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Published as doi: 10.1096/fj.08-114363.
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(The FASEB Journal. 2009;23:79-89.)
© 2009 FASEB

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-β activation

Mukundan G. Attur*, Glyn D. Palmer*, Hayf E. Al-Mussawir*, Mandar Dave*, Cristina C. Teixeira{dagger}, Daniel B. Rifkin{ddagger}, C. Thomas G. Appleton§, Frank Beier§ and Steven B. Abramson*,1

* Division of Rheumatology, School of Medicine, Hospital for Joint Diseases,

{dagger} Department of Basic Science and Craniofacial Biology, College of Dentistry, and

{ddagger} Department of Cell Biology, School of Medicine, New York University, New York, New York, USA; and

§ CIHR Group in Skeletal Development and Remodeling, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

1 Correspondence: Division of Rheumatology, New York University School of Medicine, New York University Hospital for Joint Diseases, 301 E. 17th St., New York, NY 10003, USA. E-mail: stevenb.abramson{at}nyumc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (~7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-β1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-β and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (~20%; P<0.05), which were both TGF-β and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-β and induce cartilage degradation via pathways that involve production of PGE2.—Attur, M. G., Palmer, G. D., Al-Mussawir, H. E., Dave, M., Teixeira, C. C., Rifkin, D. B., Appleton, C. T. G., Beier, F., Abramson, S. B. F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-β activation.


Key Words: extracellular matrix • chondrocyte regulation • prostaglandin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OSTEOARTHRITIS (OA) IS CHARACTERIZED by progressive loss of cartilage from the articulating surfaces of diathrodial joints. While the etiology of the disease is unknown, it is widely accepted that these degenerative changes arise from an imbalance of synthetic and degradative pathways that control cartilage extracellular matrix (ECM) metabolism (1 , 2) . This is in part because of the increased production of cytokines, inflammatory mediators, and growth factors by articular tissues, including synovium and bone as well as the articular cartilage itself (3 4 5) . In an effort to develop disease-modifying treatments for OA, many laboratories, including our own, have focused on characteristic molecular changes within the articular chondrocytes that reside in OA cartilage. During OA pathogenesis, these cells undergo a series of complex changes, including hypertrophy, proliferation, catabolic alteration, and, ultimately, death. Catabolic, matrix-degrading molecules produced by OA chondrocytes include matrix metalloproteinases (MMPs), interleukin (IL) -1, tumor necrosis factor, IL-6, IL-8, nitric oxide, prostaglandins, and leukotrienes (6 7 8) . These molecules accelerate cartilage degradation and may be considered as potential "drugable" targets for disease modification.

In addition to inhibition of cartilage degradation, there is also great interest in identifying molecules associated with chondrocyte activation and cartilage repair. OA chondrocytes exhibit altered anabolic activity, including increased proliferation and expression of hypertrophic markers associated with fetal growth-plate chondrocytes (9 10 11) . While these responses cannot ultimately restore the damaged ECM, characterization of these pathways may lead to novel, alternative treatments for OA.

TGF-β has been considered a stimulator of cartilage matrix synthesis and a potential therapeutic tool for treatment of OA (12) . In human OA, altered signaling and expression of TGF-β1 has been linked to disease progression. Immunohistochemical staining of femoral heads has demonstrated decreased expression of TGF-β1 and TGFβRII within areas of degraded cartilage (13) and increased expression within osteophytes (14) . Genetically induced mutant mice have similarly shown that loss of TGF-β or its downstream signaling molecules can result in skeletal abnormalities and cartilage pathology consistent with human OA (15 16 17) . These findings underscore the importance of understanding TGF-β regulation within the joint. The identification of molecules that regulate its synthesis and activation could have a profound impact on harnessing the potential of this molecule as an agent for cartilage repair.

In an effort to identify novel genes associated with OA, we have previously performed chondrocyte gene expression analyses on nondiseased and OA cartilage to characterize functionally molecules that are up-regulated in osteoarthritis (6 , 7 , 18 , 19) . Affymetrix microarray analysis had indicated that F-spondin was among more than 200 genes that were significantly up-regulated in OA cartilage (19) . F-spondin is a thrombospondin (TSR) family member that regulates neuronal outgrowth during floor plate development of the embryonic nervous system (20 21 22) . However, there are no reports characterizing F-spondin’s role in the ECM of bone or cartilage. In the present study, we demonstrate its coexpression with chondrocyte hypertrophic markers; functional studies indicate that F-spondin has the capacity to regulate the metabolism of articular cartilage via activation of latent TGF-β, suggesting a previously unrecognized role in the pathogenesis of osteoarthritis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All media and FBS were purchased from Life Technologies (Gaithersburg, MD, USA). IL-1, fibroblast growth factor (FGF) –2, and TGF-β1 were purchased from PeproTech (Rocky Hill, NJ, USA). Active TGF-β ELISA kit, anti-TGF-β antibody, purified recombinant human F-spondin, and human recombinant TGF-β1 were purchased from R&D Systems (Minneapolis, MN, USA). Dr. Avihu Klar (Hebrew University, Hadassah Medical School, Jerusalem, Israel) generously provided R1, an F-spondin antibody raised against the TSR domain of the molecule. FS1, FS3, and FS7 plasmids containing cDNAs that encode full-length or truncated portions of the F-spondin gene were kindly provided by Dr. Thomas C. Sudhof (Neuroscience Institute, Stanford University, Palo Alto, CA, USA). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Procurement of human cartilage
The use of all discarded human cartilage tissue was approved by the New York University institutional review board. Tibial and femoral articular cartilage was obtained from patients with advanced OA at the time of knee joint replacement surgery (age 50–70 yr). All specimens exhibited macroscopic evidence of OA, including thinning, loss of cartilage, and focal eburnation. Since the amount of OA cartilage available for analyses was limiting, we typically used all available cartilage from each specimen. Thus the cartilage in our studies was heterogeneous with respect to OA disease stage. The OA patients were free of nonsteroidal antiinflammatory and steroid drugs for at least 2 wk before surgery. Control, nonarthritic knee cartilages were obtained from autopsy patients within 24 h (NDRI, Philadelphia, PA, USA) and were within the same age range (50–70 yr) as the OA specimens. For gene expression analysis, full depth cartilage slices, ~10 x 10 mm, were harvested and immediately frozen at –150°C until RNA extraction. For cartilage explant cultures, slices were further chopped into slices of ~3–5 mm.

RNA extraction and quantitative polymerase chain reaction (QPCR)
Total RNA was extracted from harvested cartilage samples as described previously (23) . Briefly, on thawing, cartilage slices were milled into fine powder in liquid nitrogen using Freezer Miller (SPEX, Metuchen, NJ, USA). RNA was then extracted in TRI Reagent (MRC Labs, Cincinnati, OH, USA) for 4 h on a rocker, and total RNA was precipitated with equal volumes of isopropanol. The RNA pellet was further purified using Qiagen RNeasy mini kit according to the manufacturer’s RNA clean-up protocol (Qiagen, Valencia, CA, USA). Total RNA (1 µg) was primed using oligo (dT)18 primers and cDNA synthesized using the Clontech cDNA synthesis kit following the manufacturer’s directions (Clontech, Mountain View, CA, USA). Predesigned TaqMan primer sets were purchased from Applied Biosystems. Real-time PCR reactions were run on the ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA). mRNA levels were normalized by using GAPDH as a housekeeping gene, and relative expression levels of various transcripts were calculated using approximation method or 2-delta computed tomography method (24) .

Western blotting
To determine F-spondin protein levels in cartilage samples, harvested slices were ground into a fine powder using Freezer Miller, and proteins were extracted with Tris (10 mM) buffer containing lithium bromide (0.2 M), deoxycholate 1%, Triton X-100 EDTA 1 mM, and a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) for 4 h at 4°C. The extracts were centrifuged briefly to generate clear supernatants containing total protein for electrophoresis. Protein concentrations were estimated using BCA reagent (Pierce, Rockford, IL, USA), and 30 µg of each sample was electrophoresed on a 10% SDS-PAGE gel. Proteins were transferred to nitrocellulose (1 h at 100 V), and blots were probed with a rabbit polyclonal F-spondin R4 antibody and catalase monoclonal antibody (kindly provided by Dr. Paul Lazarous, Mount Sinai School of Medicine, New York, NY, USA) to correct for variations in sample loading. Anti-mouse horseradish peroxidase-labeled secondary antibody was used for detection (BD Biosciences, Franklin Lakes, NJ, USA). The blots were developed using the enhanced chemiluminescence (ECL) Western blot system (Amersham, Arlington Heights, IL, USA).

Histology and immunohistochemistry of human articular cartilage specimens
Cartilage specimens were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde in PBS (pH 7.4) at 40°C, decalcified in 4.1% disodium EDTA at 40°C for 2–4 wk, and paraffin embedded. Sagittal sectioning was performed to 4 µm thickness, and sections were deparaffinized and rehydrated for immunohistochemistry. Sections were probed for F-spondin (R1 antibody; 1:200 dilution) or type II (Chemicon International, Temecula, CA, USA) and type X collagen (Calbiochem). Secondary Ab staining was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were counterstained with Alcian blue, mounted under coverslips, and scanned on a Scan Scope GL series optical microscope (Aperio, Bristol, UK).

Rodent model of surgically induced OA
All animal experiments were performed by C.T.G.A. in the laboratory of F.B. at the University of Western Ontario (London, ON, Canada), using protocols approved by the Animal Care and Use Committee at the University of Western Ontario. OA was surgically induced in the right knees of 300–325 g Sprague-Dawley rats using anterior cruciate ligament transection (ACLT) and partial medial (PM) meniscectomy according to the methods described by Appelton et al. (25) . Knees from sham-operated animals served as controls, and left (nonoperated, contralateral) knees of ACLT/PM rats were also investigated. Rats underwent forced mobilization (3 times, 30 min/wk) on a rotarod as described (25) .

For microarray analysis, total RNA was harvested from ipsilateral, contralateral, and sham-operated knee joints 4 wk after surgery and hybridized to Affymetrix gene chips RAE230_2.0 (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocols. Data analysis was performed using Gene Spring 7.2 software (Silicon Genetics, Redwood City, CA, USA).

For histology and immunohistochemistry, rat knee joints were harvested at 2, 4, and 8 wk after surgery and processed at the Robarts Research Institute Molecular Pathology Laboratory (London, ON, Canada). For immunohistochemistry, sections were probed for either F-spondin (R1 antibody) or antibodies to collagen type X (Sigma, Oakville, ON, Canada), MMP-13 (Cedarlane Labs, Hornby, ON, Canada), and alkaline phosphatase (Abcam, Cambridge, MA, USA) and horseradish peroxidase secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Complete details are provided in ref. 25 .

Cartilage explant cultures
Explanted discs (wet weight ~100–200 mg/disc) were cut into 3 x 3 mm cubes from freshly harvested OA cartilage and placed in 24-well plates (4–6 discs/well) in serum-free (SF) Ham’s F-12 medium supplemented with L-glutamine (2 mM), gentamicin (50 mg/ml), amphotericin B (0.25 mg/ml), and human albumin (0.2%) and cultured overnight at 37°C, 5% CO2. Experiments to determine TGF-β and PGE2 levels, proteoglycan synthesis, and collagen degradation were initiated the day of harvest by addition of F-spondin in fresh SF medium with or without modulators.

Determination of active TGF-β levels
TGF-β1 levels in culture supernatants following F-spondin treatment were determined by ELISA (R&D Systems). Total TGF-β1 levels were measured following acidification of the supernatants by treatment with 0.2 M HCl according to the manufacturer’s protocol. For estimation of active TGF-β1 levels the acidification step was omitted.

Cell-based reporter assay
F-spondin-induced activation of TGF-β in explant culture supernatants was also determined using mink lung epithelial cells (TMLCs) stably transfected with luciferase cDNA driven by TGF-β-responsive plasminogen activator inhibitor-1 promoter (26) . Confluent TMLCs were incubated overnight with culture supernatants in 12-well plates and assayed for luciferase activity using luciferase assay reagent (Promega, Madison, WI, USA) and a Mini-Lum SP-0100 Luminometer (Bioscan Inc., Washington, DC, USA).

PGE2 and collagen degradation assays
Conditioned medium supernatants from explant cultures were collected 24 h after treatment with F-spondin or various modulators and assayed for either collagen degradation using a C1,2C ELISA assay kit (IBEX, Montreal, QC, Canada) or PGE2 levels by radioimmunoassay according to the method described previously (7) .

Detection of MMP-13
Conditioned medium supernatants from OA explant cultures treated with F-spondin were also assayed for secreted levels of MMP-13 using the Fluorokine E immunoassay kit (R&D Systems). Total (pro and active) secreted levels of MMP-13 were determined following pretreatment of culture supernatants with the MMP activator, p-aminophenylmercuric acetate. To measure the proportion of the active form of MMP-13 within culture supernatants, this pretreatment step was omitted.

Proteoglycan synthesis
Following treatment with F-spondin for 7 days, explants were labeled with 10 µCi/ml sodium sulfate (Na235SO4) for 4 h at 37°C in a 5% CO2 atmosphere. The explants were washed 5 times with 0.15 M NaCl, fixed overnight in 0.5% cetylpyrinium chloride/10% formalin, and solubilized in 0.5 ml of Soluene 350 (PerkinElmer, Boston MA, USA) for 72 h. Following lysis, samples were centrifuged, and clear supernatants were assayed for 35SO42– radioactivity by liquid scintillation analysis using a Beckman LS 7000 counter (Beckman Instruments, Fullerton, CA, USA).

Cell culture and transient transfection
To determine F-spondin gene expression following growth factor stimulation of human OA chondrocytes, cartilage slices were harvested from discarded knees, minced finely, and digested with collagenase for 12–16 h in Ham’s F12 medium (with 5% FBS). The cell suspension was used to establish cultures in T-175 flasks. Within 4–5 days of harvest, primary chondrocytes were replated in 6-well plates for experiments.

For F-spondin transfection experiments, C28I2 chondrocytes (a human chondrocyte cell line kindly provided by Dr. Mary B. Goldring, Hospital for Special Surgery, New York, NY, USA) were seeded in 24-well plates and transfected with 3 µg full-length F-spondin cDNA (FS1) or deletion constructs encoding various portions of the F-spondin molecule (FS3, 6, and 7) using GenePorter transfection reagent (Genlantis, San Diego, CA, USA) in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. After 12–16 h, the medium was changed to serum-free Hams F12 for a further 24 h. Supernatants were then harvested and assayed for secreted PGE2 as described above.

Statistical analysis
All data are expressed as percentage control values and SEM. Statistical analysis was conducted with Prism and GraphPad software (GraphPad, San Diego, CA, USA). The nonparametric Spearman correlation was used to measure correlations between gene expression levels in normal and OA cartilage (Fig. 1A ). Student’s t test was used for analyses in all other studies.


Figure 1
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  		Figure 1. F-spondin gene expression is elevated in OA cartilage. A) RT-QPCR of total RNA extracted from nondiseased (n=10) and OA cartilage (n=12). Graph represents expression levels from individual patient samples; inset shows the average expression level for both groups. Relative F-spondin expression was determined by calculating the {Delta}CT values from GAPDH for each sample and normalizing to the {Delta}CT average of the nondiseased group. B) Protein extracts from cartilage specimens were analyzed by Western blot using an F-spondin antibody specific for the TSR domain (R1) and a catalase antibody for a loading control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential expression of F-spondin in OA
Fig. 1A demonstrates by semiquantitative RT-PCR the up-regulation of F-spondin gene expression of individual cartilage specimens (age range 50–70 yr) in patients with OA compared to nondiseased controls. Within the OA group, F-spondin levels varied considerably; however, the average expression level was ~7-fold higher than the control group (P<0.005; Fig. 1A ). F-spondin expression was also up-regulated in OA cartilage at the protein level; immunoblot of cartilage protein extracts using a spondin domain-specific, F-spondin antibody (R1) revealed a ~110 kDa protein, which stained more intensely in OA samples compared to normal (Fig. 1B ). The protein size is consistent with the predicted glycosylated form of F-spondin, previously reported in neuronal cells (20) .

Immunolocalization of F-spondin in OA cartilage
Immunohistochemical staining was performed on tissue sections of OA cartilage samples using R1 antibody. Intense extracellular staining of F-spondin was observed toward the superficial zone of OA cartilage, with little or no staining in the middle and deep zones (Fig. 2A ). Cell-associated staining was also detected in chondrocyte clusters in proximity to the articular surface (Fig. 2B ). OA cartilage also stained positive for type II collagen, which appeared mostly within the interterritorial matrix, and type X collagen, which was mainly associated with chondrocyte clusters.


Figure 2
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  		Figure 2. Immunohistochemistry of OA knee cartilage identifies extracellular and cell-associated staining of F-spondin. OA knee cartilage, obtained at total knee replacement, was sectioned and stained for F-spondin and visualized using Vectastain reagents and protocol. Subfigures A and B represent separate OA cartilage specimens. A) Full depth cartilage specimens indicate positive F-spondin staining in the ECM of the superficial zone (original view x40). F-spondin staining is representative of 2 specimens. B) Full-thickness cartilage specimens demonstrate F-spondin cell-associated staining within chondrocyte cell clusters. Staining is representative of 2 specimens. All specimens were counterstained with alcian blue. Brown-red color indicates positive antibody staining using this protocol.

Induction of F-spondin in a rat meniscectomy model of OA
We next performed experiments to determine whether chondrocyte F-spondin up-regulation was observed in an animal model of osteoarthritis. OA was induced in Sprague-Dawley rats using ACLT with partial meniscectomy and forced mobilization according to the procedure of Appleton et al. (25) . Figure 3A shows a 7-fold increase of F-spondin gene expression in operated (ipsilateral) joints compared to nonoperated, sham controls using Affymetrix microarray of RNA obtained from articular cartilage at 4 wk after surgery. Interestingly, a modest (2-fold) up-regulation was also observed in the contralateral controls, suggesting that altered mechanics or systemic factors within the nonoperated joint may also provide a stimulus for F-spondin expression. This observation was consistent with our prior report that other OA-associated genes that were up-regulated in the nonsurgical contralateral knee compared to sham controls (25) .


Figure 3
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  		Figure 3. F-spondin is up-regulated in rat knee joint cartilage following induction of OA by surgical ACLT/menisectomy. A) F-spondin gene expression in the articular cartilage of ipsilateral (operated), contralateral, and sham-operated controls determined by microarray analysis 4 wk following surgical induction of OA. B) Representative images of histological sections of articular cartilage immunostained with antibodies to F-spondin (R1), MMP-13, Col X, and alkaline phosphatase (AP). Panels show F-spondin staining in sham-operated, contralateral, and ipsilateral OA knee joints 2, 4, and 8 wk after surgery and MMP-13, Col X, and AP staining in ipsilateral joints after 8 wk (right). Positive immunostaining is indicated by red-brown color. Bottom panels indicate cartilage degeneration in operated joints at 4 wk after surgery, shown as histological staining with Safranin O (red; cartilage) and fast green (bone)..

Histological assessment of articular cartilage at 4 wk confirmed an OA-like pathology in operated knees (Fig. 3B , bottom panels) as described earlier (25) . Surface abrasion and cartilage loss were observed in the ipsilateral (OA) but not sham-operated and contralateral operated joints. Immunohistochemistry of knee joint sagittal sections revealed positive F-spondin staining 4 and 8 wk after surgery (Fig. 3B , main panels), but no detectable staining at 2 wk. As was the case in human OA, increased F-spondin expression was detected in the superficial zone of articular cartilage and was also associated with "cloned" chondrocytes within the middle to upper regions of the tissue. Of interest, F-spondin showed similar expression as markers of chondrocyte hypertrophy and mineralization, including MMP-13, Col X, and alkaline phosphatase (Fig. 3B , right panels).

Consistent with our histological observations, F-spondin gene expression showed a significant correlation with type X collagen and alkaline phosphatase following RT-PCR of individual human OA cartilage specimens (P<0.05, Table 1 ). MMP-13 (P=0.0550) and CBFA1/Runx-2 (P=0.0568) expression levels also showed nearing significant correlation with F-spondin expression. Together these findings indicate that F-spondin is coexpressed with hypertophic markers in OA cartilage.


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  		Table 1. Association of chondrocyte markers with F-spondin in OA cartilage specimens

F-spondin induction in OA chondrocytes
While F-spondin has been reported to regulate cell function in neuronal and vascular tissues, soluble factors that regulate its synthesis have not yet been identified. To determine whether factors associated with cartilage homeostasis or degeneration regulate F-spondin synthesis, we measured its gene expression in primary cultures of OA chondrocytes from 3 patients. While TGF-β1 (10 ng/ml) had little effect on F-spondin gene expression, IGF-1 (50 ng/ml), FGF (2 ng/ml), and retinol (100 nm), all significantly increased F-spondin (P<0.005; Fig. 4 ), with retinol having the greatest effect, increasing expression 140% above controls. Conversely, incubation with the proinflammatory cytokine IL-1 (10 ng/ml) inhibited F-spondin expression ~40% (Fig. 4) . These findings suggest that F-spondin expression in OA chondrocytes is a consequence of a reparative process rather than a result of inflammatory pathway induction.


Figure 4
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  		Figure 4. FGF-2 and retinol increase F-spondin gene expression in OA chondrocyte cultures. Primary human OA chondrocytes (P1) were adapted to serum-free conditions for 24 h and stimulated with various soluble factors for 24 h. F-spondin gene expression was determined by RT-QPCR. Values are normalized to untreated controls (set to 100%) and represent means + SD for 3 individual patients. P value denotes significant change from control group. Concentrations of factors were as follows: TGF-β1, 2 ng/ml; IGF-1, 50 ng/ml; FGF-2, 25 ng/ml; Retinol, 100 nM; and IL-1, 10 ng/ml.

Addition of F-spondin to cartilage explant cultures activates latent TGF-β1
Because of its sequence homology with thrombospondin-1, we hypothesized that F-spondin functions as an activator of latent TGF-β1. Within their TSR domains, both F-spondin and thrombospondin-1 share conserved WSxW and KRFK motifs, which are required to bind and release active TGF-β1 from its latent complex (27) . Since TGF-β1 is an important regulator of chondrocyte metabolism, we investigated whether F-spondin activates TGF-β1 when added to OA cartilage explant cultures. Explants obtained from human knee joints were treated with F-spondin (1 µg/ml) for 24 h. Analysis of conditioned medium supernatants by ELISA revealed that total TGF-β1 levels (latent and active forms) did not differ significantly in untreated and F-spondin-treated cultures (11–13 ng/g cartilage; Fig. 5A , right panel). Similarly, IL-1, provided as a reference, had no significant effect on TGF-β1 levels. In contrast, active TGF-β1 levels increased ~160% in response to F-spondin addition (P<0.01 vs. control; Fig. 5A , left panel), raising culture medium levels from <0.2 ng/g cartilage to >0.5 ng/g cartilage. In cartilage explant cultures from 9 OA patients, we observed F-spondin mediated activation of TGF-β in 7; among these, the mean increase in active TGF-β1 levels ranged from 25 to 240% and was significant (r=0.86, P<0.02). Within this group, however, no significant correlation was observed for total TGF-β1 levels following F-spondin treatment.


Figure 5
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  		Figure 5. F-spondin addition to cartilage explant cultures causes activation of latent TGF-β1. Recombinant F-spondin (1 µg/ml) or IL-1 (1 ng/ml) was added to cartilage explant cultures, and after 24 h culture supernatants were analyzed for levels of TGF-β. A) Active and total (latent+active) TGF-β1 levels determined by ELISA. Control cultures were treated with 1 µg/ml human albumin only. Values represent means + SD of TGF-β1 (pg/g cartilage) for triplicate samples. B) Assay of TGF-β using MLEC luciferase reporter assay. Harvested supernatants from explant cultures treated with F-spondin, F-spondin + anti-TGF-β antibody (15 µg/ml), or IL-1 (1 ng/ml) were added to TMLC cultures, and luciferase activity was determined after 24 h. Values represent mean + SD luciferase activity (RLU/g cartilage) for triplicate samples. P values indicate significant difference from control group.

F-spondin-mediated TGF-β1 activation was also assessed using a mink lung epithelial reporter cell line (TMLC), which expresses a luciferase reporter gene under the control of TGF-β1 responsive promoter (26) . Conditioned medium supernatants from OA cartilage explant cultures treated with or without F-spondin, as above, were incubated with TMLCs and assayed for luciferase activity after 24 h. F-spondin-treated culture supernatants increased luciferase activity 25% compared to nontreated controls (P<0.05 vs. control; Fig. 5B ). Coincubation with TGF-β antibody blocked this stimulation, reducing activity to below control levels (P<0.01 vs. control), thus providing further evidence that F-spondin can act as a latent TGF-β1-activating protein.

F-spondin induces PGE2 in chondrocytes via TGF-β and the TSR domain of the protein molecule
To begin to elucidate the downstream molecular effects of F-spondin in OA chondrocyte metabolism, we investigated its effects on synthesis of PGE2, the major prostaglandin synthesized by OA cartilage (7) . While PGE2 has been primarily characterized as catabolic mediator in articular cartilage (8) , its synthesis has also been reported following TGF-β stimulation of cartilage in explant culture (28) . Following addition of F-spondin to OA cartilage explants, we observed a ~780% increase in PGE2 in medium supernatants after 24 h (Fig. 6A ; P<0.01 vs. control). A similar effect was observed in explant cultures from 4 OA patients (data not shown). Coincubation of F-spondin with a TSR domain-specific, F-spondin antibody (R1) caused a reduction in F-spondin-mediated PGE2 synthesis, from ~780% to ~170% above control levels (Fig. 6A ).


Figure 6
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  		Figure 6. F-spondin induces PGE2 synthesis in cartilage explant and chondrocyte cell cultures via pathways that involve TGF-β and the TSR domain of the F-spondin molecule. A) OA cartilage explant cultures were treated with recombinant 1 µg/ml F-spondin or human albumin (control) and assayed for secreted PGE2 levels after 24 h. Values represent mean + SD levels of secreted PGE2 in culture supernatants (ng/g cartilage) for triplicate samples. Specimens were cocultured with or without a TSR domain specific, F-spondin antibody (R1). B) Cultures were stimulated with F-spondin as above and cocultured with IgG or TGF-β antibodies (15 µg/ml). Inset: PGE2 levels in cartilage explant cultures following 24 h stimulation with 10 ng/ml TGF-β1. C) Monolayer cultures of C28I2 chondrocytes were transfected with the full-length (FS1) or truncated portions (FS3, FS7) of the F-spondin molecule and assayed for secreted PGE2. Values represent mean + SD levels of secreted PGE2 (ng/ml medium) for triplicate samples. P values denote significant difference from control groups; *P < 0.01.

To determine whether F-spondin-mediated induction of PGE2 was linked to TGF-β, we measured PGE2 synthesis in the presence of a neutralizing TGF-β antibody. In the presence of control antibody (IgG), F-spondin increased PGE2 levels by 220%, from 38 to 123 ng/g cartilage (P<0.05; Fig. 6B ). However, following TGF-β inhibition, the magnitude of PGE2 induction by F-spondin was lower, increasing only 90% (32 to 61 ng/g cartilage) above unstimulated controls. In agreement with this observation, TGF-β1 stimulated PGE2 synthesis in cartilage explants (P<0.01 vs. control; Fig. 6B , inset). Together these findings suggest that F-spondin-mediated induction of PGE2 in OA cartilage is mediated, in part, via a TGF-β-dependent pathway.

We next assayed PGE2 levels in monolayer cultures of C28I2 chondrocytes transfected with cDNA constructs encoding truncated portions of F-spondin in order to identify functionally active portions of the molecule. PGE2 synthesis in cell culture was stimulated ~950% (from ~4 to ~42 ng/ml) following transfection of FS1, the full-length F-spondin cDNA construct (Fig. 6C ; P<0.01), reproducing our observations in cartilage explants. Interestingly, PGE2-induction did not significantly change following expression of a partial cDNA encoding only the TSR domain (FS 7), suggesting that majority of PGE2-inducing activity resides within this portion of the molecule. Moreover, deletion of TSR repeats 4–6 (FS3), which encode putative TGF-β binding and activation sequences, caused a marked reduction in F-spondin induction of PGE2, from ~950% to ~80% above vector control, indicating that this region is primarily responsible for PGE2 induction by F-spondin. These findings parallel the observations in neuronal axons identifying this portion of the TSR domain as a major functional component of the F-spondin molecule (22 , 29) .

F-spondin mediates increased collagen degradation and suppression of proteoglycan synthesis in OA cartilage explant cultures
To assess collagen degradation, OA cartilage explants were treated with F-spondin for 24 h and supernatants harvested and assayed for collagen degradation by C1,2C ELISA. The addition of F-spondin increased levels of C1,2C peptide ~410% above control (Fig. 7A ; P<0.05). Since F-spondin induces PGE2 synthesis and promotes activation of TGF-β, we next investigated whether these pathways are required for collagen degradation. Addition of either TGF-β antibody or the COX-2 inhibitor, celecoxib (2 µM), blocked F-spondin-mediated induction of the C1,2C peptide, reducing levels from 410% to ~122% and 194% of control levels, respectively. In accordance with these observations, addition of either TGF-β1 (10 ng/ml) or PGE2 (10 µM) alone also promoted C1,2C accumulation (Fig. 7A ; inset).


Figure 7
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  		Figure 7. F-spondin promotes type II collagen degradation, suppression of proteoglycan synthesis, and MMP-13 induction in OA cartilage explant cultures. Cultures were treated with either 1 µg/ml FS or human albumin (control) alone or in combination with anti-TGF-β antibody (15 µg/ml) or the COX-2 inhibitor, celecoxib (2 µM), and assayed for collagen degradation or proteoglycan synthesis. Insets: explant cultures treated with TGF-β1 (10 ng/ml) or PGE2 (10 µM). A) Collagen degradation was determined as levels of C1,2C peptide fragments per gram cartilage in culture supernatants following 24 h treatment with FS or mediators. B) Proteoglycan synthesis was assessed by sulfate incorporation rate (cpm/4 h/g cartilage) of cartilage explants following exposure to FS or mediators for 7 days. C) MMP-13 levels in conditioned medium supernatants (ng/g cartilage) were determined following 24 h treatment with F-spondin (1 µg/ml). Left graph represents total secreted MMP-13 levels (pro and active forms); right graph shows active MMP-13 only. All values are expressed relative to control group (100%) and represent means + SD for 3 patients. P values denote significant difference vs. control groups.

To determine the effects on proteoglycan synthesis, 35S incorporation was performed after 7 days following treatment of cultured cartilage explants with 1 µg/ml F-spondin. F-spondin treatment led to a small (~20%) but significant reduction in 35S incorporation compared to untreated controls (P<0.05; Fig. 7B ). Again, this effect was blocked by addition of either TGF-β antibody or celecoxib (Fig. 7B ; P<0.05), and addition of either TGF-β1 or PGE2 alone had a similar effect to F-spondin, causing a ~30% reduction in proteoglycan synthesis (Fig. 7B , inset). Collectively, our findings suggest that F-spondin regulates catabolic events by activating latent TGF-β within OA cartilage and promoting COX-2 dependent PGE2 production.

Since our findings suggest that TGF-β and PGE2 dependent pathways are linked to F-spondin-mediated type II collagen degradation in cartilage explant cultures, we explored whether MMP-13 production and activation are increased following F-spondin treatment. Incubation of OA cartilage explant cultures with F-spondin (1 µg/ml) for 24 h caused a 130% increase (24 ng/g cartilage above control values) in the secretion of total MMP-13 levels (pro and active forms) in culture supernatants (P<0.05, Fig. 7C ). Secretion of the active form of MMP-13 was also increased ~198% (2 ng/g cartilage above control values) following F-spondin treatment (P<0.05, Fig. 7C ). These findings suggest that MMP-13 induction is a potential mechanism of F-spondin-mediated collagen degradation in OA cartilage.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To understand the dysregulation of chondrocyte function, phenotype, and ECM interactions in OA, we have focused on the identification of differentially expressed genes in OA cartilage (19) . In the present work we report that F-spondin, a neuronal migration factor not previously associated with cartilage or OA, is overexpressed in lesions of both human and rodent OA, where it may act in situ as a TGF-β-activating factor.

Analysis of F-spondin expression levels by QPCR demonstrated F-spondin up-regulation in OA cartilage specimens of individual patients, although considerable variation was noted. Within the OA patient population, F-spondin levels were roughly equivalent to normal controls for 3 patients and increased between ~2- and ~16-fold for the other 9. This variation may reflect disease heterogeneity in the samples obtained for analysis. For these studies whole tissue was harvested, as the amount of cartilage was often limiting. However, although all specimens exhibited advanced OA, we noted some degree of variation in the amount of cartilage degeneration. It is thus possible that regional differences with respect to OA progression may incorporate multiple chondrocyte disease states leading to local variations in gene expression.

Immunolocalization studies revealed both extracellular F-spondin staining in the superficial zone and cell-associated staining within chondrocyte clusters in proximity to the articular surface. This was true for both human OA specimens and articular cartilage from the surgically induced rat model. Interestingly, in rat OA cartilage the clusters also stained positive for markers of chondrocyte hypertrophy, including type X collagen, AP, and MMP-13. Likewise, in human OA specimens, F-spondin gene expression correlated with type X collagen and alkaline phosphatase (Table 1) . These observations suggest that F-spondin expression is associated with chondrocyte hypertrophy, a common feature of osteoarthritic cartilage in which articular chondrocytes appear to replicate the terminal differentiation process of embryonic growth plate chondrocytes during limb formation (11) . Further evidence correlating F-spondin with chondrocyte hypertrophy has been observed in the embryonic growth plates of chick tibia, where F-spondin expression is confined only to hypertrophic and mineralized zones and demonstrates a similar expression profile to alkaline phosphatase (30) . Despite these similarities, it is not clear whether F-spondin expression by OA chondrocytes reflects a reversion to an immature state or rather a dysregulated state that arises as a consequence of cell proliferation in an attempt to repair the damaged matrix.

In OA chondrocyte cultures, F-spondin gene expression was stimulated by FGF-2, retinol, and IGF-1, whereas TGF-β had no significant effect. Conversely, IL-1β inhibited its expression. These findings suggest that F-spondin expression in chondrocytes may be regulated by growth and differentiation factors. Indeed, preliminary observations from our group indicate that retinoic acid stimulates F-spondin expression during differentiation of embryonic growth plate chondrocytes (30) ; however, the significance of F-spondin induction in OA chondrocytes remains unclear. While a definitive role of FGF-2 in OA cartilage has not been established, elevated levels have been reported in synovial fluid of OA knees (31) , and increased FGF-2 staining has been reported adjacent to cartilage lesions in early-stage OA (32) . Moreover, in agarose cultures of articular chondrocytes, FGF stimulation can induce cluster formation similar to that seen in OA cartilage (32 , 33) . Thus, F-spondin may be regulated in OA tissue by FGF-2. Indeed, within the pools of OA cartilage from which we initially identified F-spondin up-regulation by Affymetrix microarray (19) , FGF-2 was also significantly increased above nondiseased controls following analysis of normalized microarray data (data not shown).

Since F-spondin shares type I TSR repeat motifs with thrombospodin-1, which have been shown to be required for thrombospondin-mediated activation of the latent TGF-β complex (27) , we investigated whether F-spondin similarly activates TGF-β in cultured cartilage explants. Our findings indicate that F-spondin supplementation raised active TGF-β1 levels to 500 pg/g cartilage (>150%), which represented ~100 pg/ml of active TGF-β in the cell culture supernatants. While this may only reflect a fraction of the total TGF-β present, work by Lafeber et al. (34) has demonstrated that active concentrations in the 10–100 pg/ml range are sufficient to modulate metabolic effects within OA cartilage tissue.

These experiments demonstrate for the first time that F-spondin can activate latent TGF-β1, an observation that may have significant implications for its biological functions in the nervous system and other noncartilaginous tissues. We have not yet established whether F-spondin-mediated activation of TGF-β1 occurs directly, by binding to the latent complex, or indirectly through stimulation of downstream activating molecules, such as plasmin (35) or reactive oxygen species (36) . The presence of a highly conserved latency associated peptide (LAP)-binding KRFK motif and 3 WxxW "docking sites" in the TSR domain of F-spondin would suggest that F-spondin activates TGF-β1 by competitively inhibiting association of LAP to the mature TGF-β domain in a manner analogous to thrombospondin-1 (37) . In support of this hypothesis, F-spondin-mediated stimulation of PGE2, which was in part TGF-β dependent, was drastically reduced by TSR domain inhibition or deletion. Deletion of TSR repeats 4–6, which contain the KRFK and 2 WssW motifs, almost completely abolished F-spondin induction of PGE2 in our transfection experiments, lending support to a LAP-binding mechanism for TGF-β activation.

Our functional data also indicate that F-spondin exerts catabolic effects on cultured cartilage explants, causing both TGF-β- and PGE2-dependent degradation of collagen and inhibition of proteoglycan synthesis. Although our studies suggest TGF-β mediated-induction of PGE2 by F-spondin, it is unclear whether the observed matrix catabolism in our cultures occurs via a TGF-β-dependent induction of PGE2 or an alternate PGE2 pathway. Since PGE2 has an established role as a mediator of cartilage degeneration (7 8) , it was not surprising to find that F-spondin-mediated catabolic effects could be blocked by COX-2 inhibition, via celecoxib treatment. However, the inhibition of F-spondin-mediated catabolism via TGF-β antibody treatment was unexpected as it implies that TGF-β pathways can promote degradation of cartilage. Indeed, within our culture system, rather than promote proteoglycan synthesis and protect explants from collagen degradation, TGF-β1 treatment paralleled the catabolic effects of F-spondin among several patients. In vivo, intra-articular administration of TGF-β1 by either recombinant protein or gene delivery has been shown to promote OA-like effects within the joint, including the depletion of extracellular matrix (38 , 39) . One possible mechanism of TGFβ-1-mediated matrix degeneration in OA chondrocytes is via the activation of MMP-13 (40) . In our studies MMP-13 was induced by F-spondin following addition to OA cartilage explant cultures. This induction may be a consequence of TGF-β1 activation, or PGE2 induction by F-spondin. Recent work by our group has demonstrated induction of MMP-13 in OA cartilage explant cultures following addition of exogenous PGE2, via the EP4 receptor (8) . Collectively, our observations identify a possible pathway of F-spondin-mediated type II collagen degradation in OA chondrocytes, initiated by activation of TGF-β1 from its latent complex, followed by induction of PGE2, which in turn stimulates secretion and activation of MMP-13. These direct and indirect actions of F-spondin in OA chondrocytes are outlined in Fig. 8 .


Figure 8
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  		Figure 8. Proposed actions of F-spondin in OA chondrocytes. Our data suggest that induction of cartilage catabolism by F-spondin occurs via pathways that include activation of latent TGF-β, induction of PGE2, and MMP-13. Non-TGF-β dependent effects may also occur via as yet unidentified receptors that bind to the intact molecule or its proteolytic fragments. The combination of these effects may culminate in extracellular matrix degeneration within focal cartilage lesions.

In summary, we demonstrate that F-spondin, previously not recognized as a product of chondrocytes, is up-regulated in the lesional zones of both human and rodent osteoarthritis, where it promotes prostaglandin production, collagen degradation, and decreased proteoglycan synthesis. Among the most intriguing observations of these studies is the demonstration that, as predicted by its TSR domain homology, F-spondin is a latent TGF-β activating protein. Since the overexpression of F-spondin in OA is localized to the superficial zone of lesional areas in OA, we speculate that it may act to promote in situ TGF-β activation and characterize events important in the pathogenesis of osteoarthritis.


   ACKNOWLEDGMENTS
 
The authors thank Jyoti Patel (Division of Rheumatology, Hospital for Joint Diseases, New York, NY, USA) for her help with harvesting cartilage. This work is supported in part by U.S. National Institutes of Health grant CA034282 (D.B.R.), R01-4R05487 and T32-AR007176 (S.B.A.). The authors also thank the William and Linda Steere Foundation and the Joseph and Sophia Abeles Foundation for their generous support.

Received for publication May 28, 2008. Accepted for publication August 14, 2008.


   REFERENCES
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 RESULTS
 DISCUSSION
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