Schematic representation of possible roles of CA IX in regulation of <i>Col10a1</i> expression in chondrocytes.

<p>Blue and red lines represent pathways already known and those indicated in this study, respectively. Arrows with equilateral-triangular heads and T-shaped bars show facilitation and suppression of the pathways, respectively. HIF-1α induces the expression of <i>Car9</i> mRNA and hence that of CA IX protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Kaluz1" target="_blank">[8]</a>. It is known that cAMP-dependent pathways inhibit hypertrophic differentiation of chondrocytes including the expression of <i>Col10a1 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Tintut1" target="_blank">[26]</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Chung1" target="_blank">[29]</a>. On the other hand, it is reported that HIF-2α, encoded by <i>Epas1</i> gene, transactivates the <i>Col10a1</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Saito1" target="_blank">[14]</a>. We propose that CA IX suppresses the expression of <i>Col10a1</i> mRNA partly via a cAMP-dependent manner based on the following observations. <i>Col10a1</i> expression was induced by introduction of <i>Car9</i> siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g003" target="_blank">Figure 3G</a>). The cAMP level was lowered by <i>Car9</i> siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8B</a>). Inhibition of PKA by H89 augmented the expression of <i>Col10a1</i> induced by <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8A</a>). In addition, inhibition of PDE by IBMX and activation of PKA by Br-cAMP suppressed the expression of <i>Col10a1</i> expression induced by <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figures 8A and 8D</a>). While <i>Car9</i> siRNA also enhanced the expression of <i>Epas1</i> mRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g004" target="_blank">Figure 4E</a>), HIF-2α does not mediate the <i>Col10a1</i> induction by <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g007" target="_blank">Figure 7C</a>). It is partly because <i>Car9</i> siRNA lowers HIF-2α protein via an unknown mechanism (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g007" target="_blank">Figure 7D</a>). <i>Epas1</i> siRNA rather enhanced the expression of <i>Col10a1</i> mRNA by an unknown mechanism (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g007" target="_blank">Figure 7C</a>). It is reported that Src kinase mediates the expression of <i>Epas1</i> and <i>Col10a1 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Bursell1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Sato1" target="_blank">[36]</a>. In this study, Src kinase inhibitor I lowered the induction of <i>Epas1</i> and <i>Col10a1</i> expressions after <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8A</a>), which may indicate a possibility that CA IX suppresses the Src kinase-mediated pathways. Augmentation of <i>Epas1</i> expression in the presence of the inhibitors of p38 MAPK (SB202190) and PI3K (LY294002) suggests a possible inhibition of <i>Epas1</i> expression by these kinases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8A</a>).