Histone acetyltransferase PCAF regulates inflammatory molecules in the development of renal injury

Kidney diseases, including chronic kidney disease (CKD) and acute kidney injury (AKI), are associated with inflammation. The mechanism that regulates inflammation in these renal injuries remains unclear. Here, we demonstrated that p300/CBP-associated factor (PCAF), a histone acetyltransferase, was overexpressed in the kidneys of db/db mice and lipopolysaccharide (LPS)-injected mice. Moreover, elevated histone acetylation, such as H3K18ac, and up-regulation of some inflammatory genes, such as ICAM-1, VCAM-1, and MCP-1, were found upon these renal injuries. Furthermore, increased H3K18ac was recruited to the promoters of ICAM-1, VCAM-1, and MCP-1 in the kidneys of LPS-injected mice. In vitro studies demonstrated that PCAF knockdown in human renal proximal tubule epithelial cells (HK-2) led to downregulation of inflammatory molecules, including VCAM-1, ICAM-1, p50 subunit of NF-κB (p50), and MCP-1 mRNA and protein levels, together with significantly decreased H3K18ac level. Consistent with these, overexpression of PCAF enhanced the expression of inflammatory molecules. Furthermore, PCAF deficiency reduced palmitate-induced recruitment of H3K18ac on the promoters of ICAM-1 and MCP-1, as well as inhibited palmitate-induced upregulation of these inflammatory molecules. In summary, the present work demonstrates that PCAF plays an essential role in the regulation of inflammatory molecules through H3K18ac, which provides a potential therapeutic target for inflammation-related renal diseases.     

The role of H3K4me3 and H3K9/14ac in the induction by dexamethasone of Per1 and Sgk1, two glucococorticoid early response genes that mediate the effects of acute stress in mammals

Glucocorticoids are known to induce or repress the expression of a wide variety of genes with roles in various biological processes such as the circadian clock and the stress response. We studied the changes in the levels of two histone H3 post-translational modifications associated with active chromatin, H3 trimethylated at lysine 4 (H3K4me3) and H3 acetylated at lysines 9/14 (H3K9/14ac), that take place in the promoters of two glucocorticoid early response genes, Per1 and Sgk1, during their induction by the synthetic glucocorticoid, dexamethasone. Sgk1 mediates the effects of acute and chronic stress on the prefrontal cortex and other parts of the brain, while Per1 is a core circadian clock gene whose expression is strongly induced by the increased levels of blood-borne glucocorticoids that accompany acute and chronic stress. Here we show that dexamethasone rapidly increases the levels of H3K4me3 and H3K9/14ac in the promoters of both genes. Furthermore, the effect of dexamethasone on these genes, regarding both mRNA levels and the abundance of H3K4me3 and H3K9/14ac in their promoters, can be inhibited by the presence of nicotinamide, a metabolic molecule which has been shown to possess anxiolytic properties.     

Adverse Effects of High Concentrations of Fluoride on Characteristics of the Ovary and Mature Oocyte of Mouse

Reproductive toxicity has been an exciting topic of research in reproductive biology in recent years. Soluble fluoride salts are toxic at high concentrations; their reproductive toxicity was assessed in this study by administering different fluoride salt concentrations to mice. Continuous feeding for five weeks resulted in damage to the histological architecture of ovaries. The expression of genes, including Dazl, Stra8, Nobox, Sohlh1, and ZP3 gene, associated with oocyte formation were much lower in the experimental group as compared with the control group. The number of in vitro fertilization of mature oocytes were also much lower in the experimental group as compared with control. Moreover, the fertility of female mice, as assessed by mating with normal male mice, was also lower in experimental compared with control groups. The expression of the oocyte-specific genes: Bmp15, Gdf9, H1oo, and ZP2, which are involved in oocyte growth and the induction of the acrosome reaction, decreased with the fluoride administration. DNA methylation and histone acetylation (H3K18ac and H3K9ac) are indispensable for germline development and genomic imprinting in mammals, and fluoride administration resulted in reduced levels of H3K9ac and H3K18ac in the experimental group as compared with the control group, as detected by immunostaining. Our results indicate that the administration of high concentrations of fluoride to female mice significantly reduced the number of mature oocytes and hampered their development and fertilization. Thus, this study lays a foundation for future studies on fluoride-induced reproductive disorders in women.     

Inhibition of Histone H3K9 Acetylation by Anacardic Acid Can Correct the Over-Expression of Gata4 in the Hearts of Fetal Mice Exposed to Alcohol during Pregnancy

Background

Cardiovascular malformations can be caused by abnormalities in Gata4 expression during fetal development. In a previous study, we demonstrated that ethanol exposure could lead to histone hyperacetylation and Gata4 over-expression in fetal mouse hearts. However, the potential mechanisms of histone hyperacetylation and Gata4 over-expression induced by ethanol remain unclear.

Methods and Results

Pregnant mice were gavaged with ethanol or saline. Fetal mouse hearts were collected for analysis. The results of ethanol fed groups showed that global HAT activity was unusually high in the hearts of fetal mice while global HDAC activity remained unchanged. Binding of P300, CBP, PCAF, SRC1, but not GCN5, were increased on the Gata4 promoter relative to the saline treated group. Increased acetylation of H3K9 and increased mRNA expression of Gata4, α-MHC, cTnT were observed in these hearts. Treatment with the pan-histone acetylase inhibitor, anacardic acid, reduced the binding of P300, PCAF to the Gata4 promoter and reversed H3K9 hyperacetylation in the presence of ethanol. Interestingly, anacardic acid attenuated over-expression of Gata4, α-MHC and cTnT in fetal mouse hearts exposed to ethanol.

Conclusions

Our results suggest that P300 and PCAF may be critical regulatory factors that mediate Gata4 over-expression induced by ethanol exposure. Alternatively, P300, PCAF and Gata4 may coordinate over-expression of cardiac downstream genes in mouse hearts exposed to ethanol. Anacardic acid may thus protect against ethanol-induced Gata4, α-MHC, cTnT over-expression by inhibiting the binding of P300 and PCAF to the promoter region of these genes.     

A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance

In fungal species, lysine 56 of newly synthesized histone H3 molecules is modified by the acetyltransferase Rtt109, which promotes resistance to genotoxic agents. To further explore how H3 K56ac contributes to genome stability, we conducted screens for suppressors of the DNA damage sensitivity of budding yeast rtt109Δ mutants. We recovered a single extragenic suppressor mutation that efficiently restored damage resistance. The suppressor is a point mutation in the histone H3 gene HHT2, and converts lysine 56 to glutamic acid. In some ways, K56E mimics K56ac, because it suppresses other mutations that interfere with the production of H3 K56ac and restores histone binding to chromatin assembly proteins CAF-1 and Rtt106. Therefore, we demonstrate that enhanced association with chromatin assembly factors can be accomplished not only by acetylation-mediated charge neutralization of H3K56 but also by the replacement of the positively charged lysine with an acidic residue. These data suggest that removal of the positive charge on lysine 56 is the functionally important consequence of H3K56 acetylation. Additionally, the suppressive function of K56E requires the presence of a second H3 allele, because K56E impairs growth when it is the sole source of histones, even more so than does constitutive H3K56 acetylation. Our studies therefore emphasize how H3 K56ac not only promotes chromatin assembly but also leads to chromosomal malfunction if not removed following histone deposition.     

Saturated fatty acid palmitate induces extracellular release of histone H3: A possible mechanistic basis for high-fat diet-induced inflammation and thrombosis

Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells toexpress the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis.     

Expression of adhesion molecules in lungs of mice infected with Paracoccidioides brasiliensis conidia

In infected tissues, leukocyte recruitment is mediated by interactions between adhesion molecules, expressed on activated vascular endothelial cells, and ligands present on circulating cells. We evaluated the inflammatory response and the expression of cellular adhesion molecules (ICAM-1, VCAM-1, CD18, LFA-1 and Mac-1) in lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia. When compared with uninfected animals, infected mice had a significant increase in the inflammatory response during the first 4 days, peaking 2-3 days post-challenge, 40.3% vs. 0.0% and 41.8% vs. 0.7%, respectively. This inflammatory infiltrate was composed mainly of neutrophils and macrophages with a few eosinophils and lymphocytes. An increase in the intensity of immunofluorescence (IF) for ICAM-1 was also observed during days 1-4. ICAM-1 was present in bronchiolar epithelium, type II pneumocytes, and macrophages, as well as on vascular endothelium. The control animals presented ICAM-1 constitutively. In infected mice, VCAM-1 was only observed on vascular endothelium during the first 2 days, with some macrophages expressing this molecule throughout the study periods. CD18 and Mac-1 but not LFA-1 were expressed with a high intensity on neutrophils and macrophages present in the inflammatory infiltrate. In addition, we observed a significant decrease in Colony forming units (CFUs) after the first 2 days post-challenge. These findings suggest that during these early stages, up-regulation of ICAM-1, VCAM-1, CD18 and Mac-1 expression occurs, participating in the inflammatory process and as such, in the pathogenesis of paracoccidioidomycosis (PCM).     

Effects of Saskatoon berry powder on monocyte adhesion to vascular wall of leptin receptor-deficient diabetic mice

Hypothesis:Atherosclerotic cardiovascular complications are the leading cause of death in diabetic patients. Monocyte adhesion is an early event for atherogenesis. Previous studies demonstrated that dark-skin berries had cardiovascular protective effects. We hypothesize that Saskatoon berry (SB) powder may reduce monocyte adhesion in leptin receptor-deficient (db/db) diabetic mice.MethodsWild-type and db/db mice were fed with chow or supplemented with SB powder. Anthocyanins in SB powder were identified using mass spectrometry. Mouse monocytes were incubated with mouse aorta. Monocyte adhesion was counted under microscopy. Inflammatory or metabolic markers in blood or tissue were analyzed using immunological or biochemical methods.ResultsSB powder significantly reduced monocyte adhesion to aorta from diabetic db/db mice compared to regular chow. The increased monocyte adhesion to aorta was normalized in db/db mice treated with ≥5% of SB powder for 4 weeks. Increased contents of Nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase-4, heat shock factor-1, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, P-selectin, tumor necrosis factor-α, plasminogen activator inhibitor (PAI)-1 and urokinase plasminogen activator in aorta or heart apex, elevated plasma PAI-1 and MCP-1 were detected in db/db mice on chow compared to wild-type mice on the same diet; 5% SB powder inhibited the increases of inflammatory, fibrinolytic or stress regulators in aorta or heart apex of db/db mice. Monocyte adhesion positively correlated with blood glucose, cholesterol, body weight, heart MCP-1, PAI-1 or ICAM-1.ConclusionThe findings suggest that SB powder attenuated monocyte adhesion to aorta of db/db mice, which was potentially mediated through inhibiting the inflammatory, stress and/or fibrinolyic regulators.     

Characterisation of cellular infiltration and adhesion molecule expression in CBA/CaH-kdkd mice with tubulointerstitial renal disease

 CBA/CaH-kdkd mice develop a spontaneous and chronic tubulointerstitial renal disease which is characterised by mononuclear cell infiltration, tubular collapse and cystic dilatation of tubules. The pathogenic mechanisms of renal injury have not been fully elucidated in this model. We have analysed the nature of infiltrating cells and the expression of MHC class II antigens, cytokines and adhesion molecules in CBA/CaH-kdkd kidneys at various disease stages. Using immunohistochemical techniques we found that kdkd kidneys are characterised by abundant macrophage and dendritic cell infiltration with fewer T cells with CD4+ and CD8+ phenotypes. Interestingly, MHC class II antigens were not induced on renal tubules. The proinflammatory cytokine, TNF-α, was markedly enhanced in kdkd kidney (up to fourfold), whereas the T cell-specific cytokine, IFN-γ, increased less (less than twofold). ICAM-1 and VCAM-1 were markedly overexpressed by injured proximal tubules. ICAM-2 and PECAM-1 were constitutively expressed on glomerular capillaries and vascular endothelium in normal kidneys and did not change in CBA/CaH-kdkd mice. In conclusion, tubulointerstitial nephritis in CBA/CaH-kdkd mice is characterised by prominent macrophage infiltration and abundant expression of ICAM-1 and VCAM-1 on injured renal tubules. The lack of MHC class II antigens on injured tubules suggests that the kd gene defect could generate a secondary renal inflammatory response which is characterised by prominent macrophage infiltration and a relative scarcity of T cells. Accepted: 10 June 1997