Differential effects of histone deacetylase inhibitors on interleukin-18 gene expression in myeloid cells

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression.     

HDAC inhibition upregulates the expression of angiostatic ADAMTS1

HDAC inhibitors are promising anticancer agents that induce cell cycle arrest and apoptosis. However, the role of HDACs in cancer progression, such as angiogenesis and metastasis, remains largely unexplored. Among various HDAC inhibitors, we demonstrate that TSA and SAHA upregulated the expression of angiostatic ADAMTS1 in A549 cells. HDAC6 inhibitor tubacin, and knockdown of HDAC6, also lead to ADAMTS1 upregulation. By reporter, DAPA, and ChIP assays, the proximal GC boxes were demonstrated to be essential for ADAMTS1 induction. Decreased binding of SP1 and HDAC6 to the ADAMTS1 promoter after TSA treatment was also seen. These data suggest the involvement of HDAC6 and SP1 in the HDACi-induced expression of angiostatic ADAMTS1.     

CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1

CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1.     

CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1

CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1.     

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E₂ without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.     

Analysis of HDAC1-mediated regulation of Runx2-induced osteopontin gene expression in C3h10t1/2 cells

Histone deacetylases (HDACs) deacetylate lysine residues of histone and non-histone proteins and thereby regulate the cell-cycle, gene expression, and several other processes. We have analyzed the effects of HDAC1 on Runx2-mediated regulation of osteopontin (OPN) promoter activation and gene expression in mesenchymal progenitor C3h10t1/2 cells and show that co-expression of HDAC1 along with Runx2 results in down-regulation of Runx2-induced OPN mRNA expression during both the proliferation and differentiation stages of C3h10t1/2 cells. Luciferase assay results revealed that HDAC1 efficiently down-regulated Runx2-stimulated OPN promoter activity in a dose-dependent manner whereas TSA relieved the HDAC1-mediated repression and up-regulated the Runx2-induced OPN promoter activity and mRNA expression. In vivo HDAC1 co-localized and physically interacted with Runx2 and associated with the OPN promoter. Thus, HDAC1 not only plays a critical role in regulation of Runx2-stimulated expression of osteogenic genes, like OPN, but also regulate the proliferation and differentiation stages of mesenchymal progenitor cells, such as C3h10t1/2.     

The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression

Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes.