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. Author manuscript; available in PMC: 2012 Jul 1.
Published in final edited form as: Arthritis Rheum. 2011 Jul;63(7):1928–1937. doi: 10.1002/art.30333

Chondrocyte AMP-activated Protein Kinase Activity Suppresses Matrix Degradation Responses to Inflammatory Cytokines IL-1β and TNFα

Robert Terkeltaub 1, Bing Yang 1, Martin Lotz 2, Ru Liu-Bryan 1
PMCID: PMC3128233  NIHMSID: NIHMS277396  PMID: 21400477

Abstract

Objective

IL-1β and TNFα stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in OA. AMPK, which regulates energy homeostasis and cellular metabolism, also exerts anti-inflammatory effects in multiple tissues. Here, we tested the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1β and TNFα.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by phosphorylation status of AMPKα Thr172 in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Pro-catabolic responses to IL-1β and TNFα such as release of GAG, NO, MMP-3 and MMP-13 were determined by DMMB assay, Griess reaction and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by siRNA approach.

Results

Normal human knee articular chondrocytes express AMPKα1, α2, β1, β2 and γ1 subunits. AMPK activity is constitutively present in normal, but is decreased in OA articular chondrocytes and cartilage, and in normal chondrocytes treated with IL-1β and TNFα. Knockdown of AMPKα results in enhanced catabolic responses to IL-1β and TNFα in chondrocytes. Moreover, AMPK activators suppress cartilage/chondrocyte pro-catabolic responses to IL-1β and TNFα and the capacity of TNFα and CXCL8 (IL-8) to induce type X collagen expression.

Conclusions

AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL-1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and pro-catabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation-induced degradation.

Introduction

IL-1β, TNFα, and certain other inflammatory cytokines stimulate chondrocyte responses that promote catabolism of collagen II and proteoglycans (PG), thereby compromising cartilage extracellular matrix integrity and tissue homeostasis in OA and inflammatory arthritides (1,2). For example, IL-1β and TNFα induce expression of MMP-3 and MMP-13, activation of aggrecanases including ADAMTS-5, and stimulate iNOS expression and the generation of NO, a suppressor of PG synthesis (1-3).

Recently, the serine/threonine protein kinase AMPK has been observed to exert anti-inflammatory effects in tissues other than cartilage, mediated in part by suppression of NF-κB activation (4-10). AMPK is a “super-regulator” of energy homeostasis and cellular metabolism (11-13). AMPK is activated by stresses that increase the cellular AMP:ATP ratio, such as nutrient deprivation, hypoxia, and exercise (11-13). Once activated, AMPK responds by phosphorylating downstream targets, such that ATP-consuming pathways are inhibited and ATP-producing pathways are activated (11-13). In this manner, AMPK allows cells to adjust to changes in cellular energy demand (11-13). Furthermore, pharmacologic AMPK activation has emerged as an experimental modality to markedly enhance skeletal muscle endurance, even without exercise, in part by promoting mitochondrial biogenesis (14,15). AMPK significance in articular cartilage has not been examined but can be hypothesized, since chondrocytes cope with a nutritionally challenged avascular and hypoxic milieu, and changing metabolic demands in response to inflammation, aging, and biomechanical stress (16-27).

AMPK exists as a heterotrimer composed of an alpha catalytic subunit and two non-catalytic regulatory beta and gamma subunits, in a ratio of 1α:1β:1γ, and all three subunits are required for the formation of a stable and fully functional AMPK complex (12,13). Each subunit has two or three isoforms (α1, α2, β1, β2, γ1, γ2, γ3) that are encoded by different genes (12,13). Each AMPK subunit possesses unique structural features that facilitate differential roles in the regulation of AMPK activity and its physiological functions in mammalian cells. Importantly, phosphorylation of threonine 172 within the catalytic domain of the α subunit is crucial for AMPK activity (11-13), and is readily assessed using AMPK-phosphospecific antibodies.

Recent studies showed that mouse growth plate chondrocytes express AMPKα1 that is activated in an HIF-1-dependent manner (28). Since AMPK centrally regulates cell metabolism, function, differentiation, and inflammation, we tested the hypotheses that IL-1β and TNFα regulate activation of AMPK in chondrocytes, and conversely that activation of AMPK regulates the responses of chondrocytes to IL-1β and TNFα in vitro. Our results, coupled with the demonstration of decreased AMPKα phosphorylation in OA chondrocytes in vitro and in situ, suggest that decreased AMPK activity could contribute to progression of OA by disrupting cartilage homeostasis.

Materials and Methods

Reagents

All chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. The AMPK activators 5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside (AICAR) and metformin were obtained from CalBiochem (San Diego, CA), and the highly selective AMPK activator A-769662 was from Tocris Bioscience (Ellisville, MO). Recombinant human and mouse IL-1β and TNFα were purchased from R&D Systems, Inc. (Minneapolis, MN). Antibodies used for Western blot analyses were phospho-AMPKα (Thr172) and total AMPKα which recognize both AMPKα1 and AMPKα2; AMPK subunits α1, α2, β1, β2, γ1, γ2 and γ3, and phospho-NF-κB p65 (Ser536) from Cell Signaling Technology, Inc. (Danvers, MA); MMP-3, MMP-13 and type X collagen from Millipore (Billerica, MA), BioVision Inc. (Mountain View, CA) and CalBiochem (San Diego, CA), respectively. Antibodies used for immunohistochemistry (IHC) analyses were phospho-(AMPKα1 + AMPKα2) (Thr172 + Thr183) and total AMPKα1 + AMPKα2 from Abcam (Cambridge, MA).

Studies of human and mouse knee articular chondrocytes

All studies were performed in compliance with institutionally reviewed and approved human subjects, animal care and protocols. The human knees from different donors were graded macroscopically according to a modified Outerbridge scale where grade I represents intact surface (normal); grade II represents minimal fibrillation (OA); grade III represents overt fibrillation (OA); and grade IV represents full thickness defect (OA) (29,30). Twenty one different donors of primary human knee chondrocytes (first passage) were used for the study (7 for grade I, 5 for grade II, 6 for grade III, and 3 for grade IV). Twelve different donors of human knee cartilage sections (3 for each grade) were used for immunohistochemistry analysis. Immature mouse primary knee articular chondrocytes were isolated from 6-8 days old mouse knees as previously described (31). Chondrocytes were cultured in Dulbecco's modified Eagle's (DMEM) high glucose medium with 10% fetal calf serum (FCS), 100 μg/ml of streptomycin, and 100 IU/ml of penicillin at 37°C, and no later than first passage chondrocytes were used for all experiments. Unless otherwise indicated, chondrocytes were transferred onto 96 well plates coated with poly-HEME with 2.5 × 105 cells per well in 250 μl of medium on the day before the experimental treatment.

Studies of mouse femoral head cartilage explants

Femoral head cartilage caps from two month old mice (C57B/L6) were isolated as described (31) and placed in 0.1 ml of the same media as for chondrocytes at 37°C in a 96-well plate for 24 hours prior to the described treatments.

Measurement of glycosaminoglycans (GAG), nitric oxide (NO) release and proteoglycan (PG) synthesis

Sulfated GAG release into conditioned medium was determined by DMB colorimetric assay, using chondroitin sulfate as a standard (32). NO production was measured in conditioned media as the concentration of nitrite in conditioned media by the Griess reaction (33), using NaNO2 as standard. PG synthesis in chondrocytes was measured quantitatively by 35S/3H incorporation assay as described previously (34).

Analyses of expression of MMP-3, MMP-13, and type X collagen

Release of MMP-3 and MMP-13 in conditioned media, and expression of type X collagen in cell lysates were assessed by SDS-PAGE and Western blot analyses, followed by enhanced chemiluminescence (Pierce, Rockford, IL).

Knockdown of AMPKα in mouse knee articular chondrocytes

Primary mouse knee articular chondrocytes were transfected with AMPKα1, AMPKα2, or AMPKα1/2 siRNAs, and their control siRNA (Santa Cruz Biotechnology), with each of the siRNAs containing pools of 3 targets specific 19-25 nucleotides. Transfection used the Amaxa Nucleofection™ system (Amaxa Inc., Gaithersburg, MD), according to manufacturer protocol. Knockdown of expression of AMPKα was examined at the protein level by SDS-PAGE/western blot analysis.

Immunohistochemistry (IHC)

Slides of human knee cartilage sections from different donors with and without OA (macroscopically graded I (normal) and II-IV (mild to severe OA) according to surface features, as previously described (29)) were firstly treated with 3% H2O2 for 10 min, then blocked with 10% goat serum for 2 hours at room temperature. After washing with TBS, rabbit antibodies to phospho-(AMPKα1 + AMPKα2) (1:50 dilution), total AMPKα1 + AMPKα2 (1:50 dilution), and the negative control rabbit IgG (1 μg/ml) were applied to the sections and incubated overnight at 4°C. Next, the sections were washed with TBS, incubated with biotinylated goat anti-rabbit IgG secondary antibody for 30 min, and then incubated for 30 minutes using the Histostain Plus kit (Invitrogen, Carlsbad, CA). Finally, the sections were washed and incubated with 3,3′-diaminobenzidine (DAB) substrate for 2-5 min.

Statistical analyses

Replicates of three or greater were performed in all explants or cell culture experiments where numerical data were obtained, and such data were uniformly expressed as mean ± SD. Statistical analyses were performed by two way ANOVA with Bonferroni post-hoc test using GraphPad PRISM 5. P values less than 0.05 were considered significant.

Results

AMPK expression in cultured primary normal human knee articular chondrocytes

Because functional AMPK is a heterotrimer (11-13), and distinctions in heterotrimers of AMPK exist between different tissues (11-13), we first determined if isoforms of all 3 AMPK subunits were expressed in articular chondrocytes. To do so, cell lysates from cultured primary normal human knee articular chondrocytes (first passage) were studied by SDS-PADE/western blotting. As shown in Figure 1, expression of AMPKα1, AMPKα2, AMPKβ1, AMPKβ2 and AMPKγ1 were detected in human articular chondrocytes from each tested donor. However, expression of AMPKγ2 and AMPKγ3 were not detected (data not shown). Similar results were observed in both bovine and mouse knee articular chondrocytes (data not shown).

Figure 1.

Figure 1

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Expression of AMPK subunits in normal human knee articular chondrocytes. Cell lysates isolated from three different donors of cultured normal, first passage human knee articular chondrocytes were studied by SDS-PAGE/western blot analyses with antibodies to AMPK subunits α1, α2, β1, β2, γ1, γ2, γ3, and β-actin. Data were representative of 3 individual experiments.

AMPK activators attenuate cartilage catabolic responses to IL-1β and TNFα

AMPK in experimental model systems is commonly activated using pharmacologic agents such as 5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside (AICAR), which is metabolized to 5-aminoimidazole-4-carboxamide-1-β-D-ribosyl monophosphate, an AMP mimic (35). Here, mouse (C57B/L6) femoral head cartilage explants were pre-treated with AICAR (1 mg/ml) for 2 hours and then stimulated with IL-1β or TNFα (10 ng/ml) for 3 days. Under these conditions, release of both GAG and NO induced by IL-1β and TNFα were blunted by AICAR (Figure 2A and 2B). In addition, AICAR attenuated IL-1β and TNFα-induced MMP-3 and MMP-13 release (Figure 2C). In parallel, cultured primary normal human knee articular chondrocytes (first passage) were pre-treated with AICAR (1 mg/ml) for 2 hours before stimulation with IL-1β (10 ng/ml) and TNFα (10 ng/ml) for 18 hours. The capacity of IL-β and TNFα to inhibit proteoglycan (PG) synthesis was reduced by AICAR (Figure 2D), and no significant toxicity in chondrocytes by AICAR was observed (data not shown). Similar to the results seen in mouse cartilage explants, AICAR attenuated release of NO, MMP-3 and MMP-13 in response to IL-1β and TNFα in normal human articular chondrocytes (Figure 2E, 2F). In addition, pre-treatment of normal human articular chondrocytes with AICAR also attenuated reduction of type II collagen and induction of MMP-13 mRNA expression in response to IL-1β and TNFα (Supplemental Figure 1).

Figure 2.

Figure 2

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AICAR attenuates cartilage explant and chondrocyte pro-catabolic responses to IL-1β and TNFα. Femoral head cartilage explants isolated from C57BL/6 mice (A,B,C), or normal human knee articular chondrocytes (D,E,F) were pretreated with AICAR (1 mM) for 2 hours, and then stimulated with 10 ng/ml of IL-1β or TNFα for 3 days and 1 day, respectively. The conditioned media were analyzed for release of glycosaminoglycans (GAG), nitric oxide (NO) (n=6 for explants, n=3 for chondrocytes), MMP-3 and MMP-13 (all data shown were representative of 3 individual experiments), by Griess reaction and Western blot analyses, respectively. PG synthesis by chondrocytes was assessed as described in the Methods. Statistics for GAG release in A, * p<0.001, # p<0.05; NO release in B, *, # p<0.001; PG synthesis in D, *p<0.01, #p<0.001; and NO release in E, *, # p<0.001, relative to none with IL-1β and TNFα stimulation, respectively.

Because AICAR is not a selective activator of AMPK, and can also activate several other AMP-sensitive enzymes (35,36), we next employed the highly selective AMPK chemical activator A-769662 (37), as well as metformin, a type 2 diabetes drug that activates AMPK (38). Similar studies were carried out with mouse femoral head cartilage explants, where both A-769662 and metformin inhibited release of NO, MMP-3 and MMP-13 in response to IL-1β and TNFα (Supplemental Figure 2). Taken together, multiple pharmacologic activators of AMPK attenuated an array of inflammatory cytokine-induced pro-catabolic responses in cartilage and chondrocytes.

IL-1β and TNFα induce dephosphorylation of AMPKα in chondrocytes

We observed constitutive phosphorylation of AMPKα in primary normal human knee articular chondrocytes that was enhanced by AICAR (Figure 3A). Conversely, IL-β and TNFα decreased phosphorylation of AMPKα in chondrocytes, an effect that was prevented by pre-treatment with AICAR (Figure 3B). In addition, phosphorylation of the NF-κB p65 subunit in response to IL-1β and TNFα was inhibited by AICAR in chondrocytes (Figure 3C). Similar results were also seen with two other AMPK activators A-769662 and metformin (data not shown).

Figure 3.

Figure 3

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AICAR enhances constitutive phosphorylation of AMPKα (A), and inhibits dephosphorylation of AMPKα (B) and phosphorylation of NF-κB p65 subunit (C) in response to IL-1β and TNFα in normal human knee articular chondrocytes. Normal human knee articular chondrocytes were treated with AICAR (1 mM) for the times indicated (A), or for 2 hours before stimulation with 10 ng/ml of IL-1β or TNFα for 18 hours (B) or 6 hours (C). Cell lysates were then studied by SDS-PAGE/western blot analyses with antibodies to phospho-AMPKα (Thr172) and total AMPKα, and phospho-NF-κB p65 (Ser536). All data were representative of 3 individual experiments.

Knockdown of AMPKα enhances inflammatory cytokine-induced pro-catabolic responses and type X collagen expression in chondrocytes

To knockdown AMPKα, mouse immature knee articular chondrocytes were transfected with siRNA for AMPKα1, AMPKα2, AMPKα1/2, and control siRNA. Significant decrease in AMPKα protein expression was achieved in chondrocytes by knockdown of either AMPKα1 or AMPKα2, particularly so with knockdown of both the AMPK α1 and α2 subunits (AMPKα1/2) (Figure 4A). Induction of NO release by IL-1β and TNFα was slightly but not significantly enhanced (27% and 10%, respectively) in chondrocytes with knockdown of AMPKα2 compared with that in the siRNA control cells. Most significantly, NO release induced by IL-1β and TNFα was increased by 110% and 70%, respectively, in chondrocytes with knockdown of AMPKα1, and by 150% and 200%, respectively, in chondrocytes with knockdown of both AMPK α1 and α2 (AMPKα1/2) (Figure 4B). MMP-13 release induced by IL-1β and TNFα was also enhanced in chondrocytes with knockdown of AMPKα1, AMPKα2 or AMPKα1/2 (Figure 4C).

Figure 4.

Figure 4

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Knockdown of AMPKα enhances chondrocyte pro-catabolic responses to IL-1β and TNFα. Mouse immature knee articular chondrocytes were transfected with mouse siRNAs for AMPKα1, AMPKα2, or AMPKα1/2, using the Amaxa Nuclefection TM system. Two days after transfection, the cells were stimulated with 10 ng/ml of IL-1β or TNFα for 18 hours, the conditioned media were analyzed for release of NO (B) and MMP-13 (C). All data were representative of 3 individual experiments. Statistics for NO release in B, *p<0.001 and **p<0.001 relative to control with IL-1β stimulation, respectively. #p<0.05 and ##p<0.001 relative to control with TNFα stimulation, respectively.

Interestingly, the basal level of expression of the chondrocyte hypertrophy marker type X collagen, as well as TNFα-induced type X collagen expression, were strongly enhanced in chondrocytes with knockdown of AMPKα1 or AMPKα1/2 (Figure 5A). Conversely, induction of type X collagen expression by CXCL8 and TNFα was inhibited in chondrocytes by AICAR (Figure 5B) and A-769662 (data not shown).

Figure 5.

Figure 5

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Knockdown of AMPKα enhances type X collagen expression in chondrocytes. Mouse immature knee articular chondrocytes were transfected with mouse siRNAs for AMPKα1, AMPKα2, or AMPKα1/2 using Amaxa Nuclefection ™ system as described in the Methods. Two days after transfection, the cells were stimulated with 10 ng/ml of TNFα for 2 days (A). Mouse immature knee articular chondrocytes were pre-treated with AICAR (1 mM) for 2 hours before stimulated with 10 ng/ml of CXCL8 or TNFα for 2 days (B). The cell lysates were studied by SDS-PAGE/western blot analyses with antibodies to type X collagen and β-actin. Data were representative of 3 individual experiments.

Decreased constitutive phosphorylation, as well as expression, of AMPKα in cultured human knee OA articular chondrocytes and in human knee OA cartilage in situ

Cultured (first passage) human knee articular chondrocytes isolated from different human donors with a range of cartilage morphology from normal to severe OA (grade I to IV) were studied for AMPKα phosphorylation by SDS-PAGE/western blot analysis. Constitutively phosphorylated AMPKα was observed in grade I and II chondrocytes, but less so in grade III and grade IV OA chondrocytes, though there was slight increase in phosphorylated AMPKα in grade IV cultured chondrocytes, compared with that in Grade III OA cultured chondrocytes (Figure 6A). Similar results were observed for total AMPKα expression in these cultured chondrocytes (Figure 6A).

Figure 6.

Figure 6

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Decreased constitutive phosphorylation, as well as expression, of AMPKα in human knee OA chondrocytes and cartilage. Cell lysates isolated from cultured primary human knee articular chondrocytes (passage 1) (A) and human knee cartilage sections (B) from different donors with a range of cartilage morphology from normal to severe OA (grade I to IV) were subjected to SDS-PAGE/western blot and immunohistochemistry, respectively, with antibodies to phospho-AMPKα and total AMPKα, as described in Materials and Methods. β-actin and rabbit IgG were used as controls for A and B, respectively. Data in A were representative of 3 individual experiments (on a total 18 different donors). Data in B were representative of 3 individual experiments on cartilage donors with each grade (total of 12 individual donors, with 3 different donors studied for each grade). Original magnification × 10 in all pictures.

Immunohistochemistry (IHC) of human articular samples demonstrated constitutively phosphorylated AMPKα in grade I human knee cartilage (Figure 6B). There was a progressive decrease in phosphorylated AMPKα in grade II and grade III OA cartilages in conjunction with a progressive decrease of AMPKα expression. Notably, both phosphorylation and expression of AMPKα were robust in chondrocyte clusters in severe OA (grade IV) (Figure 6B).

Discussion

In this study, all isoforms of AMPK α and β subunits and only one isoform of AMPKγ subunit were found to be expressed in normal human knee articular chondrocytes. In addition, constitutively phosphorylated AMPKα (Thr172) was observed in normal knee articular chondrocytes, suggesting that, as in other tissues (11-13), AMPK activity may be necessary for cartilage homeostasis.

Importantly, inflammatory cytokines IL-1β and TNFα promoted dephosphorylation of AMPKα in chondrocytes, an effect inhibited by the AMPK activators AICAR, A-769662 and metformin. Moreover, multiple cartilage /chondrocyte pro-catabolic responses (GAG, NO, MMP-3 and MMP-13 release) to IL-1β and TNFα were attenuated by each pharmacologic AMPK activator tested. Furthermore, AMPK agonists suppressed the capacity of the inflammatory cytokines TNFα and CXCL8 (IL-8) to induce type X collage expression, which, along with MMP-13, is a marker of chondrocyte hypertrophy, a differentiation state linked with OA progression (39). The anti-inflammatory effects of AMPK activity observed in chondrocytes are likely regulated in part by modulation of NF-κB activation, as the AMPK activator AICAR attenuated phosphorylation of NF-κB p65 subunit in response to IL-1β and TNFα. There is growing evidence pointing that NF-κB signaling plays a central role not only in the pro-inflammatory stress-related responses of chondrocytes, but also in the control of their differentiation program (40). Thus, modulation of AMPK activity in chondrocytes may help to regulate NF-κB signaling.

In this study, knockdown of AMPKα2 slightly, but knockdown of AMPKα1 significantly enhanced not only pro-catabolic responses to IL-1β and TNFα, but also type X collagen expression at the basal level and in response to TNFα in chondrocytes. There was further enhancement of pro-catabolic responses and type X collagen expression in response to inflammatory cytokines in chondrocytes with knockdown of both AMPK α1 and α2, suggesting synergistic function of AMPKα1 and α2 in chondrocytes. However, AMPKα1 may play a more significant role in chondrocyte.

Our results linked dephosphorylation of AMPKα with a more permissive state for cartilage pro-catabolic responses to inflammatory cytokines. In addition, our analysis of human knee OA cartilages, suggested that phosphorylation of AMPKα decreased progressively as the grade of OA increased.

Limitations of this study included activation of AMPK only by pharmacologic compounds (the selective AMPK activator A-769662, and the nonselective AMPK activators AICAR and metformin). Though this is the widely employed investigative approach (12), alternative approaches to activate AMPK, such as transfection of constitutively active AMPK mutants, have not yet been evaluated in chondrocytes. AMPK phosphorylation is known to be stimulated by kinases upstream of AMPK, including LKB1, Ca2+/calmodulin-dependent protein kinase (CaMKK), and transforming growth factor-β-activated kinase 1 (TAK1) (11-13). Furthermore, protein phosphatases 2A (PP2A) and PP2C have been shown to dephosphorylate AMPKα (Thr172) (41,42). In our hands, okadaic acid, a potent, selective and cell permeable inhibitor of protein phosphatases including PP2A, PP1, PP2B and PP2C, inhibits dephosphorylation of AMPKα induced by IL-1β and TNFα in human articular chondrocytes (unpublished observation). Hence, IL-1β and TNFα may well promote dephosphorylation of AMPKα in part by induction of protein phosphatase activity in chondrocytes. This effect, and decreased overall cartilage chondrocyte AMPKα expression observed here, could contribute to the decreased phosphorylation AMPKα that was detected in OA chondrocytes, since expression of IL-1β and TNFα increase in OA cartilage. Further investigation, beyond the scope of this study, will be of interest to determine which protein kinases and phosphatases are involved in regulation of AMPK activity in chondrocytes with and without inflammatory challenge.

AMPK helps determine cell fate in response to stress (28,43,44). For example, maturation of growth plate chondrocytes includes a phase of ATP-generating autophagy that promotes cell survival, and is promoted by AMPK (28,43,44). Autophagy appears to be a constitutive homeostatic mechanism in articular cartilage (45), and chondrocyte autophagy is activated by AMPK in a HIF-1α-dependent manner (28). Conversely, apoptosis appears increased in OA cartilage chondrocytes, and promotes cartilage tissue failure (46). In this context, AMPK silencing increases chondrocyte sensitivity to induction of apoptosis, mediated in part by effects on pro-apoptotic caspase-8 and BID activation (28). Recently, reduction in autophagy and a linked increase in apoptosis were discovered in human OA and aging knee cartilages, and surgically induced knee OA cartilage in mice (45).

The precise role of AMPK in both the loss of chondrocyte viability and progression of OA will require further study. Interestingly, AMPK expression and phosphorylation appeared robust in chondrocyte clusters in late stage human knee OA cartilage in situ. Moreover, treatment of human knee articular chondrocytes with the major chondrocyte growth factor TGFβ1 promotes phosphorylation of AMPKα (unpublished observation). Hence, we speculate that AMPK activity is involved in the induction of cartilage regeneration and repair. AMPK activation also is involved in extending longevity and delaying age-dependent diseases in several species (47-49). Although OA is not an inevitable consequence of aging, OA development is known to directly associate with aging (25).

In conclusion, this study suggests that phosphorylation of AMPK, a master regulator of cell metabolism with anti-inflammatory effects, is reduced by inflammation in normal chondrocytes and in OA chondrocytes, and in turn promotes disruption of homeostasis of the cartilage extracellular matrix and chondrocyte hypertrophy, therefore may contribute to OA progression.

Supplementary Material

Supp Fig S1
Supp Fig S2

Acknowledgments

Studies were supported by the Research Service of the Department of Veterans Affairs and NIH grants PO1 AG007996 (ML and RT), AR54135 (RT) and AR1067966 (RLB).

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