JOURNAL OF CELLULAR PHYSIOLOGY 147:403411(1991)

Effects of a Putative Prostaglandin E, Antagonist, AH6809, on Chondrogenesis in Serum-Free Cultures of Chick Limb Mesenchyme ANTHONY A. CAPEHART AND DAVID M. BIDDULPH* Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103 In the present study, we have examined the effects of a putative antagonist of prostaglandin E, (PGE,), AH6809, on chondrogenesis in serum-free cultures of mesenchyme from distal tips of stage 25 chick limb buds in order to test the hypothesis that endogenous PCE,, through receptor-linked adenylate cyclase (AC), initiates differentiation of cartilage in limb mesenchyme. Daily addition of 1OP4 M concentrations of AH6809 produced marked inhibition of chondrogenesis over a 5-day period of cell culture as evaluated by Alciari green binding to cartilage matrix components. Inhibition of chondrogenesis by this compound was further shown to be reversible and treatment of cells with the antagonist limited to periods when chondrocytes had differentiated and were actively secreting cartilage-specific matrix components had little effect. Preincubation of control M concentrations of AH6809 inhibited PGE,-induced activation of cells in AC by greater than 80% without significant (P > .05) inhibition of basal activity by the antagonist. Responses to parathyroid hormone, which increased AC activity by 7-fold, and forskolin which increased AC activity by 23-fold in control cells, were also uninhibited by preincubation i n AH6809. The results demonstrate that blockade of PGE,-AC linked receptors in prechondrogenic limb mesenchyme inhibits chondrogenesis supporting the hypothesis that endogenous PGE, concentrations in undifferentiated limb mesenchyme play an initiating role in the differentiation of cartilage.

Chondrogenesis represents a model system for the study of cytodifferentiation which has been investigated extensively in several species, but particularly within the developing chick limb (Caplan, 1981; Elmer, 1982). While much has been learned about the sequence of biochemical and morphological changes that accompany this process, the specific regulatory factors within the environment of the embryonic mesenchyme which regulate chondrogenic expression are still unknown. In the last few years, several studies have presented evidence to suggest that certain prostaglandins (PGs),through their ability to increase cyclic AMP (CAMP)levels of undifferentiated mesenchyme, may be important. Evidence in support of this hypothesis includes the observations that exogenous derivatives of cAMP stimulate chondrogenesis in both cultured limb cells (Ahrens et al., 1977; Solursh et al., 1981;Biddulph et al., 1988a) and limb bud explants (Kosher and Savage, 1980). In addition, endogenous levels of cAMP have been shown to increase in cultured limb cells prior to the onset of overt chondrogenesis (Solursh et al., 1979; Biddulph et al., 1984; 1988b). It has also been demonstrated that exogenous derivatives of cAMP stimulate the production of mRNAs coding for specific gene products of differentiated chondrocytes, including 6

1991 WILEY-LISS, INC.

both type I1 collagen and cartilage-s ecific proteoglycans (Kosher et al., 1986). Addition o exogenous PGE2 produces similar stimulatory effects on chondrogenesis (Kosher and Walker, 1983; Gay and Kosher, 1984) and has been demonstrated t o increase both adenylate cyclase (AC) activity and the endogenous cAMP levels of these cells (Biddulph et al., 1988b; Ca ehart et al., 1990). It has also been shown that P s, including PGE2, are synthesized by cultured limb mesenchyme (Chepenik et al., 1984; Gay and Kosher, 1985; Biddulph et al., 1988a) prior to overt differentiation of cartilage. Increases in PGEB synthesis in prechondrogenic mesenchyme coincide temporally with increases in endogenous concentrations of CAMP (Biddulph et al., 1988a; Biddulph and Dozier, 1989) during this same period, although evidence directly linking these prechondrogenic increases in PGEz and cAMP concentrations has not been established. In one previous study (Chepenik et al., 19841, it has been re orted that established inhibitors of PG synthesis in ibit chondrogenesis in this system. However, since these inhibitors are rela-

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Received December 5, 1990; accepted February 4,1991. *To whom reprint requestskorrespondence should be addressed.

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CAPEHART AND BIDDULPH

tively nonspecific for PG synthesis (Flower, 1982) and reversal of inhibition has not been reported by exogenous application of either PGEz or CAMP,it has not been firmly established that these inhibitory effects on chondrogenesis are due specifically to reductions in endogenous concentrations of PGEz and subsequent inhibition of the cAMP system. We have reported previously that responsiveness of the adenylate cyclase cAMP system to PGEB in cultured mesenchyme from chick limb buds is maximal during the prechondrogenic period of cell culture (Biddulph et al., 1988a; 198813). This maximally responsive state is rapidly lost, however, as differentiated chondrocytes develop on days 3-6 of culture. This transient increase followed by a loss of responsiveness to PGEz could not be accounted for by either changes in the composition of the extracellular matrix or changes in the total pool of AC activity with which the PGE, receptor can interact (Capehart et al., 19901, suggesting developmentally regulated changes in the PGE2 receptor-AC complex which could play an initiating role in chondrogenesis. In the present study we have employed a putative antagonist of PGE,, AH6809 (Coleman et al., 1985b, 1986; Eglen and Whiting, 1988), to investigate effects of PGEz receptor blockade on chondrogenesis. The rationale for this study was that if PGE, receptor-mediated activation of AC and subsequent increases in concentrations of cAMP are initiating factors in chondrogenesis rather than merely effects of initial differentiation of chondrocytes due to other mechanisms, receptor blockade t o PGE, in undifferentiated mesenchyme should inhibit chondrogenesis in this system. Our results support this hypothesis. MATERIALS AND METHODS Preparation of cell cultures Micromass culture of limb cells (Solursh et al., 1978) derived from the distal tip of chick embryo limb buds employed the methods described by Gay and Kosher (1984) with minor modification, which we have previously described in detail (Biddulph et al., 1988a; 1988b). Distal tips (approximately 0.3 mm from the distal apex) were excised from both fore- and hind-limb buds from stage 25 chick embryos, pooled, and dissociated in 0.5% trypsin in calcium- and magnesium-free phosphate buffered saline (PBS) for 30 min at 37°C.The cells were washed in complete culture medium (Hams F-12 containing 10% fetal bovine serum and antibiotics), filtered through two layers of #20 Nitex, and cell number determined with a hemocytometer. Cell concentration was then adjusted to 2 x lo7 celliml and each tissue culture dish (35 mm Corning) inoculated with eight to twelve 10 111drops. Dishes were placed in a humidified incubator, allowed 2 hr for cells to attach, and were then supplemented with 2.0 ml of either complete medium or a serum-free medium, as originally described by Paulsen and Solursh (1988), which contained 40% Dulbecco’s MEM and 60% Ham’s F-12 supplemented with 5 Kgiml insulin (Collaborative Research), 100 nM hydrocortisone (Sigma), 50 pg/ml L-ascorbic acid (Sigma), and antibiotics. Cells were fed daily by complete replacement of the medium and incubated in a humidified atmosphere of 95% air and 5% CO, maintained at 37°C. The putative PGEz antag-

onist, AH6809, generously donated by Glaxo Group, United Kingdom, was dissolved in isotonic saline containing 1% NaHCO, and added to serum-free cell cultures according to the following protocol: 1) daily replacement beginning at the time of cell plating; 2) daily replacement during only the initial two days of culture; and 3) daily replacement beginnin at the end of three days of culture. N6, 02-dibutyry adenosine 3’,5’,cyclic monophosphoric acid (dbcAMP, Grade 11, Sigma) was added to cell cultures at a final concentration of 1mM 24 hr after cell plating. Daily replacement of dbcAMP was continued with each change of media over a 5-day period. Analysis of chondrogenesis Qualitative analysis of chondrogenesis utilized both phase-contrast microscopy and Alcian green binding to sulfated cartilage matrix (Ham and Sattler, 1968) employing methods we have described previously in detail (Biddulph et al., 1988a; 1988b).Binding of Alcian green to cartilage matrix has been shown to occur coincident with the synthesis of chondroitin sulfate glycosaminoglycans, as well as with cartilage-specific proteoglycans, under the conditions we have employed in these cell cultures and is a reliable indicator of the presence of cartilage matrix components (Biddulph et al., 198813) Quantitative analysis of chondrogenesis utilized a modification of methods previously described by Paulsen and Solursh (1988) for the extraction of bound Alcian green t o cartilage proteoglycans by guanidine HC1 (GuHC1) and the spectrophotometric quantitation of the Alcian green-containing extract as well have previously described in detail (Capehart and Biddulph, 1991). At various times during culture, tissue culture medium was removed and the attached cells were washed twice with PBS. Cells were then fixed overnight in absolute ethanol containing 2% glacial acetic acid at 4°C. Cultures were rehydrated through a graded series of ethanol and stained for 1 hr in 0.5% Alcian green in 3% glacial acetic acid. Cultures were then rinsed twice with 3% glacial acetic acid and extracted overnight at 4°C in 1.5 m14 M GuHC1. The absorbance of the extract was determined spectrophotometrically at 600 nm using 4 M GuHCl as a blank. Complete extraction of Alcian green was effected by this procedure and no detectable extraction of Alcian green was observed in cultures in which PBS was substituted for GuHC1, validating the specificity of the extraction procedure. DNA, as measured by the method of Richards 11974) in the extracted cell layer of each culture, was used to normalize absorbance values. Preliminary experiments established that neither fixation nor extraction with GuHCl significantly altered DNA content of the cell layer. Cell incubations a n d measurement of adenylate cyclase activity Adenylate cyclase activity was measured by methods we have described previously in detail (Biddulph et al., 1988b). Briefly, cells at various times during culture were preincubated in either the presence or absence of l o p 4 M AH6809 for 5 min in 1 ml of Krebs-Ringer buffer, pH 7.4, containing 20 mM Tris-Base and 2 mM

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BLOCKADE OF PGE, RECEPTORS INHIBITS CHONDROGENESIS

1-methyl-34sobutylxanthine(MIX). We have shown previously that this concentration of MIX maximally inhibits phosphodiesterase activity in intact limb cells (Ballard and Biddulph, 1983) and provides a reliable measure of AC activity. PGE, (Sigma) was dissolved in absolute ethanol, diluted in incubation buffer, and added in amounts to achieve a final concentration of l o p 6 M. Equivalent amounts of ethanol, which did not exceed 0.75%, were added to control cultures. Forskolin (Calbiochem) was also dissolved in absolute ethanol and added in volumes to achieve a final concentration of M. Control incubations received the equivalent amounts of ethanol which did not exceed 0.75%. Parathyroid hormone (PTH, 150 Uimg, Sigma) was dissolved in acidified saline (pH 3.0) and added in amounts to achieve final concentrations of 10 U/ml. Equivalent amounts of saline were added to control cultures. Following additions of test substances, incubations were continued for an additional 5 min, terminated by addition of 1mllO'% TCA, and tissue extracts prepared exactly as we have described previously in detail (Biddulph et al., 1988b).Cyclic AMP was assayed in ether-extracted supernatants by the method of Gilman (1970) as modified by Tovey et al. (1974). DNA within the TCA-precipitated pellet was measured by the method of Richards (1974) and protein by the method of Lowry et al. (1951). Radioimmunoassay of PGE Endogenous concentrations of PGE, in the media of serum-free cell cultures were determined by radioimmunoassay by methods we have previously described in detail (Biddulph et al., 1988a; Biddulph and Dozier, 1989) using kits purchased from Advanced Magnetics, Inc. At various times following initial cell plating, media were removed, centrifu ed at 1500g at 4"C, lyophilized and stored at -70" . At the time of the assay, the lyophilate was dissolved in 0.5 ml of assay buffer and added to assay tubes in 100 pl volumes. The cell layer was homogenized in 5% TCA and assayed for DNA content by the method of Richards (1974). RESULTS Chondrogenesis and responsiveness of adenylate cyclase to PGE in serum-free cell cultures The ma'or objective of the present study was to examine t e effects of a putative antagonist of PGE,, AH6809 (Coleman et al., 1985b), on chondrogenesis in order to test the hypothesis that endogenous PGEz produced by undifferentiated mesenchyme (Chepenik et al., 1984; Gay and Kosher, 1985; Biddulph et al., 1988a) plays a role in initiating chondrogenesis through its effects on CAMPmetabolism (Biddulph et al., 198813; Capehart et al., 1990). Since this compound has been shown previously to bind strongly to albumin (Coleman et al., 1985a), serum-free conditions of cell cultures were considered essential to the study. Initial experiments were designed to compare the time-course of both chondrogenesis and responsiveness of AC to PGE, in serum and serum-free cell cultures. In the presence of serum, as we have previously described in detail (Biddulph et al., 1988a,; 1988b), initial deposition of cartilage matrix components under the conditions employed occurred in these cultures on day 3 and

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rapidly progressed to produce by day 6 a complete1 confluent sheet of cartilage involving the entire ce 1 layer (Fig. 1, panel A). An identical pattern in the time-course of chondrogenesis occurred in serum-free cultures; however, in contrast to serum-containing cultures, completely confluent deposition of cartilage matrix did not occur during the time periods investigated (Fig. 1, panel B). By day 6, small islands of nonchondrogenic cells still persisted, separating chondrogenic regions of the cultures. Increasing culture time to 8 days or daily addition of either dbcAMP (1 mM) or forskolin (0.1 mM) were all ineffective in inducing further differentiation of these nonchondrogenic regions. Guanidine extraction of Alcian green, normalized to DNA content, demonstrated that serumfree cultures contained, at day 6, approximately 60% of the cartilage present in serum-containing cultures (Table 1).Despite these reductions in chondrogenesis in serum-free cultures, no significant (P> .05) differences could be detected over the 6-day period examined in DNA content of cultures maintained in either the presence or absence of serum (Table 1).Protein content of cultures also did not differ except at day 3 with protein in serum-free cultures reduced relative to serum-containing cultures by approximately 15% (Table 1). Endogenous concentrations of PGEz in the media of serum-free cultures were also examined using radioimmunoassay at days 1, 3, and 6 of culture in order to determine if PGEz was being synthesized by these cells in the absence of serum. As we (Biddulph and Dozier, 1989), as well as others (Gay and Kosher, 19851, have previously reported for serum-containing distal tip cultures, PGE, concentrations were highest in prechondrogenic mesenchyme on day 1(7.0 1.0 pg/pg DNA), but declined slightly by day 3 (6.1 k 0.9 pg/pg DNA) as the initial deposition of cartilage matrix occurred. By day 6, PGEz concentrations decreased significantly (1.1t 0.3 pgipg DNA) in cultures containing fully developed chondrocytes and in several samples were undetectable. Other experiments were designed to compare the time-course of PGE,-res onsive AC activity in serum and serum-free cultures ( ig. 2).An identical pattern of response occurred under both conditions with maximal responsiveness to PGE2 observed in prechondrogenic mesenchyme (day 1) followed by a loss of responsiveness occurring in cultures containing differentiated chondrocytes on both days 3 and 6. The maximal response to PGE, on day 1, however, was blunted by approximately 40% in serum-free cultures relative to cells grown in the presence of serum, which corresponded to a similar reduction on day 6 in the amount of cartilage present in serum-free, as compared with serum-containing, cultures as previously described (Table 1). Effects of AH6809 on chondrogenesis and PGE, linked AC activity In initial experiments, AH6809 was added daily to serum-free cultures beginning at the time of cell plating at concentrations varying between 10-6-10-4M. Cultures were monitored daily with phase-contrast microscopy and stained with Alcian green for cartilage

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Fig. 1. Micromass cultures of mesenchyme derived from the distal tip of stage 25 limb buds. Whole mounts were stained for cartilage with Alcian green-metanil yellow and were cultured for 6 days in either the presence (panel A) or absence (panel B) of 10% fetal bovine serum. Note the lack of confluent cartilage matrix in serum-free cultures (panel B). Scale bar represents 1 mm for each panel.

TABLE 1. Comparison of DNA, protein and Alcian green binding in serum and serum-free cultures of chick limb mesenchyme' Group Day 0 Day 1 Serum - Serum Day 3 Serum Serum Day 6 Serum Serum

+ + ~

+ ~

DNA (pglculture)

Protein (pg/culture)

Alcian green binding (absorbancelpg DNA X 103)

0.82 f 0.11

2.01 f 0.23

-

1.01 ? 0.12 0.95 & 0.04

4.73 & 0.31 5.05 k 0.19

-

1.83 k 0.17 1.78 -t 0.07

11.83 t 1.21 9.64 t 0.22'

-

2.43 i 0.19 2.46 t 0.05

16.02 k 1.02 16.14 f 0.27

43.7 0.7 26.4 t 0.4*

0

+Serum

o Serurn-Free

+

I Cells were cultured in either the presence (+ serum) or absence (- serum) of 10%fetal bovine serum for the time intervals indicated. Extraction of Alcian green from stained cultures by guanidine HCI was determined spectrophotometricallyat 600 n m at day 6. Each value is the mean f SE of 6-10 individual samples obtained from 3-4 separate experiments. *Values differing significantly (P < .Ol) from paired serum-containing treatment

group.

-+ O L

matrix on days 4 and 5. Concentrations of M produced marked inhibition of Alcian green binding throughout the 5-day period examined (Fig. 3, panel B). Quantitative analysis of Alcian green binding on day 5 of culture utilizing GuHCl extraction revealed approximately 70% reductions relative to control cultures in which absorbance of the extract was normalized to DNA content of the cell layer (Table 2). Normalization of the extract absorbance to protein content increased inhibition to greater than 80% of control cultures. In the continuous presence of the antagonist at concentrations of M, no apparent differences could be detected in either the diameter of cultures or packing density of cells relative to control cultures, although DNA content in cultures treated with the antagonist was consistently reduced from controls by 30-40%. Morphologically, as examined by phase-contrast microscopy, cells grown in the continued presence of AH6809 maintained a mesenchymal-like appearance

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0

1

2

3 Days

I

I

I

4

5

6

Fig. 2. Changes in adenylate cyclase (AC) activity in cultured limb cells derived from the distal tip of stage 25 chick limb buds. Cells were cultured for the time intervals shown in either the presence ( 0 ) or absence ( 0 ) of 10% fetal bovine serum and incubated for 5 min in 1 O P M PGE, or 0.75% ethanol in the presence of 2 mM MIX. Each point represents the increase ( A ) in AC activity of 6-9 samples in response to PGE, from an equivalent number of control (0.75% ethanol) samples. Vertical lines represent SEM.

similar to control cells during the initial 2 days of culture without signs of cytotoxicity. Concentrations of either lop6or l o p 5M AH6809 did not produce visible differences in binding of Alcian green relative to control cultures. Concentrations greater than 2 x lo-* M appeared to be cytotoxic, initiating detachment and

BLOCKADE OF PGE, RECEPTORS INHIBITS CHONIIROGENESIS

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Fig. 3. Micromass cultures of chick limb cells derived from the distal tip of stage 25 limb buds. Whole mounts were stained for cartilage with Alcian green-metanil yellow and were cultured in serum-free medium for 5 days in the absence (panel A j or resence (panels B and C) of 1W4M AH6809 which was replaced daipy. Dibutyryl cAMP

(dbcAMP 10-3M; panel C j was added to cell cultures 24 hr after plating and was replaced daily in the presence of AH6809. Note the inhibition of chondrogenesisin AH6809-treated cultures (panel B) and reversal of inhibition by dbcAMP (panel C). Scale bar represents 1 mm for each panel.

TABLE 2. Effects of AH6809 on chondrogenesis evaluated quantitatively by Alcian green binding in cultured mesenchyme from chick limb buds 1

were also observed to be reversible. Addition of this inhibitor for the initial 2 days of culture produced reductions in Alcian green binding on day 4 (Fig. 4, panel B) which appeared to be identical to those produced by continuous treatment (Fig. 3, panel B), but by day 5, binding of Alcian green, as evaluated both qualitatively (Fig. 4, panel E) and quantitatively (Table 21, had increased considerably and was rapidly approaching control levels. By day 6 of culture, no apparent differences in cartilage matrix deposition evaluated by both phase-contrast microscopy and Alcian green staining were observed relative to control cultures. Addition of AH6809 between day 3 and day 5, the period in which active synthesis and secretion of cartilage matrix components was occurring, had a relatively small effect as com ared with continuous treatment (Fig. 4,panels C an F), indicating that the inhibitory effects produced by this compound were not simply related to inhibition of secretion of cartilage matrix components of differentiating chondrocytes. The effectiveness and specificity of AH6809 on block-

Absorbance at 600 nm per pg DNA X l o 3

X of paired

Treatment group Control AH6809 (Do-Db) AH6809 (Do-D,)

16.52 k 0.52 5.55 k 0.49* 9.52 0.43*

100.0 33.6 f 2.3 59.7 k 1.4

+

control

'Cultures of chick limb mesenchyme were grown in serum-free medium far 5 days and binding of Alcian green quantitated by extraction in guanidine HCI and spectrophotometric analysis of the extract. AH6809 (10-4M) was added daily from day O-day 6 and day 0-day 2. Control cultures received equivalent volumes of vehicle. Each value is the mean of 6-7 individual samples C S E obtained from 3 separate experiments. *Values differing significantly (P< 0.01) from controls.

lysis of cells by 2 days of culture. The marked inhibition of chondrogenesis by M concentrations of AH6809 could be largely reversed by co-addition of M dbcAMP (Fig. 3, panel C), indicating that cells treated with AH6809 could still respond to cAMP stimulation. The inhibitory effects of AH6809 on chondrogenesis

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Fig. 4. Micromass cultures of chick limb cells derived from the distal tip of stage 25 limb buds. Whole mounts are stained for cartilage with Alcian green-metanil yellow and were cultured in serum-free medium for either 4 (panels A-Cj or 5 (panelsD-F) days. Control cultures on days 4 and 5 are shown in panels A and D, respectively. AH6809 was present at a concentration of 10-4M for either the initial 2 days of

culture (panels B and El or during days 3-5 (panels C and F). Note the nearly com Iete inhibition by AH6809 of chondrogenesis on day 4 when addefbetween days 0-2 (panel B), but recovery of chondrogenesis by day 5 (panel Ej. Also note the lack of effect of AH6809 when added to cells between days 3-5 (panels C and F). Scale bar represents 1mm for each panel.

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BLOCKADE OF PGEz RECEPTORS INHIBITS CHONDROGENESIS

ade of PGE2 receptors in these cell cultures was investigated in other experiments (Table 31, with adenylate cyclase activity measured as the functional expression of AC-linked PGE, receptors. In these experiments, all cells were cultured under serum-free control conditions for 24 hr to produce maximal responsiveness of AC activity to PGE, (Fig. 2). Preincubation of cultures for 5 min in l o p 4M AH6809 inhibited greater than 80%of the PGE2activation of AC without significant (P > .05) inhibition of basal AC activity. Activation of AC by parathyroid hormone (PTH), which increased AC activity by 7-fold in control cells, was also unaffected by preincubation in equivalent concentrations of AH6809 (Table 3). Direct stimulation of AC by forskolin (Seamon et al., 1981), which increased AC activity by 23-fold in control cells (Table 31, was also not reduced by this antagonist, demonstrating that the pool of AC activity with which specific receptors such as PGE2and PTH interact was uninhibited by this antagonist.

DISCUSSION The results of the present study demonstrate that a putative antagonist of PGE,, AH6809 (Coleman et al., 1985b, 1986; Eglen and Whiting, 1988), produces marked inhibition of chondrogenesis in cultured mesenchyme from chick limb buds. These results further suggest that blockade of PGE2 receptors in prechondrogenic mesenchyme prevents normal differentiation of chondrocytes in this in vitro system, supporting the hypothesis that endogenous PGE2, produced by prechondrogenic mesenchyme (Chepenik et al., 1984; Gay and Kosher, 1985; Biddulph et al., 1988a), plays an initiating role in differentiation of cartilage. Since it has been reported (Coleman et al., 1985a) that AH6809 binds strongly to albumin, serum-free conditions of cell culture were employed in the present study. We (Biddulph et al., 1988b), as well as others (Swalla et al., 1983; Gay and Kosher, 19841, have previously characterized in detail chondrogenesis in serum-containing cultures of mesenchyme derived from the distal tip of stage 25 chick limb buds. We have also reported the changing responsiveness of PGE2mediated AC activity in these serum-containing cultures which is maximal in rechondrogenic mesenchyme during days 1 and 2 o culture, but is abruptly reduced in differentiating chondrocytes on days 3-6 of culture (Biddulph et al., 198813; Capehart et al., 19901, and we have speculated that this maximally responsive state of prechondrogenic mesenchyme may be an important prerequisite to initiation of chondrogenesis. While two previous studies have reported a limited degree of chondrogenesis to occur in serum-free conditions of more heterogeneous cultures containing a variety of cell types derived from whole limb buds (Paulsen and Solursh, 1988; Kujawa et al., 1989), chondrogenesis under serum-free conditions in the more homogeneous distal tip cultures has not to our knowledge been previously characterized. We, therefore, conducted initial experiments to determine if the time-course of chondrogenesis and the responsiveness of distal tip cultures to PGE2 were altered by serum-free conditions. No qualitative differences in either the time-course of chondrogenesis or in the responsiveness of AC activity to PGE2 were noted

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TABLE 3. Effect of AH6809 on adenylate cyclase (AC) activity in cultured mesenchyme from chick limb buds 1

Group Basal-control Basal-AH6809 PGE2-control PGE2-AH6809 PTH-control PTH-AH6809 FS-control FS-AH6809

AC activity Change from pmols/5 min/kg DNA basal 0.22 & 0.04 0.26 5 0.06 3.45 & 0.08 0.75 k 0.05* 1.48 0.14 1.67 0.21 4.94 ? 0.34 6.01 ? 0.34

% inhibition

0.0

3.23 k 0.09 0.49 k 0.08' 1.26 & 0.15 1.41 ? 0.22 4.72 ? 0.35 5.75 f 0.36

**

85.0 0.0 0.0

'All cells were cultured for 24 br in serum-free medium prior to incubations. Cell cultures were preincubatedfor 5 min in either 1 0 - 4 M AH6809 or incubation buffer (control) prior to addition of either vehicle (basal), 10-8 M PGE,, 10 Ulml parathyroid hormone (FTH), or 10-4 M forskolin (FS).Incubation6 were then continued for an additional 5 min. Values are means 2 SE of 6-8 samples obtained from 3-4 separate experiments. 'Values differing significantly (P< .01) from paired controls.

between serum-containing and serum-free cell cultures, although quantitative differences were observed in both parameters. While initial appearance of cartilage matrix was observed on day 3 of culture in both groups, a less homogeneous accumulation of cartilage occurred in serum-free cultures. In contrast to serumcontaining cultures, in which a completely confluent sheet of cartilage had formed by day 6, serum-free cultures contained scattered, narrow regions of nonchondrogenic cells which persisted through 8 days of culture, No differences in changes in DNA content of serum or serum-free cultures over a 6-day period could be detected, however, suggesting that growth of cells in the absence of serum was not inhibited. Attempts t o induce the differentiation of this nonchondrogenic cell population in serum-free cultures with either dbcAMP or forskolin, both of which stimulate chondrogenesis in serum-containing cultures (Ahrens, et al., 1977; Solursh et al., 1981; Biddulph et al., 1988a; Dozier et al., 1989), were unsuccessful, demonstrating the presence in distal tip mesenchyme of at least two populations of cells differing in their requirement for unknown serum factors in initiation of chondrogenesis. Interestingly, while changes in the responsiveness of AC activity to PGE2 did not differ qualitatively between serum-containing and serum-free cultures, maximal responsiveness of serum-free prechondrogenic mesenchyme on day 1,relative to cells grown in serum, was reduced by about the same extent as chondrogenesis on day 6 (approximately 40%) as quantitated by guanidine HC1 extraction of Alcian green. In addition, responsiveness to PTH, which we have reviously characterized in these serum-containing ce 1 cultures (Capehart and Biddulph, 1991), was also reduced in cells cultured for 24 hr under serum-free conditions. It is tempting to speculate that this reduction in both PTH and PGE2responsiveness in serum-free cultures is directly related to the presence of this population of nonchondrogenic cells, although other possibilities certainly exist as well. Marked inhibition of chondrogenesis as evaluated by Alcian green binding was roduced in serum-free cultures by daily addition o M concentrations of AH6809 over a 5-day period without indications of

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cytotoxicity. Morphologically, as evaluated by phasecontrast microscopy, cells exposed to the antagonist maintained an appearance similar to those of controls during the initial 2 days of culture without obvious differences noted in either diameter of the cultures or packing density of cells relative to control cultures during the periods examined. DNA content of these cultures after 5 days of treatment was reduced, however, relative to control cultures by approximately 3040%, suggesting some inhibition of cell proliferation by the antagonist, as well as effects on chondrogenesis. It is of interest that Kosher and Walker (1983) have previously reported that exogenous addition of PGEz to limb bud explants in vitro increased DNA content by about 40% over control explants, while also enhancing chondrogenesis, and suggested that PGE, was acting as a mitogen in this system. It has also been reported in certain other cell types that PGEz stimulates proliferation and growth of cells in culture (Karmali et al., 1979; Nolan et al., 1988; Skouteris et al., 1988). Our resent results suggesting inhibition of cell growth by t is putative PGE, antagonist support these previous observations and indicate that in addition to initiating differentiation of chondrocytes from limb mesenchyme, PGE, may also exert mitogenic effects on limb mesenchyme. Co-addition of l o p 3 M dbcAMP to cultures largely reversed the inhibitory effects of AH6809 on chondrogenesis, indicating that cells in the presence of the antagonist could still respond t o CAMP stimulation. The inhibitory effects of AH6809 were also demonstrated to be reversible. Addition of this compound for the initial 2 days of culture appeared t o completely inhibit chondrogenesis as evaluated by Alcian green staining on day 4. By day 5, however, chondrogenesis was rapidly approaching that of control cultures and by day 6 appeared to be equivalent to controls, demonstrating that once the antagonist was removed, chondrogenesis proceeded normally in these cultures. Addition of the antagonist during the period of active synthesis and accumulation of cartilage matrix components between days 3 and 5 produced minimal effects as compared with continuous treatment, demonstrating that the inhibitory effects observed by addition of this compound during the prechondrogenic period of cell culture were not merely due to inhibition of matrix secretion by differentiating chondrocytes. These results also indicate that once the differentiated state of chondrocytes is achieved, PGE, does not appear to be essential to the maintenance of synthetic activity of matrix components, which is compatible with the observed loss of PGE, responsive AC activity in differentiated chondrocytes. These results support our assertion that the maximally responsive state of prechondrogenic mesenchyme to PGE, plays a transient but important role in initiating differentiation of the mature phenotype. In the present study we have also examined the effectiveness and specificity of AH6809 in inhibiting PGEz receptor-mediated activation of AC in cultured mesenchyme. Control cells in these experiments were cultured for 24 hr to initiate the maximally responsive state to PGE, prior to incubations. Preincubation for 5 min in l o p 4 M AH6809 inhibited greater than 80% of the PGE, activation of AC in control cells without

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significant effect on basal activity. Activation of AC by PTH, which occurs through AC-linked PTH receptors on these cells (Capehart and Biddulph, 1991), was not inhibited by preincubation in the antagonist. Activation of AC by forskolin, which directly and nonspecifically activates catalytic subunit activity (Seamon et al., 19811,as we have previously demonstrated in these cell cultures (Dozier et al., 1989; Capehart et al., 19901,was also uninhibited by the antagonist, demonstrating at least a degree of specificity for blockade of AC-linked PGEz receptors by AH6809. Since direct binding of PGE, and other rostaglandins to their respective receptors was not irectly measured in this study, we cannot say with certainty that only PGE, receptors were blocked in these cells by this compound. In other cell types, however, a high degree of specificity of this antagonist for PGE, receptors has been reported (Coleman et al., 1985b; Eglen and Whiting, 19881, which is consistent with our present results. In summary, the present study provides new evidence supporting the hypothesis that endogenous PGE,, through high affinity receptors which develop in prechondrogenic mesenchyme (Capehart et al., 1990), plays an important initiating role in the differentiation of cartilage. Further studies are in progress to test in more detail this hypothesis. ACKNOWLEDGMENTS The authors express appreciation to Dr. Brian Bain, Glaxo Grou , United Kingdom, for providing the AH6809 use in this study. We are also appreciative to Ms. Joy Bauguess for secretarial assistance in the preparation of this manuscript. The work reported is part of a Ph.D. dissertation submitted by Anthony A. Capehart to the Department of Neurobiology and Anatomy in partial fulfillment of re uirements for the Ph.D. degree of the Bowman Gray Sc 001 of Medicine, Wake Forest University. This work was supported by NIH grants HD 19017 and RR-05404.

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