Class II and IV HDACs function as inhibitors of osteoclast differentiation

<div><p>Histone deacetylases (HDACs) are negative regulators of transcription and have been shown to regulate specific changes in gene expression. In vertebrates, eighteen HDACs have thus far been identified and subdivided into four classes (I-IV). Key roles for several HDACs in bone development and biology have been elucidated through <i>in vitro</i> and <i>in vivo</i> models. By comparison, there is a paucity of data on the roles of individual HDACs in osteoclast formation and function. In this study, we investigated the gene expression patterns and the effects of suppressing individual class II (<i>Hdac4</i>, <i>5</i>, <i>6</i>, <i>9</i>, and <i>10</i>) and class IV (<i>Hdac11</i>) HDACs during osteoclast differentiation. We demonstrated that HDAC class II and IV members are differentially expressed during osteoclast differentiation. Additionally, individual shRNA-mediated suppression of <i>Hdac4</i>, <i>5</i>, <i>9</i>, <i>10</i> and <i>11</i> expression resulted in increased multinucleated osteoclast size and demineralization activity, with little to no change in the overall number of multinucleated osteoclasts formed compared with control shRNA-treated cells. We also detected increased expression of genes highly expressed in osteoclasts, including <i>c-Fos</i>, <i>Nfatc1</i>, <i>Dc-stamp</i> and <i>Cathepsin K</i>. These observations indicate that HDACs cooperatively regulate shared targets in a non-redundant manner.</p></div

Suppression of <i>Hdac5</i> enhances osteoclast differentiation.

<p>Representative images of TRAP staining (A) of osteoclast cultures. <i>Hdac5</i>#1 represents <i>Hdac5</i> shRNA #1and <i>Hdac5</i>#2 represents <i>Hdac5</i> shRNA #2. Quantification of the number (B) and average size of TRAP-stained multinucleated osteoclasts (C). Representative photographs (D) and quantification (E) of demineralization activity of control and <i>Hdac5</i> shRNA-expressing osteoclast cultures grown on calcium phosphate-coated plates. Scale bar represents 200 μm. Western blot (F) of control and <i>Hdac5</i> shRNA-expressing cells with relative expression of shRNA expressing cells relative to control expressing cells indicated under the blots. Expression profile (G-J) of osteoclast genes <i>c-Fos</i>, <i>Nfatc1</i>, <i>Dc-stamp and Ctsk</i>. Data presented are the mean of three independent experiments. * p <0.05; ** p <0.01; *** p < 0.001; ns = not significant compared to control infected cells.</p

Expression pattern of class II and IV HDACs during osteoclast differentiation.

<p>Illustration of the stages of osteoclast differentiation with HDAC expression as determined by the western blots presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185441#pone.0185441.g001" target="_blank">Fig 1</a>.</p

<i>Hdac6</i> suppression does not affect osteoclast differentiation.

<p>Representative images of TRAP staining (A) of osteoclast cultures. <i>Hdac6</i>#1 represents <i>Hdac6</i> shRNA #1and <i>Hdac6</i>#2 represents <i>Hdac6</i> shRNA #2. Quantification of (B) number and (C) size of TRAP-positive MNCs. Representative photographs (D) and quantification (E) of demineralization activity of control and <i>Hdac6</i> shRNA-expressing osteoclast cultures grown on calcium phosphate-coated plates. Scale bar represents 200 μm. qRT-PCR (F) of control and <i>Hdac6</i> shRNA-expressing cells. Expression profile (G-J) of <i>c-Fos</i>, <i>Nfatc1</i>, <i>Dc-stamp</i>, <i>and Ctsk</i>. Data presented are the mean of three independent experiments. *** p < 0.001; ns = not significant compared to control infected cells.</p

<i>Hdac10</i> suppression accelerates osteoclast differentiation.

<p>Representative images of TRAP staining (A) of osteoclast cultures. <i>Hdac10</i>#1 represents <i>Hdac10</i> shRNA #1and <i>Hdac10</i>#2 represents <i>Hdac10</i> shRNA #2. Quantification of number (B) and size (C) of TRAP-positive MNCs. Representative images (D) and quantification (E) of demineralization activity of control and <i>Hdac10</i> shRNA-expressing osteoclast cultures grow on calcium phosphate-coated plates. Scale bar represents 200 μm. qRT-PCR (F) of control and <i>Hdac10</i> shRNA-expressing cells. Expression profile (G, H, I and J) of <i>c-Fos</i>, <i>Nfatc1</i>, <i>Dc-stamp</i>, <i>and Ctsk</i>. Data presented are the mean of three independent experiments. * p < 0.05; *** p <0.001; ns = not significant compared to control infected cells.</p

Suppression of <i>Hdac9</i> inhibits osteoclast differentiation.

<p>Representative images of TRAP staining (A) of osteoclast cultures infected with control or <i>Hdac9</i> shRNA-expressing lentiviruses. <i>Hdac9</i>#1 represents <i>Hdac9</i> shRNA #1and <i>Hdac9</i>#2 represents <i>Hdac9</i> shRNA #2. Number (B) and size (C) of TRAP-positive MNCs. Representative photographs (D) and quantification (E) of demineralization activity of control and <i>Hdac9</i> shRNA-expressing osteoclast cultures grown on calcium phosphate-coated plates. Scale bar represents 200 μm. Western blot (F) of control and <i>Hdac9</i> shRNA-expressing cells with relative expression of shRNA expressing cells relative to control expressing cells indicated under the blots. Expression profile (G-J) of <i>c-Fos</i>, <i>Nfatc1</i>, <i>Dc-stamp and Ctsk</i>. Data presented are the mean of three independent experiments. * p <0.05; ** p <0.01; ns = not significant compared to control infected cells.</p