The insulin-like growth factor binding proteins in uncultured human cartilage: Increases in insulin-like growth factor binding protein 3 during osteoarthritis

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<ul><li><p>ARTHRITIS &amp; RHEUMATISMVol. 46, No. 9, September 2002, pp 23582367DOI 10.1002/art.10482 2002, American College of Rheumatology</p><p>The Insulin-Like Growth Factor Binding Proteins inUncultured Human Cartilage</p><p>Increases in Insulin-Like Growth Factor Binding Protein 3 During Osteoarthritis</p><p>Teresa I. Morales</p><p>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.</p><p>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 125IIGF-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.</p><p>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 IIIII 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 </p><p>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.</p><p>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.</p><p>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(46). 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(6,812). In the latter capacity, IGFBP-3 inhibits cellularproliferation in a number of cell types.</p><p>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</p><p>Supported by a Biomedical Research Grant from the ArthritisFoundation and by a grant (R03-AG-16390) from the National Insti-tute on Aging, NIH.</p><p>Teresa I. Morales, PhD: Harvard Medical School and Mas-sachusetts General Hospital, Boston, Massachusetts.</p><p>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.</p><p>Submitted for publication August 16, 2001; accepted inrevised form May 13, 2002.</p><p>2358</p></li><li><p>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 (1517), and thatcultured OA cartilage slices release more IGFBPs intoconditioned media than normal ones (17,18).</p><p>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.</p><p>MATERIALS AND METHODS</p><p>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 2448hours 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 4663 [mean SD male age 51 7 years,mean SD female age 53 7 years]; samples were coded19). 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.</p><p>Dissection of human articular cartilage. Once in thelaboratory, the articular surface of the femoral head was wipedwith sterile gauze containing phosphate buffered saline (PBS)</p><p>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 35 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 bumpycartilage 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).</p><p>The yield of tissue from the different sites varied from0.1 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 35 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.</p><p>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.</p><p>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 4C (20)(urea buffer: 0.05M Tris maleate buffer, pH 6.0, containing</p><p>IGFBP IN OSTEOARTHRITIS 2359</p></li><li><p>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 byDEAESephacel chromatography in columns equilibrated inurea buffer (250500 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).</p><p>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 60C. These PK digests were also analyzed formatrix and cell (DNA) content. When sufficient tissue wasavailable, 200250 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 07), 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 07;n 7). Thus, hypro was routinely measured only in theresidual PK extracts.</p><p>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 manufacturers instructions. Fol-lowing SDSpolyacrylamide 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 125IIGF-2 (Amersham Pharmacia Biotech,Piscataway, NJ) as the binding ligand (0.51 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</p><p>up IGFBP-6 well (24). Air-dried membranes were exposed toHyperfilm MP (Amersham Pharmacia) at 70C.</p><p>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 antiBP-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).</p><p>IGFBP-2 radioimmunoassay (RIA). The procedure (asdirected by DSL) followed the basic principle for RIA; theIGFBP-2 in standards and unknowns competed with 125IIGFB...</p></li></ul>

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