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 2␣ (HIF-2␣)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-␣ 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-2␣ 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-2␣ 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.Articular cartilage consists of a single cell type, the chondrocyte, which is solely responsible for the synthesis and maintenance of the extracellular matrix (1). The rigid nature of type II collagen fibrils present in the matrix confers tensile strength to the tissue, and the swelling pressure caused by water-saturated aggrecan molecules creates a compressive gellike stiffness allowing the tissue to resist deformation and giving cartilage its ability to absorb shocks (1, 2). Due to its shockabsorbing and articulating functions, articular cartilage cannot afford a blood or nerve supply. The tissue is therefore dependent on diffusion of oxygen from the synovial fluid on one side and the vascularized underlying bone on the other. As a result, articular cartilage (at least in larger animals and man) is maintained in a low oxygen environment (1-5% oxygen tension) throughout life (3-5). There is now significant evidence that hypoxia is a critical parameter in promoting the chondrocyte phenotype. It has been shown that hypoxia up-regulates key cartilage transcription factor SOX9 and increases expression of the main extracellular matrix genes in bovine and human chondrocytes (6 -10).The response of cells to hypoxia is mediated by hypoxiainducible factors (HIFs), 2 heterodimeric transcription factors consisting of an ␣ and ␤ subunit (11). Both subunits are constitutively expressed at the mRNA level; however, the ␣ subunit ...

Expression of Prolyl Hydroxylases (PHDs) Is Selectively Controlled by HIF-1 and HIF-2 Proteins in Nucleus Pulposus Cells of the Intervertebral Disc

“…Taken together, these observations clearly indicate the existence of a regulatory feedback loop between PHD2, PHD3, and HIF-1␣ in the hypoxic nucleus pulposus cells. This scenario is distinct from the articular cartilage, a tissue functionally similar to the nucleus pulposus, where PHD2 also regulates HIF-2␣ degradation (41). These results highlight the unique nature and control of the HIF-PHD system in nucleus pulposus cells.…”

HIF-2α - a mediator of osteoarthritis?

“…However, placing these results in context of the published literature it must be stressed that other studies have detected elevated levels of HIF-1α in OA cartilage/chondrocytes [7], while HIF-2α was actually reported to be decreased [8]. However, all these findings must also be placed in context of the fact that HIF-2α (and HIF-1α) has a half-life of approximately 5 min in normoxia [9], and therefore the time taken to initially process and fix the tissue samples is critical and can greatly affect the results.…”

“…In contrast, in human articular chondrocytes, HIF-2α is strongly regulated by prolyl hydroxylation, and can be stabilized even in the presence of atmospheric oxygen by specific inhibition of the HIF-targeting prolyl hydroxylases (PHDs) [9]. Furthermore, such PHD inhibition in human chondrocytes enhances expression of the main cartilage matrix genes [9]. Taken together, this suggests that there can be fundamental differences in the way human and murine chondrocytes respond to the same signal, in particular if that signal is hypoxia-related.…”

“…Saito and colleagues claim that HIF-2α is acting independently of oxygen-dependent hydroxylation in murine chondrocytes [2]. In contrast, in human articular chondrocytes, HIF-2α is strongly regulated by prolyl hydroxylation, and can be stabilized even in the presence of atmospheric oxygen by specific inhibition of the HIF-targeting prolyl hydroxylases (PHDs) [9]. Furthermore, such PHD inhibition in human chondrocytes enhances expression of the main cartilage matrix genes [9].…”

Chondrocyte responses to neurovascular peptides, cytokines, and a 3D environment: focus on ADAMs

“…Bevan et al resulting hypoxic environment promotes chondrocyte differentiation, [5][6][7] in addition to maintaining the chondrocyte phenotype. 8 Cartilage is also aneural, but damaged cartilage is recognized as a potentially permissive environment for neurovascular infiltration.…”