- Email: [email protected]
The effect of prostaglandins on the cyclic AMP content of limb mesenchymal cells
Cell Differentiation, 17 (1985) 159-167
159
Elsevier Scientific Publishers Ireland, Ltd. CDF 00323
The effect of prostaglandins on the cyclic AMP content of limb mesenchymal cells R o b e r t A. K o s h e r a n d Steven W. G a y Department of Anatomy, University of Connecticut, Health Center, Farmington, CT 06032, U.S.A. (Accepted 29 March 1985)
We have been investigating the hypothesis that prostaglandins including prostaglandin E 2 (PGE2) produced during the critical condensation phase of limb chondrogenesis are involved in the regulation of cartilage differentiation by acting as local modulators of cyclic A M P (cAMP) accumulation. The purpose of the present study was to determine directly whether P G E 2 and other prostanoids which had previously been shown to stimulate in vitro chondrogenic differentiation do indeed elevate the c A M P content of limb mesenchymal cells, and to determine whether the ability of various prostanoids to increase c A M P production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that P G E 2 does indeed elicit a striking elevation in the c A M P content of subridge mesenchymal cells, indicating that the cells possess adenylate cyclase-coupled receptors for this molecule. The effect of P G E 2 on c A M P accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect phosphodiesterase inhibitors have on PGE2-stimulated in vitro chondrogenesis. The effect of P G E 2 on c A M P content is dose-dependent with a 3-fold increase seen at 10 -8 M, which is the lowest concentration at which P G E 2 effectively stimulates chondrogenesis. P G E t, which is just as effective as P G E 2 in stimulating chondrogenesis, is just as effective as P G E 2 in stimulating c A M P accumulation. PGA t, which is a much less effective stimulator of chondrogenesis than P G E 2 or PGE~, is less than half as potent as these molecules in elevating c A M P levels. PGFt~, 6-keto PGFI~, and thromboxane B2, which have little or no effect on chrondrogenesis, have little or no effect on c A M P content. Thus, the abilities of various prostanoids to stimulate c A M P accumulation by subridge mesenchymai cells does indeed correlate directly with their abilities to stimulate chondrogenesis. The labile prostaglandin, prostacyclin (PGI 2), which is one of the major prostanoids produced by limb mesenchymal cells undergoing chondrogenesis in vitro, is also a potent elevator of c A M P levels. The possibility that prostaglandins and c A M P play interrelated regulatory roles in limb cartilage differentiation is discussed. limb development; prostaglandins; chondrogenesis; c A M P
Introduction The onset of limb cartilage differentiation in vivo and in vitro is characterized by a transient cellular condensation or aggregation process in
which mesenchymal cells become closely juxtaposed to one another prior to depositing a cartilage matrix. A variety of studies indicate that during this process an intimate cell-cell interaction occurs which is necessary to trigger the
0045-6039/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland, Ltd.
160
chondrogenic differentiation of the cells (see Kosher, 1983a; Solursh, 1983, for reviews). Several studies suggest that the critical condensation process may be initiated, at least in part, by a progressive decrease in accumulation of extracellular hyaluronate (Toole, 1973; Kosher et al., 1981). The nature of the cell-cell interaction occurring during condensation is not well understood, although it has been suggested that fibronectin and type 1 collagen, which are present in relatively high amounts along the surfaces of the closely apposed cells during condensation, may be be involved (Dessau et al., 1980; Silver et al., 1981: Kosher et al., 1982; Tomasek et al., 1982). Recent studies suggest that a change in the shape of the cells from a flattened mesenchymal morphology to a rounded configuration may play an important role in the process (Archer et al., 1982; Solursh et al., 1982). Cyclic AMP (cAMP) appears to have a crucial part in the cell-cell interaction occurring during condensation (see Elmer et al., 1981; Kosher, 1983b, for reviews); for example, agents that elevate cAMP levels promote limb chondrogenesis in organ culture (Kosher et al., 1979b; Kosher and Savage, 1980) and cell culture (Ahrens et al., 1977; Solursh et al., 1981). Furthermore, an increase in cAMP content correlating with the onset of chondrogenesis in micromass culture has been reported (Solursh et al.0 1979; Ho et al., 1982; see also Elmer et al., 1981). It has been suggested that an elevation of cellular cAMP levels occurring during the condensation phase of chondrogenesis may trigger the differentiation of the cells (Kosher and Savage, 1980; Elmer et al., 1981; Solursh et al., 1981). In this regard, when limb mesenchymal cells are subjected to high density cell culture under conditions conducive to chondrogenesis, they produce several prostaglandins including prostaglandin E: (PGE 2) and prostacyclin (prostaglandin 12, PGI 2) (Chepenik et al., 1980; S.W. Gay and R.A. Kosher, in preparation). These prostaglandins are critically important local regulators of a variety of cellular processes (Samuelsson et al., 1978; Kuehl and Egan, 1980), and their regulatory effects in many systems are mediated by cAMP (Kuehl, 1974; Martin and Partridge, 1980; Hammarstrom, 1982).
Furthermore, we have recently demonstrated that exogeneous PGE 2 elicits a dose-dependent and specific stimulation of limb chondrogenic differentiation in organ culture (Kosher and Walker. 1983) and in high density cell culture (Gay and Kosher, 1984). In each of these systems, the stimulatory effect of PGE 2 is greatly potentiated by phosphodiesterase inhibitors, suggesting that its influence on cartilage differentiation is mediated by cAMP, and that the stimulatory effect of PGE 2 is considerably greater than several other prostanoids including PGA~, PGF1,~, 6-keto PGFI~~, and thromboxane B2 (TxB2) (Kosher and Walker, 1983; Gay and Kosher. 1984). On the basis of these observations, we have suggested that endogenous prostaglandins produced during the condensation phase of chondrogenesis might be involved in regulating limb cartilage differentiation by acting as local modulators of cAMP formation (Kosher and Walker, 1983; Gay and Kosher, 1984). In view of our hypothesis that the effect of prostaglandins on chondrogenesis is mediated by cAMP, it was critically important to determine directly whether PGE 2 and other prostanoids which stimulate chondrogenesis do indeed elevate the cAMP content of limb mesenchymal cells, and whether the relative effectiveness of various prostanoids in stimulating the process is directly related to their abilities to elevate cAMP content. In previous studies, we found that PGE 1 was just as effective as PGE~ in stimulating in vitro chondrogenesis, whereas PGA 1 was less than half as effective and PGF~,~, 6-keto PGFI, ,, and TxB 2 had little or no effect on the process (Kosher and Walker, 1983; Gay and Kosher, 1984). Therefore, in the present investigation we have examined the effect of these and other prostanoids on the cAMP content of the mesenchymal cells in the subridge region of the stage 25 (Hamburger and Hamilton, 1951) chick wing bud. The mesoderm directly subjacent to the apical ectodermal ridge of the wing bud at stage 25 was used in the present study not only because it was the source of cells in our previous investigations on the effect of prostaglandins on in vitro chondrogenesis, but also because the subridge mesoderm at this stage consists of a relatively homogeneous population of chondrogenic progenitor cells which have not yet
161
initiated overt differentiative changes (see Newman et al., 1981) and which uniformly undergo chondrogenesis in organ culture (Kosher et al., 1979a) and micromass culture (Gay and Kosher, 1984). We have found that the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis.
Materials and Methods
Materials The prostaglandins utilized in this study were obtained from Upjohn, and 3-isobutyl-l-methylxanthine (IBMX) was purchased from Sigma Chemical Co. All reagents utilized in the radioimmunoassay including cAMP-antiserum complex and ~25I-labeled succinyl-cAMP tyrosine methyl ester (ScAMP-TME-[125I]) were obtained in kit form from New England Nuclear.
Preparation of tissue Wing buds were removed from stage 25 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks and placed into Hanks' balanced salt solution (HBSS). Distal wing bud tips (subridge regions) composed of the subridge mesoderm capped by the apical ectodermal ridge (AER) and surrounded dorsally and ventrally by ectoderm were cut away from the stage 25 limb buds as previously described (Kosher et al., 1979a). The size of the excised subridge regions was approximately 0.3-0.4 mm from the distal apex of the tissue to the proximal cut edge (see Kosher et al., 1979a for a photograph). The A E R and dorsal/ventral ectoderm were removed following brief treatment with dilute trypsin as previously described (Kosher et al., 1979a), and the intact subridge mesoderm transferred to HBSS containing 10% fetal calf serum (FCS). The intact tissues were then washed several times with HBSS and serum-free F12 medium (GIBCO), prior to being incubated and assayed as described below. In those experiments in which dissociated subridge mesenchymal cells were utilized, following removal
of ectoderm the subridge mesoderm was washed several times with calcium- and magnesium-free HBSS (CMF-HBSS), and incubated for 20 rain at 37°C in 0.25% trypsin in CMF-HBSS. The tissue was washed three times with F12 medium containing 10% FCS, and then washed three times with serum-free F12 medium. The cells were mechanically dissociated in serum-free F12 medium by repeated pipetting through the fire-polished end of a sterile Pasteur pipette (Gay and Kosher, 1984), and the concentration of the resulting suspension determined with a hemacytometer and adjusted to 9 × 105 c e l l s / m l . Aliquots of the cell suspensions in serum-free F12 medium were incubated and assayed as described below.
Incubation conditions and radioimmunoassay of cAMP In most experiments, intact subridge mesodermal tissue or dissociated subridge mesodermal cells were preincubated in a shaking water bath for 10 min at 37°C in serum-free F12 medium containing 1.0 mM of the phosphodiesterase inhibitor, IBMX. The incubation mixtures were then supplied with various concentrations of different prostanoids by the addition of small aliquots of a concentrated stock solution of the appropriate prostanoid, and the incubations were continued for 5-15 rain at 37°C in the shaking water bath. The concentrated stock solutions of PGE 2, PGF1,, PGA 1, and TxB 2 were prepared in absolute ethanol, whereas the 6-keto PGFI~ concentrated stock was prepared in acetone. The final concentration of ethanol or acetone in the incubation mixtures never exceeded 0.4%, and equivalent amounts of these solvents were added to control incubation mixtures. In a series of control experiments we found that this concentration of ethanol or acetone had no effect on the cAMP content of subridge mesodermal tissue. In experiments in which the effect of PGI 2 (prostacyclin) was examined, the agent was dissolved in serum-free F12 medium immediately prior to being added to incubation mixtures. In all experiments, blank incubation mixtures lacking cells were set up and carried through the entire procedure to determine whether the incubation conditions, medium or subsequent ex-
162
traction procedures had any nonspecific effect on the radioimmunoassay. In no instance was any cAMP detectable in such procedure blanks, indicating a lack of any non-specific interference of the procedure with the assay. At the completion of the incubation period, the incubation mixtures were made 6% with respect to trichloroacetic acid (TCA) by the addition of an aliquot of a concentrated ice-cold TCA solution. The incubation mixtures were sonicated immediately following the addition of the TCA. The TCA-soluble and insoluble materials were separated by centrifugation. The precipitate was washed with cold 6% TCA, and utilized for D N A determination by a micromodification of the procedures of A b r a h a m et al. (1972) and Richards (1974). The TCA-soluble material was neutralized as described by Tihon et al. (1977), and the cAMP content of aliquots of the samples was determined using radioimmunoassay kits obtained from New England Nuclear following acetylation of the samples to sensitize the assay (Harper and Brooker, 1975; Frandsen and Krishna, 1976). Briefly, the samples and appropriate known standards were acetylated by the addition of acetic anhydride and triethylamine prior to being incubated overnight at 4°C in the presence of cAMP antiserum complex and ScAMP-TME-[t2sI]. The resultant antigenantibody complex was separated from unbound antigen by centrifugation following the addition of excess cold acetate buffer, and the radioactivity in the precipitate was determined with a gamma counter. The cAMP content of the unknown samples was determined by comparison with the standard curve, and the results expressed on a per unit D N A basis.
Results
In our initial experiments, we examined the time course of accumulation of cAMP by subridge mesenchymal tissue in response to P G E 2. As shown in Fig. 1, 10 -5 M P G E 2 elicits a greater than 10-fold increase in cAMP content, indicating that subridge mesenchymal cells do indeed have adenylate cyclase-coupled receptors for this molecule. Accumulation of cAMP in response to P G E 2
3628 ~ cZ o~
20
~
12
<~
04
E2
CONT
TIME (mln~
Fig. 1. Time course of cAMP accumulation by subridge mesenchymal tissue in response to 10 -5 M PGE 2. The sub-
ridge mesoderm was preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to addition of PGE 2 (E2) or vehicle (CONT). Values are the means of duplicate determinations.
is essentially complete within 5 min, with little, if any, additional increase detectable up to 15 min (Fig. 1). We next examined the effect of PGE~ on cAMP accumulation by the subridge mesoderm in the presence and absence of the potent phosphodiesterase inhibitor, IBMX. As shown in Table I, P G E 2 alone elicits about a 5-fold increase in cellular cAMP content. There is a more modest (about 2-fold) increase in cAMP in response to IBMX alone (Table I). However, the effect of these agents alone is relatively small compared to the striking elevation (30-40-fold above basal levels) in cAMP seen when the phosphodiesterase inhibitor is added along with P G E 2 (Table I). The effect of these agents is not simply additive, but clearly synergistic. These observations are of particular interest in view of our previous studies in which we found that phosphodiesterase inhibitors greatly potentiate the stimulatory effect of PGE~ on the in vitro chondrogenesis of subridge mesenchymal cells (Kosher and Walker, 1983; Gay and Kosher, 1984). The effect of various prostanoids on the cAMP content of intact subridge mesenchymal tissue is shown in Fig. 2 and summarized in Table I1. P G E 2 and PGE] are by far the most effective stimulators of cAMP accumulation that we have examined, eliciting an 11-14-fold increase in cellul a r . c A M P content above control levels (Fig. 2:
163
32
50
5.0 Z
;~ 28 ""
o E
Z
uJ I-Z
0 o
D. ~E
~ 24
2.4
-~ 22 E o. 2 0
2.2
20 18 16 14 12
--
(.3 >-
04
0 ..J 0
a 26
2.6
I0 08 06
'~
E2
28
Iz
Z w 16 F-Z 14 0
'~ 0 8
B 0.6
Fla
~ O.4 0.2 .~¢,j
02
I
A
I
i
i
i
I0-~O iO-e 10-B 10-7 10-6 I0-5 CT 6K-Fta TXB2
E
Fie AI 12 TREATMENT
E
Fig. 2. Effect of various prostanoids on the cAMP content of subridge mesenchymal tissue. The subridge mesoderm was preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to addition of PGE 2 (E2) , PGE 1 ( E l ) , PGI 2 (I2) , PGA 1 (A1), PGF]a (Fla), thromboxane BE TxB2, 6-keto PGF],~ (6K-FI,,), or vehicle (CT). Values are the means of two separate duplicate determinations + range.
Table II). The next most effective promotor of c A M P accumulation is prostacyclin (PGI2) eliciting a 5-fold increase in cAMP, followed by P G A a which promotes a 3-fold increase. In contrast, PGFa~ has little effect, and TxB 2 and 6-keto PGFt~ have no effect on cellular cAMP levels (Fig. 2; Table II). The relative potencies of these prostanoids in promoting cAMP accumulation is directly related to their ability to stimulate chondro-
r-PROSTAGIANDI N] ( M ) Fig. 3. Effect of various concentrations of P G E 2 (E2) , PGI 2 (I2), PGA 1 (A1), and PGF1,~ (FI,~) on the cAMP content of subridge mesenchymal tissue. The tissue was preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to addition of the prostanoid or vehicle control. Values are the means of duplicate determinations.
genesis (see Introduction and Discussion). The effect of various concentrations of several different prostanoids on the cAMP content of the subridge mesoderm is shown in Fig. 3. At all concentrations examined, P G E 2 is the most effective stimulator of cAMP we have tested, followed by P G I 2. P G I 2 is, in turn, more potent than P G A 1 (Fig. 3). PGFI~ has a slight effect on cAMP content only at the highest concentration examined
TA B LE II T ABLE I Effect of P GE 2 on the cAMP content of subridge mesenchymal tissue in the presence and absence of the phosphodiesterase inhibitor, IBMX Treatment None IBMX PGE2 P GE 2 + IBMX
cAMP content ( p m o l / # g DNA)
Relative increase in cAMP content over control levels in subridge mesenchymal tissue in response to various prostanoids Prostanoid
Treated ( p m o l / # g D N A ) control ( p m o l / # g D N A )
a
0.068 0.203 0.360 2.860
a Values are the means of duplicate determinations.
PGE 2 PGE 1 PGI 2 PGA1 PGF1. TxB 2 6-keto PGF1~
13.62 10.91 4.77 3.0i 1.30 1.08 1.02
164 TABLE Ill Relative increase in cAMP content over control levels in dissociated subridge mesenchymat cells in response to various prostanoids
I.-Z i,i
14
I0 r,
a,
:~ ~, 0 8
Prostanoid
Treated (pmol//*g DNA) control (pmol/ffg DNA)
PGE 2 PGE 1 PGI 2 PGA 1 PGFl~ , TxB. 6-keto PGF1,
3.69 3.82 2.75 2.60 0.94 0.99 1.06
(10 5 M). It is noteworthy that PGE 2 elicits about a 3-fold increase in cAMP at a concentration of 10 -~ M, which is the lowest concentration at which it effectively stimulates in vitro chondrogenesis (Kosher and Walker, 1983; Gay and Kosher, 1984). The preceding experiments involved examining the effect of prostanoids on the cAMP content of intact subridge mesenchymal tissue. We have also examined the effect of prostanoids on the cAMP content of suspensions of subridge mesenchymal cells prepared from subridge mesenchymal tissue that was dissociated into single cells with the aid of the proteolytic enzyme, trypsin. As shown in Fig. 4 and summarized in Table III, various prostanoids have the same relative effectiveness in elevating cAMP levels in the dissociated cells as they had in the intact tissue, with PGE 2 and PGE] being most effective, PGI 2 and PGA 1 being moderately effective, and PGFI~ , 6-keto PGF~,~, and TxB 2 having no effect. However, PGE2, PGE~, PGI 2 and PGA t, are all considerably less effective in elevating the cAMP content of the dissociated cells than they are in elevating the cAMP of cells in the intact tissue. For example, P G E 2 elicits about a 14-fold increase in the cAMP content of intact subridge mesenchymal tissue (Table II), but only about a 4-fold increase in the cAMP content of the dissociated subridge mesenchymal cells (Table III). The most likely explanation for this observation is that trypsin damages a n d / o r removes cell surface receptors for this prostaglandin (see Discussion). In this regard, it is noteworthy that
o
06
o
-~
04
> (o' ~
02 CT
6K-Fla TXB 2
Fia
AI
TREATMENT
I2
E~
J Ez
Fig. 4. Effect of various prostanoids on the cAMP content of dissociated subridge mesenchymal cells. Cells were preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to the addition of PGE 2 (E2), PGE 1 (El), PGI2(I2), PGA1(A1), PGFI~ (Flo), thromboxane B2 (TXB2), 6-keto PGF1,, (6K-F~, 0, or vehicle (CT). Values are the means of two separate duplicate determinations +_range.
PGE 2 is considerably more effective in stimulating the chondrogenesis of intact subridge mesenchymal tissue in organ culture (Kosher and Walker, 1983) than it is in stimulating the chondrogenesis of dissociated subridge mesenchymal cells in high density cell culture (Gay and Kosher, 1984).
Discussion
We have suggested that the stimulatory effect of P G E 2 on the in vitro chondrogenesis of the subridge mesenchymal cells of the chick limb bud is mediated by cAMP (Kosher and Walker, 1983; G a y and Kosher, 1984). The purpose of the present study was to determine directly whether P G E 2 and other prostaglandins which stimulate chondrogenesis do indeed elevate the cAMP content of subridge mesenchymal cells, and to determine whether the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that PGE z does elicit a striking increase in cellular cAMP content, indicating that subridge mesenchymal cells have adenylate cyclase-coupled receptors for this molecule. Furthermore, we have found that PGE 1, which is just as effective as PGE 2 in promoting chondrogenesis (Kosher and Walker, 1983; Gay and Kosher, 1984), is just as effective as
165 P G E 2 in stimulating cAMP accumulation. PGA1, which is much less effective than PGE 2 or PGE] in stimulating chondrogenesis, is less than half as potent as these molecules in elevating cAMP levels. PGFI~, 6-keto PGFI~, and TxB2, which have little or no effect on chondrogenesis, have little or no effect on cAMP content. Thus, the abilities of various prostanoids to stimulate cAMP accumulation in subridge mesenchymal cells do indeed correlate directly with their abilities to stimulate chondrogenesis. It is noteworthy that the very labile prostaglandin, prostacyclin (PGI2) is a very effective elevator of the cAMP content of subridge mesenchymal cells, whereas its stable and biologically inactive breakdown product, 6-keto PGFa~, has no effect on cAMP levels. This observation is of considerable interest, since prostacyclin, along with PGE2, appears to be one of the major prostanoids produced by limb mesenchymal cells undergoing chondrogenesis in vitro (Chepenik et al., 1980; S.W. Gay and R.A. Kosher, in preparation). These observations suggest that prostacyclin, as well as PGE 2, may play an important regulatory role in cartilage differentiation. PGE 2 and the other prostanoids are considerably less effective in elevating the cAMP content of dissociated subridge mesenchymal cells than they are in elevating the cAMP content of cells in the intact tissue. This observation is not particularly surprising, since in many systems PGE 2 and other prostanoids exert their regulatory effect by interacting with specific receptors on the external surface of the responding cells (Samuelsson et al., 1978). The trypsinization used in the present study to dissociate the subridge mesoderm into single cells may very likely have damaged or removed cell surface receptors for prostaglandins, resulting in the decreased responsiveness of the cells. Parker et al. (1981) and Ballard and Biddulph (1983) have examined the effect of PGE 2 on the cAMP content of dissociated cells of whole limb buds, with almost all of their studies being done on the cells of whole stage 24-25 limb buds or the isolated cartilage rods of stage 26 28 limb buds. Although an increase in cAMP in response to PGE 2 was observed in these cells, the studies were rather difficult to interpret for several reasons.
First of all, stage 24-25 limb buds consist of a very heterogeneous population of chondrogenic, myogenic, and fibrogenic cells in various stages of differentiation (see Kosher, 1983a, for review). Thus, determining the nature of the cell type or types responding to PGE 2 in the above studies was virtually impossible. This contrasts to the present study in which we examined the effect of various prostanoids on a relatively uniform population of chondrogenic progenitor cells which have not yet initiated overt differentiative changes (Newman et al., 1981), and which uniformly undergo chondrogenesis in vitro (Kosher et al., 1979a; Gay and Kosher, 1984). The studies utilizing isolated cartilage rods of stage 26-28 limbs were done on tissue consisting of chondrocytes which have already undergone overt differentiation. The effect of prostanoids on already differentiated chondrocytes may be, and, in fact, probably is quite different from their effect on mesenchymal progenitor cells (Malemud et al., 1982; Kosher and Walker, 1983). Parker et al. (1981) and Ballard and Biddulph (1983) did in a single experiment find that high concentrations of PGE 2 stimulate cAMP accumulation in dissociated cells of whole stage 20-21 limb buds, which consist of a somewhat more homogeneous group of cells than the whole limbs used in most of their studies. In summary, the following major pieces of evidence suggest that prostaglandins and cAMP play interrelated regulatory roles in limb cartilage differentiation. (1) A variety of studies suggest that a key event in limb chondrogenesis is a cellular condensation process during which a local cell-cell interaction occurs that triggers cartilage differentiation by elevating cAMP levels. (2) Limb mesenchymal cells undergoing chondrogenesis in vitro produce several prostaglandins including PGE 2 and prostacyclin (PGI2) which are important local mediators of cAMP accumulation in several tissues (Chepenik et al., 1980; S.W. Gay and R.A. Kosher, in preparation). (3) PGE 2 elicits a dose-dependent stimulation of in vitro limb chondrogenesis (Kosher and Walker, 1983; Gay and Kosher, 1984). (4) The stimulatory effect of PGE 2 on chondrogenesis is potentiated by phosphodiesterase inhibitors, suggesting its regulatory effect is mediated by cAMP (Kosher and Walker,
166
1983; Gay and Kosher, 1984). (5) PGE 2 and PGI 2 elicit a striking increase in the cAMP content of subridge mesenchymal cells, indicating that these cells do indeed possess adenylate cyclase-coupled receptors for these molecules. (6) The effect of PGE 2 on cAMP accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect of phosphodiesterase inhibitors on in vitro chondrogenesis. (7) The relative effectiveness of various prostanoids in stimulating cAMP accumulation in subridge mesenchymal cells directly reflects the relative potencies of these same molecules in stimulating in vitro chondrogenesis. All of these observations are consistent with the hypothesis that prostaglandins produced during the condensation phase of chondrogenesis might be involved in regulating limb cartilage differentiation by acting as local modulators of cAMP formation. We are continuing to investigate this hypothesis.
Acknowledgement This work was supported by NIH Grant HD 17794 to R.A.K.
References Abraham, G.N., C. Scaletta and J.H. Vaughan: Modified diphenylamine reaction for increased sensitivity. Anal. Biochem. 49, 547-549 (1972). Ahrens, P.B., M. Solursh and R.S. Reiter: Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60, 69-82 (1977). Archer, C.W,, P. Rooney and L. Wolpert: Cell shape and cartilage differentiation of early chick limb bud cells in culture. Cell Differ. 11,245-251 (1982). Ballard, T.A. and D.M. Biddulph: Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs. Prostaglandins 25, 471-480 (1983). Chepenik, K.P., B.M. Waite and C.L. Parker: Prostaglandin synthesis during chondrogenesis in vitro. J. Cell Biol. 87, 21a (1980). Dessau, W., H. yon der Mark, K. vonder Mark and S. Fischer: Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis. J. Embryol. Exp. Morphol. 57, 51-60 (1980). Elmer, W.A., M.A. Smith and D.A. Ede: lmmunohistochemical localization of cyclic AMP during normal and abnormal
chick and mouse limb development. Teratology 24, 215-223 (1981). Frandsen, E.K. and G. Krishna: A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues. Life Sci. 18, 529-542 (1976). Gay, S.W. and R.A. Kosher: Uniform cartilage differentiation in micromass cultures prepared from a relatively uniform population of chondrogenic progenitor cells of the chick limb budJ Effect of prostaglandins. J. Exp. Zool. 232, 317-326 (1984). Hamburger, V. and H.L. Hamilton: A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92 (1951). Hammarstrom, S.: Biosynthesis and biological actions of prostaglandins and thromboxanes. Arch. Biochem. Biophys. 214, 431-445 (1982). Harper, J.F. and G. Brooker: Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhyride in aqueous solution. J. Cyclic Nucleotide Res. 1, 207-218 (1975). Ho, W.C., R.M. Greene, J. Shanfeld and Z. Davidovitch: Cyclic nucleotides during chondrogenesis: concentration and distribution in vivo and in vitro. J. Exp. Zool. 224, 321-330 (1982). Kosher, R.A.: (1983a). The chondroblast and the chondrocyte. In: Cartilage, Vol. 1, ed. B.K. Hall (Academic Press, New York) pp. 59 85 (1983a). Kosher, R.A.: Relationship between the AER, extracellular matrix, and cyclic AMP in limb development. In: Limb Development and Regeneration, Part A, eds. J.F. Fallon and A.L Caplan (Alan R. Liss, New York) pp. 279-288 (1983b). Kosher, R.A. and M.P. Savage: Studies on the possible role of cyclic AMP in limb morphogenesis and differentiation. J. Embryol. Exp. Morphol. 56, 91-105 (1980). Kosher, R.A., M.P. Savage and S.-C. Chan: In vitro studies on the morphogenesis and differentiation of the mesoderm subjacent to the apical ectodermal ridge of the embryonic chick limb bud. J. Embryol. Exp. Morphol. 50, 75 97 (1979a). Kosher, R.A., M.P. Savage and S.-C. Chan: Cyclic AMP derivatives stimulate the chondrogenic differentiation of the mesoderm subjacent to the apical ectodermal ridge of the chick limb bud. J. Exp. Zool. 209, 221-228 (1979b). Kosher, R.A., M.P. Savage and K.H. Walker: A gradation of hyaluronate accumulation along the proximodistal axis of the embryonic chick limb bud. J, Embryol. Exp. Morphol. 63, 85-98 (1981). Kosher, R.A. and K.H. Walker: The effect of prostaglandins on in vitro limb cartilage differentiation. Exp. Cell Res. 145, 145 153 (1983). Kosher, R.A., K.H. Walker and P.W. Ledger: Temporal and spatial distribution of fibronectin during development of the embryonic chick limb bud. Cell Differ. 11, 217-228 (1982). Kuehl, F.A.: Prostaglandins, cyclic nucleotides and cell function. Prostaglandins 5, 325-340 (1974).
167 Kuehl, F.A. and R.W. Egan: Prostaglandins, arachidonic acid, and inflammation. Science 210, 978-984 (1980). Malemud, C.V., R.A. Moskowitz and R.S. Papay: Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes. Biochim. Biophys. Acta 715, 70-79 (1982). Martin, T.J. and N.C. Partridge: Prostaglandins, cancer and bone: Pharmacological considerations. Metab. Bone Dis. Relat. Res. 2, 167-171 (1980). Newman, S.A., M.P. Pautou and M. Kieny: The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells. Dev. Biol. 84, 440-448 (1981). Parker, C.L., D.M. Biddulph and T.A. Ballard: Development of the cyclic AMP response to parathyroid hormone and prostaglandin E 2 in the embryonic chick limb. Calcif. Tissue Int. 33, 641-648 (1981). Richards, G.M.: Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57, 369-376 (1974). Samuelsson, B., M. Goldylne, E. Granstrom, M. Hamberg, S. Hammarstrom and C. Malmsten: Prostaglandins and thromboxanes. Annu. Rev. Biochem. 47, 997-1029 (1978). Silver, M.H., J.-M. Foidart and R.M. Pratt: Distribution of
fibronectin and collagen during mouse limb and palate development. Differentiation 18, 141-149 (1981). Solursh, M.: Cell-cell interaction in chrondrogenesis. In: Cartilage, Vol. 2 ed. B.K. Hall (Academic Press, New York) (1983). Solursh, M., T.F. Linsenmayer and K.L. Jensen: Chondrogenesis from single limb mesenchyme cells. Dev. Biol. 94, 259-264 (1982). Solursh, M., R.S. Reiter, P.B. Ahrens and R.M. Pratt: Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro. Differentiation 15, 183-186 (1979). Solursh, M., R.S. Reiter, P.B. Ahrens and B.M. Vertel: Stageand position-related changes in chondrogenic response of chick embryonic wing mesenchyme to treatment with dibutyryl cyclic AMP. Dev. Biol. 83, 9-19 (1981). Tihon, C., M.B. Goren, E. Spitz and H.V. Rickenberg: Convenient elimination of trichloroacetic acid prior to radioimmunoassay of cyclic nucleotides. Anal. Biochem. 80, 652-653 (1977). Tomasek, J.J., J.E. Mazurkiewicz and S.A. Newman: Nonuniform distribution of fibronectin during avian limb development. Dev. Biol. 90, 118-126 (1982). Toole, B.P.: Hyaluronate and hyaluronidase in morphogenesis and differentiation. Am. Zool. 13, 1061-1065 (1973).
159
Elsevier Scientific Publishers Ireland, Ltd. CDF 00323
The effect of prostaglandins on the cyclic AMP content of limb mesenchymal cells R o b e r t A. K o s h e r a n d Steven W. G a y Department of Anatomy, University of Connecticut, Health Center, Farmington, CT 06032, U.S.A. (Accepted 29 March 1985)
We have been investigating the hypothesis that prostaglandins including prostaglandin E 2 (PGE2) produced during the critical condensation phase of limb chondrogenesis are involved in the regulation of cartilage differentiation by acting as local modulators of cyclic A M P (cAMP) accumulation. The purpose of the present study was to determine directly whether P G E 2 and other prostanoids which had previously been shown to stimulate in vitro chondrogenic differentiation do indeed elevate the c A M P content of limb mesenchymal cells, and to determine whether the ability of various prostanoids to increase c A M P production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that P G E 2 does indeed elicit a striking elevation in the c A M P content of subridge mesenchymal cells, indicating that the cells possess adenylate cyclase-coupled receptors for this molecule. The effect of P G E 2 on c A M P accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect phosphodiesterase inhibitors have on PGE2-stimulated in vitro chondrogenesis. The effect of P G E 2 on c A M P content is dose-dependent with a 3-fold increase seen at 10 -8 M, which is the lowest concentration at which P G E 2 effectively stimulates chondrogenesis. P G E t, which is just as effective as P G E 2 in stimulating chondrogenesis, is just as effective as P G E 2 in stimulating c A M P accumulation. PGA t, which is a much less effective stimulator of chondrogenesis than P G E 2 or PGE~, is less than half as potent as these molecules in elevating c A M P levels. PGFt~, 6-keto PGFI~, and thromboxane B2, which have little or no effect on chrondrogenesis, have little or no effect on c A M P content. Thus, the abilities of various prostanoids to stimulate c A M P accumulation by subridge mesenchymai cells does indeed correlate directly with their abilities to stimulate chondrogenesis. The labile prostaglandin, prostacyclin (PGI 2), which is one of the major prostanoids produced by limb mesenchymal cells undergoing chondrogenesis in vitro, is also a potent elevator of c A M P levels. The possibility that prostaglandins and c A M P play interrelated regulatory roles in limb cartilage differentiation is discussed. limb development; prostaglandins; chondrogenesis; c A M P
Introduction The onset of limb cartilage differentiation in vivo and in vitro is characterized by a transient cellular condensation or aggregation process in
which mesenchymal cells become closely juxtaposed to one another prior to depositing a cartilage matrix. A variety of studies indicate that during this process an intimate cell-cell interaction occurs which is necessary to trigger the
0045-6039/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland, Ltd.
160
chondrogenic differentiation of the cells (see Kosher, 1983a; Solursh, 1983, for reviews). Several studies suggest that the critical condensation process may be initiated, at least in part, by a progressive decrease in accumulation of extracellular hyaluronate (Toole, 1973; Kosher et al., 1981). The nature of the cell-cell interaction occurring during condensation is not well understood, although it has been suggested that fibronectin and type 1 collagen, which are present in relatively high amounts along the surfaces of the closely apposed cells during condensation, may be be involved (Dessau et al., 1980; Silver et al., 1981: Kosher et al., 1982; Tomasek et al., 1982). Recent studies suggest that a change in the shape of the cells from a flattened mesenchymal morphology to a rounded configuration may play an important role in the process (Archer et al., 1982; Solursh et al., 1982). Cyclic AMP (cAMP) appears to have a crucial part in the cell-cell interaction occurring during condensation (see Elmer et al., 1981; Kosher, 1983b, for reviews); for example, agents that elevate cAMP levels promote limb chondrogenesis in organ culture (Kosher et al., 1979b; Kosher and Savage, 1980) and cell culture (Ahrens et al., 1977; Solursh et al., 1981). Furthermore, an increase in cAMP content correlating with the onset of chondrogenesis in micromass culture has been reported (Solursh et al.0 1979; Ho et al., 1982; see also Elmer et al., 1981). It has been suggested that an elevation of cellular cAMP levels occurring during the condensation phase of chondrogenesis may trigger the differentiation of the cells (Kosher and Savage, 1980; Elmer et al., 1981; Solursh et al., 1981). In this regard, when limb mesenchymal cells are subjected to high density cell culture under conditions conducive to chondrogenesis, they produce several prostaglandins including prostaglandin E: (PGE 2) and prostacyclin (prostaglandin 12, PGI 2) (Chepenik et al., 1980; S.W. Gay and R.A. Kosher, in preparation). These prostaglandins are critically important local regulators of a variety of cellular processes (Samuelsson et al., 1978; Kuehl and Egan, 1980), and their regulatory effects in many systems are mediated by cAMP (Kuehl, 1974; Martin and Partridge, 1980; Hammarstrom, 1982).
Furthermore, we have recently demonstrated that exogeneous PGE 2 elicits a dose-dependent and specific stimulation of limb chondrogenic differentiation in organ culture (Kosher and Walker. 1983) and in high density cell culture (Gay and Kosher, 1984). In each of these systems, the stimulatory effect of PGE 2 is greatly potentiated by phosphodiesterase inhibitors, suggesting that its influence on cartilage differentiation is mediated by cAMP, and that the stimulatory effect of PGE 2 is considerably greater than several other prostanoids including PGA~, PGF1,~, 6-keto PGFI~~, and thromboxane B2 (TxB2) (Kosher and Walker, 1983; Gay and Kosher. 1984). On the basis of these observations, we have suggested that endogenous prostaglandins produced during the condensation phase of chondrogenesis might be involved in regulating limb cartilage differentiation by acting as local modulators of cAMP formation (Kosher and Walker, 1983; Gay and Kosher, 1984). In view of our hypothesis that the effect of prostaglandins on chondrogenesis is mediated by cAMP, it was critically important to determine directly whether PGE 2 and other prostanoids which stimulate chondrogenesis do indeed elevate the cAMP content of limb mesenchymal cells, and whether the relative effectiveness of various prostanoids in stimulating the process is directly related to their abilities to elevate cAMP content. In previous studies, we found that PGE 1 was just as effective as PGE~ in stimulating in vitro chondrogenesis, whereas PGA 1 was less than half as effective and PGF~,~, 6-keto PGFI, ,, and TxB 2 had little or no effect on the process (Kosher and Walker, 1983; Gay and Kosher, 1984). Therefore, in the present investigation we have examined the effect of these and other prostanoids on the cAMP content of the mesenchymal cells in the subridge region of the stage 25 (Hamburger and Hamilton, 1951) chick wing bud. The mesoderm directly subjacent to the apical ectodermal ridge of the wing bud at stage 25 was used in the present study not only because it was the source of cells in our previous investigations on the effect of prostaglandins on in vitro chondrogenesis, but also because the subridge mesoderm at this stage consists of a relatively homogeneous population of chondrogenic progenitor cells which have not yet
161
initiated overt differentiative changes (see Newman et al., 1981) and which uniformly undergo chondrogenesis in organ culture (Kosher et al., 1979a) and micromass culture (Gay and Kosher, 1984). We have found that the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis.
Materials and Methods
Materials The prostaglandins utilized in this study were obtained from Upjohn, and 3-isobutyl-l-methylxanthine (IBMX) was purchased from Sigma Chemical Co. All reagents utilized in the radioimmunoassay including cAMP-antiserum complex and ~25I-labeled succinyl-cAMP tyrosine methyl ester (ScAMP-TME-[125I]) were obtained in kit form from New England Nuclear.
Preparation of tissue Wing buds were removed from stage 25 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks and placed into Hanks' balanced salt solution (HBSS). Distal wing bud tips (subridge regions) composed of the subridge mesoderm capped by the apical ectodermal ridge (AER) and surrounded dorsally and ventrally by ectoderm were cut away from the stage 25 limb buds as previously described (Kosher et al., 1979a). The size of the excised subridge regions was approximately 0.3-0.4 mm from the distal apex of the tissue to the proximal cut edge (see Kosher et al., 1979a for a photograph). The A E R and dorsal/ventral ectoderm were removed following brief treatment with dilute trypsin as previously described (Kosher et al., 1979a), and the intact subridge mesoderm transferred to HBSS containing 10% fetal calf serum (FCS). The intact tissues were then washed several times with HBSS and serum-free F12 medium (GIBCO), prior to being incubated and assayed as described below. In those experiments in which dissociated subridge mesenchymal cells were utilized, following removal
of ectoderm the subridge mesoderm was washed several times with calcium- and magnesium-free HBSS (CMF-HBSS), and incubated for 20 rain at 37°C in 0.25% trypsin in CMF-HBSS. The tissue was washed three times with F12 medium containing 10% FCS, and then washed three times with serum-free F12 medium. The cells were mechanically dissociated in serum-free F12 medium by repeated pipetting through the fire-polished end of a sterile Pasteur pipette (Gay and Kosher, 1984), and the concentration of the resulting suspension determined with a hemacytometer and adjusted to 9 × 105 c e l l s / m l . Aliquots of the cell suspensions in serum-free F12 medium were incubated and assayed as described below.
Incubation conditions and radioimmunoassay of cAMP In most experiments, intact subridge mesodermal tissue or dissociated subridge mesodermal cells were preincubated in a shaking water bath for 10 min at 37°C in serum-free F12 medium containing 1.0 mM of the phosphodiesterase inhibitor, IBMX. The incubation mixtures were then supplied with various concentrations of different prostanoids by the addition of small aliquots of a concentrated stock solution of the appropriate prostanoid, and the incubations were continued for 5-15 rain at 37°C in the shaking water bath. The concentrated stock solutions of PGE 2, PGF1,, PGA 1, and TxB 2 were prepared in absolute ethanol, whereas the 6-keto PGFI~ concentrated stock was prepared in acetone. The final concentration of ethanol or acetone in the incubation mixtures never exceeded 0.4%, and equivalent amounts of these solvents were added to control incubation mixtures. In a series of control experiments we found that this concentration of ethanol or acetone had no effect on the cAMP content of subridge mesodermal tissue. In experiments in which the effect of PGI 2 (prostacyclin) was examined, the agent was dissolved in serum-free F12 medium immediately prior to being added to incubation mixtures. In all experiments, blank incubation mixtures lacking cells were set up and carried through the entire procedure to determine whether the incubation conditions, medium or subsequent ex-
162
traction procedures had any nonspecific effect on the radioimmunoassay. In no instance was any cAMP detectable in such procedure blanks, indicating a lack of any non-specific interference of the procedure with the assay. At the completion of the incubation period, the incubation mixtures were made 6% with respect to trichloroacetic acid (TCA) by the addition of an aliquot of a concentrated ice-cold TCA solution. The incubation mixtures were sonicated immediately following the addition of the TCA. The TCA-soluble and insoluble materials were separated by centrifugation. The precipitate was washed with cold 6% TCA, and utilized for D N A determination by a micromodification of the procedures of A b r a h a m et al. (1972) and Richards (1974). The TCA-soluble material was neutralized as described by Tihon et al. (1977), and the cAMP content of aliquots of the samples was determined using radioimmunoassay kits obtained from New England Nuclear following acetylation of the samples to sensitize the assay (Harper and Brooker, 1975; Frandsen and Krishna, 1976). Briefly, the samples and appropriate known standards were acetylated by the addition of acetic anhydride and triethylamine prior to being incubated overnight at 4°C in the presence of cAMP antiserum complex and ScAMP-TME-[t2sI]. The resultant antigenantibody complex was separated from unbound antigen by centrifugation following the addition of excess cold acetate buffer, and the radioactivity in the precipitate was determined with a gamma counter. The cAMP content of the unknown samples was determined by comparison with the standard curve, and the results expressed on a per unit D N A basis.
Results
In our initial experiments, we examined the time course of accumulation of cAMP by subridge mesenchymal tissue in response to P G E 2. As shown in Fig. 1, 10 -5 M P G E 2 elicits a greater than 10-fold increase in cAMP content, indicating that subridge mesenchymal cells do indeed have adenylate cyclase-coupled receptors for this molecule. Accumulation of cAMP in response to P G E 2
3628 ~ cZ o~
20
~
12
<~
04
E2
CONT
TIME (mln~
Fig. 1. Time course of cAMP accumulation by subridge mesenchymal tissue in response to 10 -5 M PGE 2. The sub-
ridge mesoderm was preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to addition of PGE 2 (E2) or vehicle (CONT). Values are the means of duplicate determinations.
is essentially complete within 5 min, with little, if any, additional increase detectable up to 15 min (Fig. 1). We next examined the effect of PGE~ on cAMP accumulation by the subridge mesoderm in the presence and absence of the potent phosphodiesterase inhibitor, IBMX. As shown in Table I, P G E 2 alone elicits about a 5-fold increase in cellular cAMP content. There is a more modest (about 2-fold) increase in cAMP in response to IBMX alone (Table I). However, the effect of these agents alone is relatively small compared to the striking elevation (30-40-fold above basal levels) in cAMP seen when the phosphodiesterase inhibitor is added along with P G E 2 (Table I). The effect of these agents is not simply additive, but clearly synergistic. These observations are of particular interest in view of our previous studies in which we found that phosphodiesterase inhibitors greatly potentiate the stimulatory effect of PGE~ on the in vitro chondrogenesis of subridge mesenchymal cells (Kosher and Walker, 1983; Gay and Kosher, 1984). The effect of various prostanoids on the cAMP content of intact subridge mesenchymal tissue is shown in Fig. 2 and summarized in Table I1. P G E 2 and PGE] are by far the most effective stimulators of cAMP accumulation that we have examined, eliciting an 11-14-fold increase in cellul a r . c A M P content above control levels (Fig. 2:
163
32
50
5.0 Z
;~ 28 ""
o E
Z
uJ I-Z
0 o
D. ~E
~ 24
2.4
-~ 22 E o. 2 0
2.2
20 18 16 14 12
--
(.3 >-
04
0 ..J 0
a 26
2.6
I0 08 06
'~
E2
28
Iz
Z w 16 F-Z 14 0
'~ 0 8
B 0.6
Fla
~ O.4 0.2 .~¢,j
02
I
A
I
i
i
i
I0-~O iO-e 10-B 10-7 10-6 I0-5 CT 6K-Fta TXB2
E
Fie AI 12 TREATMENT
E
Fig. 2. Effect of various prostanoids on the cAMP content of subridge mesenchymal tissue. The subridge mesoderm was preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to addition of PGE 2 (E2) , PGE 1 ( E l ) , PGI 2 (I2) , PGA 1 (A1), PGF]a (Fla), thromboxane BE TxB2, 6-keto PGF],~ (6K-FI,,), or vehicle (CT). Values are the means of two separate duplicate determinations + range.
Table II). The next most effective promotor of c A M P accumulation is prostacyclin (PGI2) eliciting a 5-fold increase in cAMP, followed by P G A a which promotes a 3-fold increase. In contrast, PGFa~ has little effect, and TxB 2 and 6-keto PGFt~ have no effect on cellular cAMP levels (Fig. 2; Table II). The relative potencies of these prostanoids in promoting cAMP accumulation is directly related to their ability to stimulate chondro-
r-PROSTAGIANDI N] ( M ) Fig. 3. Effect of various concentrations of P G E 2 (E2) , PGI 2 (I2), PGA 1 (A1), and PGF1,~ (FI,~) on the cAMP content of subridge mesenchymal tissue. The tissue was preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to addition of the prostanoid or vehicle control. Values are the means of duplicate determinations.
genesis (see Introduction and Discussion). The effect of various concentrations of several different prostanoids on the cAMP content of the subridge mesoderm is shown in Fig. 3. At all concentrations examined, P G E 2 is the most effective stimulator of cAMP we have tested, followed by P G I 2. P G I 2 is, in turn, more potent than P G A 1 (Fig. 3). PGFI~ has a slight effect on cAMP content only at the highest concentration examined
TA B LE II T ABLE I Effect of P GE 2 on the cAMP content of subridge mesenchymal tissue in the presence and absence of the phosphodiesterase inhibitor, IBMX Treatment None IBMX PGE2 P GE 2 + IBMX
cAMP content ( p m o l / # g DNA)
Relative increase in cAMP content over control levels in subridge mesenchymal tissue in response to various prostanoids Prostanoid
Treated ( p m o l / # g D N A ) control ( p m o l / # g D N A )
a
0.068 0.203 0.360 2.860
a Values are the means of duplicate determinations.
PGE 2 PGE 1 PGI 2 PGA1 PGF1. TxB 2 6-keto PGF1~
13.62 10.91 4.77 3.0i 1.30 1.08 1.02
164 TABLE Ill Relative increase in cAMP content over control levels in dissociated subridge mesenchymat cells in response to various prostanoids
I.-Z i,i
14
I0 r,
a,
:~ ~, 0 8
Prostanoid
Treated (pmol//*g DNA) control (pmol/ffg DNA)
PGE 2 PGE 1 PGI 2 PGA 1 PGFl~ , TxB. 6-keto PGF1,
3.69 3.82 2.75 2.60 0.94 0.99 1.06
(10 5 M). It is noteworthy that PGE 2 elicits about a 3-fold increase in cAMP at a concentration of 10 -~ M, which is the lowest concentration at which it effectively stimulates in vitro chondrogenesis (Kosher and Walker, 1983; Gay and Kosher, 1984). The preceding experiments involved examining the effect of prostanoids on the cAMP content of intact subridge mesenchymal tissue. We have also examined the effect of prostanoids on the cAMP content of suspensions of subridge mesenchymal cells prepared from subridge mesenchymal tissue that was dissociated into single cells with the aid of the proteolytic enzyme, trypsin. As shown in Fig. 4 and summarized in Table III, various prostanoids have the same relative effectiveness in elevating cAMP levels in the dissociated cells as they had in the intact tissue, with PGE 2 and PGE] being most effective, PGI 2 and PGA 1 being moderately effective, and PGFI~ , 6-keto PGF~,~, and TxB 2 having no effect. However, PGE2, PGE~, PGI 2 and PGA t, are all considerably less effective in elevating the cAMP content of the dissociated cells than they are in elevating the cAMP of cells in the intact tissue. For example, P G E 2 elicits about a 14-fold increase in the cAMP content of intact subridge mesenchymal tissue (Table II), but only about a 4-fold increase in the cAMP content of the dissociated subridge mesenchymal cells (Table III). The most likely explanation for this observation is that trypsin damages a n d / o r removes cell surface receptors for this prostaglandin (see Discussion). In this regard, it is noteworthy that
o
06
o
-~
04
> (o' ~
02 CT
6K-Fla TXB 2
Fia
AI
TREATMENT
I2
E~
J Ez
Fig. 4. Effect of various prostanoids on the cAMP content of dissociated subridge mesenchymal cells. Cells were preincubated for 10 min at 37°C in the presence of 1.0 mM IBMX prior to the addition of PGE 2 (E2), PGE 1 (El), PGI2(I2), PGA1(A1), PGFI~ (Flo), thromboxane B2 (TXB2), 6-keto PGF1,, (6K-F~, 0, or vehicle (CT). Values are the means of two separate duplicate determinations +_range.
PGE 2 is considerably more effective in stimulating the chondrogenesis of intact subridge mesenchymal tissue in organ culture (Kosher and Walker, 1983) than it is in stimulating the chondrogenesis of dissociated subridge mesenchymal cells in high density cell culture (Gay and Kosher, 1984).
Discussion
We have suggested that the stimulatory effect of P G E 2 on the in vitro chondrogenesis of the subridge mesenchymal cells of the chick limb bud is mediated by cAMP (Kosher and Walker, 1983; G a y and Kosher, 1984). The purpose of the present study was to determine directly whether P G E 2 and other prostaglandins which stimulate chondrogenesis do indeed elevate the cAMP content of subridge mesenchymal cells, and to determine whether the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that PGE z does elicit a striking increase in cellular cAMP content, indicating that subridge mesenchymal cells have adenylate cyclase-coupled receptors for this molecule. Furthermore, we have found that PGE 1, which is just as effective as PGE 2 in promoting chondrogenesis (Kosher and Walker, 1983; Gay and Kosher, 1984), is just as effective as
165 P G E 2 in stimulating cAMP accumulation. PGA1, which is much less effective than PGE 2 or PGE] in stimulating chondrogenesis, is less than half as potent as these molecules in elevating cAMP levels. PGFI~, 6-keto PGFI~, and TxB2, which have little or no effect on chondrogenesis, have little or no effect on cAMP content. Thus, the abilities of various prostanoids to stimulate cAMP accumulation in subridge mesenchymal cells do indeed correlate directly with their abilities to stimulate chondrogenesis. It is noteworthy that the very labile prostaglandin, prostacyclin (PGI2) is a very effective elevator of the cAMP content of subridge mesenchymal cells, whereas its stable and biologically inactive breakdown product, 6-keto PGFa~, has no effect on cAMP levels. This observation is of considerable interest, since prostacyclin, along with PGE2, appears to be one of the major prostanoids produced by limb mesenchymal cells undergoing chondrogenesis in vitro (Chepenik et al., 1980; S.W. Gay and R.A. Kosher, in preparation). These observations suggest that prostacyclin, as well as PGE 2, may play an important regulatory role in cartilage differentiation. PGE 2 and the other prostanoids are considerably less effective in elevating the cAMP content of dissociated subridge mesenchymal cells than they are in elevating the cAMP content of cells in the intact tissue. This observation is not particularly surprising, since in many systems PGE 2 and other prostanoids exert their regulatory effect by interacting with specific receptors on the external surface of the responding cells (Samuelsson et al., 1978). The trypsinization used in the present study to dissociate the subridge mesoderm into single cells may very likely have damaged or removed cell surface receptors for prostaglandins, resulting in the decreased responsiveness of the cells. Parker et al. (1981) and Ballard and Biddulph (1983) have examined the effect of PGE 2 on the cAMP content of dissociated cells of whole limb buds, with almost all of their studies being done on the cells of whole stage 24-25 limb buds or the isolated cartilage rods of stage 26 28 limb buds. Although an increase in cAMP in response to PGE 2 was observed in these cells, the studies were rather difficult to interpret for several reasons.
First of all, stage 24-25 limb buds consist of a very heterogeneous population of chondrogenic, myogenic, and fibrogenic cells in various stages of differentiation (see Kosher, 1983a, for review). Thus, determining the nature of the cell type or types responding to PGE 2 in the above studies was virtually impossible. This contrasts to the present study in which we examined the effect of various prostanoids on a relatively uniform population of chondrogenic progenitor cells which have not yet initiated overt differentiative changes (Newman et al., 1981), and which uniformly undergo chondrogenesis in vitro (Kosher et al., 1979a; Gay and Kosher, 1984). The studies utilizing isolated cartilage rods of stage 26-28 limbs were done on tissue consisting of chondrocytes which have already undergone overt differentiation. The effect of prostanoids on already differentiated chondrocytes may be, and, in fact, probably is quite different from their effect on mesenchymal progenitor cells (Malemud et al., 1982; Kosher and Walker, 1983). Parker et al. (1981) and Ballard and Biddulph (1983) did in a single experiment find that high concentrations of PGE 2 stimulate cAMP accumulation in dissociated cells of whole stage 20-21 limb buds, which consist of a somewhat more homogeneous group of cells than the whole limbs used in most of their studies. In summary, the following major pieces of evidence suggest that prostaglandins and cAMP play interrelated regulatory roles in limb cartilage differentiation. (1) A variety of studies suggest that a key event in limb chondrogenesis is a cellular condensation process during which a local cell-cell interaction occurs that triggers cartilage differentiation by elevating cAMP levels. (2) Limb mesenchymal cells undergoing chondrogenesis in vitro produce several prostaglandins including PGE 2 and prostacyclin (PGI2) which are important local mediators of cAMP accumulation in several tissues (Chepenik et al., 1980; S.W. Gay and R.A. Kosher, in preparation). (3) PGE 2 elicits a dose-dependent stimulation of in vitro limb chondrogenesis (Kosher and Walker, 1983; Gay and Kosher, 1984). (4) The stimulatory effect of PGE 2 on chondrogenesis is potentiated by phosphodiesterase inhibitors, suggesting its regulatory effect is mediated by cAMP (Kosher and Walker,
166
1983; Gay and Kosher, 1984). (5) PGE 2 and PGI 2 elicit a striking increase in the cAMP content of subridge mesenchymal cells, indicating that these cells do indeed possess adenylate cyclase-coupled receptors for these molecules. (6) The effect of PGE 2 on cAMP accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect of phosphodiesterase inhibitors on in vitro chondrogenesis. (7) The relative effectiveness of various prostanoids in stimulating cAMP accumulation in subridge mesenchymal cells directly reflects the relative potencies of these same molecules in stimulating in vitro chondrogenesis. All of these observations are consistent with the hypothesis that prostaglandins produced during the condensation phase of chondrogenesis might be involved in regulating limb cartilage differentiation by acting as local modulators of cAMP formation. We are continuing to investigate this hypothesis.
Acknowledgement This work was supported by NIH Grant HD 17794 to R.A.K.
References Abraham, G.N., C. Scaletta and J.H. Vaughan: Modified diphenylamine reaction for increased sensitivity. Anal. Biochem. 49, 547-549 (1972). Ahrens, P.B., M. Solursh and R.S. Reiter: Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60, 69-82 (1977). Archer, C.W,, P. Rooney and L. Wolpert: Cell shape and cartilage differentiation of early chick limb bud cells in culture. Cell Differ. 11,245-251 (1982). Ballard, T.A. and D.M. Biddulph: Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs. Prostaglandins 25, 471-480 (1983). Chepenik, K.P., B.M. Waite and C.L. Parker: Prostaglandin synthesis during chondrogenesis in vitro. J. Cell Biol. 87, 21a (1980). Dessau, W., H. yon der Mark, K. vonder Mark and S. Fischer: Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis. J. Embryol. Exp. Morphol. 57, 51-60 (1980). Elmer, W.A., M.A. Smith and D.A. Ede: lmmunohistochemical localization of cyclic AMP during normal and abnormal
chick and mouse limb development. Teratology 24, 215-223 (1981). Frandsen, E.K. and G. Krishna: A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues. Life Sci. 18, 529-542 (1976). Gay, S.W. and R.A. Kosher: Uniform cartilage differentiation in micromass cultures prepared from a relatively uniform population of chondrogenic progenitor cells of the chick limb budJ Effect of prostaglandins. J. Exp. Zool. 232, 317-326 (1984). Hamburger, V. and H.L. Hamilton: A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92 (1951). Hammarstrom, S.: Biosynthesis and biological actions of prostaglandins and thromboxanes. Arch. Biochem. Biophys. 214, 431-445 (1982). Harper, J.F. and G. Brooker: Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhyride in aqueous solution. J. Cyclic Nucleotide Res. 1, 207-218 (1975). Ho, W.C., R.M. Greene, J. Shanfeld and Z. Davidovitch: Cyclic nucleotides during chondrogenesis: concentration and distribution in vivo and in vitro. J. Exp. Zool. 224, 321-330 (1982). Kosher, R.A.: (1983a). The chondroblast and the chondrocyte. In: Cartilage, Vol. 1, ed. B.K. Hall (Academic Press, New York) pp. 59 85 (1983a). Kosher, R.A.: Relationship between the AER, extracellular matrix, and cyclic AMP in limb development. In: Limb Development and Regeneration, Part A, eds. J.F. Fallon and A.L Caplan (Alan R. Liss, New York) pp. 279-288 (1983b). Kosher, R.A. and M.P. Savage: Studies on the possible role of cyclic AMP in limb morphogenesis and differentiation. J. Embryol. Exp. Morphol. 56, 91-105 (1980). Kosher, R.A., M.P. Savage and S.-C. Chan: In vitro studies on the morphogenesis and differentiation of the mesoderm subjacent to the apical ectodermal ridge of the embryonic chick limb bud. J. Embryol. Exp. Morphol. 50, 75 97 (1979a). Kosher, R.A., M.P. Savage and S.-C. Chan: Cyclic AMP derivatives stimulate the chondrogenic differentiation of the mesoderm subjacent to the apical ectodermal ridge of the chick limb bud. J. Exp. Zool. 209, 221-228 (1979b). Kosher, R.A., M.P. Savage and K.H. Walker: A gradation of hyaluronate accumulation along the proximodistal axis of the embryonic chick limb bud. J, Embryol. Exp. Morphol. 63, 85-98 (1981). Kosher, R.A. and K.H. Walker: The effect of prostaglandins on in vitro limb cartilage differentiation. Exp. Cell Res. 145, 145 153 (1983). Kosher, R.A., K.H. Walker and P.W. Ledger: Temporal and spatial distribution of fibronectin during development of the embryonic chick limb bud. Cell Differ. 11, 217-228 (1982). Kuehl, F.A.: Prostaglandins, cyclic nucleotides and cell function. Prostaglandins 5, 325-340 (1974).
167 Kuehl, F.A. and R.W. Egan: Prostaglandins, arachidonic acid, and inflammation. Science 210, 978-984 (1980). Malemud, C.V., R.A. Moskowitz and R.S. Papay: Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes. Biochim. Biophys. Acta 715, 70-79 (1982). Martin, T.J. and N.C. Partridge: Prostaglandins, cancer and bone: Pharmacological considerations. Metab. Bone Dis. Relat. Res. 2, 167-171 (1980). Newman, S.A., M.P. Pautou and M. Kieny: The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells. Dev. Biol. 84, 440-448 (1981). Parker, C.L., D.M. Biddulph and T.A. Ballard: Development of the cyclic AMP response to parathyroid hormone and prostaglandin E 2 in the embryonic chick limb. Calcif. Tissue Int. 33, 641-648 (1981). Richards, G.M.: Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57, 369-376 (1974). Samuelsson, B., M. Goldylne, E. Granstrom, M. Hamberg, S. Hammarstrom and C. Malmsten: Prostaglandins and thromboxanes. Annu. Rev. Biochem. 47, 997-1029 (1978). Silver, M.H., J.-M. Foidart and R.M. Pratt: Distribution of
fibronectin and collagen during mouse limb and palate development. Differentiation 18, 141-149 (1981). Solursh, M.: Cell-cell interaction in chrondrogenesis. In: Cartilage, Vol. 2 ed. B.K. Hall (Academic Press, New York) (1983). Solursh, M., T.F. Linsenmayer and K.L. Jensen: Chondrogenesis from single limb mesenchyme cells. Dev. Biol. 94, 259-264 (1982). Solursh, M., R.S. Reiter, P.B. Ahrens and R.M. Pratt: Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro. Differentiation 15, 183-186 (1979). Solursh, M., R.S. Reiter, P.B. Ahrens and B.M. Vertel: Stageand position-related changes in chondrogenic response of chick embryonic wing mesenchyme to treatment with dibutyryl cyclic AMP. Dev. Biol. 83, 9-19 (1981). Tihon, C., M.B. Goren, E. Spitz and H.V. Rickenberg: Convenient elimination of trichloroacetic acid prior to radioimmunoassay of cyclic nucleotides. Anal. Biochem. 80, 652-653 (1977). Tomasek, J.J., J.E. Mazurkiewicz and S.A. Newman: Nonuniform distribution of fibronectin during avian limb development. Dev. Biol. 90, 118-126 (1982). Toole, B.P.: Hyaluronate and hyaluronidase in morphogenesis and differentiation. Am. Zool. 13, 1061-1065 (1973).