Regulation of 2-Deoxy-D-GlucoseTransport, Lactate Metabolism,and MMP-2 Secretion by the HypoxiaMimetic Cobalt Chloridein Articular Chondrocytes

ALI MOBASHERI,a,b NICOLA PLATT,a COLIN THORPE,a

AND MEHDI SHAKIBAEIc

aMolecular Pathogenesis and Connective Tissue Research Groups, Departmentof Veterinary Preclinical Sciences, Faculty of Veterinary Science, University ofLiverpool, Brownlow Hill and Crown Street, Liverpool, L69 7ZJ, United KingdombDivision of Veterinary Medicine, The School of Veterinary Medicine andScience, The University of Nottingham, Sutton Bonington Campus, SuttonBonington, Leicestershire LE12 5RD, United KingdomcMusculoskeletal Research Group, Institute of Anatomy,Ludwig-Maximilian-University Munich, Pettenkoferstrasse 11, 80336 Munich,Germany

ABSTRACT: Articular cartilage is an avascular tissue with significantlyreduced levels of oxygen and nutrients compared to plasma and syn-ovial fluid. Therefore, chondrocyte survival and cartilage homeosta-sis require effective mechanisms for oxygen and nutrient signaling. Togain a better understanding of the mechanisms responsible for oxy-gen and nutrient sensing in chondrocytes, we investigated the effects ofhypoxic stimulation induced by cobalt chloride treatment (a hypoxia-mimetic) on glucose uptake and lactate production in chondrocytes.We also studied the effects of cobalt chloride and glucose depriva-tion on the expression and secretion of active MMP-2. Primary cul-tures of articular chondrocytes were either maintained in 20% O2 (nor-moxia) or exposed to the hypoxia-mimetic cobalt chloride for up to24 h at the following concentrations: 15 �M, 37.5 �M, and 75 �M. Glu-cose transport was determined by measuring the net uptake of nonme-tabolizable 2-deoxy-D-[2, 6-3H] glucose into chondrocytes. Active MMP-2secretion was assayed by gelatin zymography. Lactic acid production wasassayed using a lactate kit. Exposure to cobalt chloride significantly in-creased the uptake of 2-deoxy-D-[2, 6-3H] glucose and the production

Address for correspondence: Ali Mobasheri, B.Sc., A.R.C.S. (Hons), M.Sc., D.Phil. (Oxon), Divisionof Veterinary Medicine, The School of Veterinary Medicine and Science, The University of Nottingham,Sutton Bonington Campus, Sutton Bonington, Leicestershire LE12 5RD, United Kingdom. Voice: +44-0-115-951-6449; fax: +44-0-115-951-6415.

e-mail: ali.mobasheri@nottingham.ac.uk

Ann. N.Y. Acad. Sci. 1091: 83–93 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1378.057

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of lactate. Glucose deprivation and cobalt chloride treatment increasedlevels of active MMP-2 in the culture medium. Our results suggest thatthese metabolic alterations are important events during adaptation tohypoxia. Upregulation of MMP-2 and the build-up of lactic acid willhave detrimental effects on the extracellular matrix and may contributeto the pathogenesis and progression of osteoarthritis (OA).

KEYWORDS: articular cartilage; chondrocyte; hypoxia-inducible fac-tor alpha (HIF-1�); GLUT1; GLUT3; matrix metalloproteinase-2(MMP-2); glucose; lactate; osteoarthritis

INTRODUCTION

The maintenance of oxygen and glucose homeostasis is essential for manyvital cellular functions including division, proliferation, differentiation, ex-citability, and secretion.1 Oxygen and glucose homeostasis is also critical tocell fate, senescence, and apoptosis.2,3 Cells can generally “sense” the levelsof oxygen available for oxidative phosphorylation. However, only a limitednumber of specialized cell types can switch to anaerobic glycolysis when de-prived of oxygen for extended periods of time. A significant advance in ourunderstanding of the hypoxia response stems from the discovery of the hypoxia-inducible factor 1 (HIF-1) and related oxygen-regulated transcription factors.Depending on the duration and severity of hypoxia, cellular oxygen-sensorresponses activate a variety of short- and long-term adaptations to save energyand protect cells. Transcriptional regulation of genes that maintain oxygenhomeostasis is primarily mediated by HIF-1.4 HIF-1 plays essential roles inmammalian development, physiology, and disease pathogenesis.5 HIF-1 is aheterodimer whose � and � subunits are members of the PAS family of basichelix-loop-helix (bHLH) transcription factors.6 HIF-1� primarily mediates re-sponses to oxygen deprivation and prolonged hypoxia. The levels of HIF-1�increase during hypoxia, but the protein is unstable in the presence of oxygenbecause of the presence of an oxygen-dependent degradation domain that tar-gets it for ubiquitination.5,7 HIF-1� is involved in the transcriptional controlof key genes involved in oxygen and glucose homeostasis including those en-coding erythropoietin, glucose transporters, glycolytic enzymes, and vascularendothelial growth factor.8

Articular cartilage is a vascular tissue and hence the quantities of oxygenand glucose available to chondrocytes in the extracellular matrix can fluctu-ate considerably.9–11 Therefore, chondrocytes must possess the ability to sensethe amount of available oxygen and glucose in the matrix and respond ap-propriately to shortages or surpluses by altering metabolic rate. Chondrocytesconsume less oxygen in comparison with most other cell types and conse-quently anaerobic glycolysis forms the principal source of cellular ATP incartilage. Recent studies have clearly demonstrated the importance of HIF-1�

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and its target genes in the maintenance of anaerobic glycolysis9 and cellularglucose levels12,13 in response to hypoxia and nutrient stress.10 The differen-tial expression of the two HIF isoforms, HIF-1� and HIF-1�, GLUT-1 andMMP-2 in chondrocyte-like cells of the nucleus pulposus in the intervertebraldisk have been used as a phenotypic and metabolic signature, allowing thesecells to be distinguished from fibroblast-like cells in neighboring tissues.10

In light of these findings, in this study we designed and carried out a seriesof experiments aimed at testing the hypothesis that articular chondrocytes arealso capable of adaptive metabolic responses to glucose deprivation and hy-poxia mimetics, such as cobalt chloride. Other investigators have shown thatthis adaptation occurs by HIF-1�-mediated upregulation of genes encoding thehypoxia-responsive GLUT1 glucose transporter, which may promote glycol-ysis in chondrocytes from osteoarthritic cartilage.14 Our results suggest thathypoxia mimetics (i.e., cobalt chloride) and glucose deprivation increase glu-cose transport, the production of lactic acid, and secretion of the active formof the matrix metalloproteinase MMP-2. We propose that in chronic hypoxiaand in degenerate osteoarthritic joints these metabolic alterations will haveimportant consequences for extracellular matrix turnover and may lead to theprogression of arthritis.

MATERIALS AND METHODS

Chondrocyte Isolation and Culture

Normal equine articular cartilage was obtained from the stifle joints of horseseuthanized for unrelated clinical reasons at the Philip Leverhulme Large An-imal Hospital, University of Liverpool. The study was conducted with eth-ical approval in strict accordance with local guidelines (none of the horseswere euthanized for the purpose of this study). Equine cartilage shavings wererinsed with phosphate-buffered saline (PBS), cut into small slices and incu-bated overnight with type I collagenase (EC 3.4.24.3 from Clostridium his-tolyticum) (approximately 100 collagen digestion units mL−1) in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 1,000 mg L−1 glucose,10% fetal calf serum, and 1% antibiotic/antimycotic solution. The filtered cellsuspension was washed three times in fresh DMEM and the cells were countedon a hemocytometer; cell viability was determined by trypan blue dye exclu-sion and was usually 95% or higher. The cells (2 × 106 cells mL−1) werecultured in monolayers for up to three passages as previously described.13

Monolayer cultured equine chondrocytes were used in this study because2-deoxy-D-[2, 6-3H] glucose uptake experiments can only be performed withmonolayer cultured chondrocytes because the final washes in nonradioactivemedium cannot be performed rapidly with cells in alginate beads. Chondro-cytes used for glucose uptake, lactate production, and MMP-2 assays were not

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passaged more than three times to prevent chondrocyte dedifferentiation andphenotypic instability.

Experimental Design

Cobalt chloride was used as a hypoxia mimetic to simulate hypoxia by induc-ing HIF-1� in confluent monolayers of equine chondrocytes in 24-well platesand 75-cm2 culture flasks. Net glucose transport was compared in normoxicand cobalt chloride–induced hypoxic conditions by measuring the uptake ofnonmetabolizable 2-deoxy-D-[2, 6-3H] glucose by chondrocytes and the pro-duction of lactic acid was assayed using a lactate kit. We also investigated theeffects of cobalt chloride and glucose deprivation on active MMP-2 secretionby gelatin zymography. The MMP-2 assay was carried out with cells exposedto serum-free DMEM.

2-Deoxy-D-[2,6-3H] Glucose Uptake

Glucose transport into equine chondrocytes was determined by measur-ing the uptake of nonmetabolizable 2-deoxy-D-[2, 6-3H] glucose (Amersham/Pharmacia, Little Chalfont, UK) in the presence and absence of the glu-cose transport inhibitor cytochalasin B (1 �M mL−1). Equine chondrocyteswere cultured to 95% confluence in 24-well plates in DMEM containing1,000 mg mL−1 glucose and supplemented with 4% fetal calf serum. Thewells were rinsed three times with PBS before the assay was performed for 35min at room temperature in glucose, pyruvate, and serum-free DMEM (SigmaPoole, Dorset, UK) containing 1 �Ci mL−1 2-deoxy-D-[2, 6-3H] glucose. Thewells were then washed three times with ice-cold PBS before the cells werelysed in 0.5 mL of a cell lysis solution consisting of 0.5% sodium dodecyl sul-fate (SDS) and 0.5% Triton X-100 in PBS. Aliquots of cell lysates (0.45 mL)were transferred to 5-mL scintillation vials containing 3.55 mL of NACS104scintillation cocktail for aqueous samples (Amersham/Pharmacia, Little Chal-font, UK). The uptake of the radiolabeled glucose was normalized to total cellprotein content using a Bio-Rad detergent–compatible (DC) protein assay. Alluptake experiments were carried out in triplicate and repeated under identicalconditions at least three times. The data are presented as the percentage changein total 2-deoxy-D-[2, 6-3H] glucose uptake.

Lactate Assays

Lactate assays were carried out on chondrocyte culture supernatants usinga lactate kit from Sigma Diagnostics. The assay was performed using SigmaLactate Reagent (Catalogue No. 735-10) following the manufacturer’s instruc-tions in the Sigma Lactate Assay Procedure No. 735. The optical density

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readings were obtained using a Novaspec II Visible Spectrophotometer at540 nm. A 40 mgL−1 solution of lactic acid (Sigma) was used as a standardfor calibration.

MMP-2 Zymography

The effects of cobalt chloride and glucose deprivation on the secretion ofactive MMP-2 was determined by gelatin zymography. Chondrocytes used inthese experiments were incubated with serum-free DMEM culture medium in24-well plates in the above conditions. At the end of the 24-h incubation period,aliquots of the culture medium supernatant were subjected to SDS-PAGE andzymography as recently described. The zymogram gels were scanned usinga flatbed scanner and the densities of active MMP-2 bands quantified usingScion Image for Windows (version 4.0.2).

Statistical Analysis

Comparisons between experimental and control groups were made by meansof the Student’s t-test using GraphPad Instat software (version 3.05).

RESULTS

Cobalt Chloride Treatment Increases the Net Uptakeof 2-Deoxy-D-[2,6-3H] Glucose

The hypoxia-mimetic cobalt chloride is known to stimulate the expression ofgenes responsive to reduced oxygen tension. Concentrations ranging between12 and 75 �M are tolerated by cells in vitro for up to 24 h without any cyto-toxic effects, but concentrations higher than 250 �M are toxic.15 Therefore,we used cobalt chloride at concentrations of 15, 37.5, and 75 �M and com-pared the uptake of 2-deoxy-D-[2, 6-3H] glucose uptake by untreated, controlequine chondrocytes to cells incubated for 24 h with increasing concentrationsof cobalt chloride. The net uptake of 2-deoxy-D-[2, 6-3H] glucose was signif-icantly higher in chondrocytes incubated with cobalt chloride and the highestincrease was seen with 37.5 �M cobalt chloride (FIG. 1A).

Cobalt Chloride Treatment Increases the Production of Lactate

Another cellular effect of cobalt chloride treatment is the stimulation ofglycolysis. A byproduct of increased glycolysis is elevated production of lac-tate. We assayed the production of lactic acid by monolayer cultured equinechondrocytes in 24-well plates. The production of lactate in culture super-natants of control chondrocytes was compared with cells incubated for 24 h

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(B)

FIGURE 1. (A) Effects of the hypoxia-mimetic cobalt chloride on the uptake of 2-deoxy-D-[2,6-3H] glucose by monolayer cultured equine chondrocytes in 24-well plates.The uptake of 2-deoxy-D-[2,6-3H] glucose in control chondrocytes was compared with cellsincubated for 24 h with increasing concentrations of cobalt chloride (15, 37.5, and 75 �M).The net uptake of 2-deoxy-D-[2,6-3H] glucose was significantly higher in chondrocytesincubated with cobalt chloride compared to control cells. The highest increase was seenwith 37.5 �M cobalt chloride. Error bars indicate standard errors of the means (n = 3).In cases where a statistically significant difference between an experimental group and thecontrol group was found, the bar is labeled with ∗. (B) Effects of cobalt chloride on theproduction of lactic acid by monolayer cultured equine chondrocytes in 24-well plates. Theproduction of lactate in culture supernatants of control chondrocytes was compared withcells incubated for 24 h with increasing concentrations of cobalt chloride (15, 37.5, and75 �M). Lactic acid production was higher in chondrocytes incubated with cobalt chloridecompared to control cells and the effect seemed to plateau at a concentration of 75 �Mcobalt chloride.

with increasing concentrations of cobalt chloride (15, 37.5, and 75 �M). Lac-tic acid production was higher in chondrocytes incubated with cobalt chloridecompared to control cells and the effect was maximal at a concentration of 75�M cobalt chloride (FIG. 1B).

MOBASHERI et al.: HYPOXIC REGULATION OF CHONDROCYTE METABOLISM 89

FIGURE 2. Effects of glucose deprivation and cobalt chloride on the expression of ac-tive MMP-2 secreted into the culture medium of chondrocytes. (A) A representative gelatinzymogram used for the quantitative analysis of active MMP-2 expression. (B) Glucose de-privation, exposure to 75 �M cobalt chloride, or a combination of both for periods of upto 24 h significantly increased MMP-2 production secretion by chondrocytes compared tothe control group. MMP-2 is detected as two closely migrating bands on the zymogram;the lower molecular weight band corresponds to active MMP-2 and the higher molecularweight band represents inactive MMP-2.

Cobalt Chloride and Glucose Deprivation Increase Levels of Active MMP-2

We studied the effects of cobalt chloride and glucose deprivation on theexpression of active MMP-2 secreted into the culture medium of chondro-cytes. A typical gelatin zymogram is shown in FIGURE 2A. MMP-2 is seen astwo closely migrating bands on the zymogram; the lower molecular weightband corresponds to active MMP-2. Exposure to 75 �M cobalt chloride for24 h, glucose-free DMEM (glucose deprivation), or a combination of both

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significantly increased MMP-2 production and secretion and the levels of ac-tive MMP-2 compared to chondrocytes in the control cultures (FIG. 2 B).

DISCUSSION

This study leads to the following findings: (1) Treatment of chondro-cyte cultures with the hypoxia mimetic cobalt chloride increases the glucosetransporter-mediated uptake of 2-deoxy-D-[2, 6-3H] glucose, which is sugges-tive of HIF-1� upregulation of GLUTs, presumably the hypoxia-responsiveGLUT1 and GLUT3 proteins. (2) Cobalt chloride treatment also promotesglycolysis by increasing the production of lactic acid, which accumulates inthe culture medium supernatant.

Recent research suggests that articular cartilage is a tissue routinely exposedto low oxygen tensions9 and hypoxia.16,17 Furthermore, the generation of reac-tive oxygen species (ROS) has been found to increase in joint diseases, such asOA.18,19 Studies in other tissues have demonstrated that one of the hallmarks ofhypoxia is functional activation of HIF-1�, an oxygen-sensitive transcriptionalactivator that mediates changes in gene expression in response to changes inoxygen levels. Under low oxygen tensions, cellular hypoxia results and ATPlevels are diminished. Reduced degradation and stabilization of the HIF1-�protein increase its transcriptional activity as cellular oxygen concentrationsdecrease. The net effect of this is transactivation of several dozen target genes.These include the genes encoding erythropoietin (EPO), glucose transporters(GLUT1, GLUT3), glycolytic enzymes (lactate dehydrogenase, LDH; phos-phofructokinase, PFK), and vascular endothelial growth factor (VEGF).8 Theproducts of these genes increase oxygen delivery and glycolysis to meet themetabolic demands of reduced oxygen availability.

Experimental studies from several laboratories have shown that chondro-cytes express the oxygen-regulated transcription factor HIF-1�.9,10,16 Chon-drocytes also possess multiple isoforms of the GLUT/SLC2A family of glu-cose transporters.11–13,20,21 Among these are the hypoxia-responsive GLUT1and GLUT3 transporters.12,13 We have recently proposed that the metabolic ad-justment of chondrocytes to a low-oxygen, low-glucose environment in the ex-tracellular matrix of articular cartilage involves HIF-1� as an “oxygen sensor”capable of activating hypoxia-responsive target genes involved in extracellular“glucose sensing” and uptake. GLUT1 and GLUT3 are therefore putative glu-cose sensors in chondrocytes as well as targets of HIF-1�.22,23 It is likely thatother HIF-1� gene targets concerned with anaerobic glycolysis (i.e., lactatedehydrogenase and phosphofructokinase)24–26 may also be involved in glucosesensing in chondrocytes.

Inflammation is a process that is closely integrated in the pathogenesis andprogression of osteoarthritis.27 Notably, the synovial fluid of most OA pa-tients has an increased number of mononuclear cells (mainly macrophages

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and T lymphocytes) and increased levels of immunoglobulins, complementproteins, and inflammatory cytokines. Synovial macrophages are known toaccumulate in OA joints, especially when the synovium has been activatedby proinflammatory cytokines and degraded components of the extracellularmatrix of the damaged cartilage.28 This activation of synovial macrophagesalso activates synovial fibroblasts resulting in the generation of a broad rangeof catabolic factors.29 Recent studies suggest that hypoxia alters the phenotypeof macrophages in a manner that promotes the progression of the inflamma-tory process. As commonly observed in other cell types, GLUT1 and matrixmetalloproteinase-7 (MMP-7) are upregulated by hypoxia in macrophages.29

Hypoxic upregulation of these proteins suggests that hypoxia may affect thefunctioning of synovial macrophages in OA joint tissues. The results of thisstudy suggest that hypoxic conditions also result in overexpression of MMP-2in chondrocytes, which may promote hypoxia-mediated degradation of the ex-tracellular matrix in cartilage. Inflammation can stimulate angiogenesis, andangiogenesis can facilitate inflammation by facilitating innervation and alter-ing tissue oxygenation.

In summary, our results suggest that chondrocyte adaptation to hypoxia oc-curs by metabolic alterations including enhancement of the glucose transport-ing capacity of the cells, increased glycolysis, and lactate production. Theseprocesses are likely to be HIF-1�-mediated and reversible. Furthermore, wehave shown that hypoxia and glucose deprivation increase the production oflactic acid and production of the active form of the matrix metalloproteinaseMMP-2. Upregulation of MMP-2 and the build-up of lactate will have detri-mental effects on the extracellular matrix. We propose that chronic hypoxiamay occur in structurally degenerated osteoarthritic joints and the consequentmetabolic alterations may contribute to the pathogenesis and progression ofosteoarthritis. Future studies will determine the effects of hypoxia on other keymetabolites and biochemical pathways in chondrocytes in health and disease.

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