DNMT and HDAC inhibitors modulate MMP-9-dependent H3 N-terminal tail proteolysis and osteoclastogenesis

Abstract

Background: MMP-9-dependent proteolysis of histone H3 N-terminal tail (H3NT) is an important mechanism for activation of gene expression during osteoclast differentiation. Like other enzymes targeting their substrates within chromatin structure, MMP-9 enzymatic activity toward H3NT is tightly controlled by histone modifications such as H3K18 acetylation (H3K18ac) and H3K27 monomethylation (H3K27me1). Growing evidence indicates that DNA methylation is another epigenetic mechanism controlling osteoclastogenesis, but whether DNA methylation is also critical for regulating MMP-9-dependent H3NT proteolysis and gene expression remains unknown.

Results: We show here that treating RANKL-induced osteoclast progenitor (OCP) cells with the DNMT inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) induces CpG island hypomethylation and facilitates MMP-9 transcription. This increase in MMP-9 expression results in a significant enhancement of H3NT proteolysis and OCP cell differentiation. On the other hand, despite an increase in levels of H3K18ac, treatment with the HDAC inhibitor trichostatin A (TSA) leads to impairment of osteoclastogenic gene expression. Mechanistically, TSA treatment of OCP-induced cells stimulates H3K27ac with accompanying reduction in H3K27me1, which is a key modification to facilitate stable interaction of MMP-9 with nucleosomes for H3NT proteolysis. Moreover, hypomethylated osteoclastogenic genes in 5-Aza-CdR-treated cells remain transcriptionally inactive after TSA treatment, because H3K27 is highly acetylated and cannot be modified by G9a.

Conclusions: These findings clearly indicate that DNA methylation and histone modification are important mechanisms in regulating osteoclastogenic gene expression and that their inhibitors can be used as potential therapeutic tools for treating bone disorders.

Keywords: 5-Aza-dC; DNA methylation; H3 proteolysis; MMP-9; Osteoclast differentiation; TSA.

Fig. 1

Effects of 5-Aza-CdR treatment on osteoclastogenesis and MMP-9 expression. a Mock-depleted (upper and middle panels) or MMP-9-depleted (lower panel) OCP cells were incubated with RANKL in the absence (upper panel) or presence (middle and lower panels) of 5-Aza-dC for 0, 1, 3, or 5 days. Cells were fixed with formaldehyde, stained for TRAP and photographed under a light microscope (10 ×) (left). Representative images of osteoclasts are shown (Scale bar, 100 µm). TRAP-positive multinucleated cells [TRAP(+)MNCs] containing three or more nuclei and a full actin ring were counted as osteoclasts. b After treating OCP-induced cells as in a, the levels of MMP-9 expression were measured by qRT-PCR (upper panel) with primers in Methods section and Western blotting (lower panel) with MMP-9 antibody. Lamin B antibody was used as a loading control

Fig. 2

Effects of 5-Aza-CdR treatment on MMP-9 CpG methylation and H3NT proteolysis. a Mock-depleted or MMP-9-depleted OCP-induced cells were cultured with or without 5-Aza-dC for 0, 1, 3 and 5 days. Chromatin was extracted, and the extent of H3NT proteolysis was analyzed by Western blotting with H3 C-terminal tail antibody. H2A was used as a loading control. b The sequence of the MMP-9 CpG island locus (NCBI accession: NC_000068, region: 164950229 to 164950544) containing the 16 CpGs that are written in bold and underlined. c Genomic DNA was obtained from OCP-induced cells that were grown in the presence or absence of 5-Aza-CdR for 5 days. The purified DNA molecules were subjected to sodium bisulfite conversion reaction and then analyzed by methylation-specific PCR of the MMP-9 CpG island. M, methylated; U, unmethylated. d Bisulfite sequencing analysis of the MMP-9 CpG island locus using genomic DNA prepared as in c. White circles, unmethylated CpGs; black circles, methylated CpGs

Fig. 3

Effects of 5-Aza-CdR treatment on MMP-9 transactivation function. a Mock-depleted or MMP-9-depleted OCP-induced cells were cultured in the presence or absence of 5-Aza-CdR for 5 days and subjected to ChIPac/ChIP analysis using the antibodies indicated on the top. Precipitation efficiencies were determined for the promoter (P) and coding region (CR) of Nfatc1 (P-cleaved), Lif (CR-cleaved) and Xpr1 (P + CR-cleaved) genes by qPCR with primers used in our previous study [21, 22] and Methods section. b RT-qPCR assays were performed to determine fold changes in Nfatc1, Lif and Xpr1 expression in mock-depleted or MMP-9-depleted OCP-induced cells after 5 days of culture in the presence or absence of 5-Aza-CdR

Fig. 4

Effects of TSA treatment on osteoclastogenesis and H3K27me1. a Mock-depleted or MMP-9-depleted OCP-induced cells were grown with or without TSA for 0, 1, 3 and 5 days and analyzed by TRAP staining (left panel) and total counting (right panel). b Mock-depleted or MMP-9-depleted OCP-induced cells were cultured as in (a), and chromatin was extracted and analyzed by Western blotting with the antibodies indicated on the left

Fig. 5

Effects of TSA treatment on MMP-9 transactivation function. a Mock-depleted or MMP-9-depleted OCP-induced cells were cultured in the presence or absence of TSA for 5 days and analyzed by ChIPac/ChIP-qPCR. b Mock-depleted or MMP-9-depleted OCP-induced cells were treated with TSA as in a, and relative expression levels of Nfatc1, Lif and Xpr1 genes were determined by qRT-PCR

Fig. 6

Effects of TSA and 5-Aza-CdR co-treatment on osteoclastogenesis. a OCP-induced cells were treated with the combination of 5-Aza-CdR and TSA (lower panel) or DMSO control (upper panel) for 0, 1, 3 and 5 days and stained for TRAP. b OCP-induced cells were treated as in a, and changes in MMP-9 expression were analyzed by RT-qPCR (left panel) and Western blot (right panel). Chromatin was also isolated and analyzed by Western blotting to assess the effects of the combined treatment on H3NT proteolysis (right panel)

Fig. 7

Effects of TSA and 5-Aza-CdR co-treatment on MMP-9 transactivation function. After treating with the combination of 5-Aza-dC and TSA for 5 days, OCP-induced cells were subjected to ChIPac/ChIP-qPCR (a) to measure H3NT proteolysis levels and RT-qPCR (b) to quantitate Nfatc1, Lif and Xpr1 expression levels