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Iowa Orthop J. 2000; 20: 1–10.
PMCID: PMC1888750

Damage Control Mechanisms in Articular Cartilage

The Role of the Insulin-like Growth Factor I Axis

Abstract

Articular chondrocytes maintain cartilage throughout life by replacing lost or damaged matrix with freshly synthesized material. Synthesis activity is regulated, rapidly increasing to well above basal levels in response to cartilage injury. Such responses suggest that synthesis activity is linked to the rate of matrix loss by endogenous "damage control" mechanisms. As a major stimulator of matrix synthesis in cartilage, insulin-like growth factor I (IGF-I) is likely to play a role in such mechanisms. Although IGF-I is nearly ubiquitous, its bioavailability in cartilage is controlled by IGF-I binding proteins (IGFBPs) secreted by chondrocytes. IGFBPs are part of a complex system, termed the IGF-I axis, that tightly regulates IGF-I activities. For the most part, IGFBPs block IGF-I activity by sequestering IGF-I from its cell surface receptor. We recently found that the expression of one binding protein, IGFBP-3, increases with chondrocyte age, paralleling an age-related decline in synthesis activity. In addition, IGFBP-3 is overexpressed in osteoarthritic cartilage, leading to metabolic disturbances that contribute to cartilage degeneration. These observations indicate that IGFBP-3 plays a crucial role in regulating matrix synthesis in cartilage, and suggest that cartilage damage control mechanisms may fail due to age-related changes in IGFBP-3 expression or distribution. Our investigation of this hypothesis began with immunolocalization studies to determine the tissue distribution of IGFBP-3 in human cartilage. We found that IGFBP-3 accumulated around chondrocytes in the pericellular/territorial matrix, where it co-localized with fibronectin, but not with the other matrix proteins tenascin-C and type VI collagen. This result suggested that the IGFBP-3 distribution is determined by binding to fibronectin. Binding studies using purified proteins demonstrated that IGFBP-3 does in fact bind to fibronectin, but not to tenascin-C or type VI collagen. Finally, we investigated the metabolic effects of fibronectin and IGFBP-3 in a chondrocyte culture system. These experiments showed that fibronectin enhanced the inhibitory effect that low concentrations of IGFBP-3 had on matrix synthesis. Taken together, these observations confirm that IGFBP-3-fibronectin interactions affect the IGF-I axis, and they indicate that IGF-I is stored in the chondrocyte territorial matrix through binding to a complex of IGFBP-3 and intact fibronectin. This arrangement may play an important role in cartilage damage control mechanisms. The local increase in matrix synthesis following injury could result from damage-induced IGF-I release from such pools. An age-related failure to organize this system may contribute to degenerative disease.

INTRODUCTION

The proteoglycan- and collagen-rich cartilage extracellular matrix (ECM) is well-adapted for resisting mechanical stresses imposed by weight bearing and joint motion: proteoglycans (aggrecan) serve to resist compression, while type II collagen fibers lend tensile strength.41,65 This complex structure is actively maintained by ECM-synthesizing chondrocytes which replace components lost to mechanical wear and tear or injury.12,53 Chondrocytes control the rates of synthesis and turnover of matrix proteins, stabilizing the existing ECM (maintenance and repair) or forming a new ECM with a different composition (remodeling). Maintenance and repair activities replace worn or damaged ECM components, while remodeling expands or strengthens the matrix. Together, these activities counter the effects of daily wear and tear and support cartilage adaptation to changing patterns of joint use.11,12,47,49,54,72,82 Permanent structural damage may be done when cells fail to keep pace with matrix losses, a situation that can lead to the degenerative joint disease, osteoarthritis (OA).38 The strongly age-related incidence of OA suggests that this failure to control and repair damage to the cartilage ECM is an age-dependent phenomenon;54 however, precisely how repair efficiency declines with aging is not clear.

The demand for new matrix components in cartilage depends, in part, on patterns of joint use. Animal models show that cartilage tolerates chronic, vigorous exercise, but undergoes important structural changes to maintain stability. These include increased thickness, stiffness and proteoglycan content.47,49,72 An initial increase in ECM turnover implies that both matrix loss and matrix synthesis rates are induced by mechanical stress.75 Several laboratories have demonstrated modest (~1.5-fold) stimulation of proteoglycan and/or type II collagen synthesis following exposure to cyclic stress in vitro.46,78 While the cell signaling mechanisms mediating these effects are still unknown, it has been widely assumed that stress stimulates ECM synthesis directly via stress-dependent signaling mechanisms. However, other lines of evidence indicate that chondrocytes respond to ECM depletion or damage.54 These data show ECM synthesis in organ culture is stimulated by culture-induced proteoglycan depletion,78 by proteolytic digestion of the matrix,75 or by mechanically induced ECM damage.54 Again, the specific mechanisms mediating the stimulatory effects have not been elucidated.

ECM synthesis in cartilage is tightly regulated by cytokines, such as interleukin-1b (IL-1 β) and tumor necrosis factor alpha (TNFα), which inhibit matrix synthesis, 40,79,81 and by growth factors such as insulin-like growth factor I (IGF-I), transforming growth factor beta (TGF-β), and bone morphogenic proteins (BMPs) which stimulate synthesis.62,64,79,81 IGF-I is the major endogenous inducer of matrix synthesis in normal adult cartilage. 25,52,62,79,81 IGF-I alone is capable of maintaining matrix stability in cartilage organ cultures,52 and IGF-I can reverse some of the damage to cartilage caused by cytokine treatment.80 These studies indicate that IGF-I is vital for cartilage integrity. Unfortunately, because osteoarthritic chondrocytes lose IGF-I responsiveness, 27,29,45 IGF-I treatments do not reverse OA.12 Interestingly, the actions of IGF-I in OA cartilage do not appear to be limited by the concentration of IGF-I itself, which is actually higher in OA than in normal cartilage; rather, as described below, it appears that other factors curtail the bio-availability of IGF-I.27,63,77

The IGF-I found in articular cartilage is derived from synovial fluid or is secreted by chondrocytes.25,34,55,63,79 IGF-I stimulates overall protein synthesis, cell division, glucose uptake and oxidation, and synthesis and secretion of collagen type II and aggrecan.25,55,62,79,81 Protein and ECM synthesis in bovine and ovine cartilage is stimulated 2-4 fold by physiologic doses of IGF-I.25,54,62,79 IGF-I levels are determined in part by IGF-I synthesis. Growth factors and cytokines found in the synovial compartment have been shown to affect IGF-I expression; however, activities are also controlled by regulation of IGF-I receptor density,77 or by processes that limit the bio-availability of IGF-I once it is secreted.25,26,55,67,69,76

The activity of secreted IGF-I is regulated by a class of extracellular proteins, the IGFBPs which bind IGF-I with an affinity equal to that of the cell surface IGF-I receptor. These proteins are important systemic regulators of IGF-I activities and also act on a tissue-specific basis.3,17,2 Peptides derived from IGFBPs directly affect cell metabolism,7,8,68 but the principle mechanism of action for most IGFBPs is to sequester IGF-I in the extracellular space, making the growth factor unavailable for binding cell surface receptors.23,61 Thus, an apparent loss of IGF-I sensitivity is observed even though the total number of IGF-I binding sites in the tissue increases. 13,27,45,55,56,57,67 Like IGF-I itself, IGFBPs are secreted by chondrocytes. Of the six genetically distinct IGFBPs described thus far, at least 3 (IGFBP-3, -4, -5) are expressed by human articular car tilage chondrocytes.26,27,70,77 Although IGFBPs have been found to potentiate IGF-I action in some cell types,28 only inhibitory actions have been shown in chondrocytes. Weak chondrocyte IGF-I responses have been associated with excessive IGFBP-3 expression in aging, in wasting syndromes35,56 and in OA.27,29,55,77 Increased IGFBP-3 levels in human synovial fluid are associated with expression of the OA/Rheumatoid marker, C-reactive protein (CRP).29 IGFBP expression is stimulated by IGF-I, IL-1, prostaglandin E2, and TNFa26,58,69,74,76 and inhibited by TGF-b.23

IGFBPs are themselves subject to complex regulation. 3,17,23 Though their physiologic significance is unclear, post-translational modifications such as phosphor ylation24 and glycosylation2,30 affect IGF-I binding activity. IGF-I binding affinity is reduced by specific proteolytic cleavage of IGFBPs, a mechanism that has been shown to regulate IGF-I availability in many tissues. 1,2,4,5,6,40,48,50,59,60 Proteinases that target IGFBPs include intracellular cysteine proteinases such as cathepsin D9,22 and extracellular proteinases including MMPs,31,32 plasmin8,16 and unidentified serine proteinases, 1,6,66 which cleave IGFBPs into fragments with reduced affinity for IGF-I.4,5,6 Finally, IGFBP distribution in a tissue can be regulated by specific interactions with selected ECM components, including heparan sulfate proteoglycans.7,43,44,33,71,73 Studies in our laboratory suggest that fibronectin, a quantitatively minor glycoprotein of the cartilage ECM, plays such an IGFBP-binding role. Cartilage-specific fibronectin isoforms10,14,15,20 play important roles in regulating metabolism in normal and OA cartilage.11 Numerous studies have demonstrated that fibronectin expression is increased in OA.10,18,19,20,42,51 Proteolytic fragments of fibronectin, which are abundant in OA synovial fluid, strongly induce cartilage proteoglycan degradation.39

We propose that most cartilage IGF-I is held in an inactive pool by macromolecular complexes composed of IGF-I, IGFBPs, and matrix proteins which act together to link matrix disruption and matrix synthesis by controlling the bio-availability of IGF-I. Moreover, we predict that physical and/or enzymatic ECM disruption stimulates proteolysis of IGFBPs, resulting in the release of IGF-I from the pool. The studies summarized below present three lines of evidence that fibronectin and IGFBP-3 are part of this damage control mechanism which regulates matrix synthesis in cartilage: (1) Striking colocalization of fibronectin and IGFBP-3 was observed in immunohistological studies of human tibial plateau cartilage, whereas other matrix proteins such as tenascin-C and type VI collagen, that are similarly enriched in the pericellular\territorial matrix,21,41 did not colocalize with IGFBP-3. These data suggest a physical interaction between fibronectin and the binding protein. (2) Binding assays with purified proteins showed that IGFBP-3 does indeed bind to fibronectin, but not to tenascin-C or type VI collagen, supporting our interpretation of the immunohistologic data. (3) Fibronectin strongly affected the physiologic activities of IGFBP-3, altering its effects on proteoglycan synthesis in a chondrocyte culture system.

MATERIALS AND METHODS

Human cartilage blocks were harvested from the central regions of 5 tibial plateaus obtained as surgical discards from patients (aged 37, 52, 67, 77, 84 years) undergoing total joint replacement for degenerative joint disease. Only cartilage which appeared grossly normal was used. The blocks were cryoembedded and sectioned. Ten mm-thick frozen sections were placed on gelatin-coated slides and fixed for 5 minutes in 3.5% paraformaldehyde freshly prepared in phosphate buffered saline (PBS). One slide was stained with the sulfated glycosaminoglycan-specific stain, Safranin-O,41 to confirm normal cartilage morphology. The remaining slides were then washed repeatedly with PBS and blocked with 1% bovine serum albumin (BSA fraction V) in PBS with 0.5% Tween-20 (PBST) for 30 minutes. Mouse monoclonal antibodies against fibronectin (clone HFN7.1, Developmental Studies Hybridoma Bank), type VI collagen (clone 5C6, Developmental Studies Hybridoma Bank), and tenascin-C (Clone EB2, ICN), were applied at ~10 μg/ml IgG. The rabbit anti human IGFBP-3 polyclonal antibody (Upstate Biotechnology) was applied at the same time at a 1:50 dilution. The sections were incubated with primary antibodies overnight at 4°C. The sections were washed in PBST and blocked in 10% normal goat serum for 30 minutes before the addition of secondary antibodies. Secondary antibodies consisted of a goat anti-mouse Cy2 conjugate (Jackson Immunoresearch) and a goat anti-rabbit Cy5 conjugate (Jackson Immunoresearch). These were diluted 1:400 and applied to the sections. The slides were incubated for 1.0 hour at ambient temperature. Isotype controls in which purified mouse IgG or rabbit serum (diluted 1:200) replaced the primary antibodies were included on every slide. After several PBS washes the slides were mounted using Aquamount (Lerner Laboratories) and imaged on a Biorad scanning confocal laser system using a Nikon microscope and 20X objective. Two images of the same section were recorded using an excitation wavelength of 488 nm and a 515 nm cutoff filter (for Cy2) and an excitation wavelength of 630 nm with a cutoff of 670 nm (for Cy5). Semi-quantitative image analysis was performed to determine the extent of overlap between individual stains. Paired images representing double stains for IGFBP-3 and fibronectin, IGFBP-3 and tenascin-C, and IGFBP-3 and type VI collagen were first thresholded, then combined using Boolean operators "and" and "or" (Scion Image). The areas covered when the images were combined using either the "and" or "or" functions were determined and the ratio of "and" to "or" calculated. These ratios, which are a measure of colocalization, were determined for all 5 individual samples. These data were pooled for statistical analysis.

A novel diffusion assay was developed to study interactions of selected ECM proteins with IGFBP-3. Purified proteins (200 nM) including bovine plasma fibronectin (ICN), human collagen type VI (Chemicon), bovine heparan sulfate proteoglycan (HSPG) (Sigma), and human tenascin-C (Chemicon) were mixed with 1% molten low-melt agarose (FMC Bioproducts) in Dulbecco's Modified Eagle Medium (DMEM) buffered with 10 mM HEPES. Purified IGFBP-3 at 200 nM (R&D Systems) and 125I-IGF-1 at ~5.0 nM (ICN) were added and multiple 10 ml aliquots of molten agarose were dotted on culture dishes and allowed to solidify. The agarose dots were pre-incubated for 1.0 hour at 37°C to allow intermolecular protein binding to occur. The dots were then overlaid with 100 ml DMEM and incubated at room temperature. The medium was removed at various time points (1, 5, 10, 20, 30, 60, 120, and 180 minutes) and the agarose dots extracted in 7.7 M urea. 125I-IGF-I CPM was quantitated by liquid scintillation counting and IGFBP-3 was assayed by immunoblot in both the medium and agarose extracts. Ratios of extract (agarose dot) to total (agarose + medium) 125I-IGF-I and IGFBP-3 were calculated for each time point. These data were plotted and fitted with exponential decay curves (Microcal Origin) to determine the t1/2 for protein retention in the agarose dot. Each experiment included controls consisting of 125I-IGF-I without additional proteins and 125I-IGF-I with IGFBP-3.

Cultures of rat articular chondrocytes were used to determine the effects of fibronectin on IGFBP-3 activity. Chondrocytes were isolated from cartilage pooled from the tibial plateau and femoral condyles of 6-8 one-month-old Sprague Dawley rats. The cartilage was digested overnight in DMEM with 10% fetal calf serum containing 0.5 mg/ml each type IA collagenase (Sigma) and dispase (Life Technologies) and antibiotics. The digestion was filtered through 40 mm nylon mesh and the cells counted on a hemocytometer in the presence of 0.04% trypan blue. Greater than 90% of the isolated chondrocytes were viable. The cells were pelleted and resuspended in alginate (Kelco) to a concentration of 2 x 107 cells/ml as described.36 The suspension was split into two fractions and rat plasma fibronectin (Life Technologies) was added to one fraction to a final concentration 10 μg/ml. An equal volume of the solvent (0.01 M acetic acid) was added to the other fraction. Each fraction was again divided into 4 equal fractions and IGFBP-3 was added from a freshly reconstituted 50 μg/ml solution to final concentrations of 0.01, 0.10, or 1.0 μg/ml. The 0.01 M acetic acid solvent was added to make an equal percentage (2%) of 0.01 M acetic acid in all fractions. Alginate beads were formed as described36 and were placed in 96-well tissue culture plates (1 bead per well) in 200 μl DMEM/10% FCS containing 50 μCi/ml carrier-free 35SO4(Amersham). The cultures were incubated overnight at 37°C in a humidified chamber with 5% CO2. The whole cultures (beads and medium) were extracted by adding 200 ul 8 M 2X extraction buffer (guanidine-HCl with 100 mM sodium acetate, 100 mM 6 amino hexanoic acid, 20 mM EDTA and 4% Triton X 100, pH 6.8) to each well and incubating overnight at 4°C with shaking.37 The extractions were passed over Sephadex G-50 (Pharmacia) columns to remove free 35SO4. Column eluates were mixed with scintillation cocktail and counted in a Beckman LS 3801 liquid scintillation counter. The means and associated standard deviations for 6 replicate wells for each dose group (CPM/bead) are presented as a function of IGFBP-3 concentration in Figure 4. Chondrocytes from at least one bead per dose group were not extracted but instead were recovered from the bead and counted on a hemocytometer in the presence of trypan blue. Viability averaged 88% and there were no significant differences attributable to IGFBP-3 or fibronectin treatments.

Figure 4
Effects of Fibronectin and IGFBP-3 on 35S-sulfate Incorporation in Chondrocyte Cultures

RESULTS

Double immunofluorescence studies showed that IGFBP-3 was concentrated in the pericellular/territorial matrix around chondrocytes where it appeared to colocalize with fibronectin. Typical sets of double-stained fluorescence images taken using a 10X objective (Figure 1) showed that whereas the matrix proteins fibronectin (A, green), type VI collagen (B, green), and tenascin-C (C, green) were all concentrated in the pericellular/ territorial matrix, only fibronectin showed an overall tissue distribution similar to IGFBP-3 (red). This relationship was found to hold true at higher resolution: 3-dimensional reconstructions done using a 63X objective confirmed IGFBP-3/fibronectin colocalization (not shown). Semi-quantitative analysis of cartilage samples from 5 different individuals confirmed this subjective impression (Figure 2). These data represent the extent of colocalization or overlap between red and green stains. Kruskal-Wallis one way analysis of variance with Dunn's method for multiple comparisons indicated that the IGFBP-3 co-localized with fibronectin to a significantly greater extent than colocalization with either type VI collagen or tenascin-C (p<0.05).

Figure 1
Tissue Distributions of IGFBP-3, Fibronectin, Type VI collagen, and Tenascin-C
Figure 2
Colocalization Analysis

IGFBP-3 interactions with selected ECM proteins were determined in an agarose diffusion assay. Purified proteins including fibronectin, collagen type VI, heparan sulfate proteoglycan, and tenascin-C were mixed with molten agarose. Purified IGFBP-3 and 125I-IGF-1 were added and multiple 10 ml aliquots of the mixtures were dotted on culture dishes and overlaid with DMEM. The negative control for 125I-IGF-I diffusion experiments consisted of 125I-IGF-I alone without additional proteins. The negative control for IGFBP-3 diffusion consisted of IGFBP-3 without the addition of matrix proteins. DMEM was removed at subsequent time points and the 125I-IGF-I quantitated by liquid scintillation counting of the medium and agarose extracts. IGFBP-3 was assayed by immunoblot in the same medium and agarose extracts. The ratios of the 125I-IGF-I and IGFBP-3 remaining in the agarose dot to the total present in both phases were calculated for each time point. These data were normalized to controls, plotted, and fitted with exponential decay curves to determine the t1/2 for protein retention in the agarose dot. Data from three independent assays are shown in Figure 3. 125I-IGF-I diffusion data indicated that, as expected, IGFBP-3 increased the t1/2 of 125I-IGF-I in agarose: The mean for 125I-IGF-I alone was 14.2 minutes +/- 6.5 minutes (SD), whereas the mean for 125I-IGF-I in the presence of IGFBP-3 alone was 30.7+/- 17.9 minutes (~2-fold longer). Although the binding protein retarded 125I-IGF-I diffusion in both the presence and absence of matrix proteins, matrix proteins did affect t1/2: While tenascin-C and type VI collagen reduced the retardation observed with IGFBP-3 alone, both fibronectin and heparan sulfate proteoglycan enhanced the effect. To determine if this might be due to IGFBP-3-matrix interactions, we performed assays for IGFBP-3 on the same medium and extracts used for 125I-IGF diffusion studies. These immunoblot data (Figure 3B) show that IGFBP-3 diffusion was slowed by fibronectin or HSPG (t1/2=2-4 fold greater than IGFBP-3-only). In contrast, both type VI collagen and tenascin-C had very modest effects.

Figure 3
In Vitro Diffusion Assays

A chondrocyte culture study was performed to determine the effects of fibronectin on matrix synthesis activity in the presence and absence of IGFBP-3 (Figure 4). Fibronectin and IGFBP-3 were incorporated into the alginate matrix and the cells were cultured in medium with 35SO4 and varying doses of IGF-I. 35SO4 incorporation was determined as a measure of matrix synthesis activity. Consistent with previous observations57, we found that fibronectin itself stimulated anabolic activity in the absence of added IGFBP-3. IGFBP-3 clearly inhibited 35SO4 incorporation both in the presence and absence of fibronectin; however, there were substantial fibronectin-related differences in the IGFBP-3 dose response. The means for fibronectin-treated and untreated controls were significantly different by one-way analysis of variance (p<0.05) at all IGFBP-3 doses except the 0.1 mg/ml dose. Only the highest IGFBP-3 doses (0.1 and 1.0 mg/ml IGFBP-3) consistently inhibited 35SO4 incorporation in fibronectin-free cultures whereas, in the presence of fibronectin the lowest (0.01 mg/ml) dose was inhibitory. Interestingly, incorporation was stimulated by 1.0 mg/ml IGFBP-3 in the presence of fibronectin, but not in its absence. This result may be due to fibronectin-dependent enhancement of the stimulatory effects of IGFBP-3 fragments contaminating the IGFBP-3 preparation.8

DISCUSSION

Our studies confirm that IGFBPs are part of the cartilage system for regulating IGF-I availability and support the hypothesis that IGF-I is stored in the matrix as part of a damage control mechanism. We observed striking colocalization of IGFBP-3 with fibronectin in the pericellular/territorial matrix. This relationship was not observed with tenascin-C or type VI collagen, suggesting that the colocalization with fibronectin was not merely incidental, but was due to specific binding between fibronectin and IGFBP-3. Evidence for such an interaction was sought using purified proteins in a cell-free in vitro diffusion assay. These data indicated that IGFBP-3 binds to fibronectin, and heparan sulfate proteoglycan, a protein which had previously been shown to bind IGFBP-3. Consistent with the colocalization data there was no evidence of binding to tenascin-C or type VI collagen. Finally, we sought physiologic evidence that fibronectin affects cellular responses to IGF-I in the presence of added IGFBP-3. These cell culture studies showed that fibronectin significantly altered matrix synthesis activity, particularly when IGFBP-3 was present. Taken together these findings suggest that IGF-I, IGFBP-3, and fibronectin form a strategically placed growth factor pool. We propose that this spatial arrangement maintains a constant, low level of bioavailable IGF-I in normal cartilage, consistent with the minimal need for synthesis activity. Moreover, we postulate that proteinases activated during the initial response to mechanical or chemical insult degrade IGFBP-3 and fibronectin, destabilizing IGF-I binding. This results in a rapid rise in available IGF-I around chondrocytes at the damaged site without the need for an immediate increase in local IGF-I synthesis. The resulting burst of IGF-I-driven matrix synthesis is limited to the damaged site and persists until the local ECM, and its associated IGF-I storage pool, is re-established.

Although aberrant IGF-I responses almost certainly contribute to osteoarthritis, the mechanisms of IGF-I action in normal cartilage must be understood first before events leading to degenerative disease can be recognized. The long-term goal of this effort is to develop strategies to improve the effectiveness of IGF-I as a pharmacologic agent against cartilage degeneration, perhaps by combining IGF-I and IGFBPs. Another important goal will be to determine whether disturbances in the IGF-I axis are related to aging processes, or to environmental factors such as mechanical stress, which might be avoided in at-risk individuals. These studies will also contribute to the design of engineered cartilage implant materials by identifying the molecules that regulate the IGF-I pathway.

ACKNOWLEDGEMENTS

This work was funded by a Veterans Affairs Merit Review Grant and by the Department of Orthopaedic Surgery, The University of Iowa. The authors thank Gail Kurriger and Aaron Schroeder for their technical contributions and Dr. Jeff Stevens for his astute advice and intellectual support.

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