Terkhorn S P, Bohensky J, Shapiro I M, Koyama E, Srinivas V
Department of Orthopaedic Surgery, Division of Orthopaedic Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Cell Physiol. 2007 Jan;210(1):257-65. doi: 10.1002/jcp.20873.
The overall goal of the current study was to examine the functional activity of the prolyl hydroxylases (PHDs) in maturing chondrocytes. Herein, we show for the first time that the PHDs are expressed in the maturing zone of the growth plate, and by a chondrocytic cell line. We determined if this protein and its substrate, hypoxia inducible factor (HIF)-1alpha, modulated the induction of apoptosis. Using a chondrocyte cell line that matured in culture, we inhibited HIF-1alpha expression using siRNA technology and pharmacologically blocked PHD activity. We noted that PHD suppression sensitized the cells to an apoptotic challenge with H(2)O(2). We next examined the interplay between the PHDs and HIF-1alpha by suppressing HIF-1alpha and blocking PHD activity. We noted reduced killing when the mature HIF-silenced cells were challenged with H(2)O(2). In contrast, there was limited change in the viability of immature cells. Based on these differences in chondrocyte susceptibility, it is concluded that HIF-1alpha sensitizes maturing cells to H(2)O(2)-mediated killing. We next determined if this change in the viability of the PHD-inhibited cells was linked to changes in activation of caspase-3. It was noted that there was a minimal change in enzyme activity of the PHD-inhibited HIF-1alpha suppressed cells. Finally, we found that as the chondrocytes mature, the activities of catalase and SOD were significantly reduced and that there was a decrease in the levels of Bcl-2 and Bcl(XL). This loss of protective activity together with the changes mediated by HIF would be expected to generate conditions that would favor the induction of chondrocyte apoptosis.
本研究的总体目标是检测脯氨酰羟化酶（PHD）在成熟软骨细胞中的功能活性。在此，我们首次表明PHD在生长板的成熟区以及软骨细胞系中表达。我们确定了这种蛋白质及其底物缺氧诱导因子（HIF）-1α是否调节细胞凋亡的诱导。使用在培养中成熟的软骨细胞系，我们利用小干扰RNA（siRNA）技术抑制HIF-1α的表达，并通过药理学方法阻断PHD活性。我们注意到，抑制PHD会使细胞对H₂O₂诱导的凋亡刺激更加敏感。接下来，我们通过抑制HIF-1α和阻断PHD活性来检测PHD与HIF-1α之间的相互作用。我们注意到，当用H₂O₂刺激成熟的HIF沉默细胞时，细胞杀伤作用减弱。相比之下，未成熟细胞的活力变化有限。基于软骨细胞易感性的这些差异，我们得出结论：HIF-1α使成熟细胞对H₂O₂介导的杀伤更加敏感。接下来，我们确定PHD抑制细胞活力的这种变化是否与半胱天冬酶-3激活的变化有关。我们注意到，在抑制PHD并抑制HIF-1α的细胞中，酶活性的变化很小。最后，我们发现随着软骨细胞的成熟，过氧化氢酶和超氧化物歧化酶的活性显著降低，并且Bcl-2和Bcl(XL)的水平也有所下降。这种保护活性的丧失以及HIF介导的变化预计会产生有利于诱导软骨细胞凋亡的条件。
