Purification of growth factors from cartilage

416

OTHER GROWTH

FACTORS AND GROWTH

INHIB1TORS

[40]

for instance, quantitatively converted CTAP-III to CTAP-III(des 1-15)/ NAP-2. Acknowledgments The authors are indebted to Virginia Castor for preparation of the figure and to Mary Helen Gilbert for preparation of the manuscript. The author is supported by U.S. Public Health Service Grant AR10728, the Michigan Chapter of the Arthritis Foundation, and the University of Michigan Multipurpose Arthritis Center USPHS Grant AR20557.

[40] Purification of G r o w t h F a c t o r s f r o m Cartilage B y YUKXO K A T O , K A Z U H I S A NAKASHIMA, KATSUHIKO SATO, W E I Q U N Y A N , MASAHIRO IWAMOTO, a n d F u J I O SUZUKI

Introduction Cartilage contains factors that stimulate the synthesis of large chondroitin sulfate proteoglycans by chondrocytes in culture.~-8 These cartilagederived factors (CDFs) consist of somatomedin-like2-4 and non-somatomedin growth factors. 4'6"9 They exert various biological effects in cultured chondrocytes, such as stimulation of DNA synthesis in the presence of fibroblast (FGF) and epidermal (EGF) growth factors,~°-~2 stimulation of colony formation in soft agar in the presence of 10% serum, and stimulai y. Kato, Y. Nomura, Y. Daikuhara, N. Nasu, M. Tsuji, A. Asada, and F. Suzuki, Exp. Cell Res. 130, 73 (1980). 2 y. Kato, Y. Nomura, M. Tsuji, R. Watanabe, and F. Suzuki, Biochem. Int. 1, 319 (1980). 3 y. Kato, Y. Nomura, M. Tsuji, H. Ohmae, M. Kinoshita, S. Hamamoto, and F. Suzuki, Exp. Cell Res. 132, 339 (1981). 4 y. Kato, Y. Nomura, M. Tsuji, M. Kinoshita, H. Ohmae, and F. Suzuki, Proc. Natl. Acad. Sci. U.S.A, 78, 6831 (1981). 5 y. Kato, Y. Nomura, M. Tsuji, H. Ohmae, T. Nakazawa, and F. Suzuki, J. Biochem. (Tokyo) 90, 1377 (1981). 6 F. Suzuki, Y. Hiraki, and Y. Kato, this series, Vol. 146, p. 313. 7 y. Hiraki, Y. Yutani, M. Takigawa, Y. Kato, and F. Suzuki, Biochim. Biophys. Acta 845, 445 (1985). 8 y. Eilam, A. Beit-Or, and Z. Nevo, Biochern. Biophys. Res. Commun. 132, 770 (1985). 9 V. Shen, L. Rifas, G. Kohler, and W. A. Peck, Endocrinology 116, 920 (1985). l0 y. Kato, R. Watanabe, Y. Hiraki, F. Suzuki, E. Canalis, L. G. Raisz, K. Nishikawa, and K. Adachi, Biochim. Biophys. Acta 716, 232 (1982). II y. Kato, Y. Hiraki, H. Inoue, M. Kinoshita, Y. Yutani, and F. Suzuki, Eur, J. Biochem. 129, 685 (1983). tz y. Hiraki, Y. Kato, H. Inoue, and F. Suzuki, Eur. J. Biochem. 158, 333 (1986).

METHODS IN ENZYMOLOGY. VOL. 198

Copyright © 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

[40]

CARTILAGE-DERIVED GROWTH FACTORS

417

tion of proteoglycan synthesis. In this chapter, we describe methods for isolation of non-somatomedin-like growth factors from fetal bovine cartilage.

Assays

Proteoglycan Synthesis. Chondrocytes are isolated from resting cartilage from the ribs of 4-week-old New Zealand White rabbits as described previously. ~3 Resting cartilage cells are used because the basal level of proteoglycan synthesis is lower than that of growth-plate chondrocytes. Freshly isolated chondrocytes are seeded at 1 × 104 cells/6-mm microwell in 0.1 ml of Eagle's minimum essential medium (MEM) with 10% fetal bovine serum and 60/~g/ml of kanamycin. When the cells become confluent, they are promptly preincubated for 24 hr in 0.1 ml of a 1 : 1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium with 0.3% fetal bovine serum, 32 U/ml of penicillin, and 40 /zg/ml of streptomycin (DF medium). They are then transferred for 20 hr to 0.1 ml of DF medium in the presence or absence of CDF. Three hours after the addition of CDF, 10/xl of DF medium containing 0.5/zCi of [35S]sulfate is added. Proteoglycan synthesis is assayed by measuring incorporation of [35S]sulfate into material precipitated with cetylpyridinium chloride after treatment of the medium and cell layers with pronase E. 1.6 Previous studies have shown that after cultures become confluent, they form multicell layers of well-differentiated, spherical chondrocytes surrounded by an abundant matrix, and that in these cultures added CDF does not increase proteoglycan synthesis beyond the high basal level, probably because of accumulation of endogenous CDF in the matrix. 7 Embryonic chicken 8 and rat chondrocytes 9 can also be used for CDF assay. Colony Formation by Chondrocytes in Soft Agar. Chick embryo chondrocytes are obtained from 17-day embryonic sternal cartilage. Five to six sterna are separated from soft tissues with forceps and incubated at 37 ° for 30 min in 10 ml of phosphate-buffered saline (PBS) (calcium- and magnesium-free) containing 2.5 mg/ml of trypsin. ~4 Then the sterna are washed 3 times with PBS to remove fibroblastic cells and cut into small pieces (0.5-1.0 mm 3) with a scalpel. The tissue fragments are transferred to 10 ml of PBS containing 0.3-1.0 mg/ml (depending on the batch) of crude collagenase (Sigma, St. Louis, MO, type IA) and 2.5 mg/ml of trypsin at 37 °. After 1.5 hr, the tissue fragments and cell aggregates are 13 y . Shimomura, T. Yoneda, and F. Suzuki, Calcif. Tissue Res. 19, 179 (1975). 14 y . Kato, M. lwamoto, and T. Koike, J. Cell. Physiol. 133, 491 (1987).

418

OTHER GROWTH FACTORS AND GROWTH INH1BITORS

[40]

dispersed by pipetting in a 10-ml plastic pipette, and the resulting cell suspension is filtered through a nylon sieve (pore size 120/zm). The filtrate is centrifuged at 1500 rpm for 5 min in a Hitachi clinical centrifuge, and the pellet is washed 3 times with DF medium supplemented with I0% fetal bovine serum. The cells are suspended in DF medium supplemented with 10% fetal bovine serum, 0.3% (w/v) tryptose phosphate broth, 32 U/ml of penicillin, and 40/xg/ml of streptomycin (medium A). A basal layer of 0.25 ml of 0.72% Bacto-agar (Difco, Detroit, MI) in medium A is introduced into a 16-mm petri dish. Chondrocytes (2.5 x 103) are suspended in 0.25 ml of 0.41% Bacto-agar in medium A and used as an overlayer. CDF is added 2 days after cell seeding and then every fourth day. Cultures are maintained in 5% CO2/95% air at 37 °, and after 14 days colonies are counted. A cell colony is defined as a cluster of cells of more than 0.1 mm in diameter. Radioreceptor Assays o f Insulin-Like Growth Factor 1 (IGF-I) and FGF. Chondrocytes are obtained from resting cartilage of the ribs of 4week-old rabbits. ~3 Cells are seeded at 3 x l 0 4 cells/16-mm microwell in 0.5 ml of MEM supplemented with 10% fetal bovine serum and 60/zg/ml kanamycin. When the chondrocytes become confluent, they are washed twice with MEM supplemented with 20 mM HEPES, pH 7.5, and 0.2% gelatin (buffer A) and then transferred to 0.25 ml of buffer A with various concentrations of cartilage extract or IGF-I in the presence of ~25I-labeled IGF-I (2000 Ci/mmol, Amersham, Arlington Heights, IL, 20,000 dpm in 20/A of water containing 0.03% CHAPS, 0.1% bovine serum albumin, and 0.9% NaCI). The cells are incubated for 2 hr at 4 ° and then washed 5 times with cold PBS. They are solubilized in 0.5 ml of 0.1 M NaOH/0.2% Triton X-100, and the radioactivity is determined. In experiments with J25I-labeled basic FGF, chondrocytes are incubated in 0.25 ml of buffer A with various concentrations of cartilage extract or basic FGF in the presence of ~25I-labeled basic FGF (1000 Ci/mmol, Amersham, 20,000 dpm in 20/~1 of water containing 0.1% CHAPS, 0.1% gelatin, and 0.9% NaCI). The cells are incubated for 90 min at 4 ° and then washed twice with PBS. They are then incubated at 4 ° in 0.5 ml of buffer containing l0 mM HEPES, pH 8.0, 0.1% bovine serum albumin, and 0.8 M NaCI. After 5 min, the buffer is removed by aspiration. This procedure is repeated twice. The cells are then solubilized in 0.5 ml of 0.1 M NaOH/ 0.2% Triton X-100, and the radioactivity is determined. Cartilage Fetal bovine cartilage obtained from local slaughterhouses within l0 hr of sacrifice was used in previous studies. But it became difficult to

[40]

CARTILAGE-DERIVED GROWTH FACTORS

419

Cartilage

t t

Extraction with IM guanidine/HC1 P r e c i p i t a t i o n with cold acetone(45-65%)

V

UItrafiltration l

t

UM20fraetion (20-300kDa)

U M 10fraction (10-20kDa)

Serum-protein affinity

lteparin-affinity

chromatography

chromatography

Reversed-phase HPLC

I so~leetric focusing (pit3-10)

t Reversed-phase

HPLC

26kDa CDF (CCSF)

t

MONO-S cation exchange chromatography

t t

Reversed-phase HPLC 16kDa CDF FIG. 1. Procedures for the purification of cartilage-derived factors.

obtain fresh fetal bovine months at 3 to 5 times

fetal bovine cartilage, so here we report a procedure using cartilage (articular, limb, and rib cartilage) stored for 1 to 3 20 °. The specific activity of CDF from the frozen cartilage is less than that of CDF from fresh cartilage.

Purification of 16-kDa Cartilage-Derived Factor

Cartilage Extract. Cartilage extract is prepared by homogenizing fetal bovine cartilage in l0 volumes of 1 M guanidine hydrochloride/0.1 M 6amino-n-caproic acid/20 m M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0, at 4 ° in a Polytron.l'6 The homogenate is stirred at 4 ° for 48 hr and then centrifuged at 10,000 g for 20 rain. The supernatant is fractionated with cold acetone (45-65%), as described previously 1'6 (Fig. 1). The acetone-fractionated cartilage extract (4 g) is dissolved in 3000 ml of 4 M guanidine hydrochloride/0.1 M 6-amino-n-caproic acid/20 m M Tris-HCl, pH 8.0/1 M NaCI. The solution is filtered through an Amicon (Danvers, MA) XM300 filter. The filtrate is then filtered through an Amicon UM20 filter (20,000 M r cutoff), and the resulting filtrate is filtered through a UM10 filter (10,000 M r cutoff). The fractions concentrated with UMI0 and UM20 filters are dialyzed for 3-4 days at 4 ° against distilled water and

420

OTHER GROWTH FACTORS AND GROWTH INHIBITORS

[40]

lyophilized. The lyophilized material is suspended in distilled water, and insoluble material is removed by centrifugation. Approximately 0.4 and 1 g (dry weight) of the UM 10 and UM20 fractions are obtained from 40 g of the acetone fraction of CDF (from 10 kg cartilage). Heparin Affinity Chromatography. A column of heparin-5PW (7.5 × 75 mm, Tosoh Co., Tokyo, Japan) connected to a fast protein liquid chromatography (FPLC) system (Pharmacia, Piscataway, N J) is equilibrated with 25 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCI and 0.03% CHAPS. The UMI0 fraction (9 mg soluble protein/170 mg dry weight) is applied to the column. After washing with 10 column volumes of equilibration buffer, CDF is eluted with a 0.15-3 M NaC1 gradient (30 ml) at a flow rate of I ml/min. Fractions of 2 ml are collected. Portions (1-3/zl) of each fraction are used to assay CDF. CDF activity is recovered in the fractions eluted between 0.5 and 1.2 M NaCI. The active fractions are pooled, dialyzed against distilled water, and lyophilized. The recovery of CDF activity from the column of Heparin-5PW is about 60%. About 2.64 mg of protein is obtained. Isoelectric Focusing. CDF (2.64 mg) partially purified by heparin affinity chromatography is dissolved in 0.2 ml of water. The solution is applied to a thin layer of Sephadex-IEF gel with 2% Pharmalyte, pH 3-10 (Pharmacia). Isoelectrofocusing is carried out in a flat bed apparatus (FBE 3000, Pharmacia) at 2000 V and 15 W at 1°. After 5 hr, 1.5-cm fractions are collected, a portion of each fraction is transferred to 1 ml of water and the pH measured. The rest of each fraction is scraped into a plastic column, and the gels are eluted with 2-4 ml of 0.5 M acetic acid containing 0.1% CHAPS. A portion (0.1 ml) of the eluate is mixed with 0.1 ml of 0.5% bovine serum albumin and dialyzed for 3 days against sterilized saline. Aliquots (1-5 tzl) of the dialyzed samples are used for CDF assay. CDF activity is found between pH 6.7 and 8.3 and between pH 8.7 and 9.9. The pH 6.7-8.3 fraction (1.8 mg protein) is dialyzed for 1.5 hr against distilled water at 4 °. The pH is adjusted at 7.4 with 0.2 M Na2HPO 4, and NaCI is added to a concentration of 0.15 M. Mono S Cation-Exchange Chromatography. A Mono S HR5/5 column (Pharmacia) is equilibrated with 25 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCI and 0.03% CHAPS. CDF (1.8 mg protein) partially purified by isoelectric focusing is applied to the column. After washing with 10 volumes of equilibration buffer, CDF is eluted with a 0.15-1 M NaCI gradient (60 ml) at a flow rate of 1.0 ml/min. Fractions of 2 ml are collected. Portions (1-3/xl) of each fraction are added to cultures grown in 96-well plates to assay of their activity to stimulate proteoglycan synthesis. CDF is eluted in the fractions between 0.2 and 0.5 M NaCI. The recovery of CDF activity from the column of Mono S is about 50%.

[401

c"

CARTILAGE-DERIVED GROWTH FACTORS

421

i5010011

ii

•~

y-1 l°°

,

0

?

.,I

c

~g Ot"

100

-

a 0

I 0

I

50

100

150

Elution volume (ml) Fro. 2. The active fraction eluted from the Mono S column was mixed with 0.1 volume of 1% trifluoroacetic acid and 0.1 volume of 2-propanol and loaded onto a 3.9 x 150 mm Ci8 reversed-phase column (Waters, /xBondasphere). The column was then washed until unbound materials were removed (at a flow rate of 0.5 ml/min with 10% 2-propanol). The column was developed with two linear gradients of 2-propanol (10-18% and 18-33%) in 0.1% trifluoroacetic acid in water. Fractions of 1 ml were collected. CDF activity was eluted with 30.5% 2-propanol.

Reversed-Phase H P L C . M o n o S-bound C D F (544/xg) is dissolved in 18 ml of 0.1% (v/v) trifluoroacetic acid in water and applied to a column of ~ B o n d a s p h e r e Cl8 (5/.~m, 100 A, Waters Ltd., Rochester, MI, 3.9 × 150 mm) which is equilibrated with 0.1% trifluoroacetic acid and 10% 2propanol in water. A 2-propanol gradient c o m p o s e d of 0.1% trifluoroacetic acid in w a t e r as starting buffer and 0.1% trifluoroacetic acid in 100% 2propanol as limit buffer is used. All solutions are degassed before use. The column is operated at a flow rate of 0.5 ml/min, and the effluent is collected in 1-ml fractions. A portion (100/,d) of each fraction is mixed with 0.2% bovine serum albumin, lyophilized, and dissolved in 100/zl of PBS. Portions (1-5/zl) of the solution are used to assay CDF. The p e a k of activity is eluted with 30.5% 2-propanol (Fig. 2). The protein in the active fraction is iodinated with ~25I by the chloramine-T method and analyzed by S D S - P A G E . One band corresponding to a molecular weight of 16,000 is seen in the autoradiogram in the presence and absence of 2-mercaptoethanol.

422

OTHER GROWTH FACTORS AND GROWTH INHIBITORS

[40]

Purification of Factors That Stimulate Chondrocyte Colony Formation in Soft Agar Chondrocytes are unique connective tissue cells that can be maintained in soft agar. Chondrocytes in soft agar retain their differentiated state for more than 3 weeks, I~ although cells maintained on plastic dishes lose their phenotypic characteristics after several generations. Recently, we found that a cartilage extract stimulates the growth of chick embryo chondrocytes in soft agar. The chondrocyte colony-stimulating factor (CCSF) can be partially purified by serum-protein affinity chromatography and reversed-phase HPLC. Serum Protein Affinity Chromatography. Rabbit serum is fractionated with ammonium sulfate (20-33%), and the precipitated proteins are bound to CNBr-activated Sepharose CL-4B according to the manufacturer's recommended procedure (2 mg protein/ml gel). A column (5 × 11 cm) of serum protein-bound Sepharose CL-4B is equilibrated with PBS. The UM20 fraction (200 mg protein) is applied to the column and washed with 500 ml of PBS. CCSF is eluted from the column with 100-200 ml of 4 M guanidine hydrochloride/Tris-HCl, pH 8.0. The fraction bound to serum protein is dialyzed against distilled water for 2 days at 4 ° and lyophilized. Reversed-Phase HPLC. CCSF (50 rag) partially purified by serum protein affinity chromatography is dissolved in 0.5 ml of 0.1% trifluoroacetic acid in water and applied to a column of/zBondasphere CIs (5/zm, 100 ,~, Waters, 3.9 × 150 mm) which is equilibrated with 0.1% trifluoroacetic acid in water. A 2-propanol gradient composed of 0.1% trifluoroacetic acid in water as starting buffer and 0.1% trifluoroacetic acid in 40% (v/v) 2propanol as limit buffer is used. The column is operated at a flow rate of 0.5 ml/min, and the effluent is collected in 1-ml fractions. A portion (100 /A) of each fraction is mixed with 0.2% bovine serum albumin, lyophilized, and dissolved in 100/zl of PBS. Portions (1-5/zl) of the solution are used to assay CCSF. The peak of activities is eluted with 30% 2-propanol, and the active fraction is rechromatographed on the same column. A portion of the active fraction is labeled with ~25Iand analyzed by SDS-PAGE under reducing conditions. Three bands corresponding to molecular weights of 22,000-26,000, 14,000-16,000, and 6000-10,000 can be shown by autoradiography. The protein eluted from the 22,000-26,000 band has both CCSF and proteoglycan synthesis stimulating activity. Thus, CCSF is a CDF. Figure 3 shows that CCSF, as well as a crude cartilage extract, stimulates colony formation by chick embryo chondrocytes in soft agar in a dosedependent manner. Molecular Weight of Various Cartilage-Derived Factors. The UMI0 fraction (300/zg) and the serum protein-bound fraction (300/~g) are each

[40]

C A R T I L A G E - D E R I V E D G R O W T H FACTORS

v

10

I0

E ©

2©

2nd HPLC

SPA b o u n d A

423

tone(ppt)

!

5

0.001

0.01

0.1

1

10

Protein

cone.

100

1000

(~g/ml)

FIG. 3. Dose-dependent stimulation of chondrocyte colony formation in soft agar by the acetone-fractionated cartilage extract, the UMI0 fraction, and CCSF purified by serum protein affinity chromatography and reversed-phase HPLC. Basic FGF (0.4 ng/ml) and transforming growth factor fll (TGF-fll, 3 ng/ml) also stimulated colony formation by chondrocytes in soft agar.

dissolved in 0.1 ml of 10 mM Tris-HC1 buffer, pH 8.0, containing 0.1% SDS and 20% glycerol, and incubated for 2 hr at 37°. The samples are then applied on SDS-polyacrylamide (17.8%) gels (running length 5 cm, width 2 cm, thickness 1 mm). After electrophoresis, the gels are sliced (2 mm width). Each slice is cut into three or four pieces, placed in 0.15 ml of PBS in a 1.5-ml plastic centrifuge tube, and allowed to stand for 18 hr at 4°. The gels are washed with 0.1 ml of PBS, and the washing fluid is combined with the eluate. Aliquots (1-2/~1) of the samples are used for CDF assays. For assay of CCSF activity, a portion (0.2 ml) of the samples is mixed with an equal volume of PBS containing 0.2% bovine serum albumin and dialyzed for 2 days against distilled water at 4°, because chondrocytes in soft agar are sensitive to low concentrations of SDS. Aliquots (1-2 pJ) of the samples are added to the semiliquid medium of soft agar cultures. In addition, aliquots (5/~1) of the samples are used to test IGF-I activity. On SDS-PAGE, the UM 10 fraction (10-20 kDa CDF) gives three peaks of CDF activity corresponding to molecular weights of 24,000-27,000,

424

OTHER GROWTH FACTORS AND GROWTH 1NHIB|TORS

[40]

14,000-16,000, and 6000-11,000. The protein with a mobility corresponding to 6-11 kDa also competes with J25I-labeled IGF-I in radioreceptor assays. On SDS-PAGE, the serum protein-bound fraction gives two peaks of CDF activity corresponding to molecular weights of 22,000-26,000 and 14,000-16,000. The 22-26K but not the 14K-16K protein induces colony formation by chondrocytes in soft agar. Although a crude cartilage extract shows high levels of basic FGF 15in the radioreceptor assay, no detectable basic FGF is found in the UM20 and UM10 fractions, probably because FGF is inactivated or removed during ultrafiltration. Transforming growth factor 13 (TGF-/3) is reported to be present in cartilage extract 16'~7and to stimulate proteoglycan synthesis by chondrocytes in culture. ~8,~9However, TGF-/3 alone, unlike CCSF, induces only low levels of colony formation by chondrocytes in soft agar at a maximal dose, although it increases the efficiency of colony formation in the presence of basic FGF. z° Furthermore, the UM20 and UM10 fractions have a very low level of TGF-/3 on the basis of radioreceptor assays (M. Iwamoto and Y. Kato, unpublished data). Thus the CDFs of 16K and 22-26K seem to be novel growth factors, although their amino acid sequences have not yet been determined. Acknowledgment We thank Mrs. Elizabeth lchihara for assistance in preparation of the manuscript.

15 R. Lobb, J. Sasse, R. Sullivan, Y. Shing, P. D'Amore, J. Jacobs, and M. Klagsbrun, J. Biol. Chem. 261, 1924 (1986). ~6 L. R. Elingsworth, J. E. Brennan, F. Fok, D. M. Rosen, H. Bentz, K. A. Piez, and S. M. Seyedin, J. Biol. Chem. 261, 12362 (1986). x7j. L. Carrington, A. B. Roberts, K. C. Flanders, N. S. Roche, and A. H. Reddi, J. Cell Biol. 107, 1969 (1988). 18 y . Hiraki, H. Inoue, R. Hirai, Y. Kato, and F. Suzuki, Biochim. Biophys. Acta 969, 91 (1988). 19 H. lnoue, Y. Kato, M. lwamoto, Y. Hiraki, M. Sakuda, and F. Suzuki, J. Cell. Physiol. 138, 329 (1989). 2o M. Iwamoto, K. Sato, K. Nakashima, H. Fuchihata, F. Suzuki, and Y. Kato, Biochem. Biophys. Res. Commun. 159, 1006 (1989).