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. 2010 Jan 22;285(13):9616–9626. doi: 10.1074/jbc.M109.094862

FIGURE 2.

FIGURE 2.

PTH decreases interaction of HDAC4 with Runx2 on the RD site of the MMP-13 promoter. A, UMR 106-01 cells were treated with control or PTH (10−8 m) for 30 and 120 min with or without preincubation with H89 (5 μm) for 30 min. After immunoprecipitation of the cross-linked lysates with HDAC4 antibody or with rabbit IgG as a negative control, the DNA was subjected to PCR with primers that amplify either the distal RD region or distal RD and proximal AP-1 sites of the rat MMP-13 promoter. Input DNA (1:100) is a positive control for the assay. **, p < 0.001 versus control; &&, p < 0.001 versus PTH 120 min; *, p < 0.03 versus control; &, p < 0.03 versus PTH 120 min; #, p < 0.05 versus PTH 30 min. B, UMR 106-01 cells were treated with PTH (10−8 m) for 0, 30, 60, and 120 min. After immunoprecipitation of the cross-linked lysates with anti-HDAC3, HDAC4, or HDAC6 antibodies or with rabbit IgG as a negative control, the DNA was subjected to PCR with primers that amplify the distal RD and proximal AP-1 sites of the rat MMP-13 promoter. Input DNA (1:100) is a positive control for the assay. ^, p < 0.01 versus 0 min. C, UMR 106-01 cells were treated with PTH (10−8 m) for 30 and 120 min. After immunoprecipitation of the cross-linked lysates with anti-Runx2 antibody or with rabbit IgG as a negative control, the DNA was subjected to PCR with primers that amplify either the distal RD region or distal RD and proximal AP-1 sites of the rat MMP-13 promoter. Input DNA (1:100) is a positive control for the assay. *, p < 0.03 versus control. D, DNA sequence of rat MMP-13 promoter (−210/+14). Distal runt domain site and proximal runt domain/activator protein-1 site are indicated as distal RD and RD/AP-1. Primer sequences used for ChIP assay are shown in bold and with arrow. The sequence of AP-1 mutation is indicated by underlining. E, agarose gel electrophoresis after sonication of chromatin. The chromatin length is 500–1000 bp (lane 1). F, UMR 106-01 cells were transfected with −148 rat MMP-13 promoter-Luc or −148 rat MMP-13 mutation (AP-1 mutation). After immunoprecipitation of the cross-linked lysates with anti-HDAC4 antibody or with rabbit IgG as a negative control, the DNA was subjected to PCR with specific primers that amplify distal RD and proximal AP-1 sites of exogenous rat MMP-13 promoter. Input DNA (1:100) is a positive control for the assay. %, p < 0.02 versus control.