Inhibition of hypoxia-inducible factor-targeting prolyl hydroxylase domain-containing protein 2 (PHD2) enhances matrix synthesis by human chondrocytes.
Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-alpha isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2alpha stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2alpha levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.
Thoms
Brendan L
BL
From The Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College London, 65 Aspenlea Road, London W6 8LH, United Kingdom.
Murphy
Christopher L
CL
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D002358:Cartilage, Articular / Q000166:cytology / Q000502:physiology; D002478:Cells, Cultured; D002648:Child; D019902:Chondrocytes / Q000166:cytology / Q000502:physiology*; D003094:Collagen / Q000378:metabolism; D005109:Extracellular Matrix / Q000502:physiology*; D005260:Female; D017353:Gene Deletion; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007527:Isoenzymes / Q000235:genetics; D008297:Male; D008875:Middle Aged; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors* / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D055758:SOX9 Transcription Factor / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D007527:Isoenzymes; D034741:RNA, Small Interfering; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D003094:Collagen; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases
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