Abstract
Hyaluronan is a critical component of articular cartilage and partially helps retain aggrecan within the extracellular matrix of this tissue. During osteoarthritis, hyaluronan and aggrecan loss are an early sign of tissue damage. However, our recent attempts to mimic hyaluronan loss with the hyaluronan inhibitor 4-methylumbelliferone (4MU) did not exacerbate arthritis-like features of in vitro models of arthritis, but surprisingly, caused the reverse (i.e. provided potent chondroprotection). Moreover, the protective effects of 4MU did not depend on its role as a hyaluronan inhibitor. To understand the molecular mechanism in 4MU-mediated chondroprotection, we considered recent studies suggesting that shifts in intracellular UDP-hexose pools promote changes in metabolism. To determine whether such metabolic shifts are associated with the mechanism of 4MU-mediated pro-catabolic inhibition, using molecular and metabolomics approaches, we examined whether bovine and human chondrocytes exhibit changes in the contribution of glycolysis and mitochondrial respiration to ATP production rates as well as in other factors that respond to or might drive these changes. Overexpression of either HA synthase-2 or 4MU effectively reduced dependence on glycolysis in chondrocytes, especially enhancing glycolysis use by interleukin-1β (IL1β)-activated chondrocytes. The reduction in glycolysis secondarily enhanced mitochondrial respiration in chondrocytes, which, in turn, rescued phospho-AMP-activated protein kinase (AMPK) levels in the activated chondrocytes. Other glycolysis inhibitors, unrelated to hyaluronan biosynthesis, namely 2-deoxyglucose and dichloroacetate, caused metabolic changes in chondrocytes equivalent to those elicited by 4MU and similarly protected both chondrocytes and cartilage explants. These results suggest that fluxes in UDP-hexoses alter metabolic energy pathways in cartilage.
Highlights
Hyaluronan is a critical component of articular cartilage and partially helps retain aggrecan within the extracellular matrix of this tissue
Chondrocytes under all conditions responded to stress testing, wherein the cells were forced to maximal glycolysis and maximal mitochondrial O2 consumption
Our data on the HA biosynthesis inhibitor 4MU may provide unexpected insight into these changes in phenotype and metabolism as largely due to mechanisms that are completely unrelated to HA biosynthesis
Summary
We found that the hyaluronan (HA) inhibitor 4-methylumbelliferone (4MU) blocked the transcriptional expression of MMP13, ADAMTS4, TSG6, and the HA synthase-2 (HAS2) enzyme in pro-catabolically activated chondrocytes [1]. Rescue of energy metabolic changes in chondrocytes sense, there are two properties that HAS2-OE and 4MU have in common Both utilize UDP-GlcUA, and both likely put stress on the cytoplasmic pool of this precursor. One important outcome from this study is that stress in UDP-hexose pools (the hexosamine pathway in their case) can cause a dramatic effect on cell energy metabolism. To examine whether such metabolic changes could be the common mechanistic link between 4MU and HAS2-OE, cell energy metabolism in human and bovine chondrocytes was examined as well as the changes that occur in HIF1␣, phosphoAMPK, GlcNAcylation, and cell signaling. We found that independently manipulating glycolysis provided for potent chondroprotective effects
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