Expression of HIF prolyl hydroxylase isozymes in growth plate chondrocytes: relationship between maturation and apoptotic sensitivity.
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.
Terkhorn
S P
SP
Department of Orthopaedic Surgery, Division of Orthopaedic Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Bohensky
J
J
Shapiro
I M
IM
Koyama
E
E
Srinivas
V
V
D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000201:enzymology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D010088:Oxidoreductases / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D000600:Amino Acids, Dicarboxylic; D051017:Apoptosis Regulatory Proteins; D019485:Bone Morphogenetic Proteins; D004791:Enzyme Inhibitors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D034741:RNA, Small Interfering; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D011393:Procollagen-Proline Dioxygenase; C505409:Casp3 protein, mouse; D053148:Caspase 3; C040947:oxalylglycine