Ioitmul of Orthupuedic Rewurch 1 0 3 W 8 Raven Press, Ltd., New York 0 1992 Orthopaedic Research Society

Fibronectin and Keratan Sulfate Synthesis by Canine Articular Chondrocytes in Culture Is Modulated by Dibutyryl Cyclic Adenosine Monophosphate Harry R. Leipold, Nancy Burton-Wurster, Juergen Steinmeyer, Margaret S. Vernier-Singer, and George Lust James A. Baker Institute for Animal Health, New York Stute College of Veterinary Medicine, Cornell University, Ithaca, New York, U.S.A.

Summary: The ability of cyclic adenosine monophosphate (CAMP)to maintain differentiated properties of canine articular chondrocytes in culture is reported. Treatment with 0.5 mM dibutyryl CAMP(DBcAMP) caused the cells to adopt a more rounded morphology. This change in morphology seems to have no effect on the overall biosynthetic rates of the cells. After a pulse with 3sS-methionine, there was no difference in the concentration of labeled proteins between cultures treated with DBcAMP and control cultures. A€ter 6 days, the amount of fibronectin (FN) in the media of DBcAMP-treated cultures detected by an enzyme-linked irnmunosorbent assay was specifically reduced by 30%. The amount of 35S-FN purified by gelatin-affinity chromatography decreased 33%. Moreover, the percentage of FN containing the extra domain A sequence was reduced from 19.4 ? 8.7% in control cultures to 9.6 L 4.2%. Concomitant with the decrease in FN, there was an increase in the concentration of keratan sulfate in the media of DBcAMP-treated cultures. After 6 days, treated cultures had 47% more keratan sulfate than controls did. These changes appear not to be the result of a change in the deposition of FN or keratan sulfate, because the amount of these molecules that could be extracted from the cell layer was typically below the limit of detection of the assays. Instead, it seems there is a phenotypic change in the chondrocytes pertaining to the production of FN and keratan sulfate. Key Words: Cartilage-ChondrocytesCyclic AMP-Fibronectin-Keratan sulfate.

drocyte-specific macromolecules by cultured chondrocytes can be modulated by culture conditions (65). Cultured chondrocytes tend to maintain the same phenotype as chondrocytes in vivo (i.e., a differentiated phenotype) when they are cultured in agarose (7), suspension (15), collagen gels (25,26), or organ culture (63). Alternatively, subculture (6), treatment with FN (54,70), retinoic acid (3,5-bromodeoxyuridine (51), phorbol esters (22,24,45), or viral transformation (1,27) cause cultured chondrocytes to exhibit a phenotype unlike chondrocytes in

Chondrocytes are the only cells found in mature cartilage. They synthesize a matrix composed primarily of type TI collagen and cartilage-type proteoglycans (PGs). However, there are other matrix proteins in mature cartilage, and one of those is fibronectin (FN). Phenotypic expression of chonReceived June 28, 1989; accepted April 30. 1991. Address correspondence and reprint requests to Dr. G. Lust at James A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca. N Y 14853. U.S.A.

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H . R . LEIPOLD ET AL.

vivo. We refer to this phenomenon as dedifferentiation, but do not imply that it is a reversion to a pluripotent mesenchymal state or that the phenotype cannot be reverted to a more differentiated phenotype under certain conditions. The predominant form of PG produced by differentiated chondrocytes in cartilage is the large aggregating PG. It contains a core protein with a molecular weight of approximately 200,000-300,000 (60) to which the glycosaminoglycans (GAGs) chondroitin sulfate and keratan sulfate (KS) are bound. It has been estimated that 100 chondroitin sulfatc side chains, 50 KS chains, and additionally 100 N and O-linked oligosaccharides can be attached to a single core protein (36), resulting in a PG monomer with a molecular weight of -2,000,000 (32). KS is found almost exclusively in cartilage PGs and was therefore proposed to be a good marker for cartilage-type PGs (62). The amount and type of core protein produced by chondrocytes in culture, as well as the quantity and characteristics of the GAGs attached, may each be affected by the culture conditions. Agents that induce dedifferentiation of chondrocytes can cause a shift in the type of PGs synthesized from cartilage type PGs to PGs similar in characteristics to those synthesized by nonchondrogenic cells. Chick embryo chondrocytes transformed by Rous sarcoma virus (52), treated with 5-bromodeoxyuridine ( 5 t), or phorbol esters (45) exhibit this shift. On the other hand, disruption of microfilaments with cytochalasin D (50), high-density culture (67), and inhibition of spreading (29) each cause a stimulation of sulfate incorporation into GAGs. FNs are a group of high-molecular-weight (-440) glycoproteins found in plasma, other body fluids, and many tissues and have a role in opsonization, cell adhesion, migration, and differentiation (37,73). Cartilage explants (72) and chondrocytes in culture (20) produce F N ; however, it is still contested as to whether chondrocytes in vivo synthesize FN. Presumably, differentiated chondrocytes synthesize little FN. At least 20 different polypeptides may be generated from a single FN gene by differential splicing of the pre-mRNA at three different sites (31,41,58,76). One of these differential splice sites leads to the inclusion or exclusion of a single exon that encodes a 90-amino-acid sequence termed extra domain A (ED-A) (16,42). Recently, it has been shown that less than 1-2% of the FN extracted from cartilage contains the ED-A sequence and cultured explants of cartilage synthesize little FN with the

J Orthop Res, Vol. 10, No. 1, 1992

ED-A sequence (12,14). Thus, FN synthesized by differentiated chondrocytes should not contain the ED-A sequence. FN production by chondrocytes in culture can be altered by the culture conditions. Those conditions that lead to a dedifferentiation of the chondrocytes cause the cells to produce more F N . In chick embryo chondrocytes in culture, dissociation of the cartilage matrix with collagenase and trypsin (20), conversion from floating cells in culture to chondrocytes spread on a plastic substrate (15), transformation with Rous sarcoma virus (1,27), and treatment with phorbol-12-myristate-13-acetate(24) have induced an increase in FN synthesis. Recently, it was reported that chondrocytes cultured in the presence of dibutyryl cyclic adenosine monophosphate (CAMP) (DBcAMPj (34,40,47j or factors that stimulate intracellular cAMP accumulation (35,46) synthesized more PGs and larger PGs than untreated cultures did. Also, DBcAMP (33) and prostaglandins that stimulate cAMP accumulation in limb bud mesenchymal cells (23,43) promoted limb bud chondrogenesis. Hence, cAMP may be an important mediator in the differentiation of chondrocytes. These considerations led us to test the hypothesis that culturing canine articular chondrocytes in the presence of 0.5 mM DBcAMP would promote the following differentiated properties: (a) an increase in the synthesis of KS, (b) a lower amount of F N produced, and (c) a smaller percentage of FN with the ED-A sequence when compared with untreated controls. MATERIALS AND METHODS Experimental Design A series of 14 culture experiments were performed using chondrocytes from either one (in 12 experiments) or two (in 2 experiments) dogs for each experiment. Articular cartilage free from contaminating tissues was obtained from adult canine hips, shoulders, and/or stifles as described previously (71), and chondrocytes were isolated as described below. After isolation, the number of cultures that could be set up with a density of l x lo5 cells/ml (5 x lo4 cells/cm2) was calculated. Half of the cultures were reserved for experimental treatment; the other half were left untreated to serve as controls. Each treated culture had a specific control culture to which it was paired for all comparisons.

EFFECTS OF CYCLIC AMP ON ARTICULAR CHONDROCYTES Cell Culture Chondrocytes were isolated by scquential 30-min collagenase digestions after treatment with trypsin essentially as described by Green (30). Cells were cultured at an initial density of 1 x lo5 cells/ml (5 x lo4 cells/cm2) in Ham’s F-12 media containing 25 mM HEPES (pH 7.2), 0.3 mglml L-glutamine, 0.03 mg/ml a-ketoglutarate, 0.05 mgiml ascorbate, 10 U/ml penicillin, 10 kg/ml streptomycin, and 10% fetal bovine serum (FBS) that had been depleted of FN by passing it through a gelatin-Sepharose affinity column. Addition of 0.5 mM DBcAMP or a water-soluble forskolin derivative: 7-deacctyl-7(4-methyl-piperazino)-butyryloxy dihydrochloride . H,O; catalog no. 344273 from Calbiochem, La Jolla, CA, U.S.A. (abbreviated in this text as 344273) was made at the time of initial seeding and maintained throughout the culture period. Culturing was performed at 3TC, 5% CO,, and 95% humidity. In some experiments the medium was changed after every 3 or 4 days. When medium was aspirated, one-tenth volume of a protease inhibitors solution was added (0.1 mM phenylmethylsulfonyl fluoride, 0.2 M EDTA, 500 pg/ml benzamidine hydrochloride, and 12.5 mgiml N-ethyl maleimide), and the mixture was stored at - 20°C until assayed. For experiments performed on passaged chondrocytes, the cells were allowed to grow to confluency in 75 cm2 culture flasks (1-2 weeks). Cells were then released from the flasks with trypsinEDTA solution (GIBCO, Grand Island, NY, U.S.A.) and seeded in plates at a density of 1 X lo5 cellsiml in the media described above. Medium was changed every 3 or 4 days. Viability was assessed by exclusion of the dye, erythosin B (Sigma Chemical Co., St. Louis, MO, U.S.A.). Extraction of the Cell Layer Extraction was performed in one of three ways. When FN was the only component to be analyzed, the cell layer was extracted overnight on a rotating platform at 20°C using 4 M urea in 0.05 M phosphate buffer, p H 7.2, containing 2 mM phenylmethylsulfonyl fluoride. In some experiments in which GAGs were also to be measured, the extraction solution was 4 M guanidine hydrochloride in 0.05 M sodium acetate, pH 5.8. In experiments in which labeled GAGs were being analyzed, the cell layer was digested with 0.5 mg/ml papain in a buffer, pH 6.5, containing 0.05 M sodium phos-

35

phate, 2 mM N-acetyl cysteine, and 2 mM EDTA for 2 h at 65°C. Extracts had one-tenth volume of protease inhibitors solution added and were stored at - 20°C. Metabolic Labeling with 35S-Methionineor 35S04-2 For all metabolic labeling experiments, chondrocytes were first cultured for 3 or 6 days in the same medium as described under Cell Culture. To label proteins, chondrocytes were pulsed for 4 h with 10 pCiiml 35S-methionine (Amersham, Arlington Heights, IL, U.S.A.) added directly to the media. Medium was aspirated, one-tenth volume of the protease inhibitors solution was added, and the mixture was stored at - 20°C until assayed. To label GAGS, 10 t&i/ml Na,35S04 (Amersham) was added directly to the culture media and the cells were incubated for either 2 or 8 h. Medium was aspirated, protease inhibitors solution was added, and the mixture was stored at -20°C. PCA Precipitation of 35SProteins An aliquot of medium was diluted to a volume of 500 pl. On ice, 500 t ~ of l I N perchloric acid (PCA) was added to each sample. Samples were kept on ice for at least 1 h. Precipitates were filtered through Whatman GF/C filter circles and were washed five times with cold 0.25N PCA. Filters were dried for 20 min at 60”C, and the radioactivity was quantitated using scintillation counting. Purification of 35S-FN FN was purified from media by affinity chromatography using gelatin-Sepharose as described by Engvall and Ruoslahti (21) and Pena et al. (53). After binding of the F N to the gelatin, the columns were washed with 1 M sodium chloride in 0.05 mM phosphate buffer, pH 7.2, containing 2 mM phenylmethylsulfonyl fluoride. FN was subsequently eluted with 6 M urea in 0.05 M Tris, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride. Aliquots of the urea eluate were counted in a liquid scintillation counter. Purification of 35S04-2-LabeledPGs

PGs in media were separated from free sulfate using Sephadex G-25 M (PD-10) columns (Pharma-

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H . R . LEIPOLD ET AL.

cia, Uppsala, Sweden). The columns were first equilibrated with 1 M guanidine hydrochloride and 0.05 M sodium sulfate, pH 7.0. Aliquots of media (0.25 ml) were applied to the columns in the guanidine hydrochloride buffer and then eluted with the same buffer. Fractions of 0.5 ml were collected and quantitated in a scintillation counter. PGs or GAGS in extracts of cell layers were quantitated after precipitation with potassium acetate. For the alkaline precipitation, 50 pl of extract was combined with 50 pI of bovine serum albumin (20 mgiml in water) and then added to 400 ~ 1 5 % potassium acetate in 95% ethanol. Samples were incubated overnight at 4°C in 1.5-ml microfuge tubes. The precipitates were pelleted for 10 min in a microfuge and then washed twice with 0.5 ml absolute ethanol. The final pellets were dissolved in 0.5-ml water and quantitated in a scintillation counter. Gel filtration of 35S04-2-LabeledPGs 35S04-2-labeledPGs in media were analyzed on columns of Sepharose CL-2B (0.8 cm x 100 cm) in 4 M guanidine hydrochloride. Media samples (1 ml) were applied to the column and subsequently eluted with 4 M guanidine hydrochloride, 0.1 M sodium sulfate, 0.05 M sodium acetate, 0.1% Triton X-100, pH 6.0 at a flow rate of 4 mlih. Fractions of 0.7 ml were collected and radioactivity was quantitated in a scintillation counter. ELISA for FN and FN Containing the ED-A Sequence

quence. FN from the culture media of canine fibroblast cell line A72 (American Type Culture Collection, Rockville, MD, U.S.A.) was used as a standard. It was assumed that all the FN produced by the A72 cells contained the ED-A sequence, but this is likely to be an overestimate. ELISA for KS The ELISA for KS was essentially that described by Thonar et al. (62) with minor modifications, and has been described previously (44). Briefly, samples were incubated with a monoclonal antibody to KS (1/20/5-D-4;Miles, Naperville, IL, U.S.A.) and then the mixture was transferred to 96-well flatbottomed plates (Nunc, Roskilde, Denmark) coated with bovine nasal PG monomers (a gift from Dr. B. Caterson, West Virginia University). The antibody not bound to KS in the sample was then free to bind the KS on the plate. Bound antibody was detected with a second-stage peroxidase-linked anti-mouse IgG antibody, and the color substrate o-phenylene diamine. Statistical Analysis Comparisons between treated and control groups were made using the paired t test when n > 30, the Wilcoxon signed-rank test when 30 > n > 6, and the Mann-Whitney test when n < 6 . Significance was set at p < 0.05. RESULTS Morphology

The details of the enzyme-linked immunosorbent assay (ELISA) for FN have been reported previously (71). Briefly, a polyclonal goat anti-human FN antibody was used to detect the FN in samples. Canine plasma FN was used as the standard. FN in the sample or standard was allowed to bind to gelatin-coated 96-well flat-bottomed plates (Falcon, Oxnard, CA, U.S.A.). The bound FN was detected using the polyclonal anti-human FN antibody, a second-stage peroxidase-linked anti-goat IgG antibody, and the color substrate o-phenylene diamine. The ELISA for FN containing the ED-A sequence is similar to the ELISA for FN except that a monoclonal antibody, IST-9, prepared and kindly provided by Drs. B. Carnemolla and L. Zardi of Genoa, Italy, was used to detect the ED-A se-

J Orthop Res, Vol. 10, No. I , 1992

Primary articular chondrocytes cultured in the presence of 0.5 mM DBcAMP had a noticeably different morphology compared with control cultures. Figure 1 shows a control culture and a culture treated with DBcAMP. The control cells maintained a polygonal morphology that when fully confluent resembled a cobblestone pattern. Many of the DBcAMP-treated cells, however, remained spherical and settled at the bottom of the flask, but did not attach. In some cultures the treated cells attached, but spread out in a more rounded morphology instead of the typical polygonal shape. At the end of 8 days in culture, both control and DBCAMP-treated cells were viable (94.7 3.2% versus 94.7 k 7 k 25%, n = 3 , respectively).

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EFFECTS OF CYCLIC AMP O N ARTICULAR CHONDROCYTES

FIG. 1. The morphology of chondrocytes in primary culture for 6 days with or without dibutyryl cyclic adenosine monophosphate (CAMP).Chondrocytes were released from cartilage using collagenase and then plated at a concentration of 1 x lo5 cellsirnl in Hams F-12 media. Cells in (a) are controls; cells in (b) were treated with 0.5 mM dibutyryl CAMP for the entire 6-day culture period (Bar = 100 km).

Effect of DBcAMP on FN Synthesis The concentration of FN in the media of primary chondrocytes as detected using an ELISA is shown in Table 1 . During the first 3 days of culture there was no difference in the concentration of FN in the media of control cultures versus cultures treated with 0.5 m M DBcAMP. Cells cultured for 6 days in the presence of DBcAMP without changing the media had a 30% decrease in average FN concentration (Table 1). This diffcrence was statistically significant. Cells in which the media were changed after 3 days in culture also had a significant decrease

in FN concentration between days 4 and 6 when treated with DBcAMP (Table 1). It should be noted that the concentrations of FN from days 0-3 added to days 4-6 does not equal days 0-6. The experiments in which the media were changed after 3 days were performed separately on cells isolated from different animals from the experiments in which the cells were left for 6 days without changing the media. Thus, the differences may be due simply to differences in the samples used or in the culture conditions. Alternatively, the chondrocytes cultured for 6 days without changing the media may be secreting components that effect the

TABLE 1. Effect of dibutyryl cyclic adenosine monophosphate (DBcAMP) on jibronectin (FN) concentrations in the media from primary and first-pussage chondrocyte cultures ~

FN concentrations in media from chondrocytes in culture for various periods Treatment Primary None 0.5 mM DBcAMP First passage None 0.5 m44 DBcAMP

Days 0-3 (ng/ml) S96 582

5 ?

*

331 (37) 389 (37)

1,470 1.040 (9) 1,211 2 850 (9)’

Days 0-6 (ngiml)

Days 4-6 (ng/ml)

5,192 t 2,013 (34) 3,632 t I ,518 (34)”

1,742 2 732 (24) 1,178 ? 645 (24)b 2,546 i 959 (9) 1,639 2 528 (9Ib

Results are expressed as mean 2 SD ( n ) . Concentrations of FN in the media were determined with an enzyme-linked immunosorbent assay using a polyclonal antisera to human fibronectin. Significantly different from control, p < 0.001. Significantly different from control, p < 0.005. Li

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11. R . LEIPOLD ET AL.

metabolism of thc cells. The fact that the quantities observed in the 0-6-day cultures were higher than the 0-3 and 4-6 days added together may indicate that the cells were growing at least as well as the cells that had the media changed after 3 days. The decrease in FN concentration was not due to an overall decrease in protein synthesis because the mean concentration of 35S-methionine-labeled protein in the media after a 4-h pulse was not different between control and treated cultures. In one experiment, after 3 days in culture, the average concentration in control cultures was 13,290 k 3,510 cpmi ml whereas DBcAMP-treated cultures had 14,644 2 1,163 cpm/ml (Table 2). In another experiment, after 6 days there was still no difference between the groups (41,344 2 11,985versur 40,047 +- 2,250 cpmi ml) . The concentration of labeled FN detected in these same experiments after a 4-h pulse with 3sSmethionine in the media of primary chondrocytes in culture for 3 or 6 days is shown in Table 2. Chondrocytes in culture for 3 days in the presence of 22 cpm/ml compared DBcAMP showed 1,493 with 2,130 ? 386 cpmiml in control cultures. After 6 days in culture with DBcAMP, the mean concentration of 35S-FN was 3,056 I575 cpmiml com160 cpmiml in controls (Table pared with 5,538 2). These differences were statistically significant and were reproducible. Sodium dodccyl sulfatepolyacrylamide gel electrophoresis showed the purified "S-FN had the correct apparent M , for FN (220-240) and was at least 90% free of contaminating proteins (Fig. 2). In two separate experiments, urea extracts of the cell layer had levels of F N too low to detect by

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*

ELTSA (