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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

The chondrocyte response to insulin-like growth factor 1 (IGF-1) is reduced with aging and in osteoarthritis (OA). IGF-1 signals through the phosphatidylinositol 3-kinase/Akt pathway. TRB3, a tribbles homolog, has been shown to inhibit IGF-1–mediated activation of Akt in HEK 293 cells. This study was undertaken to determine if TRB3 is expressed in chondrocytes, and whether the chondrocyte response to IGF-1 is reduced by TRB3.

Methods

Human articular cartilage was obtained from normal tissue donors and from patients with OA at the time of knee replacement surgery. TRB3 was assessed in the tissue samples by reverse transcription–polymerase chain reaction, immunoblotting, and immunohistochemistry. Overexpression of TRB3 was induced by transient transfection to determine the effects of TRB3 on cell survival and proteoglycan synthesis.

Results

TRB3 messenger RNA was detected in normal human chondrocytes. TRB3 protein levels were low in cells from normal cartilage but significantly increased in cells from OA cartilage. Incubation with 2 agents that induce endoplasmic reticulum stress, tunicamycin and thapsigargin, increased TRB3 levels in normal cells. Overexpression of TRB3 inhibited Akt phosphorylation and reduced chondrocyte survival and proteoglycan synthesis.

Conclusion

These results are the first to demonstrate that TRB3 is present in human chondrocytes, and that the level of TRB3 is increased in OA cartilage and in isolated OA chondrocytes. Because it is an inhibitor of Akt activation, elevated TRB3 production could play a role in the increased cell death and reduced response to IGF-1 observed in OA cartilage.

Aging does not directly cause osteoarthritis (OA); however, it is the most important risk factor in the development of the disease (1). A major contributing factor in the development of OA is a loss of the anabolic and catabolic homeostasis maintained by chondrocytes, leading to a loss of articular cartilage. The balance of anabolic and catabolic processes in cartilage depends on the local activity of regulatory factors such as cytokines and growth factors (2). Insulin-like growth factor 1 (IGF-1) has the ability to stimulate matrix synthesis (3–5), promote chondrocyte survival (6), and inhibit specific catabolic pathways (7, 8). There is evidence that chondrocytes have a decreased response to IGF-1 with aging and also in OA (3, 9–12), but the mechanisms involved are incompletely understood.

IGF-1 stimulates the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway as well as the Ras/Raf/MEK/ERK pathway, by acting through the IGF-1 receptor (13). The activation of the IGF-1 receptor results in the activation of Shc and members of the insulin receptor substrate (IRS) family. After the phosphorylation of IRS and Shc, both the PI 3-kinase cascade and the ERK cascade are activated. The activation of PI 3-kinase leads to the activation of Akt (also called protein kinase B [PKB]), a serine/threonine kinase involved in cell survival (14, 15), and p70 S6 kinase, a serine/threonine kinase implicated in protein synthesis (16).

Activation of PI 3-kinase, but not of ERK, by IGF-1 is required for the stimulation of chondrogenesis and proteoglycan (PG) synthesis by IGF-1 that occurs in mesenchymal cells (17), as well as for the IGF-1–mediated stimulation of PG synthesis that occurs in adult human chondrocytes (18). Although IGF-1 acts as an autocrine survival factor for chondrocytes cultured at low density (6, 16), the required role of PI 3-kinase and/or ERK in IGF-1–mediated cell survival is not clear and may depend on the experimental conditions. Inhibition of the ERK pathway has been reported to reduce survival of chondrocytes plated on collagen (19) but not on fibronectin (20). Inhibition of PI 3-kinase reduced survival of chondrocytes treated with sodium nitroprusside (17) but did not reduce survival of chondrocytes in high-density monolayer or alginate cultures (18).

It has been shown that the tribbles homolog TRB3 (also known as SKIP3, NIPK, or SINK) inhibits IGF-1 as well as insulin activation of Akt/PKB in the liver (21). TRB3 has been classified as a pseudokinase because it lacks the Asp-Phe-Gly motif in subdomain VII of the kinase domain (22). This truncated kinase domain, which lacks the adenosine 5′-triphosphate binding site, allows TRB3 to act as a negative modulator of Akt through dose-dependent inhibition of Akt/PKB phosphorylation at Ser473 and Thr308 in HEK 293 cells (21). TRB3 has also been shown to inhibit the phosphorylation of Akt at Thr308 in FGC-4 cells (23) and to block the insulin activation of p70 S6 kinase, Akt, mammalian target of rapamycin, p70 S6 ribosomal protein, and 4EBP1 in mouse hepatocytes (24). However, a contradictory study in rat hepatocytes could not find evidence that TRB3 inhibits the insulin signaling pathway (25). This discrepancy suggests that the function of TRB3 may be dependent on the cell type and/or experimental conditions.

Investigations into the regulation of TRB3 expression have focused on the role of endoplasmic reticulum (ER) stress. Agents that induce ER stress have been found to increase TRB3 messenger RNA (mRNA) levels in HepG2 cells (26) as well as in p53-null osteosarcoma cells, MCF-7 breast cancer cells, H1299 lung cancer cells, and DU145 prostate cancer cells (27). TRB3 protein levels have also been shown to be increased in conditions of increased ER stress in HepG2, HEK 293, and A375 cells (28). Whether chondrocytes express TRB3 under normal conditions or only under conditions of ER stress has not been examined.

Because of the potential of TRB3 to serve as an inhibitor of IGF-1 signaling, we sought to determine, first, if chondrocytes express TRB3, and then, whether the expression of TRB3 changes with aging or in the presence of OA. We also examined whether an increase in ER stress would increase chondrocyte expression of TRB3, and whether an increased expression of TRB3 might contribute to a decrease in PG synthesis and cell survival. Our results suggest that increased production of TRB3 in OA cartilage may play an important role in the inhibition of chondrocyte IGF-1 signaling.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Reagents.

Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, phosphate buffered saline (PBS), and antibiotics were purchased from Gibco BRL (Gaithersburg, MD), while fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Pronase was purchased from Calbiochem (San Diego, CA), collagenase P was from Boehringer Mannheim (Mannheim, Germany), Keltone LVCR sodium alginate was from Kelco (Chicago, IL), and 35S-sulfate was from Amersham Biosciences (San Francisco, CA). Phosphospecific and non-phosphospecific antibodies directed against Akt and ERK were purchased from Cell Signaling Technology (Beverly, MA). The antibody directed against TRB3 was custom-made against a peptide (sequence SRKKRLELDDNLDTERPVC) from Quality Controlled Biochemicals (Hopkinton, MA). IGF-1 was purchased from Austral Biologicals (San Ramon, CA). The TRB3- and hemagglutinin (HA)–tagged Akt plasmids were provided by Dr. Mark Montminy from the Salk Institute (La Jolla, CA). The PI 3-kinase dominant-negative plasmid (PI3KDN) was provided by Dr. Shu Chien at the University of California, San Diego. The pcDNA 3.0 empty vector was purchased from Invitrogen (Carlsbad, CA), and the red fluorescent protein (RFP) plasmid was purchased from Clontech (Mountain View, CA).

Tissue acquisition.

Human ankle (talar) articular cartilage was obtained from normal-appearing cartilage tissue, collected from donors within 48 hours of death through the National Disease Research Interchange (Philadelphia, PA) or the Gift of Hope Organ and Tissue Donor Network (Elmhurst, IL). Human OA knee articular cartilage was obtained from tissue that was removed from patients during knee replacement surgery, in accordance with institutional guidelines and review board approval. Each donor specimen was graded for degenerative changes based on the 5-point Collins scale, as modified by Muehleman et al (29). Unless stated otherwise, cells from tissue samples with a Collins grade 0 or Collins grade 1 were used in all experiments.

Protein extraction and TRB3 immunoblotting.

Full-thickness cartilage was removed from all surfaces of the tali or from the medial and lateral condyle surfaces of the femur. The cartilage slices were rinsed with cold PBS and then frozen dry at −80°C. The frozen slices were placed in liquid nitrogen and ground using a mortar and pestle. The ground cartilage powder was placed in cell lysis buffer containing 20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin. This mixture was allowed to rotate end-over-end for 4 hours at 4°C, and then was centrifuged for 10 minutes at 1,400 revolutions per minute. The supernatants (containing soluble proteins from cartilage extracts) were centrifuged twice more to remove any particulates, and total protein was measured using a bicinchoninic acid kit (Pierce, Rockford, IL).

Samples with equal amounts of total protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose for immunoblotting with the anti-TRB3 antibody or with an antibody to β-actin as a control. Immunoreactivity was detected with enhanced chemiluminescence (Amersham Biosciences). Densitometry was performed on immunoblots after scanning, with results analyzed using Kodak 1D 3.6 software (Eastman Kodak, Rochester, NY) in order to analyze the regions of interest.

Chondrocyte isolation and culture.

Full-thickness cartilage was removed from all surfaces of the tali or from the medial and lateral condyle surfaces of the femur. As a control, full-thickness tissue was collected from all surfaces of bovine metacarpophalangeal joints. Cartilage slices were digested in a sequential manner with 0.2% Pronase for 1 hour, and then digested overnight with 0.025% collagenase as described previously (30). For experiments using monolayer cultures, short-term high-density monolayers were used, in which 6-well plates were plated at a concentration of 2 ml/well, at 1 × 106 cells/ml, in DMEM with 10% FBS. The cells were incubated at 37°C in 5% CO2 until reaching confluence (usually within 5–7 days).

Cell lysates were prepared using the cell lysis buffer described above. The cells were scraped, transferred into microcentrifuge tubes, agitated end-over-end for 30 minutes at 4°C, and centrifuged at 14,000g for 10 minutes. The supernatants were then removed and measured for total protein by immunoblotting as described above.

Immunohistochemistry.

Sections of cartilage from human knee joints that were embedded in 10% formalin-fixed paraffin were deparaffinized and rehydrated. A protein block (Serum-Free Protein Block; Dako, Carpinteria, CA) was applied for 10 minutes prior to incubation with the primary antibody. Anti-TRB3 antibodies were diluted 1:500 prior to being added to the culture, and antibody-treated sections were incubated at 4°C overnight. Antibody binding was detected using biotinylated anti-rabbit link (BioGenex, San Ramon, CA), diluted 1:20, for 30 minutes at 33°C. Alkaline phosphatase label (BioGenex), diluted 1:20, was used for 30 minutes at 3°C. Vector red (Vector Laboratories, Burlingame, CA) was used for visualization, and sections were counterstained with Mayer's hematoxylin (Dako). Negative Control Supersensitive Rabbit Serum for SuperSensitive Antibodies (BioGenex) was substituted for the primary antibody for the negative control sections.

Chondrocyte stimulation and analysis of ER stress.

Confluent monolayer cultures were placed in DMEM/Ham's F-12 medium supplemented with 10% FBS, and then stimulated with 2 μg/ml tunicamycin or 2 μM thapsigargin for 6 or 24 hours. After stimulation, the medium was removed and the cells were washed once with ice-cold PBS containing 0.1 mM Na3VO4. Cell lysates were immediately prepared by solubilization in cell lysis buffer as described above.

Transfection.

Cell transfection was performed using a human chondrocyte nucleofector kit according to the manufacturer's instructions (Amaxa Biosystems, Gaithersburg, MD); a previously described nucleofection method was used (31), with some modification. Briefly, after plating in monolayers, normal human chondrocytes were allowed to recover for 72 hours. The cells were then treated with medium containing 0.2% Pronase/0.025% collagenase and 10% FBS for 3 hours to detach the cells. The medium was centrifuged and the cell pellet was washed twice with 10% serum and once with PBS. Following the wash with PBS, the cells were resuspended in the nucleofector solution, and plasmid DNA was added. Chondrocytes were routinely transfected with 5 μg of total plasmid DNA in the Amaxa Nucleofector machine, using program U-24. After transfection, the cells from each cuvette were immediately placed in 900 μl of culture medium containing 20% FBS. This solution was mixed and then placed into 1 ml culture medium containing 20% FBS per well of a 12-well plate, which was then incubated at 37°C in 5% CO2.

The cells were allowed to recover from the nucleofection process for 24 hours in 20% FBS, changed to medium containing 10% FBS for another 24 hours, and then finally serum starved overnight. The cells were treated with 100 ng/ml IGF-1 for 30 minutes, and then whole cell lysates were made as described above. For the immunoprecipitation experiments, the lysates were agitated end-over-end for 30 minutes at 4°C, and then centrifuged at 14,000g for 10 minutes. The supernatants were removed and incubated with 40 μl 1:1 ImmunoPure Immobilized Protein A (Pierce) for 1 hour at room temperature, with end-over-end agitation. The slurry was centrifuged at 5,000 rpm for 5 minutes, the supernatant was collected, and 5 μl of anti-HA antibody was added. The supernatant/antibody mixture was then incubated with gentle agitation overnight at 4°C. Forty microliters of 1:1 ImmunoPure Immobilized Protein A beads was added to the supernatant/antibody mixture, followed by gentle agitation overnight at 4°C. The slurry was centrifuged at 5,000 rpm for 5 minutes, the supernatant was discarded, and the beads were washed 6 times with radioimmunoprecipitation assay buffer. Forty microliters of Laemmli buffer was added to the beads, and the Laemmli/bead mixture was boiled for 5 minutes and centrifuged. The solubilized proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunoblot analysis using an antibody directed against phosphorylated Akt/Thr308.

Cell survival experiments.

Normal human articular chondrocytes were transfected with TRB expression plasmids, RFP as a control, a dominant-negative form of PI 3-kinase (PI3KDN), or HA-tagged Akt. Twenty-four hours after the nucleofection process, the medium was changed to DMEM/Ham's F-12 with 10% FBS. After 24 hours in medium with 10% serum, the cells were lifted from their respective wells with trypsin treatment, washed, and transferred to serum-free suspension cultures. To observe IGF-1–mediated cell survival in a low-density suspension culture, the cells were placed into alginate beads at a density of 2 × 105 cells/bead and cultured in alginate as previously described (6), with some modification. Briefly, freshly isolated cells were suspended in 0.9% NaCl containing low-viscosity alginate (1.2%) at a density of 2 × 106 cells/ml, and this was added, in a dropwise manner, through a 22.5-gauge needle into a 102-mM CaCl2 solution. Beads were allowed to gel in the CaCl2 for 10 minutes and then washed 3 times with DMEM/Ham's F12 medium. The beads were placed in serum-free DMEM/Ham's F-12 medium, and cell death was determined after 72 hours using a Live/Dead cell assay (Molecular Probes, Eugene, OR) as described previously (20). The percentage of cells that remained alive after treatment was measured in triplicate, with at least 100 cells counted for each data point.

PG synthesis experiments.

Incorporation of 35S-sulfate was used to measure PG synthesis in monolayer cultures of normal human chondrocytes, which were isolated and cultured at high density as described above. The cells were treated with IGF-1 overnight and then pulsed with 35S-sulfate for 4 hours. Incorporation of 35S-sulfate was measured in the medium as well as in the cell pellet after Alcian blue precipitation, carried out as previously described (18, 32).

Statistical analysis.

Results comparing the mean values between multiple groups were analyzed using a one-way analysis of variance with a post hoc Bonferroni correction, which was performed using Windows-based StatView software (SAS Institute, Cary, NC). Experiments assessing 35S-sulfate incorporation were performed in triplicate. P values less than 0.05 were considered statistically significant for all analyses.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Expression of TRB3 by chondrocytes.

TRB3 expression by human articular chondrocytes was initially assessed using reverse transcription–polymerase chain reaction. TRB3 mRNA was detected in normal articular chondrocytes, with the lowest mRNA levels found in cartilage tissue from an 8-year-old donor (Figure 1A). In addition, immunoblot analysis was used to determine whether chondrocytes synthesize the TRB3 protein. TRB3 protein was detected in chondrocytes isolated from either the knee or ankle joint of donors, with higher levels noted in cells from Collins grade 4 knee tissue from an 83-year-old donor as compared with grade 0 tissue from the ankle of the same donor and grade 0 knee and ankle tissue from a 28-year-old donor (Figure 1B).

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Figure 1. Expression of TRB3 by chondrocytes. A, For reverse transcription–polymerase chain reaction (RT-PCR) analysis of TRB3 messenger RNA, human chondrocytes were isolated from donors of different ages and plated in monolayer culture. At confluency, the RNA was isolated from these cells for RT-PCR analysis. The cDNA products were stained with ethidium after separation on an agarose gel; GAPDH expression was used as a control. B, For immunoblot analysis of TRB3, chondrocytes were isolated from matched pairs of knee (Collins grade 4) and ankle (Collins grade 0) cartilage from an older donor (age 83 years) and knee and ankle (Collins grade 0) cartilage from a younger donor (age 28 years). Cell lysates, after overnight incubation in serum-free medium, were used for immunoblot analysis with the anti-TRB3 antibody; immunoblotting for β-actin was used as a protein loading control. Densitometry was used to quantitate the band intensity (indicated below the blots as the ratio of TRB3 to β-actin).

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To determine whether the protein levels of TRB3 will increase with aging and/or with cartilage degeneration, TRB3 was measured in protein extracts derived from cartilage samples that had been removed from human tissue donors of different ages. In these extracts, which displayed a range of Collins grades and were derived directly from cartilage rather than in isolated cells, the TRB3 bands were fainter (Figures 2A and B) than those seen in cell lysates (Figures 1A and B and 2C). Somewhat higher TRB3 levels, quantified by densitometry and normalized to β-actin, were noted in the extracts from normal ankle cartilage of 4 older adults (mean ± SEM TRB3:β-actin 0.63 ± 0.1) when compared with normal ankle cartilage of 4 younger adults (TRB3:β-actin 0.37 ± 0.6) in extracts on the same blot (Figure 2A), but this difference in TRB3 levels between age groups did not reach statistical significance (P = 0.7 by t-test). There was no difference in TRB3 levels by Collins grade among tissue samples from older adults, whose cartilage samples ranged in Collins grades from 0 to 3 (Figure 2B).

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Figure 2. Effect of donor age, tissue grade, and osteoarthritis (OA) on TRB3 protein levels. A, Protein extracts were derived directly from ankle tissue graded 0–1 (on the Collins scale of degenerative changes) that was obtained from younger and older tissue donors without a known history of OA. Immunoblot analysis was performed using an anti-TRB3 antibody; anti–β-actin antibody was used as a loading control. B, Protein extracts were analyzed as described in A in samples of ankle cartilage obtained from older adults whose tissue displayed Collins grade 0–3 degenerative changes. The band intensity in A and B, determined by densitometry as the ratio of TRB3 to β-actin, is shown under the blots. C, Cell lysates were prepared from cultured untreated human chondrocytes from age-matched normal tissue donors (Collins grade 1) and from tissue harvested at the time of knee joint replacement from patients with OA. The lysates were used for immunoblot analysis with an anti-TRB3 antibody; anti–β-actin antibody was used as a loading control. An additional cell lysate derived from human chondrocytes transfected with a TRB3 expression vector was used as a positive control.

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These results suggest that the measurement of TRB3 by this method of immunoblotting proteins extracted from cartilage was not sensitive enough to detect significant differences related to age or grade. We therefore examined TRB3 in cell lysates, which had been prepared from cells isolated from the cartilage of age-matched normal donors and patients with OA. A striking increase in TRB3 levels was noted in the cells from OA cartilage (Figure 2C). In this experiment, a cell lysate made from normal chondrocytes transfected with a TRB3 expression construct was included as a positive control.

To confirm these results and to examine the location of the TRB3 in human cartilage, immunohistochemistry was performed on intact cartilage slices, using an anti-TRB3 antibody. Cartilage tissue from age-matched normal donors contained very low numbers of TRB3-immunopositive chondrocytes, whereas OA tissue with degenerative changes contained many TRB3-immunopositive cells (Figure 3). The majority of the TRB3 immunostaining was noted in cells in the more superficial regions, in which chondrocyte clusters were prominent and the matrix was fibrillated.

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Figure 3. Immunohistochemical staining for TRB3 in normal and osteoarthritic (OA) knee tissue. Immunohistochemistry was done on tissue from a normal knee joint (A and C) and tissue obtained at the time of joint replacement from a patient with OA (B and D). The sections were immunostained with anti-TRB3 at a 1:500 dilution. An alkaline phosphatase detection system was used, and the product was visualized with Vector red. The negative control images (not shown), in which TRB3 was not detected, were similar to the images from normal tissue. Bars in A and B = 250 μm; bars in C and D = 100 μm.

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Promotion of TRB3 expression by ER stress.

ER stress has been shown to stimulate TRB3 expression in a number of cell types (26–28). ER stress has also been shown to inhibit chondrocyte growth, down-regulate expression of the cartilage matrix proteins type II collagen and aggrecan, and induce chondrocyte apoptosis (33), suggesting that this condition might play a role in the development of OA. We induced ER stress in normal primary chondrocytes using tunicamycin, an agent that blocks protein glycosylation, and thapsigargin, an agent that depletes Ca2+ stores. GADD-153 was used as a marker for ER stress. Both treatments increased the protein levels of TRB3 (Figure 4).

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Figure 4. Role of endoplasmic reticulum (ER) stress in TRB3 expression. Normal human chondrocytes cultured in monolayer were treated with the ER stress–inducing agents tunicamycin (2 μg/ml) or thapsigargin (2 μM), or with 0.001% DMSO for the indicated times. Cell lysates were then assessed by immunoblot analysis with an anti-TRB3 antibody or with an anti–GADD-153 antibody (ER stress marker) or anti–β-actin antibody as a loading control.

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Association of TRB3 overexpression with decreased chondrocyte survival and inhibition of IGF-1 stimulation of Akt phosphorylation.

Since the inhibition of Akt has been shown to be a causal mediator of cell death (34), the blocking of Akt activation by TRB3 might induce cell death under conditions in which IGF-1 stimulation of Akt is necessary for chondrocyte survival. We had previously found that chondrocytes cultured in suspension in serum-free medium at low density required autocrine signaling by IGF-1 to survive (6); therefore, similar culture conditions were used in the present study to test the effects of TRB3.

Overexpression of TRB3 reduced the survival of normal human chondrocytes by almost 40%, as compared with that of cells transfected with a control (RFP) plasmid. Furthermore, the addition of IGF-1 to the suspension cultures of cells overexpressing TRB3 was not able to improve cell survival (Figure 5A). However, cell death induced by TRB3 overexpression could be prevented by cotransfection of a construct designed to overexpress Akt, consistent with the notion that TRB3 overexpression causes cell death through inhibition of endogenous Akt activation.

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Figure 5. Role of TRB3 overexpression in reducing chondrocyte survival and insulin-like growth factor 1 (IGF-1)–stimulated Akt phosphorylation. A, Human chondrocytes isolated from articular cartilage ankle tissue obtained from normal donors were nucleofected with plasmid constructs to express TRB3, hemagglutinin (HA)–tagged Akt, red fluorescent protein (RFP) as a control (expression set at 100%), or a dominant-negative phosphatidylinositol 3-kinase (PI3KDN). After 48 hours, cells were harvested and placed into alginate beads at 2 × 105 cells/bead and cultured in serum-free medium for 72 hours with either 50 ng/ml IGF-1 or vehicle as a control. A Live/Dead survival assay was then performed; bars show the mean and SD results from 3 experiments with cells from different donors. ∗ = P < 0.0001 versus RFP control; # = P < 0.0001 versus RFP plus IGF-1; ∗∗ = P = 0.001 versus TRB3; ∗∗∗ = P = 0.03 versus TRB3 plus IGF-1. B, Normal human chondrocytes were transfected with an HA-tagged Akt expression construct or cotransfected with HA-tagged Akt and TRB3 expression constructs. After 48 hours, cultures were serum starved overnight. Wells were treated with 100 ng/ml IGF-1 for 30 minutes, and then all wells were lysed. Immunoprecipitation was performed using an anti-HA antibody, and the lysates were immunoblotted using an anti–phosphorylated Akt antibody (P-AKT). The membrane was stripped and reprobed using an anti-Akt antibody (T-AKT). Results show a representative blot from 1 of 4 experiments.

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PI 3-kinase activates Akt in response to the activity of IGF-1. Therefore, as an additional control to support the role of IGF-1–mediated Akt activation in promoting cell survival under these conditions, we transfected chondrocytes with a dominant-negative PI 3-kinase construct, PI3KDN. After the addition of PI3KDN, we observed cell death similar to that observed in response to overexpression of TRB3 induced by the TRB3 construct.

In order to determine whether overexpression of TRB3 inhibits Akt phosphorylation, human chondrocytes were either transfected with a plasmid expressing HA-tagged Akt or cotransfected with HA-tagged Akt and TRB3 constructs, followed by stimulation with IGF-1. The HA-tagged Akt construct was phosphorylated at Thr308 in the presence of IGF-1. Cotransfection of chondrocytes with HA-tagged Akt and the TRB3 construct inhibited phosphorylation of Akt (Figure 5B).

Inhibition of IGF-1–induced PG synthesis by TRB3.

Previous work has shown that stimulation of the PI 3-kinase/Akt pathway is required for the production of PG by chondrocytes in response to IGF-1 (18). To examine whether TRB3 overexpression affects PG synthesis, normal human chondrocytes (from Collins grade 0 or grade 1 tissue) were transfected with either the TRB3 construct or the pcDNA 3.0 empty vector as a control. Because overexpression of TRB3 in low-density serum-free alginate cultures resulted in cell death, the studies of PG synthesis were performed with cells in high-density monolayer and with 5% FBS added to the medium, which promoted cell survival under these conditions. PG synthesis was measured by 35S-sulfate incorporation, and the results were normalized to DNA content in order to control for any loss of cells. In this culture system, overexpression of TRB3 that was induced by the TRB3 construct, but not the control vector, reduced the basal synthesis of PG and completely blocked the IGF-1 stimulation of PG synthesis (Figure 6).

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Figure 6. Role of TRB3 overexpression in decreasing insulin-like growth factor 1 (IGF-1) stimulation of proteoglycan (PG) synthesis. Normal human chondrocytes were transfected with 5 μg pcDNA 3.0 empty vector or 5 μg TRB3 overexpression plasmids. The cells were allowed to recover from the transfection in monolayer culture and then placed in medium containing 5% serum. IGF-1 (100 ng/ml) was added to the wells for an overnight treatment, and then 35S-sulfate incorporation (a measure of PG synthesis) was assessed as the counts per minute normalized to the DNA concentration of the samples. Bars show the mean and SEM percentage in 3 experiments with cells from different donors relative to that of the unstimulated control (set at 100%). ∗ = P = 0.02 versus control; ∗∗ = P = 0.02 versus control + IGF-1.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

This study is the first to show that chondrocytes express TRB3, a protein that has previously been identified in other cell types as an inhibitor of insulin- and IGF-induced activation of Akt/PKB (21, 23, 24). Activation of Akt by PI 3-kinase is required for the IGF-1–mediated stimulation of chondrocyte PG synthesis (18) as well as for cell survival under conditions dependent on autocrine IGF-1 signaling (6). The PI 3-kinase/Akt pathway has also been shown to be required for transforming growth factor β induction of tissue inhibitor of metalloproteinases 3 expression (35). In our study, a significantly greater amount of TRB3 was detected in OA chondrocytes than was detected in age-matched cells from normal-appearing cartilage. Overexpression of TRB3 in normal chondrocytes reduced cell survival and blocked IGF-1 stimulation of PG synthesis. Together, these findings suggest that an increased level of TRB3 in OA chondrocytes could contribute to the decreased response of OA chondrocytes to IGF-1 that has been previously observed (3, 9), and might contribute to the cell death and matrix loss seen during the development of OA.

A potential mechanism for increased expression of TRB3 is ER stress (26–28). Chondrocytes exhibit a variety of behaviors related to different stressors, including osmotic stress, nutrient deprivation, oxidative stress, and mechanical stress (36–38), which can potentially impair ER function, resulting in ER stress (39). Recently, it has been shown that ER stress can be induced in chondrocytes under certain conditions (33, 40, 41). We found that an increase in ER stress induced by either tunicamycin or thapsigargin caused an increase in chondrocyte TRB3 levels. ER stress in chondrocytes has been shown to decrease the expression of cartilage matrix genes (40), and prolonged ER stress leads to apoptosis (33, 41). The present findings demonstrating that overexpression of TRB3 can inhibit IGF-1–mediated PG synthesis and reduce chondrocyte survival suggest a potential mechanism for TRB3 as a mediator of the effects of ER stress in chondrocytes.

TRB3 is a pseudokinase that binds Akt and, thus, prevents the phosphorylation of Akt, a process that is required for Akt to be active (21). The results of our study confirmed that TRB3 blocks the IGF-1–stimulated phosphorylation of Akt in human chondrocytes, similar to previous observations in other cell types (21, 23, 24). Inhibition of Akt activation by TRB3 was associated with reduced chondrocyte survival in low-density, serum-free alginate cultures, an experimental condition in which we have previously demonstrated that IGF-1 autocrine signaling promotes chondrocyte survival (6). This is consistent with other reports showing that TRB3 caused cell death in several different cell types (28, 42, 43).

The IGF-1/PI 3-kinase/Akt pathway is a well-characterized survival pathway in many cell types (14, 15, 44, 45). The PI 3-kinase inhibitor wortmannin was previously shown to decrease the survival of rabbit chondrocytes (46). In the present study, we found that overexpression of a PI 3-kinase dominant-negative construct decreased survival of human chondrocytes. These findings suggest that increased levels of TRB3 cause cell death through inhibition of the IGF-1/PI 3-kinase/Akt survival pathway. When TRB3 was overexpressed in high-density monolayer in medium supplemented with 5% FBS, the negative effects of TRB3 on cell survival could be overcome. Using these conditions, we determined that increased TRB3 levels could inhibit PG synthesis. This is consistent with previous studies in which it was shown that the PI 3-kinase/Akt pathway was required for IGF-1 stimulation of PG synthesis by chondrocytes (18). The finding of increased levels of TRB3 in OA chondrocytes suggests that TRB3 could play a role in the reduced response to IGF-1 that has been noted previously in OA (10). It was less clear whether TRB3 levels were increased with older age in subjects whose cartilage had no OA-related changes; therefore, we do not know, at this time, whether TRB3 also contributes to the age-related decline in the IGF-1 response.

In this study, we were thus able to demonstrate TRB3 production by human chondrocytes and to show that TRB3 levels are dramatically increased in cells from OA cartilage. TRB3 blocks the IGF-1–stimulated phosphorylation of Akt in chondrocytes. Moreover, overexpression of TRB3 causes chondrocyte cell death by inhibiting the PI 3-kinase/Akt pathway in culture conditions in which this pathway is important for chondrocyte survival. Overexpression of TRB3 also inhibits the IGF-1–stimulated PG synthesis by chondrocytes. It was also seen that an increase in ER stress can up-regulate the production of TRB3 in chondrocytes. Together, these results suggest that TRB3 may play an important role in the reduced response to IGF-1 in OA chondrocytes, and therefore TRB3 may contribute to the imbalance in anabolic and catabolic activity that is characteristic of OA. Further studies will be necessary to determine whether inhibition of TRB3 during the development of OA can slow the progression of cartilage loss.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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 design. Cravero, Loeser.

Acquisition of data. Cravero, Carlson, Im, Yammani, Long.

Analysis and interpretation of data. Cravero, Carlson, Yammani, Long, Loeser.

Manuscript preparation. Cravero, Carlson, Loeser.

Statistical analysis. Cravero.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to thank Dr. Mark Montminy for supplying the TRB3 and HA-tagged Akt plasmids, Dr. Shu Chien for providing the PI 3-kinase dominant-negative plasmid, and Carol Pacione and Anne Undersander for technical assistance. We gratefully acknowledge the Gift of Hope Organ and Tissue Donor Network, the National Disease Research Interchange, and the donor families for providing the donor tissue, and Dr. David Martin for assistance in collecting the OA tissue.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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