Osteogenin (bone morphogenic protein 3) inhibits proliferation and stimulates differentiation of osteoprogenitors in human bone marrow

Differentiation ( 1994) 58: 157- I64

Differentiation

Ontogeny, Neoplasia and Differenlintion Therapy

0 Springer-Verlag 1994

Osteogenin (bone morphogenic protein 3) inhibits proliferation and stimulates differentiation of osteoprogenitors in human bone marrow JoZIleAmCdbe,I, Reine Bareillel, Franqois Rouais’, Noreen Cunningham2,Harri Reddi2, Marie-Franqoise Harmandl IINSERM-U. 306, UniversitC de Bordeaux 11, 146 rue LCo Saignat, F-33076 Bordeaux CCdex, France *Departmentof Orthopaedic Surgery, Johns Hopkins University, School of Medicine, Baltimore MD2 1205, USA Accepted in revised form: 1 July 1994

Abstract. Treatment of human bone marrow osteoprogenitors with osteogenin (BMP-3; at 1, 2.5 and 10 ng/ml) caused dose-and time-dependent inhibition of DNA synthesis and cell proliferation. Simultaneously, osteogenin stimulated type I collagen synthesis and CAMP production. Addition of osteogenin to the cell culture increased intracellular alkaline phosphatase activity and osteocalcin synthesis, with maximal stimulation at 2.5 ng/ml. Simultaneous addition of 2.5 ng/ml osteogenin and 1,25 dihydroxy vitamin D, ( M)enhanced the stimulation observed in osteocalcin synthesis. The experiments reported here demonstrate the significant “in vitro” influence of osteogenin in the stimulation of osteogenic phenotype in osteoprogenitor cells which have been isolated from human bone marrow and cloned. These results support a reciprocal relationship between cell growth inhibition and expression of osteoblast differentiation.

Introduction Demineralized bone matrix is a repository of growth and differentiation factors [ 121 which are able to induce cartilage and bone formation in vivo [ 13, 181. Implantation of demineralized diaphyseal bone matrix in intramuscular and subcutaneous sites results in local bone differentiation. The osteoinductive proteins contained in this bone matrix initiate events consisting of migration of mesenchymal cells into the implant, proliferation and differentiation of cartilage which is subsequently replaced by bone [28, 29, 351. A group of related bone morphogenetic factors has been described and includes osteogenin, bone morphogenetic protein (BMP) 2A, BMP 2B, BMP 3, and osteogenic protein 1 (OP-1) [5, 19, 38, 39, 411. Osteogenin, an osteoinductive protein derived from bovine bone matrix, was recently obtained in highly purified form [32]. The amino acid sequence of tryptic peptides [20] from osteogenin is identical to that Correspondence to: J. AmCdCe

of human BMP3. Osteogenin stimulates alkaline phosphatase activity and collagen synthesis by rat periosteal cells and calvarial osteoblasts “in vitro” [37]. Proteoglycan synthesis by fetal rat chondroblasts and rabbit articular chondrocytes is also stimulated by osteogenin addition [ 10, 371. Recently, osteogenin was demonstrated to stimulate and maintain the chondrocyte phenotype “in vitro” [ l l , 191. “In vitro” studies of rat bone marrow stromal cells have demonstrated its function in expression of osteogenic phenotype [37, 381. In the present study, we have investigated the “in vitro” action of highly purified bovine osteogenin on osteoblast precursors isolated from human bone marrow [36]. Osteogenin’s effect was determined on cell growth (DNA synthesis and proliferation) and cell differentiation (alkaline phosphatase activity and synthesis of osteocalcin, osteonectin and type I collagen response to 1,25(OH),D, human parathyroid hormone; and hPTH). The results demonstrate that osteogenin stimulates differentiation of osteoprogenitors isolated from human bone marrow.

Methods Materials. Osteogenin ( 1 ng/pl) was dissolved in 5 mM HCI, and 0.2% (w/v) bovine serum albumin (BSA). Fitton Jackson modified BGJ medium (BGJb medium) and fetal calf serum (FCS) were purchased from Gibco, (Grand Island, NY, USA). Plastic culture dishes and Lab-Tek chamber-slides were from Nunc (Inter Med SA, Paris, France). [‘HI-proline (26 Ci/mmole), and [3H]-thymidine (82 Ci/mmole) were purchased from Amersham (Les Ulis, France). The radioimmunoassay detection kit, specific to bovine osteocalcin, was from Oris-Industrie (Bagnols/Ceze, France). The polyclonal antibody specific for bovine osteonectin was a gift from P. Seguin (Oris-lndustrie, Bagnols/Ceze, France). Protein A from Staphylococcus aureus was from Sigma (St Louis Mo, USA). Cell culture. Human bone marrow was obtained by iliac aspiration from normal donors (aged 20-30 years) undergoing hip prosthesis surgery after trauma. Cells were separated into a single suspension by sequential passage through syringes fitted with a 16-, 18- and 21-gauge needle. Cells were then counted and plated into 35-mm

dishes in BGJb medium supplemented with 10% (v/v) FCS, at 105 cells/cm? and incubated in a humidified atmosphere of 95% (v/v) air and 5% (v/v) CO, at 37" C. The initial medium change was performed 3 days later and thereafter the medium was changed every 2 days. Confluence was obtained 3 weeks later, and cells were cloned by limiting dilution followed by successive subculturing, performed until the highest intracellular alkaline phosphatase activity was reached [36]. Electron microscopy. Cells derived from human bone marrow were seeded at lo3 cells/cm? in Lab-Tek chamber-slides and grown in culture for 3 days. Scanning electron microscopv (SEM).Samples were fixed for 15 min with 2% (v/v) glutaraldehyde in 0.15 M cacodylate buffer, pH 7.3. Surfaces were washed with 0.15 M cacodylate for 10 min. Samples were then dehydrated through a graded series of ethanol from 25% to 100% . The dehydrated samples were dried at critical point with liquid COz and finally coated using a gold target, before observation in a Hitachi S 2500 microscope. Mineralized areas in the section were quantitatively analysed using an energy dispersive X-ray analysis system. All analysis was carried out at 75 keV for 100 s. Transmission electron microscopy (TEM).Samples were fixed with the glutaraldehyde-cacodylate buffer for I h at 4" C. Cells were then washed with 0.15 M cacodylate. Post-fixation with 2% (v/v) OsO,-O.3 M cacodylate was carried out for 60 min. The samples were dehydrated through a 25-100% graded series of ethanol. The last dehydration was carried out with propylene oxide: Epon (1: I ) . Finally, the samples were placed in 100% fresh Epon and polymerized in a 60" C oven for 48 h. Thick sections ( 1 pm) were cut with a diamond knife and observed using a Hitachi HU 1 I E transmission electron microscope. Osteogenin treatment. At confluence, the medium was replaced with fresh BGJb medium containing 0.2% (w/v) BSA for 24 h. Thereafter, osteogenin dilutions ( I , 2.5 and 10 ng/ml) were added to each well. Controls were assessed using 5 mM HCI and 0.2% (w/v) BSA. Cells were treated for 3 days as described above. Cell replication studies. DNA synthesis was determined by incorporation of [3H]-thymidine [ 121. Human bone marrow derived cells were grown to confluence ( I 0 4 cells/cm2) in 96-well culture plates. Cells were deprived of FCS for 24 h and then treated with osteogenin solutions. At 24 h before the end of the incubation period, cells were incubated with [3H]-thymidine (5 pCi/ml) in medium containing 0.2% (wlv) BSA. Material precipitable with trichloroacetic acid was solubilized in 0.2 ml 0.3 N NaOH, and the radioactivity of the material was determined in a liquid scintillation counter (Packard). Proliferation ana1y.sis. Bone marrow stromal cells were plated at 5x103 cells/cm2 with or without 2.5 ng-ml osteogenin. Cell numbedwell (n=4) was calculated at different times (days I , 2, 3 and 6). Collagen labeling and analvsis. Collagen was biosynthetically radiolabeled and purified as described by Bonadio et al. [4] and modified by Michel and Harmand [23]. Cells were grown to confluence (2x104 cells/cm*) in 35-mm tissue culture plates. Cultures were treated with osteogenin and labeled with ['HI-proline (30 pCi/ml) for the last 24 h of the culture period. Medium was removed, the cell layer was scraped in 0.1 M PBS pH 7.0, and proteins were precipitated by the addition of one volume of a saturated solution of ammonium sulfate in PBS. Labeled material was analysed by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide (5%) slab gels according to Laemmli [ 151 , and then the gels were prepared for fluorography before being subjected to autoradiography using a high performance autoradiography film @ Max, Amersham). The radioactivity incorporated in al (I) and a 2 (11)

was estimated by cutting out and counting the corresponding polyacrylamide bands. Alkaline phosphatase activity. lntracellular alkaline phosphatase

activity was determined in scraped and sonicated cells as described [22]. Data are expressed as nmole inorganic phosphate cleaved by the enzyme in 30 min and for 104 cells. Osteocalcin synthesis. The effect of osteogenin on osteocalcin synthesis was measured by a specific radioimmunoassay with an antibody raised in rabbit against bovine osteocalcin. The detection limit for the assay is 1 ng/ml. Following exposure to osteogenin, at two concentrations 2.5-10 ng/ml, and 1,25(OH),D, at M for 3 days, the medium was removed, and the cell layer was scraped in PBS. Cells were then sonicated and proteins were precipitated with 50% (v/v) ammonium sulfate. Osteocalcin in the cell layer and secreted in the culture medium was then determined by radioimmunoassay.

Osteonectin synthesis. Cells were plated at 104 cells/cm2 in LabTek chamber-slides and grown for 8 days. At confluence, cells were treated for 3 days with 2.5 and 10 ng/ml osteogenin in the osteogenin buffer. Controls were performed using cells treated for 3 days with the same amount of osteogenin buffer. Thereafter, medium was collected, the cell layer was fixed using 100% methanol for 10 min at 4" C, and incubated overnight at 26" C with a polyclonal antibody specific to bovine osteonectin diluted at 1/200 in 0.1 M PBS pH 7.4. Fixed immunoglobulins were revealed using [1?5I]-proteinA ( 1 pCi/pg) diluted at lo5 cpm/well. After extensive washings, the radioactivity in ten wells was determined in a y counter (Packard). CAMP determination. Cells arising from the third subculturing were treated for 3 days with osteogenin (2.5 ng/ml). The medium

was removed, and the cultures were preincubated with fresh medium containing 0.5% (wlv) BSA and 5x104 M isobutylmethylxanthine (IBMX); (Sigma) at 37" C for 15 min. Thereafter hPTH (Sigma) at 100 ng/ml was administered for 5 min of incubation at 37" C and the cell layer was scraped, sonicated on ice for 30 s, then lyophylized. Lyophylized samples were assayed with a [ qcAMP assay kit (NEN) according to the manufacturer's instructions.

Results Human bone marrow derived cells

With successive cloning and subculturing, resulting cells exhibited high intracellular phosphatase activity and all the osteoblastic phenotype markers (type I collagen, osteocalcin synthesis, intracellular cAMP response to parathyroid hormone, osteonectin synthesis) [36](Fig. 1). As shown by scanning electron microscopy (Fig. 2A) cells appeared large and multipolar with many cytoplasmic extensions; they exhibited a granular cytoplasm, and a nucleus with generally two or three nucleoli. Moreover, these cells exhibited a high degree of budding (Fig. 2B). Transmission electron microscopy showed some structures (Fig. 3) which could have corresponded to matrix vesicles bounded by unit membranes. The components of the corresponding matrix were analyzed by an energy dispersive X-ray analyser (Fig. 4).The mineral deposited was mainly composed of calcium and phosphorus. The Ca/P ratio of these structures was about 2.08f0.19, the same as that of the hydroxyapatite crystal (Gift from C. Rey, INP Toulouse, France) used as a stan-

I59

A

EM cells -3H-prollno

D

C 6*

s

is

- 2

I

1 b -,

V

+

hPTH

Fig. 1A-D. Osteoblast precursors exhibit an osteoblastic phenotype. A Alkaline phosphatase (ALP) activity of cell clusters by cytochemical analysis performed as in [36]. B [3H]-Proline incorporation in cell culture: type I collagen synthesis. C Response of CAMPaccumulation to human parathyroid hormone (hPTH; 100 ng/ml) D Osteocalcin synthesis and secretion in culture medium; response to vitamin D, (1,25(OH),D,; M).

Fig. 2A, B. Scanning electron microscopy of osteoblast precursors isolated from human bone marrow. A Multilayered cells (~1000, 30 Fm). B Budding cells ( ~ 3 0 0 0 , 10 ym)

dard. This last result indicates that these human bone marrow-derived cells produce an extracellular matrix surrounding the cells which are undergoing mineralization. Effect of osteogenin on cell replication

Treatment of osteoblast precursors arising from human bone marrow with osteogenin for 3 days resulted in

dose-dependent inhibition of DNA synthesis (Fig. 5 ) . Maximal cell growth inhibition was obtained using 10 ng/ml after 2 days of treatment (84%, Pc0.001). Controls carried out with the same amount of 5 mM HCI, 0.2% BSA did not show significant change. Moreover, treatment of&teoblast pr&ursors with osteogenin at 2.5 ng/ml inhibited proliferation as early as the- first day of the incubation- period. Inhibition -was about 69% (Pc0.001) as assessed by cell counting on

160

Fig. 3. Transmission electron nnicroscopy of budding cells. The extracellular matrix exhibits colkigen fibers and electron dense inch. sions in vesicules x33 600

Livetime: 182 Deadtime: 22%

I

Ca

1

cr, I 2

2

IT

T

t4

-E g 1 n

Fig. 4. X-ray energy spectrum of mineralized areas; C carbon; 0 oxygen: P phosphorus; Co, calcium

m

D1 D2 daysinculture

D3

Fig. 5. Influence of osteogenin on DNA synthesis. Osteoblast precursors isolated from human bone marrow were cultured for 3 days with osteogenin at I (@I), 2.5 (@) and 10 (m) ng/ml) and labeled with ['HI-thymidine for the last 24 h of the incubation. [3H]-thymidine incorporation in the cells was determined as described in Methods. Data are means f S D (n=6)

day 6 when compared to a control carried out with the osteogenin buffer alone (Fig. 6). Effect o s osteogenin 011 cell differentiation

Alkaline phosphatase (ALP) is a well-known marker of bone differentiation. Addition of osteogenin to the cell cultures resulted in a prompt time-dependent increase in ALP activity (Fig. 7). The highest stimulation (220%, P
dayainculture

Fig. 6. Influence of osteogenin at 2.5 ng/ml on cell proliferation. Bone marrow stromal cells were plated at 5x10' cells/cm2 with (-) or without (-) 2.5 nglml osteogenin. Cell number/well (n=4) was calculated on days I , 2, 3 and 6. Data are means +SD

161

1 t

Table 1. Effect of osteogenin (OG) and 1,25(OH),D, on osteocalcin (OC) synthesis. Cells were treated as described above with OG (2.5-10 ng/ml) alone or in combination with 1,25(OH),D, at M for 3 days at 37" C. Controls were performed using OG buffer alone. The OC content of the culture medium and of the cell layer were measured by radioimmunoassay. Data are expressed in ng OC/lOh cells (n=6)

80

A

701

T

M

Treatment

Osteocalcin content (ng1106 cells)

Control OG (2.5 ng/ml) OG ( I0 ng/rnl) I .25(OH),D, (10-8 M ) OG 2.5 ng/ml+l,25(OH),D3 OG 10 ng/ml+ I ,25(OH),D,

Cell layer

Culture medium

2.6f0.25 4.2f0. I6 3.3f0.21 4.8f0.29 4.7f0.14 5.5f0.24

0.6f0.0 I2 0.533.01

0.9f0.03 I .2M.23 3.733. I2 I .8M.06

I

0

1

2

3

days Fig. 7. Influence of osteogenin on intracellular ALP activity. Human bone marrow cells were treated with two different concentrations of osteogenin (2.5 and 10 ng/ml; n=6) ( A ) or with 2.5 ng/ml (-) osteogenin for 3 days. ( B ) Control (-) was effected using osteogenin buffer alone. ALP activity was determined for each day (n=4). Data are expressed in nmole Pi/30 rnin/lO4 cells

gel electrophoresis (PAGE) followed by autoradiography (Fig. 8A) and quantification of the two labeled bands al (I) and a 2 (Fig. 8B) showed that osteogenin stimulated type I collagen synthesis in a dose-dependent manner; the maximum (280%, P
2.5

10

Treatment with 2.5 ng/ml osteogenin caused a greater increase of osteocalcin content ( 146%, RO.00 I ) than with 10 ng/ml ( 131 %, k O . 0 1 ). The osteocalcin content of the culture was increased by 88% when exposed to vitamin D, alone. When 1 ,25(OH),D, (10-8 M ) was combined with osteogenin (2.5 or 10ng/ml), the response in the cell layer and particularly in the culture medium was greater than that due to osteogenin alone or 1,25(OH),D, alone. The total osteocalcin content (in the cell layer and in the culture medium) of cultures simultaneously exposed to osteogenin at 2.5 ng/ml and 1,25(OH),D, was greater than that of cultures that were exposed to osteogenin at 10 ng/ml and vitamin D, (162%, P
B

A

10

a1

Fig. 8A, B. Influence of osteogenin on collagen type I synthesis. Cells were treated with osteogenin at 2.5 and 10 ng/ml for 3 days; control ( r ) was performed using a same volume of osteogenin buffer. ["IProline was added for the last 24 h and radiolabeled material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography (A). Radioactivity incorporated in the two al(1)and a2(I) bands was quantified and estimated for lo4 cells (B). Data are means +SD (n=6)

3-

2-

1-

O

L

a osteogenin concentration (ndml)

162

Table 2. Effect of osteogenin on cyclic AMP (CAMP)and osteonectin production. Cells were treated for 3 days with osteogenin at 2.5 and 10 ng/ml. cAMP production after human parathyroid hormone (hPTH) treatment during 5 min at 37” C was measured by radioimmunoassay. Results are expressed in pmol cAMP/I06 cells (n=4); ND, not detected. The osteonectin synthesis was estimated using a polyclonal antibody against bovine osteonectin revealed by [1*5I]-protein A as described in Methods. Results are expressed as cpmx 1 OVI06 cells (n= 10) ~~

Treatment

cAMP [l*SI]-proteinA (pmole/l06 cells) anti-osteonectin (cpmx 103/lO6 cells)

Control Osteogenin (2.5 ng/ml) Osteogenin ( 10 ng/ml)

2.lM.7 5.3M.6 ND

1088+280 1 185+110 107M130

osteogenin-treated cultures was increased by 152% (P
Discussion The foregoing results demonstrate that homogenous bovine osteogenin affects proliferation and stimulates the expression of the osteogenic phenotype in cells cloned from human bone marrow in a serum-free medium. The results were reproducible with different human bone marrow clones (n=6). In all cases, exposure of osteoprogenitors to osteogenin resulted in inhibition of DNA synthesis as assessed by [,H]-thymidine incorporation, and cell proliferation. Addition of osteogenin to the cell culture resulted in a dose-dependent increase of type I collagen synthesis. Osteogenin stimulates cellular alkaline phosphatase activity and osteocalcin synthesis, and synergism exists between osteogenin and 1,25(OH),D, on osteocalcin synthesis and secretion into the culture medium. Moreover cAMP stimulation by PTH was increased by osteogenin treatment. The antimitogenic response induced by osteogenin in serum-free medium suggests that osteoprogenitors arising from human bone marrow may have osteogenin binding sites or surface receptors. Little is known about how cell proliferation is regulated. In this cell type osteogenin could appear as a “negative growth regulator” [24, 401. Osteogenin from bovine bone [37, 381 and recombinant BMP-2 114, 421 have been proved ro stimulate the growth of preosteoblasts, periosteal cells and to inhibit the proliferation of established osteoblast-like cells, MC 3T3-El cells. Moreover recombinant human osteogenic protein-I (BMP-7) [33] has been shown to stimulate rat calvaria cell proliferation and differentiation “in vitro”. Osteogenin treatment seems to induce human osteoprogenitors to leave the proliferation phase and enter the GI, or GO, phase of their cell cycle where protein synthesis is particularly promoted. The relationship between cell growth inhibition and expression of osteoblast differentiation can be addressed experimental-

ly by shortening the proliferation period of the oste+ blast. Direct demonstration that the down-regulation of proliferation induces the expression of some genes that are normally expressed later in rat osteoblast develop ment, is derived from experiments in which inhibition of DNA synthesis by hydroxyurea resulted in a fourfold increase in alkaline phosphatase (ALP) mRNA levels [25]. This indicates that the premature down-regulation of proliferation induces the expression of an early marker of the osteoblast developmental sequence [ 181. Increased expression of ALP with decreased proliferative activity has similarly been observed in the osteoblastic osteosarcoma cell line (ROS 17/2.8) (21) in mouse osteoblastlike cells (MC 3T3-El) 1381 and in human osteoblastlike cells 191. Osteogenin stimulated the specific activity of cellular ALP. It has previously been shown 1371 to increase the number of ALP-positive colonies formed by bone marrow stromal cells from rats, as well as ALP activity in rat calvarial osteoblasts. The effect was time-dependent, and maximal effect was observed at the higher doses (10 ng/ml) studied. In our study, maximum effect was observed at 2.5 ng/ml, which seems to indicate that human osteoprogenitor cells are more sensitive to osteogenin than rat osteoblasts. Whether the differing maturity of these cell systems, or species differences are responsible for this higher sensitivity needs to be demonstrated. In like manner BMP-2 and BMP-4 stimulated ALP in calvarial osteoblasts and MC3T3-EI cells 114,421. In a dose-dependent manner osteogenin enhances type I collagen synthesis in human osteoprogenitors. It has previously been shown to increase the synthesis of collagenase digestible proteins by rat calvarial osteoblasts in culture 1371. BMP-4 has been reported to enhance type I collagen synthesis in osteoblast-enriched cultures 171 whereas recombinant BMP-2 has no effect on the expression of collagen mRNA or collagen synthesis by osteoblast-like cells and calvarial cells 1421. Moreover, our study shows that, for osteocalcin, synthesis and secretion by human osteoprogenitor cells are stimulated with a comparable intensity by osteogenin and 1,25(OH),D,, and that a synergic effect is detected using both osteogenin and 1,25(OH),D,. Maximum effect is obtained using 2.5 ng/ml osteogenin. It is well known that 1,25(OH),D,, the hormonally active form of vitamin D,, upregulates osteocalcin synthesis when added to cultured human osteoblasts 13, ‘8, 261 or human osteoprogenitors 126, 361. On the other hand BMP-2 has also been demonstrated to stimulate osteocalcin synthesis when added to osteoblast precursors in the presence of 1,25(OH),D, 1421. 1,25(OH),D, has been reported to increase insulin-like growth factor- 1 (IGF- 1) production by human osteoblasts 181. An increased growth factor production in the presence of vitamin D, could be responsible for the enhancement of the osteogenin effect at the cellular level. This effect could be associated with matrix mineralization events “in vitro” 1261 and “in vivo” [6]. In fact, osteocalcin appears to be regulated by a different pathway when compared with ALP. Osteocalcin has been shown to be expressed in osteoblasts later during the period of extracellular matrix mineral-

163

ization, and its appearance does not seem directly correlated to the inhibition of proliferative activity [25]. Osteocalcin, the only known bone-specific protein [ 171, is reported to appear at a late stage of osteoblast differentiation [16], whereas ALP [34], PTH receptors [31] and type I collagen [34] appear earlier, even in less-differentiated osteoblasts [ 181. The nonoccurence of osteocalcin induction in the first stages of osteoblast maturation is consistent with the concept that there is a second set of genes whose expression is not coupled directly to the down-regulation of proliferation but rather to the development of the more differentiated osteoblast in a mineralized matrix [ I]. The mineralization process may be required to signal expression of a subset of osteoblast phenotype genes, such as ALP [2] and later osteocalcin. With respect to osteogenin stimulation of the osteoprogenitor phenotype regulation, the production of cAMP in response to PTH is more than doubled in human osteoprogenitor cells following treatment with osteogenin. A comparable effect was observed using rat calvarial osteoblasts (371. It is well known that adenylate cyclase activity related to PTH stimulation is a biochemical marker of mature osteoblast differentiation “in vitro” [ 181. In this first “in vitro” study related to the effect of osteogenin on human bone cells, we demonstrated in serum-free cultures that osteogenin inhibited proliferation and stimulated differentiation and maturation of human osteoprenitors arising from bone marrow. It specifically stimulated markers of matrix synthesis (type I collagen, osteocalcin) and cellular alkaline phosphatase activity. Moreover, a synergic effect together with vitamin D, was observed on osteocalcin synthesis and secretion. PTH-mediated intracellular cAMP production was also enhanced. All these markers are associated with the bone-forming functions of mature osteoblasts, and our study demonstrates that osteogenin could play a significant role in the recruitment, differentiation and maturation of stem cells. Little is known concerning the mechanism of action and second messengers of osteogenin. The occurence of binding sites for BMP-4 in MC 3T3-El cells is known [27]. Cross-linking experiments have demonstrated two binding components at 200 kDa and 70 kDa. The effective dose range of bone morphogenic proteins is comparable, BMP-4 and osteogenin (BMP-3) exerted an effect over a dose range of 0.1-10ng/ml. The BMP-2 effect occurs at doses of 10-1000ng/ml [42], whereas the BMP-7 effective dose range is 1-40ng/ml [33]. All these proteins are members of the transforming growth factor-beta (TGF=P) superfamily, and the effective dose for TGF-PI, is 0.01-10 ng/ml. TGF-PI in serum free medium has been proved to stimulate proliferation of rat calvarial cells and synthesis of collagenase-digestible proteins. However, TGF-PI does not enhance the expression of markers characteristic of the osteoblast phenotype. In fact, significant decreases are caused by TGF-PI in ALP-specific activity, PTH-mediated cAMP production [33] and osteocalcin synthesis [ 131. The discrepancy in the effects observed “in vitro” using various bone morphogenic proteins could be the consequence of the

variety of experimental conditions used - assay systems, dose range, treatment conditions. . . Bone morphogenic proteins could act via individual specific receptors, or via a common receptor system in which competition may occur depending on the dose range used. The profound influence of osteogenin in the induction of bone in non-skeletal ectopic sites “in vivo” has been previously demonstrated; the present work shows “in vitro” a significant effect of osteogenin on osteoprogenitor cells isolated and cloned from human bone marrow. The function of osteogenin in the maintenance of the newly differentiated phenotype may be useful in a potential therapeutic approach to bone repair and metabolic bone diseases. Acknowledgments. This work was supported by E.P.R (Etablissement Public Regional) and NIH. We are grateful to Dr. J.C. Le Huec for providing the human bone marrow samples, to P. Seguin for the detection kit for osteocalcin and for monoclonal antibodies against bovine osteonectin, J. Gautreau, and M.O. Lamouret for technical assistance, M. Rouais and V. Silverio for typing the manuscript.

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