Promotion of Chondrocyte-specific Function by Inhibition of HIF-targeting Hydroxylases
Author(s)
Thoms, Brendan Lawless
Type
Thesis
Abstract
Human articular cartilage is an avascular tissue, and therefore functions in a hypoxic
environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia
to drive tissue-specific functions. Previous work in our laboratory has shown that human
chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through
Hypoxia Inducible Factor 2alpha (HIF-2α) mediated upregulation 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 hydroxylases (PHDs)
which target them for proteosomal degradation. In fact, the PHDs are the true direct oxygen
sensors since they require molecular oxygen as a co-substrate. Here we show that PHD2 is
the dominant isoenzyme regulating HIF-2α (and HIF-1α) stability in human chondrocytes.
Moreover, specific inhibition of PHD2 using RNAi-mediated depletion caused an
upregulation of SOX9 and enhanced extracellular matrix protein production. Depletion of
PHD2 resulted in greater HIF-2α levels, and therefore enhanced SOX9-induced matrix
cartilage matrix production, even when performed in hypoxia (1% oxygen) implying that
PHD2 inhibition offers a novel means to enhance cartilage repair. The need for HIF-specific
hydroxylase inhibitors was highlighted as treatment with the oxoglutarate analogue
dimethyloxalylglycine (which also inhibits the collagen prolyl hydroxylases) resulted in
reduced secretion of type II collagen, a critical matrix component.
Complimentary to these anabolic effects, hypoxia was found to mediate anti-catabolic
effects, suppressing aggrecan degradation in human cartilage explants. In isolated
chondrocytes hypoxia downregulates key matrix metalloproteinases (ADAMTS5 and
MMP13) and upregulates metalloproteinase inhibitor (TIMP3). Unlike the anabolic effects
(HIF-2α-dependent), these hypoxia induced anti-catabolic chondrocyte responses were
predominantly HIF-1α dependent.
Both anabolic and anti-catabolic hypoxia-induced responses were further enhanced by
depletion of PHD2, whose inhibition thus offers a novel means to induce cartilage repair.
environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia
to drive tissue-specific functions. Previous work in our laboratory has shown that human
chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through
Hypoxia Inducible Factor 2alpha (HIF-2α) mediated upregulation 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 hydroxylases (PHDs)
which target them for proteosomal degradation. In fact, the PHDs are the true direct oxygen
sensors since they require molecular oxygen as a co-substrate. Here we show that PHD2 is
the dominant isoenzyme regulating HIF-2α (and HIF-1α) stability in human chondrocytes.
Moreover, specific inhibition of PHD2 using RNAi-mediated depletion caused an
upregulation of SOX9 and enhanced extracellular matrix protein production. Depletion of
PHD2 resulted in greater HIF-2α levels, and therefore enhanced SOX9-induced matrix
cartilage matrix production, even when performed in hypoxia (1% oxygen) implying that
PHD2 inhibition offers a novel means to enhance cartilage repair. The need for HIF-specific
hydroxylase inhibitors was highlighted as treatment with the oxoglutarate analogue
dimethyloxalylglycine (which also inhibits the collagen prolyl hydroxylases) resulted in
reduced secretion of type II collagen, a critical matrix component.
Complimentary to these anabolic effects, hypoxia was found to mediate anti-catabolic
effects, suppressing aggrecan degradation in human cartilage explants. In isolated
chondrocytes hypoxia downregulates key matrix metalloproteinases (ADAMTS5 and
MMP13) and upregulates metalloproteinase inhibitor (TIMP3). Unlike the anabolic effects
(HIF-2α-dependent), these hypoxia induced anti-catabolic chondrocyte responses were
predominantly HIF-1α dependent.
Both anabolic and anti-catabolic hypoxia-induced responses were further enhanced by
depletion of PHD2, whose inhibition thus offers a novel means to induce cartilage repair.
Date Issued
2011-06
Date Awarded
2011-09
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Murphy, Chris
Saklatvala, Jeremy
Creator
Thoms, Brendan Lawless
Publisher Department
Medicine: Kennedy Institute of Rheumatology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)