ARTHRITIS & RHEUMATISMVol. 48, No. 5, May 2003, pp 1302–1314DOI 10.1002/art.10927© 2003, American College of Rheumatology

One of Two Chondrocyte-Expressed Isoforms ofCartilage Intermediate-Layer Protein Functions as an

Insulin-Like Growth Factor 1 Antagonist

Kristen Johnson,1 David Farley,2 Shou-Ih Hu,2 and Robert Terkeltaub1

Objective. Aging and osteoarthritic (OA) carti-lage commonly demonstrate enhanced expression ofthe large, transforming growth factor � (TGF�)–inducible glycoprotein cartilage intermediate-layerprotein (CILP) as well as enhanced extracellular in-organic pyrophosphate (PPi) that promotes the depo-sition of calcium pyrophosphate dihydrate crystals. Innormal chondrocytes, TGF� induces elevated chondro-cyte extracellular PPi. Insulin-like growth factor 1(IGF-1) normally blocks this response and reducesextracellular PPi. However, chondrocyte resistance toIGF-1 is observed in OA and aging. Because CILP wasreported to chromatographically fractionate withPPi-generating nucleotide pyrophosphatase phosphodi-esterase (NPP) activity, it has been broadly assumedthat CILP itself has NPP activity. Our objective was todirectly define CILP functions and their relationship toIGF-1 in chondrocytes.

Methods. Using primary cultures of articularchondrocytes from the knee, we defined the function ofthe previously described CILP (CILP-1) and of a re-cently described 50.6% identical protein that we desig-nated the CILP-2 isoform.

Results. Both CILP isoforms were constitu-tively expressed by primary cultured articular chon-drocytes, but only CILP-1 expression was detectablein cultured knee meniscal cartilage cells. NeitherCILP isoform had intrinsic NPP activity. But CILP-1blocked the ability of IGF-1 to decrease extracellularPPi, an activity specific for the CILP-1 N-terminaldomain. The CILP-1 N-terminal domain also sup-pressed IGF-1–induced (but not TGF�-induced) pro-liferation and sulfated proteoglycan synthesis, andit inhibited ligand-induced IGF-1 receptor autophos-phorylation.

Conclusion. Two CILP isoforms are differentiallyexpressed by chondrocytes. Neither CILP isoformexhibits PPi-generating NPP activity. But, increasedexpression of CILP-1, via N-terminal domain–mediatedinhibitory effects of CILP-1 on chondrocyte IGF-1 re-sponsiveness, could impair chondrocyte growth andmatrix repair and indirectly promote PPi supersatura-tion in aging and OA cartilage.

Marked elevation of extracellular inorganic pyro-phosphate (PPi) promotes the development of calciumpyrophosphate dihydrate (CPPD) crystal deposition inaging and osteoarthritic (OA) joint cartilage (1,2). Sev-eral growth factors and calciotropic hormones regulateextracellular PPi levels (3). For example, transforminggrowth factor � (TGF�) (3–6) stimulates increasedextracellular PPi (3–6). Insulin-like growth factor 1(IGF-1) blocks this TGF� response, and IGF-1 alonepromotes the lowering of extracellular PPi (7). However,IGF-1 may be less able to restrain extracellular PPilevels in cartilage with established OA, where chondro-cytes typically manifest varying degrees of IGF-1 resis-tance (8–16).

Supported by grants from the NIH (P01-AG-07996), NovartisPharmaceuticals, and by the Research Service of the Department ofVeterans Affairs.

1Kristen Johnson, BA, Robert Terkeltaub, MD: Departmentof Veterans Affairs Medical Center, San Diego, and University ofCalifornia, San Diego; 2David Farley, PhD, Shou-Ih Hu, PhD: Novar-tis Pharmaceuticals, Summit, New Jersey.

Dr. Terkeltaub also serves as a paid consultant to NovartisPharmaceuticals.

Address correspondence and reprint requests to Robert Ter-keltaub, MD, Veterans Affairs Medical Center, 3350 La Jolla VillageDrive, San Diego, CA 92161. E-mail: rterkeltaub@ucsd.edu.

Submitted for publication October 10, 2002; accepted inrevised form January 13, 2003.

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PPi is channeled from the cytosol to the cellexterior via the TGF�-inducible transmembrane pro-tein ANK (3,17,18). A substantial portion of the trans-ported PPi in chondrocytes appears to be generatedvia alkaline nucleotide phosphodiesterase I activity ofisozymes of the nucleotide pyrophosphatase phospho-diesterase (NPP) family (3,5,6,19,20). Human jointchondrocytic cells express the highly homologous NPPisozymes plasma cell membrane glycoprotein 1 (PC-1/NPP1), autotaxin/NPP2, and B10/NPP3 (21). Each ofthese isozymes demonstrates alkaline pH optimumnucleotide phosphodiesterase I activity that is capableof generating PPi from ATP (also termed nucleosidetriphosphate pyrophosphohydrolase [NTPPPH] ac-tivity) (3,21). But PC-1, whose expression is markedlyup-regulated by TGF� (5), plays a greater role thanthe other two NPP family isozymes in regulatingextracellular PPi in chondrocytes (6,21). Furthermore,the inherited absence of PC-1 expression profoundlylowers plasma PPi and fibroblast extracellular PPilevels (to �50% of basal levels) in vitro and in vivo(22,23).

Cartilage intermediate-layer protein (CILP), alarge secreted glycoprotein (24–27) thought to play arole in cartilage scaffolding (28), has also been claimedto have alkaline nucleotide phosphodiesterase I activity(29–32). The expression of CILP appears to be largelyrestricted to cartilage (21,24,25,30,32). CILP expressionrises substantially in association with aging in human hiparticular cartilage, and CILP is one of only a fewcartilage matrix proteins whose expression becomesup-regulated in early OA (25). In normal culturedporcine chondrocytes, TGF� induces CILP expression,whereas IGF-1 suppresses CILP expression (31). Con-cordantly, the extent of detectable CILP expression maydirectly correlate with elevations in extracellular PPi ofaging chondrocytes (31). Furthermore, the expression ofCILP in OA articular cartilage becomes elevated prin-cipally in the middle zone (“intermediate layer”) (25),the major locus in articular cartilage in which CPPDcrystals become deposited (32). In the middle zone ofhyaline articular cartilage, CILP has been detectedintracellularly in cells near crystal deposits (33) andextracellularly in both the interterritorial matrix andpericellular matrix (25,32).

CILP was initially suspected to be an NPP be-cause a fragment of the protein was found in a chro-matographic fraction enriched in NPP activity frommaterial secreted by porcine articular chondrocytes (29).CILP can be cleaved into distinct, free N-terminal andC-terminal domains at a furin endoprotease consensus

site (24,26,27,30), and it was the free �60-kd C-terminalfragment that was linked to possible NPP activity (29).However, we questioned the assignment of NPP ac-tivity to CILP because CILP is genetically unrelated tothe NPP family (24,26,27,30,34). Neither the N-terminalnor the C-terminal segment of CILP has a consensusNPP catalytic site or a divalent cation-binding site(such as the EF hand), domains required for NPPactivity (35). Furthermore, confirmation of NPP activityusing recombinant CILP has not been provided (29,30).

Our objective in this study was to directly iden-tify functions of recombinant CILP, including reg-ulation of extracellular PPi. In searching GenBank formolecules genetically related to CILP (GenBank ac-cession no. AF035408), we noted that a highly homolo-gous protein (termed “similar to CILP”; GenBank ac-cession no. AAE74453) (36) was recently identified asbeing encoded by messenger RNA (mRNA) derivedfrom a gene with the chromosomal location 9p13.11(GenBank accession no. NT_011295), as opposed to thechromosomal location 15q22 for CILP (34). In thisstudy, we designated the original CILP as the isoformCILP-1 and the novel CILP-related species as the iso-form CILP-2. We report that neither CILP isoform hasNPP activity but that CILP-1 functions as an IGF-1antagonist.

MATERIALS AND METHODS

Reagents. Human recombinant TGF�1, interleukin-1�(IL-1�), and IGF-1 were from R&D Systems (Minneapolis,MN). Long[R3] IGF-1 (LR3IGF-1) was obtained fromGroPep (Adelaide, South Australia, Australia). All otherreagents were from Sigma (St. Louis, MO), unless indicatedotherwise.

Cells and culture conditions and reverse transcription–polymerase chain reaction (RT-PCR) analyses. Articular carti-lage from the tibial plateaus and femoral condyles of normalbovine knees (Animal Technologies, Tyler, TX) was dissected,and primary articular chondrocytes were isolated by digestionwith type II collagenase (2 mg/ml; Worthington Biochemical,Lakewood, NJ) for 18 hours at 37°C. Typical yields were �50million chondrocytes per knee. Primary human knee articularchondrocytes and knee medial and lateral meniscal fibrocarti-lage cells from the central chondrocytic zone (21) were ob-tained at autopsy from normal donors, as described previously(6). Specimens containing fractures, cartilage fibrillation, orcartilage erosion were excluded from study. Individual donorswere studied in triplicate; cells from different donors were notpooled in any single experiment.

The primary bovine chondrocytes were initially platedat 80–90% confluency after isolation via collagenase digestion.Primary chondrocytes were cultured in high-glucose Dulbec-co’s modified Eagle’s medium (DMEM) supplemented with

CILP-1 ANTAGONIZES IGF-1 1303

10% fetal calf serum (FCS), 1% L-glutamine, 100 units/ml ofpenicillin, and 50 �g/ml of streptomycin (Omega Scientific,Tarzana, CA) and maintained at 37°C in the presence of5% CO2 for 7 days prior to initiation of each experiment.During the 7-day culture period, the cells adhered and estab-lished a chondrocyte-like appearance that was maintainedthroughout the experimental period. Functional studies onchondrocytes were performed in high-glucose DMEM supple-mented with 1% FCS, 1% L-glutamine, 100 units/ml of peni-cillin, and 50 �g/ml of streptomycin (Medium A), unlessindicated otherwise.

PC-1–null mice were generated as described else-where (23). Primary cultures of osteoblasts were isolatedfrom intramembranous bone (calvariae) from 0–3-day-oldmice via sequential collagenase digestion (23). The cellswere cultured in �–minimum essential medium (�-MEM;Gibco BRL, Grand Island, NY) with 10% heat-inactivatedFCS, L-glutamine (2 mM), penicillin (50 units/ml), and strep-tomycin (0.5 mg/ml).

For RT-PCR, TRIzol (Invitrogen, Carlsbad, CA) wasused to isolate total RNA from human osteoblastoid SaOS-2osteosarcoma cells, third-passage aortic smooth musclecells, primary dermal fibroblasts (Cascade Biologics, Portland,OR), and primary chondrocytes and meniscal cells, each ofwhich was cultured for 24 hours at a density of 5 � 105

cells/60-mm dish. RNA was extracted, reverse-transcribed, andanalyzed in 1 round (40 cycles) of RT-PCR, as describedpreviously (6).

To test the differential expression of CILP-1 andCILP-2, we validated the isoform selectivity (via testing ofcomplementary DNA [cDNA]) of 2 RT-PCR primer sets:for CILP-1, sense 5�-TTC-GCT-TCT-ACT-ATG-GGG-ACC-G-3� and antisense 5�-GGC-ATC-ACA-GTC-AGC-ATT-CAC-C-3� to amplify a 425-nucleotide product;for CILP-2, sense 5�-CAG-CCT-TGA-CAC-CTG-TGA-ATG-C-3� and antisense 5�-AAT-CTC-CCT-TCT-CTC-CAG-ACG-G-3� to amplify a 748-nucleotide product. Primersfor the “housekeeping gene” L30 were those described else-where (6).

CILP cDNA for expression studies, isolation of solubleCILP-1, CILP-1 fragments, and soluble PC-1, and antibodiesto CILP-1. We used a full-length human PC-1 cDNA constructin pcDNA3. The CILP-2 cDNA clone was isolated from alibrary of human OA cartilage chondrocyte cDNA (publishedas GenBank accession no. AF542080; provided by Dr. C.Kumar, Novartis, Summit, NJ) and subcloned in pCMV-SPORT6 (Invitrogen). Amino acid residues 532–1156 of thetranslated sequence were 99% identical to those derived fromGenBank sequence XP_086058 (previously named “similar toCILP”). The complete sequence of our CILP-2 clone was also99% identical to that of GenBank entry AAE74453 for “hu-man nucleotide pyrophosphohydrolase-2” (36). The onlyamino acid difference between the CILP-2 clone used in thisstudy and the GenBank entries was glycine instead of alanineat residue 768.

CILP-1 cDNA was cloned by RT-PCR from humanchondrocyte cDNA and subcloned into pcDNA3� (Invitro-gen). The variances between the cloned CILP-1 and theinitially published human CILP-1 sequence (24) (GenBankaccession no. AF035408) were as follows: intentional changesin nucleotide 2102, A to G, and nucleotide 2105, C to T,

(no amino acid change) to create a unique Hind III site.Unintentional differences, seen in 5 individual human CILP-1clones isolated and sequenced by us, were nucleotide 187,T to G (amino acid change from Leu to Trp) and nucleotide1195, C to T (amino acid changed from Thr to Ile), andthese same 2 CILP-1 nucleotide changes were recently re-ported by another group (GenBank accession no. BC035776),consistent with naturally occurring polymorphisms. TheN-terminal CILP-1 fragment cDNA (nucleotide 1–2100) wasgenerated by digestion of CILP-1 with Hind III and theC-terminal CILP-1 fragment cDNA (nucleotide 2019–3573)was generated by restriction digestion of CILP-1 with Bst EII.Polyclonal rabbit IgG antibody to CILP-1 fragments weremade to the N-terminal and C-terminal peptides generatedfrom the cDNA for these fragments expressed in Escherichiacoli.

To produce the soluble CILP-1, N-terminal CILP-1,and C-terminal CILP-1 polypeptides, each CILP-1 fragmentand the full-length CILP-1 were subcloned into the pcDNA4/Hismax vector (Invitrogen). An additional N-terminal CILP-1fragment via Bst EII digestion (nucleotide 1–2012) was alsosubcloned into the pcDNA4/Hismax vector for use as a control.The respective pcDNA4/Hismax cDNA constructs were ex-pressed in 293 cells and His-Tagged polypeptides were isolatedfrom conditioned media and cell lysates using the ProBondpurification system (Invitrogen) according to the manufactur-er’s instructions for mammalian cells. The purified polypep-tides were digested with EnterokinaseMax (Invitrogen). Solu-ble PC-1 was prepared by a modification of a previouslydescribed method (6).

Sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS-PAGE)/Western blot analyses. For SDS-PAGE/Western blotting, cell lysates were treated with 10 mMTris HCl, pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA,1% SDS, 1 mM sodium orthovanadate with 1 mM phenylmeth-ylsulfonyl fluoride, and 2 �g/ml of aprotinin, sonicated for 10seconds, and centrifuged for 20 minutes at 1,200g. The proteinconcentration of supernatants was determined with the bicin-choninic acid protein assay (Pierce, Rockford, IL). Protein(50 �g) from each sample was separated by SDS-PAGE underreducing conditions and transferred to nitrocellulose.Where indicated, CILP antibodies were used at a dilution of1:1,000, and anti–tyrosine phosphorylated–IGF-1 receptor(Cell Signaling, Beverly, MA) was used at a dilution of 1:1,000.Washed nitrocellulose membranes were incubated with horse-radish peroxidase–conjugated secondary antibody in blockingbuffer for 1 hour, washed again, and immunoreactive productswere detected by using the enhanced chemiluminescencesystem (Pierce).

Transfection studies. For transfection of cDNA ex-pression constructs (2 �g of DNA) into primary mouseosteoblasts, we used Lipofectamine Plus (Life Technologies,Grand Island, NY), with transfection efficiency �40%, asdescribed elsewhere (37). For transfection of bovine artic-ular chondrocytes, we plated 4 � 105 cells/60-mm dish, andtransfected the cells (under adherent conditions) withthe indicated constructs using a modified FuGENE6/hyaluronidase method (38,39). The procedure included a15-minute incubation of 2 �g of DNA with FuGENE 6and the addition of 4 units/ml of hyaluronidase in Medium Ato this complex for 8 hours. Transfection efficiency was �40%,

1304 JOHNSON ET AL

as determined by control �-galactosidase transfection andstaining. Where indicated, cell culture medium waschanged to fresh Medium A containing 10 ng/ml of IGF-1 orLR3IGF-1.

To study cells under conditions where extracellularPPi was up-regulated via ascorbate treatment (40), we de-tached cells using 0.2M EDTA, pH 8.0, and transferred themto 96-well plates (1 � 105 cells/well) coated with 10% poly-HEME in 95% ethanol, (nonadherent cell culture) (39).We supplemented Medium A with 50 �g/ml of ascorbate (40)and 1 mM sodium phosphate. To verify up-regulation ofeach CILP-1 construct following transfection, conditionedmedia were collected and the protein was immunoprecipitatedusing an anti–His-Tag antibody, separated by SDS-PAGE,and then probed with the antibodies described above. Inexperiments using soluble CILP-1 proteins, addition of 400 ngof the purified protein was substituted for the transfectionstep.

PPi, NPP, alkaline phosphatase (AP), cellular DNA,and nitric oxide assays. PPi was determined radiometricallyand equalized per DNA concentration, as described previously(6). We assayed the specific activity of NPP and AP asdescribed previously (6,23), with units of NPP and AP desig-nated as micromoles of substrate hydrolyzed per hour (permicrogram of protein). Nitric oxide release was assayed by theGreiss reaction (39).

Cell proliferation and sulfated proteoglycan (PG)synthesis assays. Following transfection, primary bovinechondrocytes were plated in 96-well plates in monolayerculture (1 � 104 cells/well) and stimulated with IGF-1 orLR3IGF-1 as indicated. After 48 hours at 37°C, conditionedmedia and dead cells were removed by 2 washes, and cells werefixed and stained with 50 �l of 0.05% crystal violet, 0.37%formaldehyde in 20% ethanol for 10 minutes at 23°C. Cells inwashed plates were methanol-lysed, and cell numbers werequantified at an optical density of 562 nm and correlated to astandard curve of chondrocytes (0–100,000 cells/well). Tomeasure sulfated PG synthesis, aliquots of 1 � 105 cells/well ofa 96-well plate coated with polyHEME were cultured, and cellswere treated for 48 hours with agonists in Medium A with 20�Ci/ml of 35S-labeled sodium sulfate. Conditioned media werecollected and cells washed once with phosphate bufferedsaline, placed into 0.5M NaOH, and mixed for 48 hours at 4°C.Media and cell extracts were eluted from Sephadex G-25MPD-10 columns (Amersham Pharmacia, Piscataway, NJ) using4M guanidine HCl, and eluates were analyzed by scintillationcounting.

Statistical analysis. Statistical analyses were per-formed using the Student’s t-test (paired 2-sample testing formeans). Values are expressed as the mean � SD.

RESULTS

Comparison of CILP isoform expression andstructure. Both CILP isoforms were constitutively ex-pressed by cultured normal hyaline cartilage articularchondrocytes, but not by osteoblasts, fibroblasts, orvascular smooth muscle cells (Figure 1). Only CILP-1

expression was detectable in normal knee meniscalfibrocartilage chondrocytic cells.

Sequence alignment and domain analyses ofhuman CILP-1 and CILP-2 polypeptides using theBLAST and PROSITE programs are shown inFigure 2. CILP-1 and CILP-2 were 50.6% identical and�66% conserved on an amino acid basis. Their cDNAwere 51.5% identical. CILP-1 can be cleaved into dis-tinct, free N-terminal and C-terminal domains at a furinendoprotease consensus site (24,26,27,30) that washighly conserved in CILP-2 (Figure 2). The primarystructure of the CILP-1 C-terminal domain, which isencoded by a single exon and was the CILP domainreported to have NPP activity (30), was particularlyhighly conserved in CILP-2. The CILP-1 and CILP-2N-terminal domains each had a substantially conservedthrombospondin type I repeat domain (41–44) and animmunoglobulin C-2 type domain (Figure 2). How-ever, putative motifs for the aldehyde dehydrogenase–active site (45) and an ATP-binding site (46) in theN-terminal domain of CILP-1 were not shared inCILP-2 (Figure 2).

Differential NPP-independent effects of trans-fected CILP-1 and CILP-2 on IGF-1 responsiveness.CILP-1 has been detected both intracellularly and extra-cellularly in chondrocytes in OA and chondrocalcinoticcartilage (25,32,33). Thus, to thoroughly examine thedirect and potentially indirect effects of “gain-of-function” of CILP-1 on NPP activity and PPi, we firstperformed transfection studies. We used a full-lengthCILP-2 cDNA construct and CILP-1 cDNA constructs

Figure 1. Tissue expression of cartilage intermediate-layer protein 1(CILP-1) and CILP-2. We assessed CILP-1 and CILP-2 expression byreverse transcription–polymerase chain reaction (RT-PCR) in kneearticular chondrocytes (lane 1), human aortic smooth muscle cells(lane 2), human osteoblastoid SaOS-2 osteosarcoma cells (lane 3),dermal fibroblasts (lane 4), and primary meniscal chondrocytic cellsfrom a normal knee (lane 5) (see Materials and Methods). Cells(5 � 105) were grown for 24 hours in a 60-mm dish and then total RNAwas collected, reverse transcribed, and subjected to 40 cycles of PCRunder conditions where the cDNA for CILP-1 and CILP-2 served aspositive controls (lane �). Results are representative of 3 experimentswith each cell type.

CILP-1 ANTAGONIZES IGF-1 1305

shown in Figure 3A. The parental CILP-1 constructincluded minor polymorphisms described in the Materi-als and Methods section and an engineered Hind III sitethat we used to separately express and study the freeCILP-1 N-terminal domain (1–2,100 bp). The

C-terminal domain–containing fragment of CILP-1(2,019–3,573 bp) was generated by digestion of CILP-1with Bst EII (Figure 3).

We transfected expression constructs encodingfull-length CILP-1 and the CILP-1 N-terminal and

Figure 2. Schematic of the sequence and specific domain alignment of human cartilageintermediate-layer protein 1 (CILP-1) and CILP-2 polypeptides using the BLAST program. A,The CILP-1 and CILP-2 N-terminal domains each had a substantially, but not totally, conservedthrombospondin (TSP) type I repeat domain and Ig C-2 type domain, as delineated in B. Asillustrated, CILP-2 did not share putative aldehyde dehydrogenase and ATP-binding motifs inthe N-terminal domain of CILP-1 (where the conserved consensus residues are indicated bycapital letters). The CILP-1 C-terminal domain, which is encoded by a single exon and was theCILP domain previously suspected to have nucleotide pyrophosphatase phosphodiesteraseactivity, was highly conserved in CILP-2, as was the furin endoprotease consensus site at whichCILP-1 is cleaved into distinct, free N-terminal and C-terminal domains.

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C-terminal domains into primary cultures of normalbovine knee articular chondrocytes. Using SDS-PAGEand Western blot analysis, we confirmed that theserespective transfections increased the expression of full-length CILP-1 and selectively up-regulated the expres-sion of the CILP-1 N-terminal and C-terminal domains(Figure 3B). In doing so, we noted the sizes of the majorimmunoreactive CILP-1 peptide fragments processedand secreted by chondrocytes were �60 kd for thepeptides seen in the media after transfection ofboth the C-terminal and the N-terminal CILP-1 frag-ments (Figure 3B). In addition, we noted secretion ofboth N-terminal and C-terminal immunoreactive pep-tide fragments of �60 kd after transfection of the

full-length CILP-1 (Figure 3B). Thus, both N-terminaland C-terminal peptide domain fragments of CILP-1were released from chondrocytes. Because the expres-sion of the CILP-1 N-terminal polypeptide was supe-rior using the Hind III–digested cDNA, we primarilyused this construct, but we also separately verified (datanot shown) comparable results with the CILP-1N-terminal Bst EII–digested cDNA for each result pre-sented below.

Background NPP activity of PC-1 could the-oretically mask the direct effects of CILP isoforms onNPP activity. Hence, we transfected both CILP iso-forms into primary osteoblasts of PC-1–null mice, whichhave �50% lower NPP and extracellular PPi levels

Figure 3. A, Schematic for cartilage intermediate-layer protein 1 (CILP-1) cDNA con-structs used, and B, verification of up-regulated CILP-1 polypeptide expression by trans-fection. A, The parental CILP-1 construct included an engineered Hind III site that allowedus to express and study the free CILP-1 N-terminal domain (1–2,100 bp) and C-terminaldomain (2,019–3,573 bp, generated by digestion with Bst EII). nt � nucleotide. B,Expression constructs encoding full-length CILP-1 and the CILP-1 N-terminal andC-terminal domains were transfected into primary cultures of normal bovine knee articularchondrocytes (see Materials and Methods). Forty-eight hours after transfection, theconditioned media were collected and polypeptides were immunoprecipitated for 18 hoursat 4°C using anti–His-Tag antibody. The immunoprecipitates were separated by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis and then analyzed by Western blotting,using N-terminal (top) and C-terminal (bottom) CILP-1–specific antibodies (see Materialsand Methods). The major immunoreactive CILP-1 peptide fragments of anti-His immuno-precipitates that had undergone secretion and processing by the chondrocytes are demon-strated.

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than do wild-type controls (23). Neither CILP-1 norCILP-2 significantly affected the activity of NPP (mea-sured by alkaline nucleotide phosphodiesterase Iassay) or the PPi-degrading ectoenzyme AP, which isa member of the same metalloenzyme family asPC-1 (20) (Figure 4). In contrast, transfection of thepositive control PC-1 markedly elevated NPP activity(Figure 4).

Transfection of full-length CILP-1, of theCILP-1 N-terminal and C-terminal domains alone, orof CILP-2 in primary bovine knee chondrocytes alsodid not significantly affect cell-associated NPP or APactivity (Figure 5A). Furthermore, CILP isoforms didnot demonstrate significant direct effects on extracellu-

lar PPi in chondrocytes (Figure 6). As a positive control,we transfected PC-1, which significantly elevated cell-associated NPP and extracellular PPi (Figure 6). PC-1,but not the CILP isoforms, also significantly elevated

Figure 4. Lack of significant effect of cartilage intermediate-layerprotein 1 (CILP-1) or CILP-2 transfection on nucleotide pyrophos-phatase phosphodiesterase (NPP) or alkaline phosphatase (AP)activity in primary bovine chondrocytes. We cultured primary bovinechondrocytes (1 � 105 cells/well in a 96-well plate) under nonadherentconditions for 48 hours following transfection (see Materials andMethods). Cell lysates were collected and assayed for A, NPP and B,AP specific activity as described in Materials and Methods. Valuesare the mean and SD of 5 experiments, each performed in quadrupli-cate. � � P � 0.05 versus vector transfection alone. N-CILP-1 �N-terminal CILP-1; C-CILP-1 � C-terminal CILP-1; PC-1 � plasmacell membrane glycoprotein 1.

Figure 5. Failure of CILP-1 and CILP-2 to increase NPP activity inprimary calvarial osteoblasts from PC-1–knockout mice (PC-1–/–

mice). Primary calvarial osteoblasts from PC-1–/– mice (3 � 105

cells/35-mm culture dish) were transfected and then incubated for 48hours at 37°C (see Materials and Methods). Cell lysates were collectedand assayed for NPP and alkaline phosphatase (AP) activity asdescribed in Materials and Methods. Values are the mean and SD of3 experiments each performed in triplicate. � � P � 0.05 versus controlcells. See Figure 4 for other definitions.

Figure 6. Blocking of the ability of insulin-like growth factor 1 (IGF-1)and Long[R3] IGF-1 (LR3IGF-1) to lower extracellular inorganic pyro-phosphate (PPi) levels by transfection of cartilage intermediate-layerprotein 1 (CILP-1) but not CILP-2. Following transfections, primarybovine chondrocytes were cultured under nonadherent conditions (1 �105 cells/well in a 96-well plate coated with polyHEME). Cells were thenincubated for 48 hours at 37°C with 10 ng/ml of IGF-1 or LR3IGF-1 for48 hours (see Materials and Methods). The conditioned media werecollected, and the PPi concentrations were determined as described inMaterials and Methods. Values are the mean and SD of 5 experiments,each performed in triplicate. � � P � 0.05 versus vector transfectionalone. N-CILP-1 � N-terminal CILP-1; C-CILP-1 � C-terminal CILP-1;PC-1 � plasma cell membrane glycoprotein 1.

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intracellular PPi (data not shown). Although CILP-1 wassecreted, extracellular NPP activity also was unaffectedby CILP isoforms (data not shown).

To test for possible indirect effects of CILPisoforms on extracellular PPi levels, we treated primarycultured bovine chondrocytes with IGF-1 (10 ng/ml).IGF-1 was confirmed to significantly diminish extracel-lular PPi relative to resting cells (7) (Figure 6). IGF-1treatment decreased extracellular PPi without affectingtotal NPP or AP activity (data not shown). IGF-1treatment also suppressed the ability of transfected PC-1to elevate extracellular PPi (without affecting total NPPactivity) (Figure 6). Under these conditions, transfectedfull-length CILP-1 and the N-terminal domain ofCILP-1 blocked the capacity of IGF-1 to lower extracel-lular PPi (Figure 6). In contrast, neither the transfectedC-terminal domain of CILP-1 nor full-length CILP-2significantly affected PPi levels or blocked the ability ofIGF-1 to lower extracellular PPi (Figure 6). The effectsof CILP-1 on IGF-1 responsiveness were selective, sincetransfected CILP-1 did not interfere with the capacity ofTGF� to elevate extracellular PPi (3–6) (data notshown).

Chondrocytes express IGF binding protein(IGFBP) family members that modulate IGF-1 re-sponsiveness (8–14,16). Thus, we also used LR3IGF-1(Figure 6), an 83–amino acid IGF-1 receptor–bindinganalog of human IGF-1 that bears a substitution ofarginine for glutamine at the third amino acid of IGF-1,as well as the addition of 13 amino acids to theN-terminus (10). These changes impart markedly de-creased binding capacity for all known IGFBP familymembers (10,47) and allow delineation of IGF-1 signal-ing and biologic activity for a given cell type indepen-dently of potentially variable effects of IGFBPs onIGF-1 responsiveness (10,47). LR3IGF-1 and IGF-1comparably suppressed extracellular PPi levels in chon-drocytes (Figure 6). The effects of LR3IGF-1 on extra-cellular PPi also were blocked by transfected CILP-1 andthe CILP-1 N-terminal domain, but not by the CILP-1C-terminal domain or full-length CILP-2. Transfectionof CILP-1 and the free N-terminal CILP-1 domain (butnot the C-terminal CILP-1 domain or full-lengthCILP-2) inhibited the capacity of IGF-1 and LR3IGF-1to stimulate PG synthesis and proliferation of chondro-cytes (Figure 7).

NPP-independent effects of soluble CILP-1N-terminal domain–containing polypeptides on IGF-1responsiveness in chondrocytes. We concluded thestudy by focusing on functional analysis of CILP-1

without potential limitations of transfection artifacts,and with the goal of assessing whether exogenous extra-cellular CILP-1 polypeptides could block IGF-1 re-sponsiveness. To do so, we prepared soluble recombi-nant full-length CILP-1 and N-terminal and C-terminaldomain polypeptides. These soluble CILP-1 polypep-tides were confirmed to lack NPP and AP activity

Figure 7. Antagonism of the capacity of IGF-1 and LR3IGF-1 tostimulate proteoglycan (PG) synthesis and proliferation by transfectedCILP-1 but not CILP-2. A, Primary bovine chondrocytes were culturedunder nonadherent conditions following transfection and then incu-bated for 48 hours at 37°C in medium supplemented with 20 �Ci/ml of35S-labeled sodium sulfate and 10 ng/ml of IGF-1 or LR3IGF-1 (whereindicated) (see Materials and Methods). The conditioned media andcells were collected, extracted, and eluted from Sephadex G-25MPD-10 columns. Values are the mean and SD of 6 experiments, eachperformed in triplicate. � � P � 0.05 versus vector transfection alone.B, Following transfections, primary monolayer-cultured bovine chon-drocytes (1 � 104 cells/well in a 96-well plate) were incubated for 48hours at 37°C in medium supplemented with 10 ng/ml of IGF-1 orLR3IGF-1 (where indicated) (see Materials and Methods). Prolifera-tion of crystal violet–stained adherent cells was quantified spectropho-tometrically at an optical density of 562 nm. Values are the mean andSD of 6 experiments, each performed in replicates of 8. � � P � 0.05versus vector transfection alone. See Figure 6 for other definitions.

CILP-1 ANTAGONIZES IGF-1 1309

(Figure 8). Soluble full-length CILP-1 and the freeN-terminal CILP-1 domain both significantly inhibitedthe capacity of IGF-1 and LR3IGF-1 to stimulate PGsynthesis and proliferation of chondrocytes (Figure 9).Nitric oxide inhibits ligand-induced IGF-1 receptorphosphorylation in chondrocytes (10), but CILP-1 didnot induce changes in nitric oxide production in chon-drocytes (data not shown). Addition of full-lengthCILP-1 and the free N-terminal CILP-1 polypeptide, butnot the C-terminal CILP-1 polypeptide, to chondrocytesblocked the rapid LR3IGF-1–induced IGF-1 receptortyrosine autophosphorylation response (Figure 10).

DISCUSSION

CILP-1 has been reported to be an NPP enzyme(29–32). In this study, we extensively tested recombinant

CILP-1 for NPP activity (as well as PPi-degrading APactivity). We determined that CILP-1 (and the closelyrelated isoform CILP-2) does not possess intrinsic NPP

Figure 9. Inhibition of the capacity of both IGF-1 and LR3IGF-1 tostimulate proteoglycan (PG) synthesis and proliferation by solubleCILP-1 and the free N-terminal CILP-1 domain polypeptide. A, Primarybovine chondrocytes were cultured under nonadherent conditions asdescribed in Materials and Methods and were then incubated for 48 hoursat 37°C in the presence or absence of 400 ng/ml of the indicatedHis-Tagged CILP-1 polypeptide and 10 ng/ml of IGF-1 or LR3IGF-1(where indicated), and sulfated PG synthesis was quantified (see Materi-als and Methods). Values are the mean and SD of 6 experiments, eachperformed in triplicate. � � P � 0.05 versus control cells. B, Primarymonolayer-cultured bovine chondrocytes were incubated for 48 hours at37°C in medium containing 400 ng/ml of the identified CILP-1 polypep-tide and 10 ng/ml of IGF-1 or LR3IGF-1 (where indicated), and prolif-eration was measured (see Materials and Methods). Values are the meanand SD of 6 experiments, each performed in replicates of 8. � � P � 0.05versus vector transfection alone. See Figure 6 for other definitions.

Figure 8. Direct assay of soluble cartilage intermediate-layerprotein 1 (CILP-1) polypeptides to determine nucleotide pyrophos-phatase phosphodiesterase (NPP) or alkaline phosphatase (AP)activities. Purified His-Tagged soluble polypeptides (CILP-1, andthe free N-terminal and C-terminal domain polypeptides N-CILP-1and C-CILP-1, respectively) were incubated at the indicated concen-trations for 1 hour at 37°C to determine A, NPP activity and B, APactivity (see Materials and Methods). Soluble human plasma cellmembrane glycoprotein 1 (PC-1) and calf intestine AP served as thepositive controls for NPP and AP activity, respectively. Values are themean and SD pooled results of 3 experiments, each performed intriplicate.

1310 JOHNSON ET AL

(or AP) enzymatic activity. The results were in sharpcontrast to our control findings with the NPP familymember PC-1, a major regulator of plasma PPi and ofextracellular PPi in cartilage and certain other tissues(3,5,6,22). We further described concomitant chondro-cyte expression of both CILP-1 and CILP-2 in vitro. Wediscovered that the CILP isoforms had differential ef-fects on chondrocyte function, specifically, CILP-1 wasas an IGF-1 antagonist.

We demonstrated here that both N-terminal andC-terminal peptide domain fragments of CILP-1 werereleased from chondrocytes. The CILP-1 C-terminaldomain, which is liberated by endoproteolysis at aconsensus furin site and is encoded by a single exon(26,27,30), had previously been reported to possess NPPactivity (29,30), but the primary structure of the CILP-1C-terminal domain was highly conserved in CILP-2.Furthermore, the free CILP-1 C-terminal domain andfull-length CILP-2 both lacked NPP activity and func-tional IGF-1 antagonism.

The primary function of CILP-1 (and CILP-2)may be to modulate the architecture of the cartilagematrix (28), consistent with the predominant localizationof CILP-1 in the interterritorial matrix of the middle

zone of articular cartilage (25,32). However, pericellular(and intracellular) localization of CILP-1 is also ob-served in articular cartilage (32,33). It is possible thatantibodies to the C-terminal of CILP-1 (32,33) may havepreviously detected the highly homologous C-terminaldomain of CILP-2 at some cartilage loci in situ. How-ever, physiologic significance remains likely for ourfindings that full-length CILP-1 and the free N-terminalCILP-1 domain polypeptide attenuated ligand-inducedIGF-1 receptor signaling and interfered with the capac-ity of IGF-1 to lower extracellular PPi and to promotechondrocyte PG synthesis and growth.

IGF-1 resistance in OA and aging cartilage ap-pears to be multifactorial (8–10). First, chondrocytesexpress several members of the IGFBP family, whichbind IGF-1 with high affinity and whose central struc-tural feature is conserved cysteine-rich domains (48).Increased levels of the IGFBPs 2, 3, and 4, and alteredIGFBP complex formation with IGF-1 and the acid-labile subunit (11) could play a role in chondrocyteIGF-1 resistance, since IGF-1 receptor levels do notgenerally decrease in OA chondrocytes (16). Second, thecapacity of nitric oxide to suppress ligand-induced auto-phosphorylation of the IGF-1 receptor and to inhibit PG

Figure 10. Inhibition of ligand-induced autophosphorylation of the IGF-1 receptor bysoluble full-length CILP-1 and by the N-CILP-1 polypeptide. Primary bovine chondrocyteswere plated under nonadherent conditions (1 � 105 cells/well in a 96-well plate coated withpolyHEME, as described in Materials and Methods) and incubated for 18 hours; themedium was then changed to 200 �l/well of serum-free, high-glucose Dulbecco’s modifiedEagle’s medium containing 400 ng/ml of the indicated His-Tagged CILP-1 polypeptide.After a 1-hour incubation at 37°C, 10 ng/ml of LR3IGF-1 was added for 1 minute. The cellswere immediately removed, lysed, and aliquots of 50 �g of protein were separated by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, andprobed with antibody to tyrosine phosphorylated IGF-1 receptor (anti-phospho-IGFR)(top) and, as controls, antitubulin antibody (middle) and antiphosphotyrosine antibodyPY99 (bottom), for the entire array of cell proteins. Results are representative of 5 separateexperiments. Molecular weight markers are shown on the left. See Figure 6 for otherdefinitions.

CILP-1 ANTAGONIZES IGF-1 1311

synthesis also may be significant modulators of IGF-1action in OA, in which cartilaginous nitric oxide produc-tion is up-regulated (10). However, CILP-1 did notinduce nitric oxide production by chondrocytes.

Increasing severity of OA in a non-human pri-mate model was associated with reduced responsivenessto intact IGF-1 but not des(1-3) IGF-1, which only weaklybinds IGFBP family members (8). Hence, increasedproduction of inhibitory IGFBPs was suggested to play apredominant role in chondrocyte IGF-1 hyporesponsive-ness in OA (8). In that same study, cells from olderanimals had a reduced response to both full-length anddes(1-3) IGF-1, which indicated that the mechanism ofthe reduced IGF-1 responsiveness with aging might beIGFBP-independent (8). In the current study, CILP-1and the CILP-1 N-terminal domain polypeptide antag-onized chondrocyte responsiveness to not only IGF-1,but also LR3IGF-1, another IGF-1 analog that onlyweakly binds IGFBP family members (47). As such,IGF-1 antagonism by CILP-1 did not appear to be directlymediated by IGFBP-dependent effects in chondrocytes.CILP-1–induced IGF-1 antagonism might be more signif-icant in cartilage aging than in OA, where up-regulation ofcertain IGFBPs and of nitric oxide production could playlead roles in IGF-1 antagonism (8–10).

IGF-1 resistance can be produced by low-affinity

binding to IGF-1 of IGF-1 antagonists, such as connec-tive tissue growth factor (CTGF), that do not belong tothe IGFBP family (48). The capacity of IGF-1 and/orIGFBPs to bind a variety of other molecules (includingfibronectin, transferrin, and cell-surface heparin-likemolecules) also can modulate IGF-1 responsiveness(49–51). It will be of interest to determine whether thecapacity of CILP-1 to attenuate ligand-induced IGF-1receptor signaling reflects a direct effect of CILP-1 onIGF-1 binding to its receptor or an indirect effect. Forexample, it is possible that CILP-1 may act by modulat-ing the localization, conformation, and/or expression ofa matrix or cell-surface molecule interacting with IGF-1or the IGF-1 receptor.

The lack of IGF-1 antagonism by CILP-2, inassociation with distinctions in CILP-1 and CILP-2N-terminal domain structure, suggests a potential rolein IGF-1 antagonism of one or more partially distinctdomains in the CILP-1 N-terminal domain. Thesemight include the putative aldehyde dehydrogenase–active site (45) and ATP-binding motifs (46) in CILP-1.Alternatively, distinct features of the thrombospondintype I repeat domain or immunoglobulin C-2 typedomain of CILP-1 may be significant. With respect tothe thrombospondin type I repeat, this motif has thepotential to modulate CILP-1 anchoring to matrix con-

Figure 11. Model for the consequences of regulated cartilage intermediate-layer protein1 (CILP-1) expression in physiologic control of extracellular inorganic pyrophosphate (PPi)in normal cartilage and pathologically increased extracellular PPi in aging and osteo-arthritic (OA) cartilage: This paradigm ties the observations in the current study on theeffects of CILP-1 on insulin-like growth factor 1 (IGF-1) responsiveness and control ofextracellular PPi levels to the known regulation of both CILP-1 expression and extra-cellular PPi by transforming growth factor � (TGF�) and IGF-1. CPPD � calciumpyrophosphate dihydrate.

1312 JOHNSON ET AL

stituents, including glycosaminoglycans (41–44). Indeed,the heparin-binding and TGF�-binding capacity withinsome thrombospondin type I repeat domains may me-diate antiadhesive and antiproliferative effects, includ-ing binding to TGF� antagonism of cell activation byTGF� (43). But, it was noteworthy in the current studythat CILP-1 did not antagonize the effects of TGF� onchondrocytes under conditions in which IGF-1 respon-siveness was suppressed. With respect to immunoglobu-lin domains, they are found in hundreds of proteinsother than antibodies, including receptor protein ki-nases, and such immunoglobulin domains can mediateprotein–protein interactions (52,53).

Our results support a role for dysregulatedCILP-1 expression in IGF-1 resistance in the pathogen-esis of cartilage aging and OA. The aforementionedassociations of aging with both up-regulated CILP-1expression and chondrocalcinosis suggest a new mecha-nism contributing to cartilage PPi supersaturation inaging and OA cartilage (Figure 11). Specifically, in-creased cartilage PPi generation occurs in response toTGF� (3–6) and is normally antagonized by IGF-1 (7).But TGF� induces CILP-1 expression (31). By interfer-ing with IGF-1 lowering of extracellular PPi, up-regulated CILP-1 expression can promote cartilage su-persaturation with PPi and subsequent CPPD crystaldeposition in aging and OA cartilage.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Daphne Szeto,Novartis, for help in characterizing the CILP-2 cDNA clone,and the helpful comments by Dr. Martin Lotz, ScrippsResearch Institute (La Jolla, CA) and Dr. Robert Crowl,Novartis.

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