin. Prompt deacetylation of new histones does not appear to be a prerequisite for successful deposition processes. Initial enzymatic studies indicate that not only do the butyrate-resistant cells show an increased deacetylase activity (on a per cell basis), but also the enzyme is less sensitive to sodium butyrate under in vitro assay conditions. In contrast to control HTC cells in 6 mM butyrate in which dexamethasone induction of tyrosine aminotransferase is inhibited, the butyrate-resistant variant cells are capable of tyrosine aminotransferase induction even in the presence of butyrate. The implications of these observations are discussed.
... It has also been reported to cause changes in cell morphology and physiology in many normal and transformed mammalian cells (14,(20)(21)(22)(23)(24). For example, it induces differentiation of Friend erythroleukemia cells (16) and embryonal carcinoma cells (21) and inhibits hormone-mediated protein synthesis, such as the induction of egg white proteins by estrogen (40) and that of tyrosine aminotransferase by dexamethasone (41). In these cases, the actions of sodium butyrate were associated with alterations in histone acetylation. ...
The lung-colonizing ability of low-metastatic Lewis lung carcinoma cells (P-29) was enhanced by their in vitro treatment with butyric acid and its sodium salt, sodium butyrate. Of the short chain fatty acids tested, butyric acid was the most effective in enhancing the lung-colonizing ability of P-29 cells; propionic acid and valeric acid were slightly effective, but acetic acid and caproic acid were ineffective. The enhancing effect of butyric acid on the lung-colonizing ability of P-29 cells was reversible, indicating that the result was the consequence of epigenetic alterations. Treatment of P-29 cells with butyric acid resulted in enhancement of secretion of plasminogen activator, cellular cathepsin B activity, and cellular adhesiveness. The phenotypes of cells treated with butyric acid were compared with those of cells treated with dimethyl sulfoxide, which was reported to enhance the lung-colonizing ability of P-29 cells. Significant differences were found in the phenotypes, especially that of cellular adhesiveness; that is, butyric acid enhanced mainly homotypic aggregation of the cells, while dimethyl sulfoxide enhanced mainly heterotypic adhesion, such as adhesion to monolayers of endothelial cells. In addition, butyric acid reversibly caused hyperacetylation of core histones in P-29 cells, while dimethyl sulfoxide did not.
... periment, a hybrid contained about 2% -positive cells following culture for 34 days in the absence of butyrate. This hybrid was subsequently cultured for an additional 28 days in the presence of butyrate; the frequency of -positive cells at this point was less than 1% (Fig. 4C). These data suggest that -ABA cannot reactivate silenced -globin genes. 25, 32, 33). Since -ABA decreases the rate of globin gene switching in the hybrids, we wished to test whether glucocorticoids would have the opposite effect, i.e., acceleration of the -to-switch. Dexamethasone sodium phosphate, which is an analog of glucocorticoid steroids that is easily used in tissue culture, was applied. Since glucocorti ...
Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that alpha-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the gamma- to beta-globin gene (gamma-to-beta) switch in the HFE x MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on gamma-globin expression is due to inhibition of gamma gene silencing rather than to induction of gamma gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the gamma-to-beta switch in HFE x MEL hybrids was examined. Dexamethasone strikingly accelerated the gamma-to-beta switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the gamma-to-beta switch of the HFE x MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.
... Sodium butyrate was used as a general activator of viral gene expression, which is known to modulate expression of a number of viral promoters (Luka et al., 1979;Long et al., 1980;Bohan et al., 1987Bohan et al., , 1989Radsak et al., 1989;Contreras-Salazar et al., 1990;Golub et al., 1991;Tanaka et al., 1991;Laughlin et al., 1993;Oster et al., 1993) and cellular promoters (Kruh, 1982;Plesko et al., 1983;Takano et al., 1988;Foss et al., 1989). Despite these reports little is known of its mechanism of action. ...
The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.
... 15 In spite of its ability to activate a number of genes, such as the`dormant' fetal g-globin gene, 16 it was also noted that butyrate inhibits the transcriptional activation of several steroid hormone-dependent genes. An inhibitory effect of butyrate was shown for estrogen-mediated gene expression in the chicken oviduct, 17 for glucocorticoiddependent genes in HTC cells, 18,19 in cultured mammary gland fragments 20,21 and for thyroid-mediated gene expression in rat pituitary tumor cells. 22 Interestingly, in rat glioma C6 cells, butyrate was found to block the glucocorticoid-induced increase in glycerol phosphate dehydrogenase 23 but had no effect on glucocorticoid induction of glutamine synthase activity; 24 in HeLa cells the induction of alkaline phosphatase by dexamethasone was, however, potentiated by butyrate. ...
In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.
... Treatment with histone deacetylase (HDAC) 1 inhibitors, which results in hyperacetylation of histones, can decrease rather than increase transactivation at some promoters or result in no change at others (12). The HDAC inhibitor sodium butyrate inhibits transactivation of the ovalbumin promoter by estrogen receptors (ERs) (13), and of the tyrosine aminotransferase promoter (14) and the MMTV-LTR by glucocorticoid receptors (GRs) (15). Furthermore, Deckert and Struhl (16) have recently shown that in Saccharomyces cerevisiae both acetylated and deacetylated histones H3 and H4 can be associated with transcriptionally active promoters. ...
Acetylation of lysines in histones H3 and H4 N-terminal tails is associated with transcriptional activation and deacetylation
with repression. Our studies with the mouse mammary tumor virus (MMTV) promoter in chromatin show significant levels of acetylation
at promoter proximal and distal regions prior to transactivation. Upon activation with glucocorticoids or progestins, promoter
proximal histones become deacetylated within the region of inducible nuclease hypersensitivity. The deacetylation lags behind
the initiation of transcription, indicating a role in post-activation regulation. Our results indicate a novel mechanism by
which target promoters are regulated by steroid receptors and chromatin modification machinery.
... As the magnitude of the inhibition was large when dibutyryl-cAMP was used, it might be likely that butyrate, the breakdown product of dibutyryl-cAMP, is also acting to modify the cytokine induction of GCHI. The biological effects of butyrate on cellular signal transduction systems [26] or mRNA expression [27] have been reported. The possible roles of BH 4 synthesized in vascular endothelial cells have been investigated in several laboratories. ...
We studied the effects of cAMP on cytokine (interferon-gamma plus tumor necrosis factor-alpha)-induced stimulation of tetrahydrobiopterin (BH4) synthesis in human umbilical vein endothelial cells (HUVEC). The cytokine mixture caused a marked increase in the biosynthesis and release of BH4 by HUVEC. Dibutyryl-cAMP produced a dose-dependent inhibition of this cytokine-induced stimulation of synthesis and release of BH4 by these cells. 8-Bromo-cAMP also caused a significant inhibition, although the effects were less marked than those of dibutyryl-cAMP. Both forskolin and the stable analog of prostacyclin, iloprost, caused cAMP accumulation and a concomitant diminution of the cytokine-induced BH4 synthesis in HUVEC. Dibutyryl-cAMP and iloprost also significantly inhibited the cytokine-induced stimulation of GTP cyclohydrolase I (GCHI) activity and mRNA production. We concluded that the suppression by the cAMP messenger system of cytokine-induced stimulation of synthesis and release of BH4 by HUVEC can be attributed to the inhibition of the activity of GCHI, the rate-limiting enzyme in BH4 biosynthetic pathway, in HUVEC. The data also suggest that the cAMP-mediated reduction in the GCHI mRNA level may at least partially explain the decline in GCHI activity. It is reasoned that under inflammatory conditions, cAMP-elevating agents such as prostacyclin exert regulatory effects on circulation by inhibiting cytokine-induced synthesis and release of BH4 by HUVEC.
... The acetylation and deacetylation of core histones have been extensively investigated, with a general pattern of acetylation correlating with actively transcribed chromatin (35, 85). There is, however, a growing body of evidence to suggest that for any individual gene, more complex relationships between transcription and acetylation exist (3, 10, 19, 50, 54, 57, 59, 68, 71). In the present work, we show that the HAT activity of the SAGA complex is required for the repression of the ARG1 gene in rich medium. ...
Transcriptional regulation of the Saccharomyces cerevisiae ARG1 gene is controlled by positive and negative elements. The transactivator Gcn4p is required for activation in minimal medium,
while arginine repression requires the ArgR/Mcm1 regulatory complex, which binds to two upstream arginine control elements.
We have found that the coordinated regulation of ARG1 requires components of the SAGA chromatin-remodeling complex. Using gcn5 deletion strains and a Gcn5 protein carrying the E173Q mutation in the histone acetyltransferase (HAT) region, we show that
the HAT activity of Gcn5p is required for repression of ARG1 in rich medium. Similar increases in expression were seen upon deletion of other SAGA components but not upon deletion of
the ADA-specific component, Ahc1p. Chromatin immunoprecipitations using antibodies to acetylated H3 confirmed that a decrease
in the level of acetylated histones at the ARG1 promoter correlated with increased ARG1 expression. Up-regulation of ARG1 in the absence of Gcn5p also correlated with increased binding of TATA-binding protein to the promoter. The analysis of promoter
deletions showed that Gcn5/Ada repression of ARG1 was mediated through the action of the ArgR/Mcm1 regulatory complex. In addition, studies with minimal medium demonstrated
a requirement for the Ada proteins in activation of ARG1. This suggests that SAGA has a dual role at ARG1, acting to repress transcription in rich medium and activate transcription in minimal medium.
... Alternatively, acetylation may directly alter the conformation of nucleosome and thereby facilitate the accessibility of transcription factors. Hyperacetylation of core histones has been shown to enhance the binding of transcription factors [7,10,33]. ...
We have previously reported that androgen receptor (AR) expression is inversely correlated to its promoter methylation and is regulated by sex steroids. As chromatin structure plays an important role in transcriptional regulation, the effect of sex steroids on DNaseI accessibility of chromatin of AR promoter was examined in the brain cortex of adult and old mice of both sexes. Nuclei were digested with different concentrations of DNaseI and the extracted DNA was further cleaved by PstI and analyzed by Southern hybridization with DIG-labeled 695-bp AR promoter. With 50 U DNaseI, the intensity of PstI-specific 1.45-kb band was lower in intact female as compared to male groups, suggesting increased nuclease accessibility in female than male. Although gonadectomy increased DNaseI accessibility remarkably in male and female of both ages, testosterone decreased the accessibility in adult but increased in old male. Estradiol, on the other hand, decreased DNaseI accessibility in both adult male and old female but increased in old male and adult female. Thus, these findings suggest that the chromatin conformation of AR promoter varies with age and sex and its accessibility to DNaseI is reduced by testosterone and estradiol in the brain cortex of adult male mice.
... Furthermore, the GRE-dependent reporter activity was reduced in the S49.1 thymoma cell line that expressed the antisense SRG3. In an inhibitor study of Plesko et al. using the HDAC inhibitor, sodium butyrate, HDAC activity was required for the GRE-dependent tyrosine aminotransferase gene expression in hepatoma cells (Plesko et al., 1983). Recent studies of Ito et al. have shown that GR represses NFκB-mediated HAT activity by a combination of the direct inhibition of CBP-associated HAT activity and the recruitment of HDAC2 to the NF-κB activation complex, p65-HAT complex (Ito et al., 2001). ...
The major function of the thymus is to eliminate developing thymocytes that are potentially useless or autoreactive, and select only those that bear functional T cell antigen receptors (TCRs) through fastidious screening. It is believed that glucocorticoids (GCs) are at least in part responsible for cell death during death by neglect. In this review, we will mainly cover the topic of the GC-induced apoptosis of developing thymocytes. We will also discuss how thymocytes that are fated to die by GCs can be rescued from GC-induced apoptosis in response to a variety of signals with antagonizing properties for GC receptor (GR) signaling. Currently, a lot of evidence supports the notion that the decision is made as a result of the integration of the multiple signal transduction networks that are triggered by GR, TCR, and Notch. A few candidate molecules at the converging point of these multiple signaling pathyways will be discussed. We will particularly describe the role of the SRG3 protein as a potent modulator of GC-induced apoptosis in the crosstalk.
Most of the carbohydrates of the fibre fraction are extensively broken down by the microflora when they reach the large intestine; this metabolic process results in the production of gas and short chain fatty acids (SCFA; essentially acetic, propionic and butyric acids). In non-ruminant mammals, including humans, the large intestine is the major site of SCFA production and they are extensively absorbed. The mechanisms for SCFA absorption depend on conditions prevailing in the intestinal lumen; basically, the SCFA always diffuse along the concentration gradient, mainly the non-ionised (protonated) form. However, SCFA absorption may involve facilitated transfers: when the lumen pH is close to neutral, SCFA absorption is parallel to a net secretion of bicarbonate (Hoverstad, 1986). This process contributes to water and mineral recovery from the large intestine. Absorption capacity from the human colon has been estimated at 200 to 700 mmol/24 hours (Cummings, 1984), which is in accordance with an estimated breakdown of 30 to 60 g carbohydrates in the colon. There is also a minor production of some other SCFA (isobutyrate, n-valerate, isovalerate), the iso C4 and C5 monocarboxylates chiefly arise from the de-amination of branched chain amino acids.
Sodium butyrate exerts a pronounced inhibition on the gibberellic acid-induced synthesis and secretion of α-amylase by aleurone cells of barley seeds. This inhibition, which is reversible and non-competitive with cespect to gibberellic acid, is concentration dependent, with virtually total inhibition being accomplished between 4 and 5 mM sodium butyrate. The pattern of inhibition of α-amylase formation correlates well with a decrease in the accumulation of its messenger RNA. The addition of butyrate 12 h after the addition of gibberellic acid to half-seeds, has no effect on the formation and secretion of α-amylase. It has been shown in earlier studies that the synthesis of α-amylase mRNAs takes about 12 h for completion. The conclusion that butyrate interferes with some step in the transcriptional process is supported by a decrease observed in the RNAs that hybridize with a cloned α-amylase cDNA. The results of in vitro translation confirm the inhibition of the formation of several translatable mRNAs. Further, immunological probing detected only trace amounts of α-amylase proteins in the secretion of butyrate-treated seeds. Translation of functional mRNAs, post-translational modifications and the secretion α-amylase are not affected by sodium butyrate. It is concluded that butyrate selectively inhibits the transcription of several genes that are under the influence of gibberellic acid. This report is the first one documenting the inhibitory effect of sodium butyrate on a hormone-induced synthesis and accumulation of mRNAs in a plant system.
Background
KDACis impair GR transactivation of the MMTV promoter, but their impact on cellular target genes is unknown.
Results: KDACi or KDAC depletion suppresses transactivation of about 50% of GR target genes.
Conclusion: KDAC1 is required for efficient GR transactivation in a gene-selective fashion.
Significance: Because KDACs facilitate GR transactivation, clinical KDACi use may have a major impact on GR signaling.
Nuclear receptors use lysine acetyltransferases and lysine deacetylases (KDACs) in regulating transcription through histone acetylation. Lysine acetyltransferases interact with steroid receptors upon binding of an agonist and are recruited to target genes. KDACs have been shown to interact with steroid receptors upon binding to an antagonist. We have shown previously that KDAC inhibitors (KDACis) potently repress the mouse mammary tumor virus promoter through transcriptional mechanisms and impair the ability of the glucocorticoid receptor (GR) to activate it, suggesting that KDACs can play a positive role in GR transactivation. In the current study, we extended this analysis to the entire GR transcriptome and found that the KDACi valproic acid impairs the ability of agonist-bound GR to activate about 50% of its target genes. This inhibition is largely due to impaired transcription rather than defective GR processing and was also observed using a structurally distinct KDACi. Depletion of KDAC1 expression mimicked the effects of KDACi in over half of the genes found to be impaired in GR transactivation. Simultaneous depletion of KDACs 1 and 2 caused full or partial impairment of several more GR target genes. Altogether we found that Class I KDAC activity facilitates GR-mediated activation at a sizable fraction of GR-activated target genes and that KDAC1 alone or in coordination with KDAC2 is required for efficient GR transactivation at many of these target genes. Finally, our work demonstrates that KDACi exposure has a significant impact on GR signaling and thus has ramifications for the clinical use of these drugs.
5-Azacytidine and sodium butyrate, two DNA methylation-disrupting agents, were tested for radioprotective properties on V79A03 cells. Both compounds can activate genes not previously expressed (e.g. metallothionein). 5-Azacytidine treatment (3 microM, 24 h) caused a 50% decrease in the 5-methylcytosine content of V79A03 DNA whereas sodium butyrate treatment (1 mM, 24h) resulted in a 700% increase in 5-methylcytosine content. Additionally, 5-azacytidine treatment resulted in the increased survival of V79A03 cells, with treatment 24 h prior to exposure to gamma radiation providing a dose reduction factor of 1.8. Sodium butyrate treatment did not result in a significant increase in survival. These results indicate that the hypomethylation of genomic DNA prior to exposure to gamma radiation correlates with an increase in survival of V79A03 cells, possibly due to the activation of the enzymes involved in repair.
We have identified DNA elements In the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound ‘in vivo’ by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue
specific expression of the gene. in vitro DNase I footprintlng demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE
and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. ‘in vivo’ DNase I footprlnting demonstrated that the BSEJCRE, NF1/ CCAAT, and three additional sites are bound by protein in H4IIE
cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome
binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential
methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine
residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated
in HTC DNA; including the cytosine at position -90 within the BSE/CRE. Only one cytosine residue, at position -90, was methylated
in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the -90 position yet this was insufficient
to allow synthesis of a detectable amount of PEPCK mRNA
Three populations (1, 3 and 6) of bone cells, derived from rat calvaria by sequential enzymatic digestion, were cultured with [3H]glucosamine and [35S]sulfate, in the presence or absence of transforming growth factor-beta (TGF-beta) or bone-derived matrigenin activity. Population 6 synthesized a chondroitin sulfate proteoglycan (PG) and responded to the addition of the factors by increased rates of synthesis of hyaluronic acid (HA) and PG and an increase in the size of the HA. Comparisons of populations 1, 3 and 6 showed an ordered, spontaneous increase in HA and PG synthesis. However, the addition of matrigenin activity resulted in a much greater stimulation of PG, but not HA, synthesis in population 1 compared to population 6, suggesting a cellular organization in the calvarium whose net effect would be to direct PG synthesis towards the periphery of the tissue.
We have generated rat insulinoma (RIN) sublines AlGh, m5F, A12, A13 and AhGh with increasing surface expression of the A2B5 ganglioside, a marker of differentiation. We asked whether the capacity of the sublines to differentiate was related to their stage of differentiation, as is characteristic of cells within the normal beta-cell lineage. To answer this, we measured the effect of the differentiation inducer sodium butyrate (NaB, 1 mM) on proliferation, insulin content, secretion and biosynthesis, and the expression of A2B5 and 3G5 gangliosides by the sublines. Six days after exposure to NaB, cell numbers/dish ranged from (1-3) x 10(6) compared to (4-6) x 10(6) in control cultures. By day 2, AlGh, m5F, A12, A13 and AhGh cells exposed to NaB contained 1.5-, 1.4-, 1.4-, 1.2- and 1.0-fold higher amounts of insulin, respectively, and by day 6, 3.6-, 2.3- and 1.0-fold higher, and 1.2- and 2.4-fold lower, amounts of insulin, respectively, than control cells. After 2 days, insulin secretion from AlGh, m5F, A12, A13 and AhGh cells was 1.7-, 1.0-, 1.5-, 1.0- and 1.0-fold higher, respectively, and the rate of (pro)insulin biosynthesis 1.7-, 2.3-, 1.3-, 1.0- and 1.0-fold higher, respectively, than control cells. After 6 days, A2B5 ganglioside expression was increased 3-, 1.9- and 2-fold on m5F, A12 and A13 cells, respectively, but was not significantly altered on AlGh and AhGh cells. 3G5 ganglioside expression was increased 1.5- and 8.4-fold, respectively, on AlGh and m5F cells, but was unaltered on A12, A13 and AhGh cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Our laboratory has previously developed cell lines derived from mouse NIH 3T3 fibroblasts and C127 mammary tumor cells that stably express mouse mammary tumor virus (MMTV) long terminal repeat fusion genes in bovine papillomavirus-based episomes. Glucocorticoid hormone strongly activates transcription from episomes and induces the disruption of a single nucleosome in an array of phased nucleosomes on the MMTV promoter. Sodium butyrate inhibits the glucocorticoid hormone-dependent development of a nuclease-hypersensitive site that is due to the displacement of this nucleosome, and inhibits induction of RNA transcripts from episomes. Saturation binding studies show that butyrate treatment does not significantly affect the amount or the hormone-binding affinity of the glucocorticoid receptor. In a transient transfection assay, glucocorticoid hormone can activate transcription from a MMTV long terminal repeat-driven luciferase gene construct equivalently in untreated and butyrate-treated cells, indicating that the soluble factors necessary for transactivation of the MMTV promoter are unaffected by butyrate. The differential effect of butyrate on the induction of stable chromatin templates and transiently expressed plasmids suggests that butyrate prevents nucleosome displacement and represses transcription by inducing a modification of chromatin.
The human hepatoma cell line (Li-7A) possesses a high concentration of epidermal growth factor (EGF) receptors and exhibits ectoATPase activity in the presence of either MgATP or CaATP (Knowles: J. Cell. Physiol., 134:109-116, 1988). Growth for 96 hours in the presence of both EGF and cholera toxin or another cyclic AMP elevating agent induced an ectoATPase activity which was more active with CaATP and resistant to inhibition by the sulfydryl reagent, p-chloromercuriphenylsulfonate (pCMPS) (Knowles: Arch. Biochem. Biophys., 263: 264-271, 1988). In contrast, treatment of cells with butyrate, a short chain organic acid which can be derived from the analogue, dibutyryl cyclic AMP, resulted in a 4-7-fold increase of an ectoATPase which was more active with MgATP and highly sensitive to pCMPS inhibition. Maximal induction by butyrate required 48 hours and was dependent on butyrate concentration, but was independent of EGF and cyclic AMP elevating agents. Of six organic acids tested, butyrate was most effective in the induction of the ectoMg2(+)-ATPase. The increase in the ectoMg2(+)-ATPase activity could be prevented with actinomycin D and cycloheximide, indicating that both transcription and translation were necessary for induction. In addition to the induction of the ectoMg2(+)-ATPase, butyrate induced alkaline phosphatase activity, but had no effect on a third ectoenzyme 5'-nucleotidase. These data further support our proposal that two distinct ectoATPases exist in the plasma membrane of Li-7A hepatoma cells.
Sodium butyrate has been reported to induce cellular differentiation and reduce the tumorigenicity of certain tumor cells. We have examined the effects of butyrate on alpha-fetoprotein (AFP) gene expression in 7777 and McA-RH8994 rat hepatoma cells and have found that nontoxic concentrations of the drug decrease AFP mRNA levels in both cell lines. However, McA-RH8994 requires a 10-fold lower concentration (0.5 mM) of butyrate to affect a 50% reduction in AFP mRNA levels within 48 h. At 2 mM, sodium butyrate reduces AFP mRNA levels in McA-RH8994 cells by at least 90% after 48 h, while having little effect on the expression of either the 7S RNA or Harvey-ras genes. Time-course studies show that the effect of butyrate on McA-RH8994 AFP mRNA levels is immediate and is accompanied by an accumulation of cells in the G1/G0 phase of the cell cycle. Sodium butyrate was found to reduce AFP mRNA levels in both dexamethasone-treated 7777 and McA-RH8994 cells; dexamethasone decreases AFP mRNA levels in the former cell line and increases AFP mRNA levels in the latter. Therefore, it is unlikely that butyrate acts simply by reducing the dexamethasone receptor concentration in 7777 cells.
Treatment of tumor cells that have little if any metastatic potential with certain drugs that have little or no mutagenic activity has been found to result in marked phenotypic alterations of the cells, including development of a metastatic potential. We found that polar compounds and butyric acid, which are known to alter the expressions of normally silent genes, enhanced the lung-colonizing ability of cloned low-metastatic Lewis lung carcinoma cells. This change was accompanied by increases in the activities of degradative enzymes such as glycosidases, cathepsin B, and plasminogen activator; adhesion of the cells to culture dishes, monolayers of endothelial cells, and a subendothelial matrix; and homotypic aggregation. The effects of these drugs in enhancing the lung-colonizing ability of the cells was found to be reversible, suggesting that it was due to epigenetic alterations. Other investigators have shown that treatment of nonmetastatic tumor cells with 5-azacytidine, which causes hypomethylation of DNA and activates normally silent genes, results in the emergence of a small number of clones with a heritable but unstable metastatic phenotype. These findings suggest that epigenetic mechanisms are involved in rapid cellular phenotypic diversification and tumor progression.
n-Butyrate was previously found to increase the epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. We show here that butyrate and dexamethasone synergistically modulate the surface expression of the EGF receptors. The butyrate-induced enhancement of high-affinity EGF binding was only slight in the absence of glucocorticoid, but was strongly and dose-dependently amplified by dexamethasone. Butyrate counteracted the inhibition by insulin of the dexamethasone-induced increase in EGF binding. The results indicate that the glucocorticoid has a permissive effect on a butyrate-sensitive process that determines the surface expression of the high-affinity class of EGF receptors.
In mouse 3T3-L1 preadipocytes, the glucocorticoid dexamethasone has been shown to promote a switch in beta-adrenoceptor subtype expression from beta 1 to beta 2 and to increase the total number of beta-adrenoceptors. The present study demonstrates that sodium butyrate also modulates beta-adrenoceptor expression in these cells. Incubation of preadipocytes with 2-10 mM butyrate for 24-48 h promoted a dose- and time-dependent switch in beta-adrenoceptor subtype from a near equal mixture of beta 1 and beta 2 to greater than 85% beta 2 and caused an approximate doubling of the receptor number. beta-Adrenoceptors were assayed in membranes prepared from 3T3-L1 cells using the radiolabeled antagonist [125I]iodocyanopindolol and the beta 2-selective antagonist ICI 118.551. Other short chain acids were not as effective as butyrate in promoting changes in beta-adrenoceptor expression. Cycloheximide (1.0 microgram/ml) inhibited the effects of butyrate on both beta-adrenoceptor subtype and number. Alterations in beta-adrenoceptor phenotype promoted by either butyrate or dexamethasone were functionally correlated with cAMP accumulation in these cells. Comparison of the effects of butyrate and dexamethasone on beta-adrenoceptor expression suggests that these two agents regulate beta-adrenoceptors by different mechanisms.
We have studied the effects of sodium butyrate (SB) on 1,25(OH)2D3 receptors in the pig kidney cell line (LLC-PK1). In this system, we have shown that 1,25(OH)2D3 induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity is dependent on the level of 1,25(OH)2D3 receptors. Treatment of confluent cells with SB (5 mM/48 h) caused an approximately 50% decrease in total receptors, whereas the affinity for 1,25(OH)2D3 was unchanged. At 5 mm, the action of SB on these receptors required more than 24 h to be detected. The effect of the decrease in receptors on the functional response to the hormone was studied by measuring the 1,25(OH)2D3 induction of 24-hydroxylase activity after treatment with SB. The induction of 24-hydroxylase activity at higher doses of hormone paralleled the reduction in receptors and was diminished by 25-50%. At low doses of hormone, the cells appear to be more sensitive to 1,25(OH)2D3 induction, exhibiting an unexplained increase in 24-hydroxylase activity compared to cells not exposed to SB. An additional effect of SB was also noted: SB decreased cell proliferation and inhibited [3H]thymidine incorporation by 75% when added to cells prior to confluence. At confluence, SB caused a less drastic effect on protein and DNA synthesis. Therefore, most binding experiments were conducted at confluence when the SB effect on cell proliferation was less. Other short chain fatty acids in addition to SB were also tested. The action to decrease 1,25(OH)2D3 receptors was more specific upon exposure to SB. We have previously demonstrated up-regulation of 1,25(OH)2D3 receptors in LLC-PK1 cells after treatment with various vitamin D metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)
The hormone-responsive enzymes tyrosine aminotransferase and glycerol-3-phosphate dehydrogenase were studied with respect to current models of the mechanism of glucocorticoid/cAMP interaction during the induction of enzyme activity in responsive cell hybrids between rat C6 glioma cells and rat FU5AH hepatoma cells. The results of experiments involving protein and mRNA synthesis inhibitors, sequential addition of inducers, and the assay of cyclic-AMP-dependent protein kinase could not be adequately explained by any one model of inducer interaction. Comparison of the hybrid clones revealed the presence of factors that may modify induction but that are not essential for synergistic induction.
We have determined that sodium butyrate and, to a lesser extent, dimethylsulfoxide (DMSO) and 3-aminobenzamide (3-AB) preserve aspects of the differentiated phenotype of primary cultures of adult rat hepatocytes. The histone deacetylase inhibitor, butyrate, inhibits the increase in gamma-glutamyltranspeptidase (GGT) activity and the decrease in basal tyrosine aminotransferase (TAT) activity normally observed when hepatocytes are cultured under appropriate conditions. The effects of butyrate on GGT and TAT activities are accompanied by parallel changes in GGT and TAT mRNA levels. The poly(ADP)ribose-synthetase inhibitor, 3-aminobenzamide, has effects similar to butyrate on GGT activity and mRNA levels, while both 3-AB and DMSO increase basal TAT activity in cultured hepatocytes. Under appropriate conditions all three agents--butyrate, 3-AB, and DMSO--extend the length of time cultured hepatocytes can be maintained as confluent monolayers. However, under all the conditions studied, butyrate extended the length of time hepatocytes could be maintained as monolayers more than any other treatment used. Butyrate-treated hepatocytes maintained ultrastructural features that were more similar to those of hepatocytes in vivo than hepatocytes treated with any other of the agents tested. Histone acetylation levels of primary cultures of adult rat hepatocytes declined concomitant with the loss of the differentiated phenotype of the cells. These results suggest that histone acetylation may play a role in the changes in gene expression observed when hepatocytes are placed in culture.
It is axiomatic that the extraordinary diversity in form and function of cells, tissues, and organs within a higher organism results not from differences in their genomic composition, but from the finely tuned selective expression of portions of their common genetic repertoires. The fact that steroids act as gene-regulating molecules, and that they do so, for the most part, by acting as allosteric modifiers of their respective receptor (R) proteins, has focused much attention on the manner in which steroid—receptor (S—R) complexes act to regulate gene expression, particularly at the level of gene transcription.
The expression of metallothionein genes (MT-I and MT-II) was shown to be enhanced within 2 h of addition of 2.5-5 mM sodium butyrate to cultures of teratocarcinoma cells. Both undifferentiated stem cells (F9 and OC15) and differentiated cells (PSA5E and OC15 END) reacted similarly to butyrate by increased accumulation of MT mRNAs. As expected, all of the teratocarcinoma cells that were tested also responded to Zn2+ and Cd2+ by 5- to 10-fold increases in MT mRNA accumulation within 2-24 h of metal addition to the culture media. Surprisingly, MT genes in cells pretreated with butyrate were hypersensitive to metal induction, and this was demonstrated by accumulated transcript levels and by synthesis of MT protein. The maximal metal response was obtained by exposure of cells to butyrate for around 5-8 h together with 10 microM heavy metals. Metal additions to culture media over a range of concentrations and times only induced half the levels of MT mRNA that were achieved by butyrate plus metals. Butyrate enhanced the rate of accumulation of MT mRNA in response to metals, increased the sensitivity of the MT gene to metals, and protected cells from toxic effects of high concentrations of metals. The butyrate and metal ion responses were selective in that no accumulation of c-myc, c-fms, HSP-70, or AFP mRNA was detected. However, c-fos mRNA accumulated in cells exposed to toxic concentrations of metals (50 microM and higher) and this was also potentiated by butyrate treatment. These results suggest that butyrate alters the chromatin conformation of both the MT-I and MT-II genes leading to an accentuated transcriptional response to metals.
Replicative DNA synthesis in primary cultures of adult rat hepatocytes is increased by the addition of the histone deacetylase inhibitors, propionate or butyrate. DNA synthesis was increased by the addition of 0.5 or 1.0 mM butyrate; in contrast 5.0 mM butyrate inhibited replicative DNA synthesis. Replicative DNA synthesis was increased only when low levels of butyrate were added as the hepatocytes entered the S phase. The observed apparent increase in replicative DNA synthesis was real, and not owing to changes in the specific activity of the dTTP precursor pool. The effects of butyrate and propionate on DNA synthesis appear to be related to their effects on histone acetylation.
The cell line 4IC6, adapted for growth in 6 mM sodium butyrate from Hepatoma Tissue Culture cells [R. Chalkley, and A. Shires (1985) J. Biol. Chem. 260, 7698-7704], exhibits a fourfold increase in histone acetate turnover. The 4IC6 cells were about 25 times more resistant to butyrate relative to the parental cell line as measured by cloning efficiency. This line also maintains a flatter and more extended morphology when growing in the presence of 6 mM sodium butyrate relative to the parental line. Both cell lines maintain similar intracellular butyrate levels and incorporate [1-14C]butyrate into lipids to similar extents when incubated in medium containing high levels of the fatty acid. These results show that 4IC6 cells have not attained butyrate resistance through acquiring the ability to metabolize butyrate more efficiently or in a significantly different manner when compared with the parental cell line. The membrane lipid composition was nearly identical between the two cell types. Thus the different morphologies exhibited by each cell line were not a consequence of altered membrane lipid composition. The resistant line, 4IC6, maintains about 10-fold higher cholesterol ester levels and half the level of triglycerides found in the parental line. The butyrate-resistant cells also synthesize cholesterol at about a 1.8-fold higher rate than do the parental cells. This difference in de novo synthesis is reflected by a difference of a similar factor in the amount of radioactive cholesterol the two cell lines accumulate over 12 generations. These results are discussed with respect to models for equilibration of serum lipoprotein-derived and newly synthesized cholesterol.
We have studied the effect of butyrate and other short-chain fatty acids on thyroid hormone nuclear receptors in C6 cells, a rat glioma cell line. Exposure of C6 cells to butyrate leads to increased levels of L-triiodothyronine (T3) in the nuclear and extranuclear compartments. The rise in nuclear binding is not merely a reflection of the higher cellular hormone content, and Scatchard analysis of T3 binding to isolated nuclei reveals that butyrate increases receptor number without changing affinity. The effect on the receptor is quantitatively important: a 48-h incubation with 2 mM butyrate increases nuclear binding by 2-3-fold, and 5 mM butyrate by 3-5-fold. Other short-chain fatty acids were found to similarly influence both nuclear receptor and extranuclear T3 levels with the following potency: butyrate > valerate > propionate > acetate. On the contrary, ketone bodies were ineffective. Butyrate increases receptor levels by decreasing receptor degradation, since the apparent t( 1/2 ) of receptor disappearance increased by approximately 3-fold in cells incubated with 2 mM butyrate for 48 h. The regulation of receptor number might be secondary to an action of butyrate on regions of the chromatin to which the receptor associates. We then examined the effect of butyrate on histone acetylation. The fatty acid had little effect in increasing the level of multiacetylated forms of H3 and H4 histone when studied in acid-urea gels, but it markedly inhibited the turnover of [3H]acetate from the histone fraction. There was a striking similarity in the dose-response of butyrate for increasing receptor levels and inhibiting histone deacetylation. Furthermore, a very close correlation between receptor levels and [3H]acetate release was also found when different short-chain fatty acids were used. We thus conclude that the effect of butyrate on the receptor could be explained by a modification of the chromatin structure of C6 cells secondary to acetylation.
A derivative of mouse mammary tumor virus (MTV) DNA, LTL, was constructed in vitro and introduced into the genome of mouse L cells. Transcription of LTL was stimulated by dexamethasone, a glucocorticoid hormone. Two features of LTL chromatin structure are altered upon hormone treatment. First, "moderate" DNAase I sensitivity of the entire LTL element increases following addition of dexamethasone; this alteration persists after hormone withdrawal, when LTL transcription is shut off. Second, a discrete DNAase I-hypersensitive region is induced with a time course that closely parallels the rate of increasing transcription from the MTV promoter; this structure disappears upon hormone removal. The induced hypersensitive region coincides with a segment of the MTV long terminal repeat sequence that specifically binds purified glucocorticoid receptor in vitro and functions as a hormone-dependent enhancer element in vivo. We suggest that specific glucocorticoid receptor-DNA interactions may alter the configuration of DNA or chromatin in the vicinity of the binding sites, thereby creating an active transcriptional enhancer.
Glucocorticoids control the expression of a small number of transcriptionally active genes by increasing or decreasing mRNA concentration. Either effect can result from a transcriptional or a post-transcriptional mechanism. Induction of mouse mammary tumour virus RNA results from a stimulation of transcription initiation and depends on the presence of defined regions in proviral DNA. These regions bind the glucocorticoid receptor and behave functionally as proto-enhancers. Glucocorticoid-inducible genes can retain their sensitivity to the hormone after transfer to a heterologous cell by transfection techniques. Non-inducible genes can become inducible when linked to the promoter region of an inducible gene. The mechanisms by which the receptor-steroid complex stimulates or inhibits transcription or influences mRNA stability are unknown. Receptor binding to nucleic acids appears to be a necessary but not sufficient condition. It is likely that the receptor also interacts with chromatin proteins. This might lead to a catalytic modification of these proteins, resulting in a modulation of gene expression. Development of glucocorticoid-sensitive, biochemically defined, cell-free transcription systems should provide a tool to delineate the molecular determinants of this essential regulatory mechanism.
Effect of sodium butyrate on DNA synthesis and the induction of ornithine decarboxylase (EC 4.1.1.17), a rate-limiting enzyme of polyamine biosynthesis, was studied in phytohemagglutinin (PHA)-stimulated bovine lymphocytes. Millimolar concentrations of butyrate completely inhibited the incorporation of [3H]thymidine into the acid-insoluble fraction and reversibly suppressed the induction of ornithine decarboxylase. Other short-chain fatty acids were much less active than butyrate. These results suggest that the suppression of ornithine decarboxylase activity may be one of the reasons for the inhibition of DNA synthesis with butyrate in bovine lymphocytes, because our previous experimental results have shown that the induction of ornithine decarboxylase closely correlates with the DNA synthesis in growth-stimulated cells.
We have examined the effect of sodium butyrate on the levels of histone acetylation, the pattern of protein synthesis and the inducibility of heat shock polypeptides (hsps) in cultured trout fibroblasts. Maximal levels of histone acetylation are achieved upon treatment of these cells with 5 mM butyrate for 24 h. No significant changes in the pattern of protein synthesis, as detected by two-dimensional gel electrophoresis, are apparent under these conditions, although changes in the levels of three polypeptides are seen at shorter times of exposure to butyrate. Heat shock polypeptides are inducible at normal levels in butyrate-treated cells. This is in contrast to the ability of butyrate to inhibit the activation of steroid-inducible genes in some systems.
We have shown that ethanol, propanol and butanol (at 0.5-2%) and salts of butyric and propionic acids (at 8-40 mM) all cause a major reduction in heat-shock protein (hsp) synthesis when present in the growth medium of Drosophila cultured cells (Kc and SL2) subjected to either increased temperature or chemical stressors. Inhibition of normal protein synthesis in unstressed cells was comparatively slight, and the usual suppression of synthesis of non-heat-shock proteins in stressed cells was unaffected. Maximum suppression of hsp synthesis occurred only if inhibitors were added before initiation of the stress response, an observation that eliminates the possibility that these findings are due to non-specific, toxic effects. Suppression was accompanied by severely reduced levels of both hsp70 mRNA and active heat-shock factor (HSF). We conclude that the inhibitors act by suppressing the initiation of transcription of heat-shock genes.
Sodium butyrate is a small, naturally occurring molecule with demonstrated activity on cell growth and differentiation. However, its effect on smooth muscle cells had not been examined. We have found that sodium butyrate and its more stable in vivo analogue tributyrin are potent DNA synthesis and cell proliferation inhibitors. The inhibitory activity of sodium butyrate was not mediated by an elevation of endogenous cAMP levels, a known pathway involved in SMC growth-arrest and maintenance of the contractile phenotype. Consistent with the concept that its activity is mediated by a cAMP-independent pathway, butyrate was able to augment the maximum DNA synthesis inhibitory effect of various agents that elevated intracellular cAMP levels. Additionally, butyrate present for just the initial 8 h or present as late as 16 h after serum addition was able to inhibit DNA synthesis. By contrast, the cAMP analogue, 8Br-cAMP had to be present throughout the entire G0 to S phase of the cell cycle to effectively inhibit DNA synthesis. These results indicated that sodium butyrate inhibited SMC growth through a cAMP-independent mechanism. We also found that sodium butyrate was unable to abrogate the expression of the serum-inducible genes c-fos, c-myc, Ki-Ras and PS4, but was able to directly stimulate the expression of PS4 and thrombospondin. These results indicate that a number of important early G1 events initiated by serum growth factors are unaltered by sodium butyrate and that this compound is able to directly stimulate the expression of certain genes normally associated with SMC proliferation.
Butyrate (5 mM), Trichostatin A (1 microM) or Trapoxin A (30 nM) increased choline acetyltransferase (ChAT) activity in cultured rat sympathetic neurons 3- to 8-fold in 2 days. On the contrary, the three drugs decreased ChAT activity in human CHP126 cells. Butyrate had little effect on ChAT mRNA level in these cells, suggesting post-transcriptional mechanisms for the decrease in ChAT activity. However, transient transfection experiments using CHP126 cells revealed that the M promoter, but not the R promoter, of human ChAT gene was activated 20- to 130-fold by the three hyperacetylating agents. A butyrate-responsive element was localized in the 1 kbp region upstream of exon M. Constructs containing in addition the genomic segment between exons M and 1 displayed maximal basal activity and inducibility by butyrate, suggesting the presence of butyrate-activated promoter/enhancer elements in this region. The stimulatory effects of butyrate and Trichostatin A were also observed in stably transfected CHP126 clones, suggesting that the chromatin environment was not preventing the induction of the endogenous ChAT gene by butyrate. Rather, the data suggest different chromatin organizations for the stable transgene and the endogenous ChAT gene.
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