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| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
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Departments of Internal Medicine and Department of Integrative Biology, Pharmacology and Physiology, The University of Texas Medical School at Houston, Houston, Texas.
Correspondence to Dr. Bruce C. Kone, Departments of Internal Medicine and of Integrative Biology and Physiology, The University of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030. Phone: 713-500-6873; Fax: 713-500-6882; E-Mail: Bruce.C.Kone{at}uth.tmc.edu
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Abstract |
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B (NF-B) element promoter were assessed in transient transfection assays. Gel shift and supershift assays were used to identify NF-B in nuclear extracts. Protein-protein interactions were assayed by co-immunoprecipitation and GST pull-down assays. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and overexpression of HDAC isoforms were used to assess the impact of acetylation status on iNOS and NF-B element promoter activity. TSA inhibited induction of endogenous NO production and iNOS as well as NF-B element promoter activity in response to interleukin–1
(IL-1) or lipopolysaccharide (LPS) + interferon-
(IFN-) in both cell types without altering NF-B DNA binding activity. Overexpression of specific HDAC isoforms enhanced cytokine induction of both the iNOS and the NF-B element promoter. HDAC2 and NF-B p65 co-immunoprecipitated from mesangial cell nuclear extracts, and in vitro translated HDAC2 specifically interacted with an NF-B p65 GST fusion protein. Hyperacetylation diminishes cytokine induction of iNOS transcription activity, at least partially, by limiting the functional efficacy of NF-B. The specific recruitment of HDAC2 to NF-B at target promoters and the consequent effects on acetylation status may play an important role in regulating iNOS as well as other NF-B–dependent genes involved in inflammation. |
Introduction |
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(4), nuclear factor–B (NF-B) (5), cAMP (4), JNK, and p38 MAPK (6), have been implicated in iNOS gene activation in mesangial cells, whereas transforming growth factor– (TGF-) (7), and interleukin-13 (IL-13) (8) are known to inhibit iNOS activation in these cells. Deletion and mutation analysis of the murine iNOS promoter transfected into various cell types has identified the functional importance of several transcription factors and their cognate binding elements on the iNOS promoter, including B sites (9), a binding site for ESE-1 (a novel member of the ETS transcription factor family) (10), a C/EBP box (11), and an interferon (IFN) regulatory factor-1 (IRF-1) site (12), and an IFN-–activated site (GAS) (13). Transcriptional regulation of genes is often governed by chromatin acetylation or by acetylation of transcription factor complexes. Unwinding of the closed DNA structure during active transcription of genes permits accessibility to transcription factors and allows them to exert regulatory control. Enhanced transcription of a restricted set of genes is associated with increased histone acetylation, governed by histone acetyltransferases (HAT), whereas gene silencing is commonly correlated with histone hypoacetylation, the result of histone deacetylase (HDAC) activity (14). HDAC are a family of proteins highly conserved during evolution (15). The class I HDAC family, comprising HDAC1, -2, -3, and -8, is similar to the yeast transcriptional regulator Rpd3p and has been associated with transcriptional corepressors, such as Sin3 and NCoR. Class II deacetylases HDAC4, -5, -6, and -7 share greater similarity to yeast Hda1p than to Rpd3, and knowledge of their biochemistry is currently limited. Both class I and class II HDAC share a common catalytic motif, but they are differentiated by unique N-terminal sequences found only in the class II enzymes (16,17).
Although it is generally believed that transcriptional activity is correlated with histone acetylation (18), recent studies indicate that transcriptional activation is not necessarily associated with increased histone acetylation and suggest a more complex picture of HDAC activity and transcription with regard to individual genes and promoters. Indeed, in the case of the mouse mammary tumor virus (MMTV) promoter, HDAC activate gene transcription (18), and HDAC inhibition has been shown to attenuate cytokine induction of C/EBP and C/EBP
(19). Recent studies have also established that HATs and HDAC can be recruited to specific promoters and transcription factors to serve as transcriptional regulators. For example, reversible deacetylation of the inducible transcription factor NF-B RelA by HDAC3 promotes effective binding to IB
and nuclear export of the complex to regulate target gene transcription (20). Moreover, HDAC1 directly associates with the Rel homology domain of NF-B p65 to alter its NF-B activity in cultured cells. Although HDAC2 has not been previously shown to directly interact with NF-B, it can regulate NF-B activity through its association with HDAC1 (21). In addition to histones and transcription factors, acetylases are known to influence nuclear import factors and -tubulin (22).
In this report, we show that HDAC2 can directly interact with NF-B p65 and that specific HDAC isoforms can accentuate LPS and cytokine activation of iNOS gene transcription in mesangial cells and RAW 264.7 macrophages. This novel effect of HDAC to augment rather than restrict gene expression may represent an important cell-specific mechanism to synergistically upregulate the inflammatory response and facilitate maximal NO production.
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Materials and Methods |
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(10 ng/ml), or LPS (1 µg/ml) + IFN- (100 U/ml) was added to the cells as indicated in the text and figure legends. Mouse recombinant IL-1 and IFN- were from R & D Systems (Minneapolis, MN) and BioSource (Camarillo, CA), respectively. Polyclonal antibodies recognizing NF-B p65 and p50 were from Santa Cruz Biotechnologies (Santa Cruz, CA). Oligonucleotides were custom synthesized by Genosys (The Woodlands, TX). Lipofectamine 2000 reagent was from Invitrogen (Carlsbad, CA). The Dual-Luciferase Reporter Assay System and the luciferase vectors pGL3-Basic and pRL-SV40 were from Promega (Madison, WI). The BCA protein estimation kit was from Pierce Chemical (Indianapolis, IN). Glutathione-Sepharose 4B beads, pGEX-5X-3 vector, and ECL reagents were from Amersham Pharmacia Biotech (Piscataway, NJ).
Plasmids
The NF-B reporter construct, p36B(-)(NF-B)3-luc, which contains three tandem copies of the B binding element (GGGGACTCTCCC) upstream of the SV40 early promoter sequence and fused to the coding sequence forthe luciferase gene (23), was provided by Dr. Bharat Aggarwal (University of Texas MD Anderson Cancer Center, Houston, TX). piNOS-luc, which contains the murine iNOS promoter/enhancer and a portion of exon 1 (nucleotides -1486 to +145) in pGL3-Basic, has been previously characterized (11). The FLAG-tagged HDAC2 expression plasmid pME18S-FLAG-HDAC2 (24) was from Dr. Edward Seto (University of South Florida, Tampa, FL). FLAG-tagged mammalian expression plasmids pBJ-HDAC1, pBJ-HDAC4, pBJ-HDAC5, and pBJ-HDAC6 (25) were from Dr. Stuart L. Schreiber (Harvard University, Cambridge, MA). To generate fusion with GST, cDNA encoding murine NF-B p65 was amplified by RT-PCR from mesangial cell RNA and subcloned into pGEX-5X-3 at the EcoRI and NotI sites to maintain the appropriate reading frame and verified by DNA sequence analysis.
Nitrite Assays
Mesangial cells and RAW 264.7 cells were seeded in 24-well plates for 24 h, transiently transfected in some experiments, and then stimulated with IL-1 or LPS + IFN- for 24 h. The medium was then collected and the nitrite concentration determined with the Griess Reagent System (Promega) according to the manufacturer’s protocol.
Transient Transfections
Mesangial cells or RAW 264.7 cells were seeded in 24-well plates and grown to 90 to 95% confluency in complete medium without antibiotics and transfected the next day using the LipoFectamine 2000 reagent according to the manufacturer’s protocol and a total of 1 µg/well of plasmid DNAs. The amount of transfected DNA was kept constant by addition of appropriate amounts of the parental empty expression vector. Transfection efficiencies were normalized by cotransfection with 20 ng/well of the Renilla luciferase expression plasmid pRL-SV40. For trans-repression experiments, 0.5 µg of piNOS-luc, p36B(-)(NF-B)3-luc, or promoterless expression vector was cotransfected with 0.5 µg of pME18S-FLAG-HDAC2, pBJ-HDAC1, pBJ-HDAC4, pBJ-HDAC5, or pBJ-HDAC6 or insertless expression vector along with 0.02 µg pRL-SV40. Twenty-four hours after transfection, complete medium was added with vehicle, IL-1, or LPS + IFN-. Twenty-four hours later, cell lysates were prepared and firefly and Renilla luciferase activities in 100 µl of lysate samples were measured as described previously in our laboratory (11). In some experiments, as indicated in the text and figure legends, trichostatin A (TSA; 200 nM) was added after transfection for 24 h before cell lysates were prepared. This concentration of TSA was chosen on the basis of its effectiveness in inhibiting HDAC activity in numerous published studies (20,21,24).
Preparation of Nuclear Extracts
Nuclear extracts were prepared from time-paired vehicle-, IL-1-, or LPS + IFN-–treated mesangial cells as detailed in our earlier work (11,26).
Electrophoretic Mobility Shift Assays (EMSA)
NF-B element double-stranded oligonucleotides from the murine iNOS promoter for use as probes and for competition studies (sense strand shown; consensus binding element italicized; "u" and "d" represent "upstream" and "downstream") were: NF-Bu - 978 5'-TGCTAGGGGGATTTTCCCTCTCTC-3' -955; NF-Bd -92 5'-CCAACTGGGGACTCTCCCTTTGGG-3' -69. The probes were end-labeled with [32P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase. Binding reactions were performed in 20 µl of solution for 30 min at room temperature by incubating 10 µg of nuclear extract protein with duplex DNA probe (
2 x 105 cpm) in reaction buffer (13 mM HEPES, pH 7.9, 65 mM NaCl, 0.14 mM EDTA, 1 mM MgCl2, 1mM dithiothreitol, 8% glycerol, and 50 µg/ml poly[dl-dC]). For supershift assays, antibodies (2 µg) specific for NF-B p65 or p50 were added to the binding reaction and incubated on ice for 10 min before the addition of labeled probe. Aliquots of the reactions were resolved on 5% native polyacrylamide gels in 0.5x Tris borate-EDTA buffer. The gels were dried and exposed to x-ray film with an enhancing screen at -70°C to detect the DNA-protein and DNA-protein-antibody complexes. Experiments were replicated a minimum of three times, as indicated in the figure legends.
Immunoprecipitation and Western Blot Analyses
Stimulated mesangial cells were harvested and lysed in nuclear extract buffer (20 mM Tris-HCl, pH 8.0, 1.5 mM MgCl2, 0.2 mM EDTA, 25% Glycerol, 0.5 mM PMSF). These lysates were then precleared by incubating with 20 µl/ml protein A/G agarose beads (Santa Cruz) for 1 h at 4°C. After brief centrifugation, the supernatant was added to anti-HDAC2 antibody (0.2 µg/ml) or control IgG in nuclear extract buffer overnight at 4°C, followed by the addition of 20 µl of protein A/G agarose beads and incubation for 1 h at 4°C. Immunoprecipitates were washed four times in nuclear extract buffer, resuspended in sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, and analyzed on 8% SDS-PAGE gels. The proteins were electrophorectically transferred to PVDF membranes (Hybond ECL, Amersham) and probed with an anti–NF-B p65 antibody (0.2 µg/ml) overnight at 4°C as indicated in the Results section and figure legends. The blots were washed extensively with a solution containing 50 mM Tris, pH 8.0, 138 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20. The antigen-antibody complexes were detected by the ECL protocol using horseradish peroxidase–conjugated donkey anti-rabbit IgG as secondary antibody.
In Vitro Translation and GST Pull-Down Assay
A cDNA encoding HDAC2 with an upstream T7 promoter sequence was generated by PCR using the HDAC2 expression plasmid pME18S-FLAG-HDAC2 as a template. Using this cDNA, HDAC2 was transcribed and translated from the presence of [35S]methionine using the TNT T7 Quick for PCR DNA kit (Promega) according to the manufacturer’s protocol. A GST-fusion protein constructed to contain full-length NF-B p65 was purified from sonicates of isopropyl -D-thiogalactoside–induced DH5 bacterial cells according to the manufacturer’s instructions (Amersham Pharmacia Biotech) and incubated with 50 µl of glutathione-Sepharose 4B beads for 1 h at 4°C. After centrifugation, the pellets was collected and resuspended in lysis buffer (PBS containing protease inhibitor cocktail). For in vitro binding reaction, 20 µl of purified GST or GST-NF-B p65 was incubated in protein binding buffer (20 mM Tris, pH 8.0, 150 mM KCl, 1 mM EDTA, 4 mM MgCl2, 0.2% NP-40, 10% glycerol) with 10 µl of [35S]methionine-labeled full-length HDAC2 translation product at 4°C overnight. The samples were then washed four times in binding buffer, boiled in SDS sample buffer, and analyzed by SDS-PAGE gel and autography.
Statistical Analyses
Quantitative data are presented as mean ± SEM and were analyzed by ANOVA. Significance was assigned at P < 0.05.
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Results |
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or LPS + IFN- or LPS + IFN- was measured in the presence of vehicle or TSA, a potent and specific HDAC inhibitor. iNOS is the only NOS isoform appreciably expressed in these cell types and induced by these stimuli (reference 27 and unpublished observations). IL-1 stimulated mesangial cells that had been treated with TSA exhibited a 79% lower nitrite levels compared with IL-1–stimulated controls, whereas LPS + IFN-–treated mesangial cells exposed to TSA exhibited nearly 72% lower levels of nitrite production compared with LPS + IFN-–treated controls (Figure 1A). Similarly, in RAW 264.7 cells, TSA inhibited LPS + IFN-–stimulated nitrite production by 50% (Figure 1B).Thus, TSA exerts prominent effects on cytokine induction of NO production by the endogenous iNOS gene.
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, or LPS + IFN-, and the induction of iNOS reporter gene activity was measured. As seen in Figure 2A, mesangial cells treated with IL-1 or LPS + IFN- in the absence of TSA exhibited the expected induction of iNOS promoter-luciferase activity. In contrast, mesangial cells treated with 200 nM TSA exhibited a 35% reduction in the fold-induction of iNOS promoter activity in response to IL-1 or LPS + IFN-. Similarly, in RAW 264.7 cells, TSA inhibited by 40% iNOS promoter activity stimulated by LPS + IFN- (Figure 2B). These data suggest that hyperacetylation decreases cytokine induction of iNOS promoter activity in mesangial cells and macrophages.
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or LPS + IFN- or LPS + IFN- (Figure 3A). HDAC1 had no discernible effect on iNOS promoter activation, whereas HDAC2 promoted a 35% superinduction and HDAC6 a 30% superinduction of promoter activity in response to IL-1 and LPS + IFN-. HDAC4 overexpression resulted in negligible effects in response to IL-1, but it exerted a 70% increase in iNOS promoter activity over controls in response to LPS + IFN-. HDAC5 was similarly more potent in superinducing the iNOS promoter in response to LPS + IFN- compared with IL-1. In RAW 264.7 cells, overexpression of HDAC1, -5, and -6, but not HDAC2 or HDAC4, significantly increased iNOS promoter activity (Figure 3B). Thus there are clear, cell type-specific differences between mesangial cells and RAW 264.7 cells in HDAC isoform action on iNOS gene transcription.
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B, a known stimulator of iNOS gene transcription. Transfection of increasing amounts of HDAC2 into mesangial cells resulted in a threshold effect with increased IL-1 or LPS + IFN-–stimulated iNOS promoter activity evident beginning at 0.5 µg of transfected DNA (Figure 4).
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B–Responsive Promoter in Response to IL-1B. This transcription factor is known to interact with HDAC1, -2, and -3 (21,28,29) and to be influenced functionally by changes in acetylation (21). We accordingly hypothesized that effects on NF-B or its interaction with cognate binding elements might mediate the reciprocal effects of TSA treatment and HDAC overexpression to modulate iNOS promoter activity. Specifically, augmented activation of NF-B or its coregulatory proteins might lead to more prominent iNOS gene activation. The effects of TSA treatment and of HDAC2 overexpression on the activity of an NF-B promoter were accordingly examined in transfection experiments in mesangial cells. As shown in Figure 5A, TSA caused a 60% decrease in IL-1–stimulated NF-B promoter activity compared with vehicle-treated controls. In accordance with these findings, overexpression of HDAC2 augmented induction of the NF-B promoter by nearly 40% in response to IL-1 (Figure 5B). These results indicate that acetylation alters the functional activity of NF-B.
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B-dependent DNA-protein complexes under basal conditions in nuclear extracts harvested from mesangial cells (Figure 6). The higher molecular weight complex was markedly upregulated after IL-1 treatment. Supershift/antibody competition assays showed that anti-NF-B p65 greatly diminished the intensity of this higher molecular weight complex and produced a supershift of the complex, whereas anti-p50 outcompeted the lower molecular weight complex (Figure 6). These data are consistent with the basal expression of NF-B p50/p50 homodimers and the inducible expression of NF-B p65/p65 homodimers. Comparable results were found when either the NF-Bu or NF-Bd probes were used. TSA treatment of thecells did not significantly affect NF-B DNA binding activity in nuclear extracts prepared from stimulated mesangial cells (Figure 6). In the aggregate, these results suggested that acetylation status influences the transactivation potential of NF-B, rather than its DNA binding properties, in its interaction with NF-B responsive promoters.
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B p65 Interact In Vitro and in Mesangial Cells In Vivo and nuclear extracts were prepared for coimmunoprecipitation assays. Co-immunoprecipitations were performed with an anti-HDAC2 antibody, and proteins present in the immunoprecipitates were revealed by immunoblotting with anti-NF-B p65 antibodies. As seen in Figure 7A, HDAC2 and NF-B p65 coimmunoprecipitated from cells treated with IL-1 but not untreated cells. The lack of co-immunoprecipitation in the untreated cells (lane 1) serves as a negative control. Treatment of the cells with TSA did not appreciably alter the abundance of the co-immunoprecipitated proteins from the IL-1–treated cells (Figure 7A). Importantly, the two endogenous proteins co-immunoprecipitated, and overexpression was not required. In agreement with these findings, radiolabeled HDAC2 transcribed and translated in vitro was specifically retained by a GST-NF-B p65 fusion protein but not by GST alone (Figure 7B).
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Discussion |
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B p65 and lead to accentuation of iNOS promoter activation in response to proinflammatory stimuli. We show that deacetylase inhibition with TSA inhibits cytokine induction of endogenous NO production, which is mediated by iNOS, and the activity of an iNOS promoter construct (Figures 1 and 2), as well as a heterologous promoter containing a three-repeat palindrome of NF-B elements (Figure 5A). These effects occur without a change in NF-B binding activity (Figure 6), suggesting that the transcription factor’s transactivation potential or interactions with other coregulatory proteins is changed. In agreement with the inhibitor data, overexpression of HDAC2, -4, -5, and -6 but not HDAC1 (no other HDAC isoforms were tested) resulted in superinduction of the activity of an iNOS promoter construct (Figure 3) and the NF-B promoter construct (Figure 5B).Finally, we demonstrate for the first time that HDAC2 and NF-B p65 directly interact in activated mesangial cells in vivo by co-immunoprecipitation and in vitro by GST pull-down assays (Figure 7). In the aggregate, the data suggest that changes in cellular acetylation status alter NF-B signaling and the transcriptional response of the iNOS gene.
The binding of NF-B to HDAC2 presumably reflects the recruitment of HDAC2 to specific NF-B target promoters. The domains of HDAC2 and NF-B that interact remain to be identified. It is unlikely that this interaction involves the DNA binding domain of NF-B, because TSA treatment did not significantly interfere with DNA binding (Figure 6). This result differs from studies of HIV-1 viral protein Tat in which acetylated NF-B p50 and p50/p65 bound with higher affinity to DNA to effect increased rates of transcription (30). Moreover, TSA treatment did not appreciably alter the abundance of the co-immunoprecipitated HDAC2 and NF-B p65 proteins from the IL-1–treated mesangial cells (Figure 7A); the acetylation status of NF-B p65 itself does not therefore appear to be required for, or to substantially influence, the protein-protein interaction. The fact that recombinant NF-B p65 and in vitro-translated HDAC2 interacted in vitro supports this conclusion. However, it remains possible that acetylation alters the transactivation function of NF-B p65. For example, phosphorylation by oncogenic forms of H-Ras (31) and the serine/threonine kinase Akt (32) have been shown to stimulate the transactivation function of NF-B p65. This potentially results from modulation of the interaction of NF-B p65 with coactivator and corepressors proteins. The transcriptional activation we observed with deacetylation might be related to enhanced binding of CBP or other transcriptional coactivators to NF-B p65 or to evictionof corepressors from this factor. The interaction also appears to be direct, given the ability of in vitro-translated HDAC2 to interact stably with GST-NF-B p65 in the absence of other accessory molecules (Figure 7B). We did not test the effects of acetylation status on other transacting factors important for iNOS induction, but it is possible that specific factors might be regulated by acetylation and thereby alter iNOS induction. One obvious candidate would be C/EBP, expression of which in intestinal epithelial cells is downregulated by TSA (19) and activity of which is important for iNOS induction in mesangial cells (33) and macrophages (26).
Acetylation has been shown to regulate many transcription factors other than NF-B, such as p53 (34), E1A (35), GATA-1 (36), and MyoD (37). Acetylation of transcription factors alters their function in several ways. For example, acetylation promotes protein-protein interactions between Rch1 and importin- (38), affects the conformation of HNF-4 (39), and alters the half-life of E2F (40). Only a few transcription factors have been found to interact with HDAC without intermediate coregulatory molecules. Retinoblastoma protein interacts directly with HDAC1 (41), and SP1 also interacts with HDAC2 (42). YY1-induced transcriptional repression is the result of direct interaction with HDAC2 (43). HDAC1 and HDAC2 also interact directly with DNA topoisomerase II to modify topoisomerase activity (44). Myocyte enhancer factor 2 (MEF2) was shown to interact directly with HDAC4 and HDAC5 (45). In addition, GATA-2 couples with HDAC3 and HDAC5 (46). Our finding of a direct interaction between HDAC2 and NF-B p65 adds to this list. The fact that the reciprocal effects of HDAC inhibition and overexpression also influenced the activation of the NF-B–responsive minimal promoter suggests that other NF-B–responsive genes may be influenced by these mechanisms in mesangial cells. It is intriguing to note that the specific HDAC isoforms that activated the iNOS promoter in mesangial cells differed to an extent from those in macrophages, suggesting that additional cell-specific mechanisms influence acetylation or the interaction of the HDACs with NF-B p65.
A major question that arises from these results is why acetylation influences the function of NF-B on target promoters in an opposite manner depending on the cell type and promoter context. In most examples, including previous studies of NF-B in other cell types, acetylation activates, rather than inhibits, gene transcription. Specifically, HDAC1 and -2 have been shown to inhibit NF-B–dependent gene activation (21,28). A recent study of the MMTV promoter suggests that these opposite effects may result from the effects of acetylation on specific combinatorial interactions required for efficient transcription of individual genes. TSA treatment inhibited transcription of the MMTV promoter by mechanisms that "evicted" the transcription factor NF-1 from the promoter. In addition, TSA downregulated chromatin remodeling proteins and coregulatory molecules known to participate in the activation of the promoter—effects that occurred in the absence of histone acetylation of the local promoter chromatin structure (47). The effects ofmanipulated acetylation were greater on the induction of the iNOS promoter than on the heterologous promoter containing a three-repeat palindrome of NF-B elements. TSA treatment of mesangial cells inhibited cytokine induction of the iNOS promoter activity by 65% (Figure 2A) but inhibited the NF-B promoter by only about 40% (Figure 5A). In agreement with this disparity, HDAC2 overexpression augmented induction of iNOS promoter activity by roughly 65% (Figure 4) but produced only a 40% augmentation of induced NF-B promoter activity (Figure 5B). These results suggest two possibilities: either the local promoter context influences differences in acetylation between the NF-B elements in the iNOS promoter and those concatamerized in the heterologous NF-B promoter, or acetylation is altering other transcription factors that influence induction of the iNOS promoter but that cannot bind to the heterologous NF-B promoter.
The ability of acetylation status to influence the activity of the iNOS gene in mesangial cells and macrophages lends further complexity to the intricate regulation of this gene. Given the potent effects of high-output NO during inflammation and injury in the glomerulus, this multilevel control may facilitate the tight control of NO production. NO is a key mediator in inflammation, eliciting both proinflammatory and antiinflammatory effects. NO promotes vasodilation and functions in nonspecific immune responses. It alters the function of key enzymes and proinflammatory genes. However, NO also contributes to the resolution of inflammation by inhibiting the activity and expression of iNOS, cell adhesion molecules, cytokines, and chemokines (3). In mesangial cells, NO has been shown to inhibit NF-B activity by augmenting IB levels and to limit iNOS and COX-2 expression in late phases of cytokine activation. The interaction of HDAC isoforms and NF-B proteins to alter acetylation reported here provides the potential to fine-tune the expression of iNOS and other downstream target genes contributing to inflammatory responses.
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Acknowledgments |
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References |
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