ARTHRITIS & RHEUMATISMVol. 46, No. 9, September 2002, pp 2358–2367DOI 10.1002/art.10482© 2002, American College of Rheumatology

The Insulin-Like Growth Factor Binding Proteins inUncultured Human Cartilage

Increases in Insulin-Like Growth Factor Binding Protein 3 During Osteoarthritis

Teresa I. Morales

Objective. To assess changes in the insulin-likegrowth factor binding proteins (IGFBPs) in unculturedcartilage during stages of osteoarthritis (OA), and todetermine if OA cartilage is capable of autocrine secre-tion of IGFBPs.

Methods. Articular cartilage was dissected fromfibrillated and nonfibrillated sites of 11 human femoralheads, and extracted in buffer containing 8M urea.IGFBPs were identified by immunoprecipitation andsubsequent analysis by 125I–IGF-2 Western ligand blot-ting (WLB), radioimmunoassay, or 2-site immunoradio-metric assay (IRMA). IGFBPs were assessed in carti-lage extracts by WLB. IGFBP-3 content was determinedby IRMA and synthesis by metabolic labeling with35S-cysteine in organ cultures.

Results. Sample grouping into 3 distinct OAstrata was supported by gross pathology of the femoralheads, histologic grading of cartilage slices, and bio-chemical analysis of the glycosaminoglycan and proteincontent of the extracts. Group I was normal/mild OA,group II was intermediate OA, and group III was severeOA. IGFBP-2 was present in all samples, IGFBP-4 insporadic samples, and BP-3 in group II–III samples. ByIRMA, group I had a mean � SD of 6.26 � 2.6 ngIGFBP-3/mg soluble protein (IGFBP-3) (n � 6), groupII had a mean � SD 14 � 7.5 IGFBP-3 (n � 10), andgroup III had a mean � SD 17.03 � 8.94 IGFBP-3 (n �

6). Analysis of variance showed group differences(F[3,19] � 3.84, P � 0.04), and post hoc tests revealedthat IGFBP-3 levels were higher for group III versusgroup I (P � 0.04). OA cartilage synthesized IGFBP-3.

Conclusion. Increases in net cartilage content ofIGFBP-3 occurred in intact OA cartilage, reachingstatistically significant elevation in severe disease.There was autocrine IGFBP-3 production in OA carti-lage.

Insulin-like growth factors (IGFs) were first dis-covered by their “sulfation” action on cartilage (1). Sincethen, compelling evidence has accumulated to show thatIGFs are key players in the regulation of growth plateactivity during skeletal growth and of matrix homeosta-sis in adult articular cartilage (2,3). In turn, a family of atleast 6 IGF binding proteins (IGFBPs) flexibly andprecisely governs the signaling activities of the IGFs(4–6). Several of the binding proteins can either aug-ment or inhibit IGF actions, depending on their modi-fications and molecular interactions. For example, ma-trix or cellular binding sites in the tissue of residence canmodify the affinity of the IGFBPs for the IGF ligandand/or help locate them at a distance from or near IGFsignaling receptors on the cell (7). IGFBP-3 is one of thebest studied of the IGFBP family of proteins. Interest-ingly, there is now strong evidence in support of thestriking versatility of action of this protein; IGFBP-3 canact as a modulator of IGF action, and can also act as anindependent ligand to promote intracellular signaling(6,8–12). In the latter capacity, IGFBP-3 inhibits cellularproliferation in a number of cell types.

Osteoarthritis (OA) afflicts articular cartilagewith marked cellular alterations: proliferative activityoccurs as part of an attempted repair process, but cellsremain cloned and do not disperse in the matrix. In

Supported by a Biomedical Research Grant from the ArthritisFoundation and by a grant (R03-AG-16390) from the National Insti-tute on Aging, NIH.

Teresa I. Morales, PhD: Harvard Medical School and Mas-sachusetts General Hospital, Boston, Massachusetts.

Address correspondence and reprint requests to Teresa I.Morales, PhD, Massachusetts General Hospital, Department of Or-thopaedic Surgery, Jackson 1223, 55 Fruit Street, Boston, MA 02114.E-mail: tmorales@partners.org.

Submitted for publication August 16, 2001; accepted inrevised form May 13, 2002.

2358

addition, loss of cells accompanies and exacerbatespathology (13). Another hallmark of the disease is lossof metabolic steady state in the cartilage matrix, whichleads to progressive, irreversible loss of tissue andexposure of the underlying bone. In addition to main-taining the cartilage matrix steady state during normalcircumstances, IGF-1 counteracts inflammatory agentsthat induce cartilage breakdown, such as interleukin-1(14). Thus, it is reasonable to consider that changes inthe regulatory proteins of the IGF axis may play a directrole in OA. Indeed, there is provocative evidence fromculture studies indicating that chondrocytes excised fromOA cartilage produce more IGFBPs than their counter-parts isolated from normal cartilage (15–17), and thatcultured OA cartilage slices release more IGFBPs intoconditioned media than normal ones (17,18).

A current proposal is that an increase in IGFBPsrenders OA cartilage insensitive to IGF action (16). Thepossible central role of this mechanism in the molecularpathology of articular cartilage underlies the importanceof determining if IGFBP alterations during OA aremanifested in fresh tissue. This study examines theIGFBPs directly in extracts of fresh (uncultured) humanarticular cartilage with different degrees of OA degen-eration, and demonstrates for the first time that statis-tically significant, specific increases in IGFBP-3 proteinoccur during severe disease. In addition, it is shown thatthere is autocrine production of this protein by OAcartilage.

MATERIALS AND METHODS

Harvesting of human articular cartilage. The humanstudies described here were approved by the institutionalreview board of Massachusetts General Hospital (MGH). Thefemoral heads of 2 deceased male organ donors, ages 37 yearsand 47 years, were harvested on site by orthopedic fellows forthe MGH Bone Bank (Boston, MA). The femoral head (cut atthe neck) was covered with gauze impregnated with sterilesaline to prevent drying of the articular surface and placed ina sterile bag. It was kept cold and dissected within �24–48hours of the time of death. The donor codes used by the bonebank were maintained (nos. 355 and 403). Cartilage fromsurgical donors was derived from subjects undergoing hipreplacement surgery for OA at the MGH (9 subjects, 4 malesand 5 females, ages 46–63 [mean � SD male age 51 � 7 years,mean � SD female age 53 � 7 years]; samples were coded1–9). After removal of the femoral head from the patient, thespecimen was extensively washed in the operating room withsterile saline and transported to the laboratory as described,but within 5 minutes.

Dissection of human articular cartilage. Once in thelaboratory, the articular surface of the femoral head was wipedwith sterile gauze containing phosphate buffered saline (PBS)

and proteinase inhibitors (PI-saline) to help remove residualblood or synovial fluid (1 tablet of proteinase inhibitor cocktail[Roche Molecular Biochemicals, Indianapolis, IN]/50 ml). Thefemoral head was visually inspected, sketched, and the grosspathology recorded. In all of the surgical specimens fromsubjects diagnosed with clinical OA, there was a frank ulcerwith denuded bone. Soft cartilage with vertical clefts andfissures visible to the naked eye (fibrillated) was present in therims of the ulcers, as expected, and �3–5 mm of the tissue allthe way around the ulcer site was dissected to provide theprimary source of fibrillated cartilage. Sometimes small rem-nants of tissue within the ulcers, or patches of fibrillated tissuedistant from the ulcers, were pooled together. In the organdonor samples, frank ulcers were not seen, but each femoralhead had a patch of fibrillated tissue, which was dissectedseparately from the rest. The more damaged sites (ulcer rimand/or other fibrillated sites) were labeled F. During dissec-tion, the cartilage surface on the femoral head was kept moistby frequent irrigation with PI-saline; the cartilage slices weretransferred to a petri dish containing PI-saline and maintainedcold. The more distal cartilage (labeled D) was dissectedseparately, avoiding areas that looked fibrillated or calcified tothe naked eye. In 2 cases where the distal sites appearedpatchy, distinct sites were dissected and pooled separately, asfollows. For sample 6, areas of relatively thick and “bumpy”cartilage were distinct from thinner, more discolored sites(6D1 and 6D2, respectively), and there were small spots offibrillated tissue that were pooled with 6F. For sample 7, anarea of thin, yellowish cartilage with a rubbery texture wasdifferent from relatively thick cartilage that was vascularized inthe deep zone (7D1 and 7D2, respectively).

The yield of tissue from the different sites varied from�0.1 gm (wet weight) for very fibrillated sites (F sites) to 1.9gm for healthier tissue (D sites). After the cartilage harvest,the slices were further diced into pieces of �3–5 mg (wetweight) (as necessary). The diced cartilage from the F and Dpools was then transferred to sterile gauze, blotted dry, andpieces were taken from each pool for histologic study; thesewere fixed in 10% buffered formalin. The cartilage pieces forhistology were selected at random, but an effort was made toselect full-depth samples that represented the rest of the pool;for example, if the gross pathology described the site as thinand yellowish, then thickness and color were the criteria forselection. The criteria for evaluation of OA severity includedcartilage structure (integrity), cellularity, and Safranin O stain-ing intensity as originally described by Mankin et al (19),except that tidemark integrity was not evaluated. The rest ofthe tissue from each site was transferred to a preweighed petridish containing PI-saline and weighed.

Twenty-four samples were obtained from the 11 fem-oral heads; 2 sites (F and D) were obtained from mostspecimens, except as noted above for samples 6 and 7 (3 siteseach) and sample 1, which did not have sufficient tissue aroundthe ulcer (F) for harvesting, so only the distal site was used.This site had a tightly adherent viscous layer on the surface,which was dissected separately from the deeper tissue to assessif there were any differences in IGFBP content.

Cartilage extraction and processing of extracts. Ureaextracts. The diced cartilage was stirred in 15 volumes (ml/gmwet weight) of urea buffer for at least 48 hours at 4°C (20)(urea buffer: 0.05M Tris maleate buffer, pH 6.0, containing

IGFBP IN OSTEOARTHRITIS 2359

0.3M NaCl, 8M urea, 0.5% CHAPS, and proteinase inhibitors[5 mM phenylmethylsulfonyl fluoride, 3 mM o-phenanthroline,6.5 �M pepstatin, and 9.5 �M leupeptin]). The urea extractswere used for IGFBP analysis (as described below), andaliquots were also analyzed for matrix and cell content asfollows: glycosaminoglycan (GAG) by the dimethylmethyleneblue dye binding procedure (21), protein by the bicinchoninicacid assay (Pierce, Rockford, IL), and DNA content by theHoechst dye binding method (22). For analysis of IGFBPs inthe urea buffer extracts, proteoglycans were removed byDEAE–Sephacel chromatography in columns equilibrated inurea buffer (250–500 �g GAG/ml of gel, depending on theDEAE batch). Under the chromatographic conditions, onlyvery highly anionic molecules such as proteoglycans bound tothe column. It was confirmed that the protein yields from thesecolumns were �100% (unbound � bound fractions). Theunbound proteins were dialyzed versus acidic solution (4 mMHCl containing 0.1 mM phenylmethylsulfonyl fluoride [PMSF],1 mM o-phenanthroline [o-phe]), 0.15 �M pepstatin, 0.2 �Mleupeptin) and concentrated by speed vacuum centrifugation(20). Protein yields from the dialysis step were also nearlycomplete (mean � SD 101 � 11%; n � 7).

Proteinase K (PK) extracts. Following urea buffer ex-traction, the tissue was washed several times with PBS. Thefluid was removed, the cartilage blotted on sterile gauze, andthen dried in a speed vacuum centrifuge to a constant weight.The dry weight was recorded and the tissue was then dispersedby overnight digestion with 60 volumes (ml/gm dry weight) of0.5 mg/ml PK at 60°C. These PK digests were also analyzed formatrix and cell (DNA) content. When sufficient tissue wasavailable, �200–250 mg (wet weight) of cartilage was directlyextracted by PK digestion (bypassing the urea buffer extrac-tion) (15 volumes PK [ml/gm wet weight]). For 7 samples(histologic OA grades 0–7), the GAG and protein content(�g/mg dry weight) of the direct PK extracts were a mean �SD of 115 � 43% and 82 � 15% of the combined urea and PKvalues, respectively. Thus, taking into account the inherentvariabilities of the assays, the sequential extraction provided areliable measure of total GAG and protein. Hydroxyproline(hypro) assay of dried acid hydrolyzates was used as a measureof collagen (23). Hypro in the direct PK extract was similar tothat in the residual extract (mean � SD 94 � 8%, grades 0–7;n � 7). Thus, hypro was routinely measured only in theresidual PK extracts.

IGFBP analysis. Western ligand blots (WLBs). Briefly,10% Bis-Tris NuPAGE gels were used for electrophoresis withMOPS sodium dodecyl sulfate (SDS) running buffer (Invitro-gen, Carlsbad, CA). Twenty-five micrograms of each driedDEAE-purified sample was resuspended in NuPAGE samplebuffer and subjected to electrophoresis (under nonreducingconditions) according to the manufacturer’s instructions. Fol-lowing SDS–polyacrylamide gel electrophoresis, the proteinswere transferred to nitrocellulose membranes using NuPAGEtransfer buffer. The proteins remaining in the polyacrylamidegels after the transfer (mostly of large size) were stained; thishelped to verify equal loading and transfer of samples within agel. The nitrocellulose membranes were washed and thendeveloped using 125I–IGF-2 (Amersham Pharmacia Biotech,Piscataway, NJ) as the binding ligand (0.5–1 million counts perminute in 20 ml buffer) (20,24). IGF-2 was used as the ligandbecause it detects all the IGFBPs, while IGF-1 does not pick

up IGFBP-6 well (24). Air-dried membranes were exposed toHyperfilm MP (Amersham Pharmacia) at �70°C.

IGFBP-3 2-site immunoradiometric assay (IRMA). As-say tubes in the kit (Diagnostic Systems Laboratories [DSL],Webster, TX) contained immobilized antibody (goat poly-clonal) to IGFBP-3. Briefly, 50 �l of sample (DEAE-purifiedcartilage sample or IGFBP-3 standard) and 200 �l of 125I-labeled anti–BP-3 (goat polyclonal) were added to the tubes.The samples were incubated overnight at room temperature toallow the bridge reaction of IGFBP-3 to the bound and solubleantibodies to occur, the tubes were washed and drained severaltimes, and the cpm of bound 125I-labeled antibody was mea-sured in a gamma counter. The standard curve was linear on alog-log scale. The other 5 IGFBPs were not detectable by thisassay, except at concentrations 4 orders of magnitude higherthan required for IGFBP-3 detection (DSL literature).

IGFBP-2 radioimmunoassay (RIA). The procedure (asdirected by DSL) followed the basic principle for RIA; theIGFBP-2 in standards and unknowns competed with 125I–IGFBP-2 for binding to rabbit anti–IGFBP-2, and the immunecomplexes were precipitated with goat anti-rabbit gammaglobulin serum and polyethylene glycol. Anti–IGFBP-2 did notreact with 25-fold higher than optimal concentrations ofIGFBP-3, -4, -5, and -6 for IGFBP-2 detection (DSL). Theassay was linear on a log-linear scale.

Identification of IGFBPs by immunoprecipitations fol-lowed by WLBs. Immunoprecipitations were as described(20,24) using 70 �g of DEAE-purified cartilage protein persample (1 �g/�l buffer). The antibodies were rabbit poly-clonals raised against recombinant human (rHu) proteinsIGFBP-3, -4, and -5 (Upstate Biotechnology, Lake Placid, NY)or rHuIGFBP-6 (Austral Biologicals, San Ramon, CA). Anti-rat IGFBP-2 polyclonal antibody and the preimmune serumfor the same rabbit were provided by Dr. B. Peterkofsky (25).Other autologous nonimmune serum was obtained from GibcoBRL (Grand Island, NY). Antibodies were diluted �10–20-fold in the reaction mixtures.

Deglycosylation. Samples were heated for 2 minutes at100°C prior to treatment with N-glycanase (Glyko, Novato,CA) in 0.02M sodium phosphate buffer, pH 7.5, containing0.02% (weight/volume) sodium azide, 0.05M EDTA, 1.5 mMo-phenanthroline, 1 mM PMSF, 6.5 �M pepstatin, 9.5 �Mleupeptin, and 0.5% SDS. Enzymatic digestions were per-formed overnight at 37°C.

Statistical analysis. One-way analysis of variance(ANOVA) was conducted to examine differences in IGFBP-3/mg protein levels, and for chondroitin sulfate, hypro, protein,and DNA (each per mg dry weight) among the 3 histologicallystratified groups of OA samples. Tukey’s honest significantdifference (HSD) post hoc tests were also conducted toexamine differences between disease strata. Confidence levelswere set at � � 0.05. All statistical analysis was performed withSPSS version 10 (SPSS, Chicago, IL).

RESULTS

Validation of human cartilage extraction proce-dure. The urea buffer extracted approximately the sameproportion of protein from normal or OA cartilage

2360 MORALES

slices, a mean � SD of 26 � 5% of the total (n � 24; thetotal protein included the insoluble collagen fraction).This allowed comparison of IGFBPs in urea extracts ofnormal versus OA cartilage.

Assessment of OA disease stage of human sam-ples. Gross pathology and histologic evaluation. The car-tilage samples obtained from the 11 human femoralheads covered the range from apparently normal tosevere OA, as determined by gross visual inspection ofthe femoral heads and by grading histologic slices by thecriteria of Mankin et al (19). The histologic scoresassigned by one blinded and by one unblinded observer(TIM) were all identical or within one OA grade, andwere averaged when needed. The gross pathology andhistologic grading were most distinct for samples thatwere apparently normal or showed signs of only verymild disruption (gross pathology: relatively thick,smooth, intact surface or only slight surface fibrillationvisible to the naked eye; histologic OA scores 0–3) andthose that had severe disease (very thin, soft cartilagewith deep vertical clefts and fissures; OA scores �8).The samples between these 2 extremes were hetero-geneous by gross inspection (presence or lack of fibril-lation visible to the naked eye, color, degree of softnessto the touch of the scalpel, thickness), and had OAscores of 3.5–7.

Cell and matrix assays. Cartilage extracts wereassayed for GAG, hypro, DNA, and protein content andnormalized to mg dry weight. The results were groupedin accordance with the observations of the gross pathol-ogy and histologic scoring into normal to mild OA (I),intermediate or moderate OA (II), and severe OA (III).Figure 1 shows a decrease in GAG content with increas-ing disease severity in the OA groups, in contrast to anincrease in protein levels. Hypro and DNA levels did notdiffer by disease severity and DNA levels did not change(Figure 1).

One-way ANOVA was conducted to examinedifferences in the parameters listed above for the 3 OAgroups. Tukey’s HSD post hoc tests were also conductedto examine differences between groups. One-wayANOVA revealed significant differences for GAG/mgdry weight (F[2,19] � 14.08, P � 0.001). There were nosignificant between-group differences for hypro (F[2,19]� 1.10, P � 0.353) and DNA (F[2,17] � 0.970, P �0.399). However, the differences for protein were signif-icant (F[2,19] � 4.43, P � 0.030). The post hoc testsrevealed that GAG was significantly different for allgroups (group I versus II, P � 0.029; group II versus III,P � 0.015; group I versus III, P � 0.001). Proteindifferences were significant only between grades I and

III (P � 0.025). Thus, gross observations of cartilage onthe femoral head, histologic grading, GAG assay ofextracts, and protein assay of extracts all supported themarked differences between groups I and III; in addi-tion, the GAG assays showed significant differencesamong all 3 groups. Both the correlation between OAstage and GAG content and the heterogeneity of thedisease, particularly during an “attempted repair” stage,are well known and widely supported (13). Thus, thesamples analyzed in this investigation can be reasonablyclassified into the 3 OA stages, I–III.

Identification of IGFBPs in cartilage extracts.The first objective was to identify the major IGFBP speciesin cartilage extracts. Samples with different OA gradeswere immunoprecipitated and the resulting pellets wereresuspended and analyzed by WLB in order to identify theproteins by 3 independent criteria: immunoreactivity, size,and ability to bind to IGF-2. Antibodies against IGFBP-2to -6 were tested (see Materials and Methods). Figure 2(left panel) shows that the IGFBP-2 antibody reactedstrongly with a protein band of �30–31 kd in all samplesexamined with different degrees of OA. The protein(s) in

Figure 1. Cell and matrix parameters in histologically stratified osteo-arthritis (OA) groups. Proteoglycan, collagen, protein, and cell contentwere estimated from glycosaminoglycan (GAG), hydroxyproline(hypro), protein, and DNA values, respectively. For all samples, boththe urea extracts and the proteinase K extracts were evaluated andvalues added. Data are the mean and SD in each group, with OAgroups I, II, and III from left to right. For GAG, hypro, and protein,n � 6 in group I, n � 10 in group II, and n � 6 in group III. (Valuesfor sample 1 from the superficial and deep zones were added to reflectthe same values used for Figure 6, and the value for sample 403F wasnot included for the same reason [insufficient amounts of sampleremained for the insulin-like growth factor binding protein 3 analysisshown in Figure 6]. For DNA analysis, fewer samples were left forassay in the severe OA group.) For DNA, n � 4 in group III.

IGFBP IN OSTEOARTHRITIS 2361

this size range also reacted weakly with antibodies toIGFBP-3 (Figure 2, right panel), -5, and -6 (results notshown), but the most immunoreactive IGFBP with itscorresponding antibody was definitely IGFBP-2. On theother hand, the IGFBP-3 antibody immunoprecipitated aprotein doublet of �39–42 kd from samples with frank OA(3F and 3D), but not from the organ donor samples withmilder degeneration (355F and 355D).* A protein of�25–26 kd was seen in some of the direct WLBs, includingsample 4F (score � 6), which gave a positive reaction withanti–BP-4 (results not shown). Positive controls showedthat all of the antibodies reacted strongly with 3 ng of thematching rHuIGFBP standard (results not shown). On theother hand, the IGFBP proteins in the human cartilageextracts did not react with preimmune sera from the rabbitproducing anti–IGFBP-2, or with nonimmune autologousserum (Figure 2). This supported the specificity of theimmune sera.

Since previous work had identified large amounts

of IGFBP-6 in bovine articular cartilage by Westernimmunoblotting (20), the human samples were alsoimmunoblotted. Anti–rHuIGFBP-6 resulted in a posi-tive signal for the bovine IGFBP doublet of �21.5–24kd, but human samples run in parallel failed to reactwith this antibody (results not shown). In summary, thepresent results support the presence of IGFBP-2, -3, and-4 in human OA cartilage. RIA for IGFBP-2 confirmedthat the cartilage extracts contained substantial amountsof this binding protein, with a mean � SD of 17 � 3 ngIGFBP-2/mg soluble protein (OA scores of 0–8; n � 5).The presence of the IGFBP-3 doublet was confirmed byWestern immunoblot (at 1:150–1:250 dilution of anti-body [results not shown]), by 2-site IRMA, and byenzymatic deglycosylation (see below).

Deglycosylation reactions. IGFBP-3 is glycosy-lated at asparagine residues (N-glycosylated) both inserum and within tissues (5,6). The occupancy of 2 or 3of the potential N-glycosylation sites results in a highmolecular weight doublet such as the 39–42-kd proteinfound in the cartilage extracts. To verify N-glycosylationof the latter, human OA cartilage samples were treatedwith N-glycanase. This enzyme removes N-linked oligo-

* Sample 355 did not have an ulcer, only a small fibrillated site(F) surrounded by vast amounts of very healthy cartilage (D). Thecriteria for scoring of the F site included the fissuring of tissue, cloningof cells close to these clefts, and loss of Safranin O staining, mostly inthe superficial zone.

Figure 2. Identification of insulin-like growth factor binding proteins (IGFBPs) by immunopre-cipitation followed by Western ligand blotting (WLB). The human sample extracts were subjectedto immunoprecipitation with IGFBP-2 or IGFBP-3 polyclonal antibodies (see Materials andMethods), and the pellets resuspended in an equal volume of 2� sodium dodecyl sulfate samplebuffer and heated at 100°C for 5 minutes to solubilize the antigen–antibody complex bound toprotein A–agarose. After cooling, the samples were centrifuged at 10,000g for 5 minutes and thensubjected to WLB. Molecular weight standard (STD) sizes in kd are shown on the left. Numbersbelow each lane refer to the patient code for human samples run on electrophoresis gels; letters For D refer to the site used for the preparation of the extract (fibrillated or distal; see Materials andMethods). For the nonimmune control, the antibody was substituted with rabbit preimmune serumfrom the same rabbit (IGFBP-2), or with nonimmune rabbit serum (Gibco BRL). OA �osteoarthritis.

2362 MORALES

saccharides by cleavage at their anchoring site, i.e.,between the acceptor asparagine moiety in the proteinand the terminal N-acetyl glucosamine in the oligosac-charide. Figure 3 shows that N-glycosidase treatmentindeed reduced the size of the �39–42-kd doublet to�33 kd, and that the size of this digest coincided withthat generated by N-glycosidase treatment ofrHuIGFBP-3. This confirms the identification of theprotein doublet in human OA cartilage samples asN-glycosylated IGFBP-3.

Changes in IGFBP levels in WLBs with OAprogression. Figures 4A and B show WLBs from se-lected specimens comparing the ulcer rim (F) and distal

(D) sites dissected from the same femoral head. Thiswas done routinely to provide a direct comparison ofsamples with different OA severity from the samedonor, such that some of the potential variables regulat-ing the IGFBP-3 protein output of the cartilage, such asage, sex, medications, or lifestyle, were controlled. Thesamples shown in Figure 4 were selected from the 24analyzed to illustrate that there was little or no detect-able IGFBP-3 in the normal sample group (scores 0–3),but this protein was evident in samples with OA. Twospecimens with very focal disease are shown, samples 9and 5 (these had severe degeneration at the ulcer sitebut apparently normal cartilage in distal sites, particu-larly sample 5, which had large amounts of thick, white,intact cartilage).

For sample 9, the ulcer site (F) had advanced OA(score � 10) and the extract from this site showedprominent IGFBP-3 and IGFBP-2 bands, while thesample from the distal site scored within the normalrange (1.5) and had decreased IGFBPs (Figure 4A).Likewise, in sample 5 both IGFBPs were most promi-nent in the F site (score � 7), compared with the distalsite (score � 0) (Figure 4B). In fact, the latter site (D)lacked detectable glycosylated IGFBP-3. In contrast,samples 8 (Figure 4A) and 4 (Figure 4B) had scores �4in ulcer and distal sites and all had significant IGFBP-3bands. To further confirm these trends, Figure 5 shows acomposite blot from 15 of 24 samples with differentdegrees of OA. Note that the samples in the normalgroup (scores of 0–3) do not show evident IGFBP-3;there are variable levels of activity in the intermediatesamples with scores of 4–6 (with moderate to high levelsin the samples with scores of 4); and all samples in the

Figure 3. Identification of glycosylated IGFBP-3 by enzymatic degly-cosylation and WLB. A set of replicate tubes containing recombinanthuman IGFBP-3 glycosylated standard (3 ng BP-3 � 10 �g bovineserum albumin) or 20 �g of human cartilage protein (7F) wasdenatured and treated with or without N-glycanase (see Materials andMethods). All incubated samples (20 �l) were adjusted by the additionof 4� sodium dodecyl sulfate sample buffer and subjected to WLB.See Figure 2 for definitions and further details.

Figure 4. Comparison of IGFBPs in paired samples from the same donor (F and D sites) by WLB.See Figure 2 for definitions and details.

IGFBP IN OSTEOARTHRITIS 2363

severe group (scores �8) displayed a significantIGFBP-3 band.

Measurement of IGFBP-3 by 2-site IRMA. Theassay values depended on protein concentration in car-tilage extracts; experimental values were 92–126% ofcalculated values. The IRMA accurately detected freeIGFBP-3 as well as IGFBP-3 complexes to IGF-1.Activity levels for the cartilage extracts were calculatedby reference to the nonglycosylated standards providedin the assay kit, but recombinant human glycosylatedIGFBP-3 was recognized with �50% of the sensitivity ofthese standards (Table 1).

Most of the samples examined by WLB were ana-lyzed by IGFBP-3 IRMA. Sample 1D showed an atypical

highly viscous superficial layer and therefore the tissue wasinitially dissected, prepared, and assayed as separate super-ficial and deeper layers. Since the IRMA results were veryclose (33 versus 29 ng IGFBP-3/mg soluble protein [BP-3/mg] for superficial versus deep, respectively) and thepatterns on WLBs were similar, the results were averagedand shown as a single bar in Figure 6A.

The average IGFBP-3 value for each individualsample, charted by OA group, is shown in Figure 6A.The vertical bars on the left side of the figure show thesamples that fell into the normal and severe OA groups(I and III; see Figure 1 for disease staging). It can beseen that 5 of 6 normal samples had �10 ng IGFBP-3/mg soluble protein, while all the samples in the severe

Figure 5. Composite WLBs of human cartilage samples. Samples with different degrees of OAdegeneration were run side by side, and the 2 gels shown run in parallel to directly compare IGFBPlevels by the WLB procedure. The numbers between the 2 gels are the molecular weight of the14C-standards. Scores of 4 are highlighted with asterisks. See Figure 2 for definitions and details.

Table 1. Insulin-like growth factor binding protein 3 (IGFBP-3) immunoradiometric assay validation*

Test sample Assay, vs.Calculated

value, ng/mlExperimental value,

ng/ml (% of calculated)

Glycosylated BP-3 Nonglycosylated 30 16 (53)IGFBP-3/IGF-I complex Free IGFBP-3 19 22 (115)Human cartilage sample

3F, concentrated 3F, 2� dilution 4.5 5.7 (126)5D, concentrated 5D, 3� dilution 0.94 0.92 (98)6F, concentrated 6F, 3� dilution 1.69 1.55 (92)

* For the test of glycosylated versus nonglycosylated IGFBP-3, Upstate Biotechnology’s recombinanthuman IGFBP-3 (rHuIGFBP-3) was compared with the nonglycosylated rHuIGFBP-3 standard includedin the kit obtained from Diagnostic Systems Laboratories. The nominal molecular weight of 47,000 versus28,500 and the concentrations of IGFBP-3 were provided by the manufacturers and used to calculate theexpected results. For the test of free rHuIGFBP-3 (Upstate Biotechnology) versus bound rHuIGFBP-3,the complexes were formed by incubating the components at a 1:2 molar ratio of IGFBP-3:IGF-1(incubated 1 hour at room temperature or overnight at 4°C). Recombinant human IGF-1 was obtainedfrom R&D Systems (Minneapolis, MN). Each experimental value represents the average of 2–4 replicates.

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OA group had near or �10 ng IGFBP-3/mg protein (upto �30 ng/mg). The samples in the intermediate group(II) showed variable, but generally elevated levels ofactivity compared with the normal group. Overall, Fig-ure 6A illustrates increases in the IGFBP-3 content ofthe OA samples, with some of the samples showingsharp, 2–3-fold increases in immunoreactivity. To betterunderstand the significance of these observations, the

samples were grouped into the disease strata shown inFigure 1, and their individual values were averaged.

Figure 6B shows a very clear and striking differ-ence between the normal/very mild OA group (I) andthe severe OA group (III), with the heterogenous inter-mediate group (II) falling between these extremes.One-way ANOVA revealed significant differencesamong the groups for IGFBP-3/mg soluble protein(F[2,19] � 3.84, P � 0.040). The post hoc tests showedthat BP-3 levels were significantly higher for histologicgroup III compared with group I (P � 0.04). Effect sizesand observed power were calculated for BP-3 (seeFigure 6), GAG, and protein tests (see Figure 1). Effectsizes (�2) were 0.288, 0.597, and 0.308, respectively, andobserved powers (�) were 0.624, 0.995, and 0.669, re-spectively, suggesting that even with a small sample size,ANOVAs were sufficiently sensitive to detect differ-ences in these measures when stratified into 3 OAgroups.

It is also worth noting that trends were observedwithin the intermediate OA group for BP-3 values, suchthat there was a clustering of high values in a very tight OAscore subgroup (3.5–4) (Figure 6, intermediate group II,first 5 bars from the top) followed by a relative decline(scores 5–7) (histologic scores 3.5–4: mean � SD 19 � 7.3BP-3/mg [n � 5] versus scores 6–7: mean � SD 9 � 2.88BP-3/mg [n � 5]). The data suggest that a peak of BP-3may occur in a subset of samples during early to mid-OA.However, uncertainties in staging OA within such anextremely tight region by histologic criteria need to benoted, and when the samples were grouped into 4 groupsinstead of 3 (by subdividing group II), ANOVA showedsignificant group differences, but the post hoc tests re-vealed no significant differences between the 2 intermedi-ate subgroups. Thus, the significance of the IGFBP-3clustering in the mid-OA region is unknown.

Autocrine production of IGFBP-3 in cultured OAcartilage slices. Since several investigators have reportedthat IGFBP-3 levels were increased in the synovial fluidof OA patients (26), and several of the OA samples hadsevere fibrillation or fragmentation of the cartilagesurface, the question of whether the binding protein isderived from infiltration of synovial fluid and its com-ponents into cartilage was pertinent. The ability of OAcartilage to synthesize IGFBP-3 was tested in a samplethat had very advanced OA (sample 8, see Figure 4A forWLB). This sample contained high levels of totalIGFBP-3 measured by IRMA (18 and 30 ng BP-3/mgprotein for the F site and D site, respectively), andcartilage from both sites was pooled and metabolically

Figure 6. Insulin-like growth factor binding protein 3 (IGFBP-3)content in human samples with varying degrees of osteoarthritis (OA).IGFBP-3 values were based on the standard curve, with the nonglyco-sylated IGFBP-3 provided by the manufacturer, and were normalizedto total protein present in the assayed extract (see Materials andMethods). Each sample was tested in replicates of 2 or more. A, Meanof replicate evaluations for each individual sample. B, Mean and SDvalues by OA severity group. Twenty-two samples were assayed (6normal, 10 intermediate OA, 6 severe OA). � � P � 0.04 versus group I.

IGFBP IN OSTEOARTHRITIS 2365

labeled with 35S-cysteine. Figure 7 shows the presence of35S-protein bands in the position of the IGFBP-3 dou-blet in the SDS gels of the immunoprecipitates of thecartilage extracts. 35S-labeled IGFBP-3 was also seen inthe conditioned media (results not shown).

DISCUSSION

Previous work by other investigators made astrong case for IGFBP messenger RNA and proteinincreases in chondrocyte monolayer cultures from OAcartilage compared with their normal counterparts, butthe influence of the culture conditions on the resultscould not be ruled out (15–17). Increased release ofIGFBPs from cultured articular cartilage slices was alsodemonstrated, but tissue levels were not assessed(17,18). Further, these early studies did not correlateIGFBP levels with disease stage. Correlative studies of

this nature are inherently difficult due to the lack of anyone specific marker or group of markers that accuratelydefine progressive OA stages. Thus, there is a need touse criteria such as histologic grading, which bringstogether an array of cell and matrix changes that accom-pany OA, but may overlap with aging and possiblytraumatic events (13).

Based on the knowledge that the molecular pa-thology of OA is most pronounced at the ulcer or ulcersites but not in distal sites (27), these sites were dissectedseparately for this investigation. Additional gross criteriawere used to segregate broad categories of apparentlynormal/very mild, intermediate, and severe OA groups.Histologic scores supported these groupings. To obtainquantitative measures, tissue extracts from the dissectedsites were assayed for cell and matrix content. Differ-ences in GAG and protein content between the extremegroups were observed and confirmed by statistical ana-lysis, which also showed differences in GAG contentamong all 3 groups. Thus, taken together, the gross,histologic, and matrix quantitation assays providedstrong support for the grouping of samples into stagesI–III of OA.

The 3 groups were evaluated for IGFBP-3 contentby WLBs and quantitative IRMAs, and it is shown thatthere are significant increases in the net content ofIGFBP-3 in fresh (uncultured) cartilage extracts fromsevere OA compared with apparently normal cartilage.The increase in IGFBP-3 in OA is superimposed on anychanges in total protein because the specific activity ofIGFBP-3 (ng/mg soluble protein) is increased during OA.

It is also worth noting that there was a clusteringof very high levels of IGFBP-3 in an early diseasesubgroup of stage II, but uncertainties in staging OAwithin such a narrow range preclude any conclusionsabout this finding. To obtain more precise staging of thedisease process, it will be necessary to use animal modelsor wait until more accurate markers for human OAdisease progression are available. However, it is cer-tainly possible that transient traumatic and/or inflamma-tory episodes during early to mid-OA cause spikes ofIGFBP-3 activity, and that despite relatively quiescentperiods, the overall progression is toward sustained highlevels of the binding protein.

Although little is known about the function ofIGFBP-3 in cartilage, it is interesting that it coprecipi-tates with a band of �54 kd (Figure 7). The size of thisprotein is similar to a breast cancer cell protein of 55 kdthat has a high specificity and affinity for IGFBP-3 andmay be a signaling receptor (8,10), and thus the cartilageprotein is worthy of future investigation.

Figure 7. Autocrine production of IGFBP-3 in short-term organcultures of severe OA cartilage. Cartilage slices from the F and D sitesof donor 8 were combined to have enough tissue, 240 mg (wet weight)placed in culture overnight in Dulbecco’s modified Eagle’s medium(DMEM) containing 10% fetal calf serum (FCS), then the mediumwas replaced with low-cysteine/methionine DMEM (4.8 mg/liter cys-teine and 3 mg/liter methionine) containing 2% FCS and 230 �Ci of35S-cysteine precursor (Amersham Pharmacia Biotech). The next day,the medium was removed, the cartilage extracted in urea buffer, andthe extract dialyzed and dried. The sample (�0.5 million 35S-cpm and290 �g of protein) was subjected to immunoprecipitation with anti–IGFBP-3 (Upstate Biotechnology) as described in Materials andMethods. Sodium dodecyl sulfate–polyacrylamide gel electrophoresiswas carried out on 10% NuPAGE gels, and the gels were treated withEnhance (New England Nuclear, Boston, MA) and subjected toautoradiography. See Figure 2 for other definitions.

2366 MORALES

In summary, the measurement of the specificactivity of IGFBP-3 directly in articular cartilage extractsand the finding that this protein is significantly increasedduring severe OA are completely novel contributionsthat nonetheless fit well with the general findings fromcultured chondrocytes. In addition, this study presentsthe first demonstration of autocrine production ofIGFBP-3 directly at the protein level in intact cartilage.The data place IGFBP-3 center stage for future investi-gations of growth factor action in the pathogenesis andprogression of OA.

ACKNOWLEDGMENTS

The author thanks Judith Miao for the capable tech-nical execution of most of the experiments in this project, andCarol Trahan for expert assistance with the preparation andblinded grading of histologic samples. Drs. William Tomford,William Harris, and Harry Rubash of Harvard Medical School/MGH provided the human cartilage samples and helpfulcomments. Dr. Chris McGibbon’s (MGH/MGH Institute ofHealth Professions) invaluable assistance in the performanceand interpretation of statistical analysis is gratefully acknowl-edged.

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