To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1).
To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1).
Chondrocytes isolated from normal articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50–100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis, and immunoblotting of cell lysates was performed to analyze cell signaling. Confocal microscopy was performed to measure nuclear translocation of Smad4.
Chondrocytes isolated from the articular cartilage of tissue donors ranging in age from 24 years to 81 years demonstrated an age-related decline in proteoglycan synthesis stimulated by IGF-1 and IGF-1 plus OP-1. Induction of oxidative stress inhibited both IGF-1– and OP-1–stimulated proteoglycan synthesis. Signaling studies showed that oxidative stress inhibited IGF-1–stimulated Akt phosphorylation while increasing phosphorylation of ERK, and that these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation, which resulted in phosphorylation of Smad1 at the Ser206 inhibitory site and reduced nuclear accumulation of Smad1. Oxidative stress also modestly reduced OP-1–stimulated nuclear translocation of Smad4.
These results demonstrate an age-related reduction in the response of human chondrocytes to IGF-1 and OP-1, which are 2 important anabolic factors in cartilage, and suggest that oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling.
The development of osteoarthritis (OA) has been closely linked to aging, but the mechanisms responsible are incompletely understood. The loss of articular cartilage that occurs during the development of OA is related to an imbalance in anabolic and catabolic activity of resident chondrocytes ([1-3]). Because growth factors such as insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1) are proanabolic and anticatabolic ([4, 5]), reduced responsiveness to growth factor stimulation could play a key role in the link between aging and OA.
There is evidence of an age-related decline in the chondrocyte response to IGF-1 in bovine (), rat ([7, 8]), and monkey chondrocytes (). However, there is a lack of data for human chondrocytes, most likely due to the difficulty in obtaining sufficient numbers of samples from the normal cartilage of young and old individuals. A previous study demonstrated an age-related decline in the mitogenic response of human chondrocytes to IGF-1, but the anabolic response was not measured (). However, IGF-1 is the major growth factor responsible for the stimulation of proteoglycan synthesis by serum, and the ability of 10% serum to stimulate sulfate incorporation by human chondrocytes in explant culture was shown to decrease with donor age ().
Growth factors do not act alone in tissue such as articular cartilage that contains multiple growth factors acting in concert to regulate cell function. In addition to IGF-1, OP-1 (also known as bone morphogenetic protein 7 [BMP-7]) is an important anabolic factor in adult articular cartilage ([12, 13]). Previous studies have demonstrated that chondrocytes isolated from either normal or OA cartilage have better survival and produce more matrix when stimulated with the combination of IGF-1 and OP-1 as compared with cells treated with either growth factor alone ([4, 14]). Likewise, the combination of IGF-1 and OP-1 was more effective than either growth factor alone at inhibiting chondrocyte matrix metalloproteinase production in response to interleukin-1β (IL-1β) or fibronectin fragments (). However, chondrocyte expression of OP-1 declined with increasing age, and this was accompanied by a decrease in the levels of endogenous OP-1 in cartilage ().
Our group previously showed that IGF-1–stimulated phosphorylation of the cell signaling protein Akt is necessary for proteoglycan synthesis in normal chondrocytes, and that oxidative stress inhibits this induction in both OA and normal chondrocytes ([16, 17]). It is not known how aging might alter the articular chondrocyte signaling response to IGF-1 or OP-1 that mediates matrix synthesis. The purpose of the present study was to determine whether aging affects the response of normal adult human articular chondrocytes stimulated with IGF-1 and OP-1, alone and in combination, and to determine whether altered cell signaling due to oxidative stress plays a role. The results support an age-related decline in the human chondrocyte growth factor response that may be attributable to altered cell signaling mediated by oxidative stress.
Normal cartilage was obtained from the ankle joints of tissue donors, through the Gift of Hope Organ and Tissue Donor Network (Elmhurst, IL) or the National Disease Research Interchange (Philadelphia, PA). The joints of 33 individual donors were acquired. The donors had no known history of joint disease, and each joint was graded on a modified Collins scale for gross evidence of damage, as previously described (). A Collins grade of 0 or 1 was considered to represent normal cartilage.
Chondrocytes were isolated enzymatically from cartilage slices removed from the joints, as previously described (). After isolation, viability and cell count were determined using trypan blue exclusion, and then the cells were cultured either in alginate beads or in monolayers. Alginate beads were used for the experiments shown in Figure 1, and all other experiments were performed using high-density monolayers. For the alginate bead experiments, cells were resuspended in sodium alginate (2 × 106 cells/ml); the alginate beads were produced as previously described, resulting in ∼20,000 cells per bead (). The beads were cultured in 24-well plates (8 beads/well) in serum-free Dulbecco's modified Eagle's medium (DMEM)–Ham's F-12 (1:1; 0.5 ml/well) supplemented with 1% mini-ITS+ (insulin–transferrin–selenium), which contains 5 nM insulin (“mini”-dose insulin so that the IGF-1 receptor is not stimulated), 2 μg/ml transferrin, 2 ng/ml selenous acid, 25 μg/ml ascorbic acid, and bovine serum albumin/linoleic acid at 420/2.1 μg/ml (). For high-density monolayer culture experiments, isolated cells were plated in 6-well plates at 2 × 106/well in DMEM–Ham's F-12 supplemented with 10% fetal bovine serum and antibiotics. Confluent primary cultures were changed to serum-free medium and used the next day for stimulation experiments.
To examine the effect of donor age on the response to IGF-1 and OP-1, proteoglycan synthesis assays were performed in cells cultured in alginate beads, as detailed above. The wells were treated with 100 ng/ml of recombinant human IGF-1 (a gift from Chiron Corporation or purchased from Austral Biologicals), 100 ng/ml of recombinant human OP-1 (provided by Stryker Biotech), or with 100 ng/ml of each growth factor. Triplicate wells were used for each condition. The medium was changed every 48 hours, with the addition of fresh growth factor to the treated wells. Proteoglycan synthesis was measured on day 7 using a sulfate incorporation assay, with correction for cell numbers by an assay for total DNA, as previously described ().
The experiments examining the effect of oxidative stress on proteoglycan synthesis were performed using confluent monolayer cultures. Cells were changed to serum-free medium for 6 hours and then pretreated for 30 minutes with 25 μM tert-butyl hydroperoxide (Sigma) followed by the addition of 100 ng/ml IGF-1 or OP-1. After an overnight incubation, proteoglycan synthesis was measured using a sulfate incorporation assay as previously described ().
Cell signaling studies were performed as previously described in detail (). Briefly, serum-free confluent monolayer cultures were treated with 50 ng/ml of IGF-1, OP-1, or IGF-1 plus OP-1. For oxidative stress experiments, cells were pretreated for 30 minutes with 250 μM tert-butyl hydroperoxide prior to the addition of growth factors. MAPK inhibitor studies used 10 μM MEK inhibitor U0126 to block ERK, 10 μM SB203580 to block p38, and 20 μM SP600125 to block JNK; these inhibitors were added 30 minutes prior to tert-butyl hydroperoxide. Cultures were lysed at the indicated time points after the addition of growth factors, and the lysates were used to analyze cell signaling proteins by immunoblotting with phosphospecific antibodies and with antibodies to total protein (non-phosphospecific) as a control for protein loading. Phospho-Akt (Ser473), Akt, phospho–ERK-1/2 (Thr202/Tyr204), ERK-1/2, phospho-p38 (Thr180/Tyr182), p38, phospho-Smad1 (Ser463/Ser465)/Smad5 (Ser463/Ser465)/Smad8 (Ser426/Ser428), phospho-Smad1 (Ser206), and Smad1 antibodies were obtained from Cell Signaling Technology. The phospho-JNK (Tyr183/Tyr185) and JNK-2 antibodies were obtained from Invitrogen. The cytosolic and nuclear samples were prepared from cell lysates using a NE-PER kit (Pierce). Densitometry was performed using Eastman Kodak 1D 3.6 image analysis software.
Analysis of Smad4 activation was performed by measuring the translocation of Smad4 from the cytoplasm into the nucleus, using confocal microscopy. We used a previously published protocol (), except the secondary antibody was an anti-rabbit antibody conjugated with Alexa Fluor 488 (Invitrogen), and nuclear staining was performed with TO-PRO-3 Iodide (Invitrogen). Approximately 100 cells were counted on each slide, and cells in the green (Smad4) and red (nuclear stain) overlay images that had a bright yellow fluorescence were counted as positive for nuclear translocation of Smad4.
Each experiment was performed at least 3 times with cells from independent tissue donors. Unless indicated otherwise, results are shown as the mean ± SD. Statistical analysis was performed using StatView software (SAS Institute). Simple linear regression was used to analyze the relationship between age and sulfate incorporation. The other results were analyzed by analysis of variance.
Articular chondrocytes isolated from the tissue of 13 normal donors were stimulated during culture in alginate beads. The donors ranged in age from 24 years to 81 years (mean ± SD 49.7 ± 16 years). Independent of age, proteoglycan synthesis was greatest in cultures treated with the combination of IGF-1 and OP-1 (average 429% change from control) followed by OP-1 alone (247%) and IGF-1 alone (24%) (Figure 1A). With increasing age, there was a significant decline in the response to IGF-1 (r = −0.63, P = 0.02) (Figure 1B) and to IGF-1 plus OP-1 (r = −0.74, P = 0.004) (Figure 1D), with a trend toward a decline in response to OP-1 alone (r = −0.51, P = 0.08) (Figure 1C).
We previously reported that oxidative stress increased with increasing age in normal human articular chondrocytes (), and that induction of oxidative stress in vitro impaired IGF-1–stimulated signaling in chondrocytes (). To determine whether oxidative stress could reduce proteoglycan synthesis in response to growth factors, chondrocytes were treated with tert-butyl hydroperoxide, a glutathione peroxidase substrate that induces oxidative stress by decreasing the cell content of reduced glutathione and increasing the amount of oxidized glutathione (). Treatment with tert-butyl hydroperoxide reduced both IGF-1–stimulated (Figure 2A) and OP-1–stimulated (Figure 2B) proteoglycan synthesis. In these experiments, we did not have sufficient cells to include tert-butyl hydroperoxide with IGF-1 plus OP-1 but would expect a similar reduction in response based on the signaling studies described below.
We examined the effects of treatment with tert-butyl hydroperoxide on the cell signaling response to the growth factors in order to determine the mechanism for proteoglycan synthesis inhibition. We previously demonstrated that proteoglycan synthesis stimulated by IGF-1 required activation of Akt, while ERK activation was inhibitory ([16, 17]). In preliminary experiments, we did not detect any stimulation of Akt or ERK in cells treated with OP-1 and therefore focused the first set of experiments on IGF-1. As we previously reported (), tert-butyl hydroperoxide pretreatment inhibited IGF-1–stimulated Akt phosphorylation. Here, we extended those findings to determine the effects of age. For these experiments, we chose age 50 years to separate younger and older adults, because OA becomes more common after age 50 years (), and the response to growth factors shown in Figure 1 declined at ∼50 years of age.
Relative to chondrocytes from older adults, those from younger adults had a higher level of Akt phosphorylation in response to IGF-1 and were much less affected by tert-butyl hydroperoxide (Figures 3A and B). In contrast, ERK phosphorylation in response to tert-butyl hydroperoxide treatment was greater in cells from older adults compared with younger adults, both in cells treated with tert-butyl hydroperoxide alone and in those treated with tert-butyl hydroperoxide prior to treatment with IGF-1 (Figures 3A and B). These results were consistent with increased susceptibility to oxidative stress with age resulting in an imbalance in Akt and ERK activity.
Next, we examined the effect of oxidative stress on phosphorylation of Smads 1, 5, and 8, which are upstream in the canonical signaling pathway stimulated by OP-1 (). We first tested an antibody that recognizes a phosphorylation site in all 3 of the Smads and therefore cannot distinguish Smad1 from Smad5 or Smad8, which run at the same location of gels, while the antibody to total Smad used as a control is specific for Smad1. The phosphorylation site is in the conserved SSXS motif in the C-terminal region of all 3 Smads and is associated with BMP signals that increase Smad activity (). As expected, we detected phosphorylation at this site in chondrocytes treated with OP-1 but not in cultures treated with IGF-1 or tert-butyl hydroperoxide (Figures 4A and B). The response to IGF-1 plus OP-1 was similar to the response to OP-1 alone, indicating that IGF-1 did not promote OP-1 signaling via phosphorylation of Smad1, Smad5, or Smad8. Treatment with tert-butyl hydroperoxide did not alter phosphorylation of Smads 1, 5, or 8 induced by OP-1 or IGF-1 plus OP-1 (Figures 4A and B).
There are additional phosphorylation sites in Smad1, in what has been termed the linker region due to its location between the MH-1 and MH-2 globular domains. Phosphorylation at these sites, which is mediated by MAPKs, has been shown to inhibit the nuclear accumulation and the transcriptional activity of Smad1 (). Immunoblotting of chondrocyte lysates (from a time course experiment) with an antibody that recognizes Ser206 phosphorylation of Smad1 in the linker region revealed increased phosphorylation in chondrocytes treated with tert-butyl hydroperoxide as early as 15 minutes after stimulation and in cultures treated with tert-butyl hydroperoxide prior to OP-1 (Figure 4B). The same lysates were immunoblotted for phosphorylation of Smad 1, 5, and 8 at the active site, which was strongest with OP-1 alone at 30 minutes and was not significantly altered by pretreatment with tert-butyl hydroperoxide (Figure 4B).
Additional experiments performed at the 30-minute time point revealed a consistent increase in phospho-Smad1 (Ser206) in cells treated with tert-butyl hydroperoxide prior to OP-1 when compared with IGF-1 or OP-1 alone (Figures 4C and D). These results demonstrated that tert-butyl hydroperoxide–induced oxidative stress increased the level of phosphorylation of Smad1 at an inhibitory site in the linker region without altering phosphorylation at the active site.
We next examined which of the MAPKs was responsible for Smad1 linker region phosphorylation. Treatment of chondrocytes with tert-butyl hydroperoxide resulted in a similar increase in phosphorylation of p38, ERK, and JNK (Figure 5A). When chondrocytes were pretreated with MAPK inhibitors, only the p38 inhibitor blocked tert-butyl hydroperoxide–induced Smad1 linker region phosphorylation (Figure 5B). As noted above, Smad1 linker region phosphorylation reduced nuclear accumulation of Smad1, which also occurred in cells treated with tert-butyl hydroperoxide prior to treatment with OP-1 (Figure 5C). Pretreatment with the p38 inhibitor blocked this effect of tert-butyl hydroperoxide.
Because the activation and translocation of Smad4 into the nucleus represent another important step downstream in the OP-1 signaling pathway, we determined whether oxidative stress induced by tert-butyl hydroperoxide altered this step. For these experiments, we used an anti-Smad4 antibody and confocal microscopy to measure nuclear translocation. Treatment of chondrocytes with OP-1 for 30 minutes resulted in a significant increase in nuclear Smad4 that was partially inhibited by tert-butyl hydroperoxide (Figure 6).
This study is the first in which primary cultures of human articular chondrocytes were used to demonstrate an age-related reduction in the ability of IGF-1 and IGF-1 plus OP-1 to stimulate proteoglycan synthesis. These 2 growth factors are important in the maintenance of cartilage homeostasis, and a loss of chondrocyte responsiveness could contribute to the development of age-related OA. Consistent with a previous study in which matrix production was measured after 21 days in alginate culture (), we observed that the combination of IGF-1 and OP-1 was more potent than either growth factor alone in stimulating proteoglycan synthesis, independent of donor age. The results of our current study, using normal chondrocytes from young and old tissue donors, combined with our previous work using chondrocytes from normal and OA joints () suggest that the reduced anabolic response to IGF-1 and to OP-1 can be attributable to disrupted cell signaling caused by oxidative stress.
Oxidative stress resulting from elevated levels of reactive oxygen species (ROS) relative to levels of antioxidants is thought to be a major contributor to many of the chronic diseases associated with aging (), including OA ([26, 27]). Using cartilage obtained from tissue donors, we previously observed an age-related decrease in the amount of reduced relative to oxidized glutathione, which is consistent with age-related oxidative stress (). There is evidence for other changes in the antioxidant capacity of chondrocytes with aging, including a decrease in catalase noted in rat cartilage () and a decrease in the mitochondrial superoxide dismutase 2 noted in human chondrocytes (). Similar to our findings of increased oxidative stress induced by tert-butyl hydroperoxide in chondrocytes from older adults, Jallali et al () demonstrated that ROS levels induced by menadione were higher in chondrocytes from older rats compared with younger rats.
In addition to causing damage to proteins, lipids, and DNA, elevated levels of ROS can alter the activity of specific cell signaling pathways that are critical to maintaining normal cell function (). Intracellular signaling pathways activated by IGF-1 via the IGF-1 receptor include the IRS-1/phosphatidylinositol 3-kinase/Akt pathway and the Shc/Grb2/Sos/Ras/Raf/MEK/ERK pathway. We previously demonstrated that the ability of IGF-1 to stimulate chondrocyte matrix production, including proteoglycans and type II collagen, requires activation of Akt, while ERK is inhibitory ([16, 17]). Induction of oxidative stress with tert-butyl hydroperoxide was shown to alter the balance of the Akt-to-ERK phosphorylation ratio to favor ERK, which results in the inhibition of proteoglycan and collagen production. Here, we show that when compared with chondrocytes from younger donors, chondrocytes from older donors exhibited lower levels of Akt phosphorylation in response to IGF-1, which correlates with the reduced proteoglycan synthesis. In addition, cells from older donors treated with tert-butyl hydroperoxide exhibited greater inhibition of IGF-1–stimulated Akt phosphorylation accompanied by increased ERK phosphorylation, consistent with the older chondrocytes being more sensitive to oxidative stress induction.
Although we observed a significant decline with age in proteoglycan synthesis in chondrocytes treated with IGF-1 and IGF-1 plus OP-1, we noted only a trend (P = 0.08) toward a reduced response with OP-1 alone. Chondrocyte proteoglycan synthesis has been reported to decline with age in response to the related BMP family members BMP-6 () and transforming growth factor β (), but OP-1 had not been previously studied. We did observe that induction of oxidative stress using tert-butyl hydroperoxide inhibited OP-1–stimulated proteoglycan synthesis.
Unlike IGF-1, little is known about ROS-regulated BMP signaling. There is some evidence that ROS generated by NADPH oxidases can promote BMP-2–stimulated gene transcription in bone and vascular tissue ([32, 33]), but effects on specific BMP pathway signaling proteins have not been investigated, and we are not aware of previous studies that have examined an effect of ROS on OP-1 signaling. We could not detect a significant effect of oxidative stress on Smad1/5/8 phosphorylation at the BMP-activating site but, interestingly, did detect increased phosphorylation at the Smad1 linker region at Ser206, which has been found to inhibit Smad1 accumulation and activity in the nucleus (). Phosphorylation at this site has been shown to be mediated by members of the MAPK family, including ERK (), which we observed to be activated in chondrocytes by oxidative stress (Figure 3), and is a major contributor to inhibition of IGF-1–stimulated proteoglycan synthesis (). However, although tert-butyl hydroperoxide induced phosphorylation of ERK as well as p38 and JNK, inhibitor experiments revealed that p38 mediated Smad1 linker region phosphorylation, and this was associated with reduced nuclear accumulation of Smad1 in response to OP-1. In previous work, IL-1β was shown to inhibit chondrocyte OP-1 signaling via linker region phosphorylation mediated by JNK and p38 (). These results indicate that MAPK activation resulting in Smad1 inhibition through phosphorylation in the linker region is an important mechanism for inhibition of chondrocyte OP-1 activity by oxidative stress and cytokines.
In addition, we measured a modest (14%) decrease in Smad4 translocation into the nucleus in chondrocytes treated with tert-butyl hydroperoxide, suggesting that a second mechanism for oxidative stress–induced inhibition of OP-1 may be involved. This level of inhibition by itself may not be sufficient to explain the almost complete inhibition of OP-1–stimulated proteoglycan synthesis by tert-butyl hydroperoxide but may be sufficient when combined with inhibition of Smad1 activity. The Smads function as a complex to regulate gene transcription, although Smad1 can regulate some transcriptional activity on its own ().
In summary, using articular chondrocytes isolated from the normal joints of young and older adult tissue donors, we observed an age-related reduction in the ability of IGF-1 and IGF-1 plus OP-1 to stimulate proteoglycan synthesis, with a trend toward a reduced response to OP-1 alone. Chondrocytes from older donors were more susceptible to the effects of oxidative stress on IGF-1 signaling, resulting in an imbalance in Akt and ERK phosphorylation. Oxidative stress was also shown to inhibit OP-1 activity, likely through p38-mediated phosphorylation of Smad1 in the inhibitory linker region, resulting in reduced nuclear accumulation of Smad1 combined with reduced Smad4 nuclear translocation. These findings support the hypothesis that oxidative stress resulting in a reduced growth factor response may be an important contributing factor to the development of age-related OA.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Loeser had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Loeser, Gandhi, Long, Yin, Chubinskaya.
Acquisition of data. Loeser, Gandhi, Long, Yin, Chubinskaya.
Analysis and interpretation of data. Loeser, Gandhi, Long, Yin, Chubinskaya.
We would like to thank National Disease Research Interchange (Philadelphia, PA), the Gift of Hope Tissue and Organ Donor Network (Elmhurst, IL), and the donor families for providing human donor tissue. We also thank Dr. David Rueger (Stryker Biotech) for providing OP-1.