JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 12, 1991 Mary Ann Liebert, Inc., Publishers

Effects of Acid and Basic Fibroblast Growth Factor and Heparin on Resorption of Cultured Fetal Rat Long Bones HOLLIS A. SIMMONS',' and LAWRENCE G. RAISZ'

ABSTRACT We tested acid and basic fibroblast growth factor (aFGF and bFGF), members of the heparin binding FGF family, for their ability to stimulate bone resorption as measured by the release of previously incorporated ' T a from cultured fetal rat long bones in the presence and absence of heparin. Purified low-molecularweight heparin (LMW heparin) at 5-125 pg/ml had no direct stimulatory effect. There was little effect from aFGF (10-"-10-8 M) alone, but increased resorption was observed in the presence of LMW heparin. With bFGF, increased bone resorption was observed at M but not at lo-*M. The stimulatory effects of aFGF M), and bFGF in the presence of LMW heparin were not blocked by the addition of indomethacin which blocks prostaglandin production, or hydroxyurea M), which blocks DNA synthesis. However, pretreatment with aphidicolin (3 x M), a potent inhibitor of DNA synthesis, blocked the effect of acid FGF and diminished the effect of bFGF. These results indicate that both aFGF and bFGF can stimulate bone resorption by a prostaglandin-independent mechanism, particularly in the presence of heparin. The activation of FGF-mediated bone resorption by heparin could play a role in producing the osteoporosis that has been described with heparin therapy and mastocytosis.

INTRODUCTION (aFGF and bFGF) are members of a family of heparin binding growth factors with potent mitogenic and angiogenic activity.(I+) Heparin binding not only appears to protect these factors from inactivation but also potentiates the biologic activity of aFGF. Both aFGF and bFGF are potent mitogens for bone cells that could act as autocrine growth regulator~.''-'~)In initial studies, aFGF was not found to have any significant effect on bone resorption in organ culture,'') but more recently Shen et al."') reported an increase in resorption in cultured long bones treated with aFGF that appeared t o be dependent on endogenous prostaglandin production. Heparin has been reported both to enhance parathyroid hormone (PTH) effects('*' and directly stimulate bone resorption in mouse calvarial organ culture systems(I3)and to inhibit bone collagen synthesis in fetal rat calvariae.(13' These effects could play a role in the

A

CID AND BASIC FIBROBLAST GROWTH FACTOR

development of osteoporosis in patients undergoing prolonged heparain therapy(ls) and in mastocytosis."6) The present study was undertaken to examine the interactions of FGF and heparin using recombinant aFGF and bFGF and a purified low-molecular-weight heparin (LMW heparin, enoxaparine) on the resorptive response of cultured fetal rat long bones as measured by the release of previously incorporated 'Ta. LMW heparin and aFGF had no significant effects on resorption when added alone, but the combination produced a dose-related increase in the release of previously incorporated 4sCa. There was a resorptive response to bFGF alone, but this was enhanced by LMW heparin at lower concentrations of bFGF.

MATERIALS AND METHODS The following materials were used: LMW heparin (enoxaparine, a purified low-molecular-weight fraction of porcine intestinal heparin; Rhone-Poulenc Sante, Nan-

'Division of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington 'Present address: Pfizer Central Research, Groton, Connecticut.

1301

SIMMONS A N D RAISZ

1302

terre, France), recombinant bovine aFGF purified from a ments were pooled by calculating individual values as a bacterial expression system'") (kindly provided by Dr. percentage of the mean control for that experiment. StatisKenneth A. Thomas, Merck, Sharp and Dohme Research tical analysis was carried out by analysis of variance and Laboratories, Rahway, NJ), recombinant human bFGF significance of differences determined by post hoc testing purified from a bacterial expression system(181and more using Bonferroni's method.(*'' than 99% pure (kindly provided by Drs. John Fiddes and Stewart Thompson, California Biotechnology, Mountain RESULTS View, CA), bovine synthetic parathyroid hormone [bPTH(1-34), Bachem, Torrance, CA), and modified BGJ culIn preliminary experiments, bones were cultured with ture medium (GIBCO, Grand Island, NY). Prostaglandin E, (PGE,), indomethacin, hydroxyurea (HU), aphidicolin LMW heparin at concentrations of 5, 25, and 125 wg/ml (APC), and bovine serum albumin (BSA, radioimmunoas- with and without aFGF. There were no significant effects say, RIA grade) were from Sigma (St. Louis, MO). 45Ca of either heparin or aFGF on 45Carelease at 5 days when M aFGF plus 5 pg/ml of LMW hepaand [3H]thymidine were from New England Nuclear (Bos- given alone, but rin produced a significant increase in ' T a release (56 f 7 ton, MA). Bone resorption was measured as the release of previ- versus 29 + 2% for LMW heparin alone, P < 0.01). With ously incorporated 45Ca as described previ0us1y.(~~)higher concentrations of LMW heparin, a resorptive reBriefly, timed pregnant Sprague-Dawley rats (Charles sponse was observed with concentrations of FGF as low as M (Table 1 and Fig. 1). The resorptive effect was sigRiver Farms, Wilmington, MA) were injected with 200 pCi of 45Ca on day 18 of gestation and sacrificed on day 19. nificant at 48 h only for the combination of LMW heparin M, 16 ng/ml). The fetal radius and ulna were dissected free of connective (125 pg/ml) and aFGF In contrast to aFGF, there was a significant effect of tissue and cartilage and precultured for 18-24 h in BGJ. and M (Table 1 Indomethacin, HU, and APC were added during precul- bFGF in the absence of heparin at ture. Bones were then transferred to fresh BGJ with BSA and Fig. 1). LMW heparin significantly increased the re( 1 mg/ml) and with and without test materials. Cultures sponse to lo-' M bFGF, but the response to lo-' bFGF was were maintained for 5 days with a change in medium at 2 decreased, although not significantly. In the presence of LMW heparin both aFGF and bFGF days. 45Ca in medium and trichloracetic acid (TCA) exM) significantly increased the incorporation of tracts of bone was determined by liquid scintillation count- ( ing, and the cumulative percentage of 45Carelease was cal- [3H]TdR in fetal rat long bone cultures (Table 2). The efculated. For measurement of incorporation of [3H]thymi- fects of bFGF were the same with or without heparin, but dine, bones were cultured with appropriate agonists for 24 aFGF caused a smaller increase in ['HITdR incorporation h and [3H]thymidine (5 pCi/ml) added for the last 2 h.c2O) in the absence of heparin. Heparin alone did not affect TCA-soluble and insoluble radioactivity was measured by TdR incorporation significantly, although the values were liquid scintillation. There was no change in TCA-soluble somewhat lower. To test whether the effects of aFGF and bFGF might be counts with FGF or heparin. Data from multiple experi-

TABLE1. EFFECTS OF ACID(AFGF) AND BASIC(BFGF) FIBROBLAST GROWTH OF 45CAFROM FETALRAT LONGBONESCULTURED WITH OR FACTORON RELEASE WITHOUTLMW HEPARIN (HEP, 125 ~ G / M L ) ~ Vo 45Careleased 2 Days

Control aFGF aFGF aFGF Control 10- l o bFGF bFGF lo-* bFGF

N

-

Hep

37 29 35 23 42 23 36 14

17 f 1 18 f 1

18 19 18

f

+ + +

1

1 1 18 1 21 f 1

28 f 3b

5 Days

+

Hep

16 + 1 19 f 1 24 f I c - d 21 f 1 17 f 1 18 f 1 26 + lcvd 22 + 2

-

Hep

26 f 2 27 f 2 31 f 2 36 f 4 30 f 2 29 + 1 47 f 3c 62 f 7c

+ 26 39 50 44 31 39 66 53

Hep 2 3b 4c.d f 5c f 2 f 4 f 4c.d f 5c f f f

aValues are means f SEM for the indicated number of cultures. Significant effect of FGF compared to the appropriate control without FGF is indicated. bP < 0.05. CP < 0.01. dsignificant effect of LMW heparin, P < 0.05.

1303

FGF, HEPARIN, AND BONE RESORPTION

TABLE3. EFFECTS OF INDOMETHACIN (INDO, M), HYDROXYUREA (HU, 1 MM), A N D APHIDICHOLIN M) ON THE RELEASE OF "CA IN (APC, 3 X M) A N D BASIC(BFGF, RESPONSE TO ACID(AFGF, lo-' M) FIBROBLAST GROWTHFACTORFROM FETALRAT LONGBONESCULTURED WITH LMW HEPARIN (125 ~ G / M L ) ~ To ' T a released -9

-10

-8

0

Lop [aFGF. MI

-10

-9

8

Log [bFGF],M

FIG. 1. Effects of acid (aFGF) and basic (bFGF) fibroblast growth factors in the presence and absence of heparin (125 pg/ml) on 45Ca release from fetal rat long bones at 5 days. Points are means and vertical lines SEM for 14-42 cultures. Significance of differences is indicated in Table 1.

M) AND BASIC TABLE 2. EFFECTOF ACID(AFGF GROWTHFACTORON (BFGF lo" M) FIBROBLAST 1NCORPORATION OF ['HITHYMIDINE (TDR) INTO FETAL RAT LONGBONESA T 24 H CULTURED WITHA N D WITHOUT LMW HEPARIN HEP, 125 ~ G / M L ) ~

/'H]TdR incorporation ~~

Control aFGF bFGF

~

~

+

Hep

76 179 162

+

2 Days

5 Days

42 35 36 25 24 13

17 24 26

+ + + 18 + 23 +

31 f 2

15 10 10

17 11 10

16 16 14

23 15 19 25 15 16 25 14 14 17

f f

+

1 Ib

Ib

1 2b 3 I 1

f

4

+

1

+

1 2b

+ f

*

1 1 1

so f 4b 66 39 63

+Z

f

+

4b 4 6b 8c

58

f

20 52

f

1

+

5c

61 f 14b 20 f 1

26 + 3 56 f 7b 19+ I 18 f 1 29 f 5 c

*

Hep

~~

loof 9 140 f 17 163 f 1 9 ~

Control + aFGF + bFGF Indo + aFGF + bFGF HU + aFGF + bFGF HU + Indo + aFGF + bFGF APC + aFGF + bFGF

N

1

* 23b

+27~

aValues are mean + SEM for 18-22 cultures expressed as percentage of control. Significant effect of bFGF compared to the appropriate control Hithout FGF is indicated. h f < 0.01. ' P < 0.0s.

due to their ability to stimulate endogenous prostaglandin production,'") the bones were cultured in the presence and absence of indomethacin at M, a concentration that completely blocks PGE, production in cultured bone (Table 3).(*3)The responses to both aFGF and bFGF M) in the presence of LMW heparin were not blocked by indomethacin (lo-" M). To assess the role of cell replication in these responses, bones were precultured with either HU and APC at concentrations that decreased [3H]thymidine incorporation by more than 90%. In the presence of HU there is a persistent stimulation of resorption by both aFGF and bFGF, but the response to aFGF was abrogated and the response to bFGF markedly diminished after treatment with APC. In addition, the responses to LMW heparin plus aFGF at lo-' and lo-'' M were blocked by APC (data not shown). Since we have previously found that HU can convert the resorptive responses t o epidermal growth factor (EGF) and transforming growth factor CY (TGF-U) in fetal rat long bones from prostaglandin independent to

aValues are mean SEM for the indicated number of cultures. Significant effect of FGF compared to the appropriate control without FGF is indicated. bf < 0.01. 'f < 0.05.

prostaglandin dependent,'," 2 4 ) bone were treated with HU and indomethacin together. Under these circumstances the response to aFGF was blocked but not the response to bFGF. To test whether the effect of heparin was specific for heparin binding growth factors, studies were carried out with insulin, which does not stimulate bone resorption, and TGF-(r, which does (Table 4). Insulin had no effect on 45Ca release, but TGF-u stimulated bone resorption to a similar extent in both the presence and absence of heparin. T o test for interactions with other bone resorbers, aFGF and heparin were added to cultures treated with submaxima1 concentrations of PTH or PGE, (Table 5 ) . The resorptive responses were not altered. In separate experiments we found that the response to PTH was not altered by higher concentrations of heparin (25 and 125 pg/ml) or by addition of bFGF (data not shown).

DISCUSSION These results showed that both aFGF and bFGF can act as stimulators of resorption in cultured fetal rat long bones, particularly in the presence of heparin. Since heparin alone was inactive even at the high concentration used in this study, it appears that the agonist was FGF and that heparin was acting to enhance the response to these growth

SIMMONS AND RAlSZ

1304

to prostaglandin dependent. This did not appear to be the case with bFGF, which still stimulated bone resorption in the presence of HU and indomethacin. With aFGF, as with TGF-a, bone resorption was inhibited by APC, a potent inhibitor of DNA synthesis. This suggests that cell replication is essential for the resorptive response but could be due to an unrelated pharmacologic effect of APC. On Yo ' T a released the other hand, the response to bFGF was not abrogated - Hep + Hep by APC, although it was markedly diminished. Except for the effect of heparin, the observed differences between Control 40f4 30 f 4 aFGF and bFGF are difficult to explain; however, bFGF 32 6 31 f 4 Insulin appears to be the more potent resorber. Further studies in 14 17 14 + 7b TGF-a other culture systems, including isolated osteoclasts and osteoclast-osteoblast mixtures, are needed to define the "Values are means * SE for 12 bones cultured mechanisms of FGF action on resorption more clearly. for 5 days. bSignificant effect of growth factor, P < 0.01. That earlier studies did not show stimulation of resorption by aFGF could be due to the absence of heparin.I4) The recent report of Shen et a l . l l l lof increased resorption in long bones treated with aFGF differs from our observaTABLE5. EFFECTSOF ACID FIBROBLAST GROWTHFACTOR tions in that the response was prostaglandin dependent. M) ON RELEASE OF 4sCA FROM FETALRAT Moreover, the preparation used in that study was obtained (FGF, LONGBONESCULTURED FOR 5 DAYSWITH PARATHYROIDby purification from bovine brain tissue and thus differed HORMONE (PTH, 6.25 NG/ML)OR PROSTAGLANDIN E, from the recombinant human preparation used here. (PGE,, M) WITHOR WITHOUTLMW HEPARIN The relevance of these observations to the pathogenesis (HEP, 5 ~ G / M L ) ~ of the osteoporosis observed with prolonged heparin therapy and in systemic mastocytosis is at present unknown. Yo of "Ca released Bone resorption appears to be increased in both disN - FGF + FGF orders.'LS.16)Moreover, bone extracts contain both aFGF and bFGF,(9) and bovine bone cells can synthesize bFGF Control 54 35 f 2 41 f 3 and store it in their extracellular matrix.(L01Hence activaHeP 29 29 2 4 4 f 4 tion of these factors by heparin could result in increased 17 72 f 6b 68 f 8b resorption. On the other hand, heparin may be interacting PTH PTH + H E P 12 69 f 9b 86 7b with other stimulators of resorption or growth factors to 12 63 f 6b 72 f 5b produce osteoporosis. PGE, TABLE4. EFFECTS OF INSULIN M), AND TRANSFORMING GROWTHFACTORa (TGF-a, 30 NG/ML)ON RELEASE OF "CA FROM FETALRAT LONGBONESWITH AND WITHOUTLMW HEPARIN(HEP, 125 ~ G / M L ) ~

* *

*

"Values are means f SEM for the indicated number of cultures. Wgnificantly different from control, P < 0.05.

ACKNOWLEDGMENTS factors. The mechanism of heparin enhancement of FGF responses is probably complex. There is evidence that heparin can both enhance direct cellular effects and decrease the degradation of aFGF." 25 2 b 1 In many culture systems heparin has marked effects on the response to aFGF with little effect on the response to bFGF.1z,',9)Heparin may also increase bFGF effects, however, either by preventing degradation or by enhancing its diffusion in Heparin could also inhibit FGF responses if it competed for binding to cell r e c e p t o r ~ . (This ~ . ~ ~may ~ explain the apparent decrease in the response to a high concentration of either aFGF or bFGF in the presence of heparin in the present experiments. Although FGF can stimulate prostaglandin production in cultured neonatal mouse calvariae, the stimulation of resorption in long bones appears to be largely prostaglandin independent. An apparent prostaglandin-mediated response was observed in cultures treated with aFGF, LMW heparin, and HU. In these circumstances, as we have previously observed with TGF-a and EGF,(20.z4) the resorptive response can be converted from prostaglandin independent

This work was supported by Grants AR-18063 and 28933 from the National Institutes of Health and by Merck, Sharp and Dohme Research Laboratories. We thank Dr. Kenneth A. Thomas for his advice and criticisms and Ms. Lisa Godin and Ms. Lynn Limeburner for their careful preparation of the manuscript. A preliminary report of this work was presented at the combined Annual Meetings of the American Society for Bone and Mineral Research and the International Conferences on Calcium Regulating Hormones, Montreal, Canada, 1989.

REFERENCES Gospodarowicz D, Ferrara N , Schweigerer L, Neufeld G 1987 Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8:95-114. Thomas KA 1987 Fibroblast growth factors. FASEB J 1: 434-440.

Burgess WH, Maciag T 1989 The heparin-binding (fibroblast) growth factor family of proteins. 58575-606. Canalis E, Lorenzo J , Burgess WH, Maciag T 1987 Effects

FGF, HEPARIN, AND BONE RESORPTION

5.

6.

7

8

9

10.

II.

12. 13.

14.

15.

16. 17.

of endothelial cell growth factor on bone remodelling in vitro. J Clin Invest 79:52-58. Rodan SB, Wesolowski G, Thomas K, Rodan GA 1987 Growth stimulation of rat calvaria osteoblastic cells by acidic fibroblast growth factor. Endocrinology 121:1917-1923. Canalis E, Centrella M, McCarthy T 1988 Effects of basic fibroblast growth factor on bone formation in vitro. J Clin Invest 81:1572-1577. Globus RK, Patterson-Buckendahl P, Gospodarowicz D 1988 Regulation of bovine bone cell proliferation by fibroblast growth and transforming growth factor p. Endocrinology 123:98-105. McCarthy TL, Centrella M, Canalis E 1989 Effects of fibroblast-growth factors on deoxyribonucleic acid and collagen synthesis in rat parietal bone cells. Endocrinology 125:21182127. Hauschka PV, Mavrakos AE, lafrati MD, Doleman SE, Klagsbrun M 1986 Growth factors in bone matrix. Isolation of multiple types by affinity chromatography on heparinsepharose. J Biol Chem 261:12665-12674. Globus RK, Plouet J , Gospodarowicz D 1989 Cultured bovine bone cells synthesize basic fibroblast growth factor and store it in their extracellular matrix. Endocrinology 124: 1539- 1547. Shen V, Kohler G , Huang J , Huang SS, Peck WA 1989 An acidic fibroblast growth factor stimulates DNA synthesis, inhibits collagen and alkaline phosphatase synthesis and induces resorption in bone. Bone Miner 7:205-219. Goldhaber P 1965 Heparin enhancement of factors stimulating bone resorption in tissue culture. Science 147:407-408. Wolinsky I , Cohn DV 1970 The stimulating by heparin and parathyroid hormone of bone resorption in tissue culture. Isr J Med Sci 6:691. Hurley MM, Kream BE, Raisz LG 1990 Effects of heparin on bone formation in cultured fetal rat calvaria. Calcif Tissue Int 46:183-188. Megard M, Cuche M, Grapeloux A, Bojoly C , Meunier PJ 1982 Heparin osteoporosis: Histomorphometric analysis of bone biopsy. Nouv Presse Med 11:261-264. McKenna MJ, Frame B 1985 The mast cell and bone. Clin Orthop 200:226-233. Linemeyer DL., Kelly LJ, Menke JG, Gimenez-Gallego G, DiSalvo J. Thomas K 1987 Expression in Escherichiu coli of a chemically synthesized gene for biologically active bovine acidic fibroblast growth factor. Biotechnology 5:960.

1305 18. Thompson SA, Protter AA, Bitting L, Fiddes JC, Abraham J A 1990 Cloning, recombinant expression and characterization of basic fibroblast growth factor. Methods Enzymol 198:96-116. 19. Raisz LG, Niemann I 1969 Effect of phosphate,,calcium, and magnesium on bone resorption and hormonal responses in tissue culture. Endocrinology 85:446-452. 20. Lorenzo JA, Quinton J , Sousa S, Raisz LG 1986 Effects of DNA and prostaglandin synthesis inhibitors on the stimulation of bone resorption by epidermal growth factor in fetal rat long-bone cultures. J Clin Invest 77:1897-1902. 21. Godfrey K 1985 Comparing the means of several groups. N Engl J Med 313:1450-1456. 22. Feyen J H M , Kuntzelmann GMM, Mackie EJ 1990 Basicand acidic fibroblast growth factor stimulate prostaglandin production and bone resorption in neonatal mouse calvaria (abstract 135). Calcif Tissue Int 46(Suppl. 2). 23. Raisz LG, Simmons HA, Fall PM 1989 Biphasic effects of nonsteroidal antiinflammatory drugs on prostaglandin production by cultured rat calvariae. Prostaglandins 37:559-565. 24. Lorenzo JA, Sousa SL, Centrella M 1988 Interleukin-l in combination with transforming growth factor-a produces enhanced bone resorption in vitro. Endocrinology 123:21942200. 25. Damon DH, Lobb RR, D’Amore PA, Wagner .IA 1989 Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life. J Cell Physiol 138:221226. 26. Mueller SN, Thomas KA, Di Salvo J , Levine EM 1989 Stabilization by heparin of acidic fibroblast growth factor mitogenicity for human endothelial cells in vitro. J Cell Physiol 140: 439-448. 27. Flaumenhaft R, Moscatelli D, Rifkin DB 1990 Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor. J Cell Biol 111:1651-1560.

Address reprint requests to:

Lawrence G. Raisz, M.D. Division of Endocrinology and Metabolism University of Connecticut Health Center Farmington, CT 06030 Received for publication November 29, 1990; in revised form February 13, 1991; accepted June 12, 1991.