Inhibition of hypoxia-inducible factor-targeting Prolyl Hydroxylase Domain-containing Protein 2 (PHD2) enhances matrix synthesis by human chondrocytes

Abstract

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 2α (HIF-2α)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-α 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-2α stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interferencemediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2α 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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.