HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation

Transforming growth factor-β (TGF-β) signaling plays a key role in excessive fibrosis. As a class IIa family histone deacetylase (HDAC), HDAC5 shows a close relationship with TGF-β signaling and fibrosis. However, the effect and regulatory mechanism of HDAC5 in hypertrophic scar (HS) formation remain elusive. We show that HDAC5 was overexpressed in HS tissues and depletion of HDAC5 attenuated HS formation in vivo and inhibited fibroblast activation in vitro. HDAC5 knockdown (KD) significantly downregulated TGF-β1 induced Smad2/3 phosphorylation and increased Smad7 expression. Meanwhile, Smad7 KD rescued the Smad2/3 phosphorylation downregulation and scar hyperplasia inhibition mediated by HDAC5 deficiency. Luciferase reporter assays and ChIP-qPCR assays revealed that HDAC5 interacts with myocyte enhancer factor 2A (MEF2A) suppressing MEF2A binding to the Smad7 promoter region, which results in Smad7 promoter activity repression. HDAC4/5 inhibitor, LMK235, significantly alleviated hypertrophic scar formation. Our study provides clues for the development of HDAC5 targeting strategies for the therapy or prophylaxis of fibrotic diseases.     

PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.     

Ten-eleven translocation 1 functions as a mediator of SOD3 expression in human lung cancer A549 cells

Superoxide dismutase (SOD) 3, one of the SOD isozymes, plays a pivotal role in extracellular redox homeostasis. The expression of SOD3 is regulated by epigenetics in human lung cancer A549 cells and human monocytic THP-1 cells; however, the molecular mechanisms governing SOD3 expression have not been elucidated in detail. Ten-eleven translocation (TET), a dioxygenase of 5-methylcytosine (5mC), plays a central role in DNA demethylation processes and induces target gene expression. In the present study, TET1 expression was abundant in U937 cells, but its expression was weakly expressed in A549 and THP-1 cells. These results are consistent with the expression pattern of SOD3 and its DNA methylation status in these cells. Moreover, above relationship was also observed in human breast cancer cells, human prostate cancer cells, and human skin fibroblasts. The overexpression of TET1-catalytic domain (TET1-CD) induced the expression of SOD3 in A549 cells, and this was accompanied by the direct binding of TET1-CD to the SOD3 promoter region. Furthermore, in TET1-CD-transfected A549 cells, the level of 5-hydroxymethylcytosine within that region was significantly increased, whereas the level of 5mC was decreased. The results of the present study demonstrate that TET1 might function as one of the key molecules in SOD3 expression through its 5mC hydroxylation in A549 cells.     

Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12

We have investigated the ability of dexamethasone to regulate interleukin-1beta (IL-1beta)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10(-10) M) repress IL-1beta-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10(-7) M) and IL-1beta (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1beta targeted K8 and K12. Low concentrations of dexamethasone (10(-10) M), which do not transactivate, repressed IL-1beta-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1beta-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1beta nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.     

Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC–MEF2 complexes

Histone deacetylase (HDAC) inhibitors are promising new epi‐drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II‐specific HDAC inhibitor MC1568, we separated class I‐ and class II‐dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4–HDAC3–MEF2D complex, and (iii) paradoxically, by inhibiting differentiation‐induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue‐selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2–HDAC complexes in a repressed state. Our results suggest that HDAC class II‐selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.     

Mycobacteria inhibition of IFN-gamma induced HLA-DR gene expression by up-regulating histone deacetylation at the promoter region in human THP-1 monocytic cells

Infection of macrophages with mycobacteria has been shown to inhibit the macrophage response to IFN-gamma. In the current study, we examined the effect of Mycobacteria avium, Mycobacteria tuberculosis, and TLR2 stimulation on IFN-gamma-induced gene expression in human PMA-differentiated THP-1 monocytic cells. Mycobacterial infection inhibited IFN-gamma-induced expression of HLA-DRalpha and HLA-DRbeta mRNA and partially inhibited CIITA expression but did not affect expression of IFN regulatory factor-1 mRNA. To determine whether inhibition of histone deacetylase (HDAC) activity could rescue HLA-DR gene expression, butyric acid and MS-275, inhibitors of HDAC activity, were added at the time of M. avium or M. tuberculosis infection or TLR2 stimulation. HDAC inhibition restored the ability of these cells to express HLA-DRalpha and HLA-DRbeta mRNA in response to IFN-gamma. Histone acetylation induced by IFN-gamma at the HLA-DRalpha promoter was repressed upon mycobacteria infection or TLR2 stimulation. HDAC gene expression was not affected by mycobacterial infection. However, mycobacterial infection or TLR2 stimulation up-regulated expression of mammalian Sin3A, a corepressor that is required for MHC class II repression by HDAC. Furthermore, we show that the mammalian Sin3A corepressor is associated with the HLA-DRalpha promoter in M. avium-infected THP-1 cells stimulated with IFN-gamma. Thus, mycobacterial infection of human THP-1 cells specifically inhibits HLA-DR gene expression by a novel pathway that involves HDAC complex formation at the HLA-DR promoter, resulting in histone deacetylation and gene silencing.     

Histone acetyltransferase p300 regulates the expression of human pituitary rumor transforming gene （hPTTG）

The human pituitary tumor transforming gene （hPTTG） serves as a marker for malignancy grading in several cancers, hPTTG is involved in multiple cellular pathways including cell transformation, apoptosis, DNA repair, genomic instability, mitotic control and angiogenesis induction. However, the molecular mechanisms underlying hPTTG regulation have not been fully explored. In this study, we found that overexpression of histone acetyltransferase （HAT） p300 upregulated hPTTG at the levels of promoter activity, mRNA and protein expression. Moreover, the HAT activity of p300 was critical for its regulatory function. Chromatin immunoprecipitation （CHIP） analysis revealed that overexpression of p300 elevated the level of histone H3 acetylation on the hPTTG promoter. Additionally, the NF-Y sites at the hPTTG promoter exhibited a synergistic effect on upregulation of hPTTG through interacting with p300. We also found that treatment of 293T cells with the histone deacetylase （HDAC） inhibitor Tfichostatin A （TSA） increased hPTTG promoter activity. Meanwhile, we provided evidence that HDAC3 decreased hPTTG promoter activity. These data implicate an important role of the histone acetylafion modification in the regulation of hPTTG.