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Biochemical and Biophysical Research Communications 274, 467–471 (2000)

doi:10.1006/bbrc.2000.3167, available online at http://www.idealibrary.com on

ge-Related Effects of TGF-b on Proteoglycan Synthesisn Equine Articular Cartilage

aved Iqbal,1 Jayesh Dudhia, Joseph L. E. Bird, and Michael T. Baylissepartment of Veterinary Basic Sciences and Farm Animal and Equine Medicine and Surgery,oyal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom

eceived June 27, 2000

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The synthesis of proteoglycans was measured in nor-al equine articular cartilage of ages 9 months to 20

ears and the effect of TGF-b1 on this activity wasnvestigated. The rate of incorporation of [35S]Na2SO4

ecreased with age as did the responsiveness of theissue to the growth factor. The enhanced synthesis ofroteoglycan induced at all ages by TGF-b1 was down-egulated by IL-1b and retinoic acid. The expression ofRNA for TGF-b1, 2, and 3 was also measured, and

lthough the level of TGF-b1 was highest at all ages,he expression of each growth factor decreased withge. © 2000 Academic Press

Key Words: cartilage; TGF-b; proteoglycan; mRNA.

The TGFb superfamily has a wide range of activitiesuring development, growth, and repair. This family isomposed of at least 25 members which retain 7 cys-eine residues within the conserved region of the pro-ein. With the exception of BMP-1 (bone morphogeneticrotein-1), BMPs 2-9, and 13, TGF-b1-5 and cartilageerived morphogenetic protein (CDMP1 and CDMP2)re members of this superfamily. TGF-b1 was firstdentified by its ability to induce colony formation ofodent cell lines in soft agar. Subsequently, TGF-b1as found to act as a potent growth inhibitor of aariety of cell lines. It has also been proposed thatGF-bs effect tissue repair, wound healing, bone for-ation, and embryonic development and abnormali-

ies in TGF-b function have been implicated in bothmmunosuppression and cellular transformation. Ofhe five isoforms, only TGF-b1, 2, and 3 have beendentified in mammals.

Articular cartilage has a limited capacity for self-enewal. Anabolic factors that are able to increase

Abbreviations used: TGF-b, transforming growth factor beta; IL-1,nterleukin-1.

1 To whom correspondence should be addressed. Fax: 0207 388027. E-mail: jiqbal@rvc.ac.uk.

467

roteoglycan (PG) synthesis and decrease PG degrada-ion may stimulate cartilage repair in arthritic joints.n this respect, transforming growth factor-b (TGF-b)s a promising candidate. TGF-b1 will stimulate PGynthesis by articular chondrocytes in vitro and in vivo1, 2, 3). In addition, TGF-b1 is able to counteract IL-1bnduced suppression of cartilage PG synthesis (4) andas the capacity to reduce IL-1b induced PG depletionn vitro (4, 5, 6). The latter effect appears to be theesult of TGF-b-induced down-regulation of the syn-hesis of proteolytic enzymes and up-regulation of theroduction of enzyme inhibitors (6, 7, 8). Furthermorets injection into the mouse knee joint leads to in-reased proteoglycan synthesis in the articular carti-age (9).

The two main aims of the present study are:

(a) To investigate the effects of TGF-b1 on proteinynthesis in explants of equine cartilage from horses ofifferent ages; and(b) To study the pattern of expression (at messengerNA and protein levels) of TGF-b1, 2, and 3 in therticular cartilage of horses of different ages.

Although RNA has been successfully extracted fromanine articular cartilage for analysis by Northern hy-ridization (10), these studies required large amountsf tissue. The sensitivity of reverse transcriptase-PCRRT-PCR) makes this technique particularly suitableor the analysis of gene expression in equine articularartilage, where the cells account for only 1–2% of theotal tissue mass. In our laboratory a competitive RT-CR method has been developed for measuring genexpression using RNA extracted directly from smallmounts of articular cartilage. The results show thathe responsiveness of equine cartilage to TGF-b1 di-inishes with age. Furthermore, all three forms ofGF-b exhibit reduced expression with age, but theroportion of TGF-b1 is always greater than TGF-b2nd TGF-b3.

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 274, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ATERIALS AND METHODS

Culturing and radio-labelling procedures. Articular cartilageas obtained at postmortem from the metacarpophalangeal joints oforses aged 9 months to 20 years. All the cartilage samples wereacroscopically normal. The cartilage was diced, washed three times

n serum-free Dulbecco’s modified Eagles Medium (DMEM: Gibco,aisley, UK) and maintained in DMEM culture medium at 37°Cnder 95% air and 5% CO2 for 24 h before the start of any experi-ent. The culture medium was then changed every 24 h (all culturesere carried out in triplicate).

Effect of TGF-b1 on proteoglycan synthesis. Cartilage explantstriplicate samples) were treated in the following ways: (1) DMEMontaining 0.1% bovine serum albumin (controls); (2) DMEM con-aining 1 ng/ml recombinant human, rhTGF-b1; (3) DMEM contains0 ng/ml rhTGF-b1; (4) DMEM containing 1 ng/ml rhTGF-b1 and 10g/ml IL-1b; and (5) DMEM containing 1 ng/ml rhTGF-b1 and 1 mMetinoic acid (RA).Explants were pulse-labelled for 4–5 h in 1 ml of Dulbecco’s mod-

fied Eagle’s medium (DMEM) containing 10 mCi [35S]Na2SO4/ml andere stored at 220°C prior to digestion with 30 mg of papain in 100M sodium acetate, pH 6.0, containing 10 mM cysteine hydrochlo-

ide and 2.4 mM EDTA, in a total volume of 0.5 ml. Quantification ofadiolabel incorporated into glycosaminoglycan (GAG) was per-ormed using an Alcian Blue microtitre plate assay (11). The cellu-arity of the tissue was determined as the DNA content of papainigests and DNA was quantified using the bisbenzimidazole fluores-ent dye technique (12).

Preparation of competitor RNAs and quantitation of TGFb iso-orms. Equine RNA was prepared directly from full thickness car-ilage from the metacarpophalangeal joint as previously described16). Briefly, approximately 200 mg of finely diced cartilage waslaced directly into 0.5 ml of solution D (4 M guanidiniumsothiocyanate/25 mM sodium citrate/0.5% (w/v) N-lauroylsarcosine/.1 M 2-mercaptoethanol) and total RNA extracted using RNeasyolumns (Qiagen, Crawley, UK). Oligonucleotide primers (Table 1)ere designed for TGF-b1 from equine sequence (13) and TGF-b2nd -b3 from the human sequences (14, 15) to amplify cartilageRNA by reverse-transcription PCR (RT-PCR). Quantification ofRNA was measured using synthetic DNA (for TGF-b1) or RNA (for

Primer Sequences Used for Quantitative Analysis of

Target mRNA

Forward primersTGF-b1 (T1): AGG CTC AATGF-b2 (TF2C) GTG TGC TGTGF-b3 (TF3C) CTC CAA GC

Reverse primersTGF-b1 (T2) CTG GAA CTTGF-b2 (TF2D) CCC TCC GTGF-b3 (TF3D) TCA CTG TC

Primers for incorporation of EcoRI siteTGF-b1 (TF1A) (Forward) 5TGA TGT GTGF-b1 (TF1B) (Reverse) -CCA GTG GTGF-b2 (TF2A) (Forward) AGA TCCGTGF-b2 (TF2B) (Reverse) TGC TCAGTGF-b3 (TF3A) (Forward) CAC TGTGTGF-b3 (TF3B) (Reverse) TCA CGCG

Note. Nucleotide positions are from published sequences for equineCR product sizes for the wild-type sequences are 251 bp for TGF-b1,

nto competitor molecules is shown underlined in bold type.

468

GF-b2 and -b3) templates as competitors prepared as described16). Briefly, oligonucleotide primers (Table 1) were used to amplifyartilage mRNA by RT-PCR. An internal EcoRI restriction site wasntroduced exactly in the middle of the amplified product by overlapCR. Purified products were cloned into the pCRII-TOPO or pZerolasmid vectors (Invitrogen) to prepare sense competitor transcriptsorresponding to each TGF-b mRNA and used for quantificationsing a modification of our previous studies (16). Briefly, a singletep RT-PCR was carried out using the Access system (Promega,nc), which is a one tube, two-enzyme assay utilising AMV reverseranscriptase (AMV RT) for first strand synthesis and the thermo-table Tfl DNA polymerase for second strand DNA synthesis andmplification. Aliquots (5 ml) of sample RNA were titrated with 2 foldilutions (in terms of copy number) of each competitor. Final reactiononditions were 5 ml AMV/Tfl reaction buffer (Promega Inc.), 0.2 mMNTPs, 1 mM of each primer, 2 mM MgSO4, 4 units of RNAsinPromega Inc.), 5 units of AMV RT and 5 units of Tfl DNA polymer-se in a total volume of 50 ml. In control reactions, the AMV RT wasmitted. Reverse transcription was carried out at 48°C for 45 minollowed by denaturation at 94°C for 2 min. PCR was carried out for0 cycles as follows: 94°C for 30 s denaturation, 60°C for 1 minnnealing, 68°C for 2 min extension. A final extension step at 68°Cor 7 min was then carried out. To each 50 ml PCR reaction was added

ml of EcoRI reaction buffer (Gibco-BRL) and 12.5 units of EcoRI,igested for 2 h and then separated on 3% (w/v) NuSieve agarose gelsFMC BioProducts, Cambridge, UK) containing ethidium bromide.he equivalence point where competitor copy number equaled targetRNA was determined from DNA fluorescence quantified using theio-Rad Gel Doc 1000 system and Molecular Analyst software (Bio-ad). Results were normalised by expressing against the DNA con-

ent (mg DNA/mg cartilage) of each specimen.

ESULTS

he Effect of Age on the Response of Equine ArticularCartilage to TGF-b1

The incorporation of [35S]Na2SO4 into equine articu-ar cartilage explants in the absence and the presencef TGF-b1, was taken as a measure of the rate of

NA and for the Synthesis of Competitor Sequences

Sequence (59–39) Primer position

TTA AGC GTG GA- 489–508GCG CTT TTC TGC 478–496CAA CGA GCA 742–761

AAC CCG TTG AT 739–758TCG CCT TC 711–723

ACA CCT TTG AAT 1009–1029

TTC CAC TGG AGT CGT GCG GCA- 608–632TTC ACA TCA AAG GAC AGC CAT- 596–618TTC TGA GCA AGC TGA AGC TCA 595–618TTC GGA TCT GCC CGC GGA TGG 587–610TTC GCG TGA ATG GCT CTT GCG 877–900TTC ACA GTG TCA GTG ACA TCA 880–903

F-b1 (13), human TGF-b2 (14), human TGF-b3 (15) mRNAs. Finalbp for TGF-b2, and 287 bp for TGF-b3. The EcoRI site incorporated

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Vol. 274, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

roteoglycan synthesis by the tissue. There was ateady decrease in the measured rate of synthesis ofroteoglycan of normal equine articular cartilage withncreasing age (Fig. 1). Furthermore, the rate of syn-hesis continued to decrease after skeletal maturity.lthough the presence of TGF-b1 (10 ng/ml) stimu-

ated the incorporation of [35S]Na2SO4 at all ages, theffect was much more pronounced after 4 years of age.he lower stimulation of synthesis in immature carti-

age was also supported by experiments in which dif-erent concentrations of TGF-b1 were included in theulture medium. Enhanced stimulation of proteoglycanynthesis, at a concentration of TGF-b1 (10 ng/ml),nly occurred in the 14-year-old cartilage (Fig. 2). It islso interesting that pro-inflammatory agents such asL-1b and retinoic acid could ameliorate the enhance-ent of synthesis induced by TGF-b1 at all ages.

ge-Related Changes in the Expressionof TGF-b1, -b2, and -b3 mRNA

TGF-b is a growth factor that is synthesised by thehondrocytes and is concentrated in the extracellular

FIG. 1. Effect of TGF-b1, IL-1b, and retinoic acid on proteogly-an synthesis by equine articular cartilage of different ages. Ex-lants of equine cartilage were cultured in DMEM containing

35S]Na2SO4 and the respective agents. 1, Control (0.1% BSA). 2, 1g/ml rhTGF-b1; 3, 10 ng/ml rhTGF-b1; 4, 1 ng/ml rhTGF-b1 1 10g/ml IL-1b; 5, 1 ng/ml rh TGF-b1 1 1 mM all-trans-retinoic acid.

469

atrix of bone and immature cartilage. TGF-b is alsoynthesised in three forms that influence the chondro-ytes in different ways depending on the source andge of the tissue. We, therefore, measured the relativeoncentration of each form in equine articular cartilagesing a quantitative RT-PCR technique. In prelimi-ary experiments, we validated the use of a DNA com-etitor for quantitation of TGF-b1 by comparing DNAompetitors for TGF-b2 and -b3 with RNA competitorolecules. In both cases, the equivalence point deter-ined as DNA or RNA was almost identical (data not

hown). These findings, therefore, validated the use ofDNA competitor for TGF-b1 analysis.Individual competition reactions were performed for

ach TGF-b mRNA (Fig. 3) to determine the equiva-ence points. From this analysis, the highest concen-ration of all three forms was observed in the 7-month-ld foetus (Fig. 4). The level of mRNA then decreasedramatically and remained constant until at least 4ears of age, when a further decline was noted for all ofhe TGF-b mRNAs. At all ages analysed, TGF-b1 waslways the highest expressed form. It was .6-foldigher than TGF-b2 and TGF-b3 in the 7-month-oldetal specimen and 8- to 10-fold higher at all other agesFig. 4).

FIG. 2. Age-related effects of TGF-b1 on stimulation of proteogly-an synthesis. Equine cartilage from horses aged 3 months to 14 yearsas cultured in the absence (closed squares) or presence (open squares)f TGF-b1 and the rate of [35S]SO4 incorporation was measured.

FIG. 3. Competitive RT–PCR for TGF-b1, 2, and 3 mRNAs inquine articular cartilage. Total RNA was isolated directly from equinerticular cartilage in order to determine the quantity of each mRNA byompetitive RT–PCR. A typical example of the gel electrophoresis ishown for cartialge from a 4-year-old specimen. Product bands arendicated by (T), target gene product; (C), EcoRI digested competitorroduct; (H), Heteroduplex formed between T and C (not visible in allanes). Lanes 1, 2, and 3 represent dilutions of competitor.

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Vol. 274, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ISCUSSION

The data presented in this manuscript supports theypothesis that the response of equine articular chon-rocytes to TGF-b depends on the age of the animal.here is also an age-related decrease in the expressionf mRNA for TGF-b1, 2, and 3, although the abundancef TGF-b2 and TGF-b3 is far less than that of TGF-b1.t is likely, therefore, that maintenance of the ho-eostasis of the extracellular matrix of normal artic-lar cartilage depends on the relative amounts of thehree isoforms that are present and that changes inheir concentration will dictate the extent to which theissue can repair itself following injury. These resultsre in accordance with the findings of Nixon et al. (17),ho has also reported that the expression of TGF-b1NA is increased in young adult equine cartilage and

hat it then declines as the cartilage ages. In theirtudy, RNA levels were at a maximum in horses aged.7 to 1 year of age with declining expression in oldernimals (2.5 and 5.5 years of age). Similarly, a contin-ous age-related decline in the proliferative responsef human articular chondrocytes to different growthactors, including TGF-b1 has been reported (18).GF-b1 may also have a role in the osteoarthritic pro-ess as it can induce osteoarthritis-like changes in mu-ine knee joints by intra-articular injection (19).

FIG. 4. Age-related changes in the expression of TGF-b1, 2, and. Densitometric scans of DNA products from competitive RT-PCRssays were measured and expressed as arbitrary units of RNA. TGF1 (closed squares), TGF b2 (open squares), TGF b3 (hatchedquares).

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Other workers (20) have shown a decrease in mRNAevels of biglycan and increased mRNA levels forecorin in response to TGF-b, as human cartilage ages.hese age-related changes in mRNA levels were alsoccompanied by corresponding changes in the concen-ration of the protein. TGF-b binding to biglycan andecorin can neutralise the growth factor and suggestshat the requirement of articular cartilage for decorins greatest in the adult, whereas, the requirement foriglycan is greatest in juvenile tissue. Serum levels ofGF-b1 in rabbits decrease during maturation and are

ncreased in cartilage trauma cases (21) and it could behat the higher TGF-b1 concentrations found in thenee joints of younger animals may be a reason forheir increased capacity to heal cartilage defects. Inor-anic pyrophosphate (PPi) levels have also been re-orted to be elevated in response to TGF-b1 in adulthondrocytes (22). Many of the anabolic and prolifera-ive activities of TGF-b1 on chondrocytes can be inhib-ted by IL-1b and TGF-b1 (23–26). Our data showinghe inhibitory effects of retinoic acid and IL-1b on theGF-b1 stimulation of proteoglycan synthesis, extendhese observations. It is also interesting to note thatollowing gene delivery by adenoviral vectors, TGF-b1enes were able to restore proteoglycan synthesis toontrol levels in the presence of IL-1b (27). Thus, theres a complex inter-relationship between TGF-b andL-1b that has major implications for cartilage repair.

CKNOWLEDGMENTS

J.I. and M.T.B. thank Action Research for their financial assis-ance. M.T.B. and J.D. also thank the Arthritis Research Campaignnd J.B. thanks the Home of Rest for Horses for their generousupport.

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