Actions of bFGF on mitogenic activity and lineage expression in rat osteoprogenitor cells: effect of age

Molecular and Cellular Endocrinology 150 (1999) 1 – 10

Actions of bFGF on mitogenic activity and lineage expression in rat osteoprogenitor cells: effect of age Hiroshi Tanaka a,b, Hiroyoshi Ogasa a, Janice Barnes a, C. Tony Liang a,* a

Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dri6e, Baltimore, MD 21224 -6825, USA b Department of Orthopedic Surgery, Yamaguchi Uni6ersity School of Medicine, 1144 Kogushi Ube, Yamaguchi, 755 -8505, Japan Received 14 August 1998; accepted 18 February 1999

Abstract Rat osteoprogenitor cells were used to examine the effects of bFGF on DNA synthesis and the expression of osteoblast (OB)-related genes. bFGF, as low as 0.1 ng/ml, stimulated DNA synthesis. bFGF also increased the mRNA level of osteopontin (OP) and decreased that of type I collagen (COL I). When cultures were grown in dexamethasone (DEX) to induce OB lineage commitment, the expression of COL I, alkaline phosphatase (AP) and OP was greatly enhanced. Subsequent incubation with bFGF partially negated the stimulatory effect of DEX on AP and COL I mRNAs. bFGF also inhibited the expression of osteocalcin mRNA in cells grown in 1,25(OH)2D3 and DEX. Combined effects of bFGF with IGF-I or PDGF on DNA synthesis and OP expression were examined. bFGF +IGF-I, but not bFGF +PDGF, was more effective than PDGF alone. By comparing cells from adult and old animals, we found that bFGF-induced mitogenic activity was reduced significantly with age. In contrast, the effect of bFGF on the expression of OB genes was not significantly altered by age. These findings suggest that bFGF plays a dual role as a local positive and negative regulator on proliferation and osteogenic lineage expression, respectively, in osteoprogenitor cells, and that the mitogenic activity in response to bFGF was impaired in aging. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: bFGF; IGF-I; PDGF; Bone marrow osteoprogenitor cells; Proliferation; Gene expression

1. Introduction bFGF was first isolated from brain and pituitary (Gospodarowicz, 1990), however, it has now been demonstrated in a variety of tissues and is known to be multifunctional (Goustin et al., 1986). FGF family is comprised of eight multifunctional proteins found in normal and/or malignant tissues (Sporn and Todara, 1980; Goustin et al., 1986; Gospodarowicz, 1990). The prototype members of the FGF family, aFGF and bFGF, have been suggested to play a role in wound

* Corresponding author. Tel.: +1-410-5588468; fax: + 1-4105588317. E-mail address: [email protected] (C.T. Liang)

repair, inflammation, transformation, and angiogenesis (Shing et al., 1984; Gimenez-Gallego et al., 1985; Goustin et al., 1986; Gospodarowicz, 1990). The two forms of FGF have similar biological effects and share approximately 55% sequence homology and interact with the same cell surface receptors. The mineralized matrix of skeletal tissue is known to contain various growth factors including aFGF and bFGF (Hauschka et al., 1986). Recent studies showed that cultured bovine bone cells synthesized bFGF and stored it in extracellular matrix (Globus et al., 1989). In addition, bFGF has been reported to enhance DNA and collagen synthesis in rat osteoblast cultures and reduce alkaline phosphatase (AP) activity (Canalis et al., 1988). Therefore, it is likely that bFGF may play a prominent role in the regulation of bone remodeling.

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The marrow stromal system, which is associated with the soft connective tissues of marrow and bone surface, contains stem cells capable of giving rise to several cell types including fibroblasts, adipocytes and bone cells (Owen and Friedenstein, 1988). Current concepts propose that these adherent stromal cells are enriched in osteoprogenitor cells that are involved in bone formation. It has been shown that fibroblast-like cells derived from bone marrow have the ability to produce a bonelike tissue in diffusion chambers (Ashton et al., 1980; Bab et al., 1986) and in collagen-hydroxyapatite implants (Goshima et al., 1991; Quarto et al., 1995) in vivo. Recently it has been demonstrated that bone marrow stromal cells, when cultured in the presence of b-glycerophosphate, ascorbic acid, and dexamethasone (DEX), can induce the formation of mineralized bonelike nodules in vitro as characterized by the appearance of collagen, osteocalcin (OC) and osteonectin and the hydroxyapatite nature of the deposited mineral (Maniatopoulos et al., 1988). In aging, bone formation activity was significantly reduced (Liang et al., 1992). It has been postulated that a deficiency in osteoprogenitor cells plays a major role in the decline of bone induction activity in senesense (Quarto et al., 1995). Proliferation and osteogenic lineage expression of bone progenitor cells are regulated by local and systemic growth factors. It is quite possible that changes in the availability of growth factors in old bones or the response of osteoprogenitor cells from old bones to growth factors may reduce the number of progenitor cells and osteoblasts in old bones and contribute to the decline in bone formation activity. Indeed, we have shown that reduced expression of IGF-I (Tanaka et al., 1996), impaired response of osteoprogenitor cells from old animals to IGF-I and PDGF (Slootweg et al., 1990; Tanaka et al., 1994; Tanaka and Liang, 1995) are the possible causes for impaired bone formation activity in old animals. In the present study, we extended our study to examine the response of osteoprogenitor cells to bFGF, an important regulatory growth factor for bone remodeling. Results obtained from this study can strengthen the general concept that the impaired response of osteoprogenitor cells to growth factors may contribute to the insufficient bone formation activity commonly observed in elderly people.

(adult) and 24-month (old) male Wistar rats (the Animal Facility, Gerontology Research Center, National Institute on Aging), with Coon’s modified Ham’s F-12 medium (NIH Media Unit, Bethesda, MD) supplemented with 10% fetal calf serum (Biofluids, Rockville, MD). Single cell suspension was obtained by passage through 18- and 23- gauge needles. Cell suspension was diluted with saline for cell counting. After a brief incubation in 1% Zapoglobin (Curtin Matheson Scientific, Houston, TX) to lyse red blood cells, the number of nucleated cells were counted with a Coulter Counter (Coulter Corporate Communications, Hialeah, FL). Cells were plated out into 60-mm plates (Falcon Becton Dickinson, Oxnard, CA) at a density of 1× 106 nucleated cells/cm2 and cultured in Coon’s modified Ham’s F-12 medium with 10% fetal calf serum, 50 mg/ml L-ascorbic acid, 100 i.u./ml penicillin and 100 mg/ml streptomycin (NIH Media Unit, Bethesda, MD) at 37°C in 5% CO2 in a humidified incubator. Media were changed after 4–5 days and the non-adherent cells were discarded. Thereafter, media were changed twice weekly until the cultures were confluent. Confluent primary stromal cells were detached with 0.05% (w/v) trypsinEDTA (NIH Media Unit, Bethesda, MD) and pooled from three rats for subsequent experiments examining [3H]thymidine incorporation or gene expression. The experimental protocol used in this study was reviewed and approved by the Animal User and Care Committee at the Gerontology Research Center, National Institute on Aging.

2.2. [ 3H]thymidine incorporation in osteoprogenitor cells Pooled primary progenitor cells were plated at 50 000 cells/well in 24-well dishes in the same media described above for primary culture. After 3 h of incubation, the media were replaced with fresh media without serum. After serum depletion for 20 h, cells were treated with growth factors; human recombinant bFGF (BACHEM, INC., Torrance, CA), human recombinant IGF-I (BACHEM, INC., Torrance, CA), human recombinant PDGF (BACHEM, INC., Torrance, CA), for 24 h in serum-free media and pulse labeled with [3H]thymidine during the last 3 h. Cells were then fixed in 10% cold TCA, lysed in 0.1 N sodium hydroxide, and assayed for acid insoluble [3H]thymidine incorporation.

2. Materials and methods

2.3. Preparation of RNA 2.1. Primary cultures of rat bone marrow osteoprogenitor cells A suspension of marrow stromal cell was prepared as described previously (Tanaka et al., 1994). Marrow cells were flushed from the midshafts of femurs of 6-

Pooled primary cells were plated at a density of 4.5×103 cells/cm2 in the same medium as primary culture. DEX and 1,25(OH)2D3 were added to selected dishes at a final concentration of 10 − 8 M. The media were changed twice weekly. When cells were confluent

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(approximately 8–10 days), growth factors were added and incubated for 2 days. Total RNA from progenitor cells was prepared with RNA STAT-60 (Tel-Test, Friendswood, TX) following the instructions provided by the supplier. The purity of RNA was monitored by the ratio of absorbencies of the samples at 260 and 280 nm.

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groups. Statistical difference between two groups was determined by paired and unpaired t-test.

3. Results

3.1. Effect of bFGF on [ 3H]thymidine incorporation into DNA

2.4. Northern analysis Northern blot analysis was carried out as described previously (Liang et al., 1992). Briefly, total RNA (20 mg) was denatured, separated on a 1% agarose gel (FMC Co., Rockland, ME) and transferred to Gene Screen paper (New England Nuclear, Boston, MA). The cDNA probes used were rat AP (0.6 kb) (Noda et al., 1987), rat procollagen a1 (I) (1.3 kb) (Genovese et al., 1984), rat OC (0.5 kb) (Yoon et al., 1988) and rat osteopontin (OP) (1.3 kb) (Yoon et al., 1987). 32P-labeled probes were prepared with a multiprime labeling kit (Amersham, Arlington Height, IL). Blots were hybridized at 42°C for 24 h in 50% formamide. After hybridization, blots were washed with 2× SSC (0.15 M NaCl and 0.015 M Sodium citrate) and 0.1% SDS twice at room temperature, 1 ×SSC and 0.1% SDS at 50°C, and 0.5 × SSC and 0.1% SDS at 60°C. For AP and OC, the last wash was carried at 57°C. Autoradiogram was exposed at −70°C with an intensifying screen. A single band of 0.6, 1.4 and 2.5 kb was labeled with probes for OC, OP and AP, respectively. Multiple transcripts of 5.1 and 5.7 kb were labeled with procollagen a1 (I) probe.

The effect of bFGF on DNA synthesis in osteoprogenitor cells was assessed by measuring the incorporation of [3H]thymidine into DNA. bFGF stimulated DNA synthesis in progenitor cells in a dose-dependent manner between 0.1–100 ng/ml (Fig. 1). Stimulation of 0.9–fold was obtained at 0.1 ng/ml of bFGF. A 5.5fold increase was achieved with bFGF at 100 ng/ml.

3.2. Effect of bFGF on the expression of osteoblast-related markers Confluent progenitor cells were treated with different doses of bFGF for 2 days. The levels of OP, type I collagen (COL I) and AP mRNAs were determined by dot blot analysis and normalized to total poly(A) + RNA. Treatment of progenitor cells with bFGF stimulated mRNA expression of OP in a dosedependent manner (Fig. 2A). Increase of 1.1–4.0-fold was obtained with 10–250 ng/ml of bFGF. In contrast, bFGF at doses higher than 50 ng/ml decreased COL I mRNA significantly. The AP mRNA was not affected. The stimulatory effect of bFGF on OP mRNA was confirmed by Northern blot. A representative Northern blot (Fig. 2B) showed a clear increase in OP mRNA.

2.5. Dot blot analysis Total RNA was applied onto Gene Screen membrane with a Minifold apparatus (Schleicher & Schuell, Waburn, MA). The membrane was baked at 80°C for 2 h to immobilize RNA and hybridized with various cDNA probes, as described above. A polythymidylate probe was used for determination of poly(A) + RNA (Tanaka et al., 1996). Hybridization and washing of the dot blots followed the procedures described above. Radioactivity of each dot on the filter was measured with a Betascope 603 Blot analyzer (Betagen Corp., Waltham, MA). The levels of mRNAs were expressed as the ratio of each mRNA to total poly(A) + RNA. This normalized mRNA level was designated to be 1.0 for control cells. In experiments using DEX-treated cells, the normalized mRNA level for DEX-treated cells was designated to be 1.0.

2.6. Statistics Data shown are the mean9 S.E. An ANOVA test was performed to determine the difference between

Fig. 1. Effect of bFGF on [3H]thymidine incorporation into DNA in rat bone marrow progenitor cells. Primary progenitor cells from three rats were pooled and plated at 5×104 cells/well in 24-well dishes. Serum-starved cells were incubated with growth factor at the concentration shown. Details of the experimental protocol are described in Section 2. Values shown are means9S.E. for a representative experiment with triplicate determinations. The experiment was repeated twice with similar results. P value for ANOVA analysis is shown in the figure. For comparison between two groups: *P 90.05 vs control, paired t-test.

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sion of osteoblast markers. Cells were grown to confluence with DEX and subsequently treated with bFGF for 2 days. In agreement with our previous report (Tanaka and Liang, 1995), DEX treatment increased the level of OP, COL I and AP mRNAs 10-, 2.2- and 1.5-fold, respectively (Fig. 3A). Incubation of confluent DEX-treated cells with bFGF at 50 ng/ml did not affect the levels of OP mRNA. In contrast, bFGF treatment decreased the elevated levels of AP and COL I mRNAs 33% and 41%, respectively. Northern blot analysis confirmed the inhibitory effect on AP and COL I mRNAs in DEX-treated cells (Fig. 3B).

Fig. 2. Effect of bFGF on the expression of osteoblast markers in bone marrow progenitor cells. Primary progenitor cells from three rats were pooled and plated at a density of 4.5 × 103 cells/cm2. Confluent cultures were treated with bFGF for 48 h at the concentration shown. RNA was prepared and analyzed by dot blot hybridization and Northern blot analysis. (A) The mRNA levels were quantitated by dot blot analysis and expressed as the ratio to poly(A) + RNA. Data shown are means9S.E. for four independent experiments. P value for ANOVA analysis is shown in each figure. For comparison between two groups: * PB 0.05 vs control, paired t-test. (B) A representative Northern blot analysis of osteopontin mRNA is shown. Total RNA (20 mg) was applied in each lane. Ethidium bromide staining of ribosomal RNA in the same gel is also shown. Northern analysis was repeated twice with different samples with similar results.

Messages for COL I and AP were barely visible in Northern blot (data not shown).

3.3. Effect of bFGF in DEX-treated cells Previously, it has been shown that treatment of progenitor cells with DEX can induce the differentiation pathway toward osteoblasts and stimulate the expres-

Fig. 3. Effect of bFGF on mRNA expression in DEX-treated cells. Pooled primary progenitor cells were grown in DEX (10 − 8 M)-supplemented media until confluent and treated with 50 ng/ml of bFGF for 48 h. (A) The mRNA levels were quantitated by dot blot analysis and expressed as the ratio to poly(A) + RNA. Levels of mRNAs in DEX-treated cells were designated to have 1 unit. Data shown are means 9S.E. for four independent experiments. P value for ANOVA analysis is shown in each figure. For comparison between two groups: c PB0.01 vs control (-DEX), * P B0.05 vs DEX-treated control ( + DEX), paired t-test. (B) A representative Northern blot analysis of osteoblast markers is shown. Total RNA (20 mg) was applied in each lane. Ethidium bromide staining of ribosomal RNA in the same gel is also shown. Northern analysis was repeated twice with different samples with similar results.

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adult and old animals. As shown in Fig. 6, the stimulation of DNA synthesis was observed at 0.1 ng/ml of

Fig. 4. Effect of bFGF on the expression of OC mRNA. Pooled primary progenitor cells were grown in media supplemented with DEX (10 − 8 M) and 1,25-(OH)2D3 (10-8 M). Confluent cultures were treated with 50 ng/ml of bFGF for 48 h. Expression of OC was analyzed by Northern blot. Total RNA (20 mg) was applied in each lane. Ethidium bromide staining of ribosomal RNA in the same gel is also shown. The experiment was repeated twice with similar results.

The effect of bFGF on the mRNA level OC was also examined. The expression of OC was minimal in control cells and can be induced several fold in cells maintained with DEX and 1,25(OH)2D3. As shown in Fig. 4, treatment of confluent cultures, maintained in DEX and vitamin D, with bFGF at 50 ng/ml reduced OC mRNA level more than 50%.

3.4. Interactions of bFGF with IGF-I or PDGF on DNA synthesis and the expression of OP To determine whether bFGF interacts with the responses of osteoprogenitor cells to other local growth factors, DNA synthesis and the expression of OP were examined in cells treated with bFGF and PDGF or bFGF and IGF-I. As shown in Fig. 5A, bFGF, PDGF and IGF-I increased [3H]thymidine incorporation 4.6-, 7.2- and 1.2-fold, respectively, over the control. However, the combination of PDGF and bFGF yielded no further stimulation above that observed with PDGF alone. In contrast, DNA synthesis was stimulated 8fold in cells treated with bFGF and IGF-I which was greater than the sum of the individual responses (4.6and 1.2-fold, respectively, for bFGF and IGF-I). Parallel experiments were carried out to determine whether bFGF can interact with IGF-I or PDGF on the expression of OP mRNA. bFGF, PDGF and IGF-I at 50 ng/ml stimulated OP mRNA expression 2.6-, 2.0and 0.2-fold over the control, respectively (Fig. 5B). The combined treatment of PDGF and bFGF resulted in no stimulation above that observed with PDGF or bFGF alone. Treatment of cells with IGF-I and bFGF stimulated OP expression 4.5-fold which was higher than the sum of individual responses (2.6- and 0.1-fold, respectively, for bFGF and IGF-I).

3.5. Effect of age on DNA synthesis in osteoprogenitor cells DNA synthesis was compared in cells derived from

Fig. 5. Effect of bFGF in combination with IGF-I or PDGF on DNA synthesis and OP expression in stromal cells. (A) Serum-starved bone marrow progenitor cells were incubated with growth factors at 100 ng/ml. Details of the experimental protocol are described in Section 2. Values shown are means9S.E. for a representative experiment with triplicate determinations. The experiment was repeated twice with similar results. P value for ANOVA analysis is shown in the figure. For comparison between two groups: * PB 0.05 vs control, c P B0.05 vs +bFGF or +IGF-I, paired t-test. (B) Confluent progenitor cells were treated with 50 ng/ml of growth factors for 48 h. The mRNA levels for OP were quantitated by dot blot analysis and expressed as the ratio to poly(A) + RNA. Data shown are means 9S.E. for four independent experiments from different animals. P value for ANOVA analysis is shown in the figure. For comparison between two groups: * PB 0.05 vs control, c PB 0.05 vs + bFGF or +IGF-I, paired t-test. A representative Northern blot of OP mRNA is shown. Total RNA (20 mg) was applied in each lane. Ethidium bromide staining of ribosomal RNA in the same gel is also shown.

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Fig. 6. Effect of age on bFGF-stimulated DNA synthesis in osteoprogenitor cells. DNA [3H]thymidine incorporation was determined as described in the legend to Fig. 1. Data shown are means 9 S.E. for quadruple measurements. The experiment was repeated three times with similar results. *PB0.05 vs cells from adult animals, unpaired t-test.

and osteoblasts in old animals and lead to impaired bone formation activity. The present study was undertaken to gain better insight into the role of bFGF on replication and the progressive phenotype expression of rat bone marrow stromal cells which are believed to contain osteoprogenitor cells. We then examined the possible defects in these responses in aging. Initially, we examined the effect of bFGF on DNA synthesis and the expression of osteoblast markers in progenitor cells derived from adult rats (6-month old). In cultures of marrow progenitor cells, treatment with bFGF as low as 0.1 ng/ml for 24 h stimulated incorporation of [3H]thymidine into DNA. Similar results have been reported in previous studies in cultured fetal rat calvariae cells (Gospodarowicz et al., 1987; Canalis et al., 1988; McCarthy et al., 1989) and bone marrow

bFGF in cells from both age groups. However, the magnitude of stimulation by bFGF in cells derived from old animals was approximately half of that obtained for cells from adult animals.

3.6. Effect of age on the expression of osteoblast-related genes The expression of osteoblast-related genes was examined in cells derived from adult and old animals. The mRNA levels of OP, COL I and AP, measured as ratio to poly(A) + RNA, were not different for cells from adult and old bones (legend to Fig. 7). bFGF stimulated the expression of OP, inhibited that of COL I and did not significantly activate the expression of AP in cells from both age groups (Fig. 7). Age did not affect these responses significantly. One exception was that the stimulation of OP expression was higher in cells from old animals.

4. Discussion Recently, it has been summarized in a review that growth factors and cytokines play an important role in bone cell metabolism (Canalis et al., 1991). Considerable interest has also been focused on the effect of these regulators on proliferation and phenotype expression of osteoprogenitor cells along the osteogenic pathway. In addition, question arises whether the deficiency in the response of osteoprogenitor cells to growth factors may contribute to the insufficient number of progenitor cells

Fig. 7. Effect of age on bFGF-induced changes in gene expression in osteoprogenitor cells. The expression of osteoblast-related genes was determined as described in the legend to Fig. 2. Equal amount of RNA samples obtained from two age groups were used in the same dot blot analysis. The basal level of mRNA expressed as ratio to poly(A) + RNA was not significantly different between adult and old animals. Data shown are means 9 S. E. for four independent experiments. *PB 0.01 vs cells treated with 50 ng/ml of bFGF, paired t-test.

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stromal cells from very young rats (Noff et al., 1989; Pitaru et al., 1993). Based on its effect on the growth of ostoblasts, bFGF is considered as a positive regulator of bone formation in adult animals. The effect of bFGF on the expression of osteoblastrelated genes was also examined in adult progenitor cells. bFGF at doses higher than 50 ng/ml significantly reduced the level of COL I mRNA in adult progenitor cells. In contrast, AP mRNA was not affected by bFGF. Osteoprogenitor cells can be induced to commit and differentiate along the osteoblast lineage by treatment with DEX (Leboy et al., 1991). Both COL I and AP are considered as markers for preosteoblasts and their expression are detected in the early phase of active proliferation of osteoblasts (Owen et al., 1990; Liang et al., 1992). In the present study, we showed that incubation of confluent DEX-treated progenitor cells with 50 ng/ml of bFGF for 2 days reduced the elevated levels of COL I and AP mRNAs. The inhibitory effect of bFGF on COL I expression may not be exclusively associated with phenotype development of osteoprogenitor cells. It has been shown that bFGF inhibits collagen synthesis by a transcriptional mechanism and the COL I gene promoter contains DNA sequences which mediate the inhibitory effect by bFGF (Hurley et al., 1991). The effect of bFGF on the expression of COL I and AP has been examined previously using cells from different sources. In rat osteosarcoma cells, bFGF inhibits the expression of AP and COL I (Rodan et al., 1989). However, bFGF decreases AP activity, but increases the level of COL I mRNA in cultures enriched in parietal derived preosteoblasts and osteoblasts (McCarthy et al., 1989). In bone marrow stromal cell cultures treated with DEX, ascorbic acid and b-glycerophosphate, bFGF increases both AP activity and [3H]proline incorporation into collagen (Pitaru et al., 1993). It is not clear whether the discrepancy is the result of differences in culture condition, cell type, the stage of cells along the osteogenic pathway, the age of the animals that these cells were derived from or other unknown parameters. Our results clearly showed that bFGF increased OP mRNA in a dose-responsive manner. The stimulatory effect can be obtained as low as 10 ng/ml of bFGF. In DEX-treated cells, the expression of OP mRNA was stimulated 10-fold. The elevated level of OP mRNA in DEX-treated cells was not enhanced further by bFGF treatment. The expression of OC was not detectable in cultured progenitor cells. Combined treatment with DEX and vitamin D enhanced the expression of OC mRNA more than 10-fold. Subsequent incubation with 50 ng/ml of bFGF significantly reduced the level of OC mRNA. The significance of the finding that bFGF regulates the expression of OP and OC is not clear. Although OP and OC are postulated to be involved in bone formation, the precise role of these proteins in

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bone remodeling have not been defined. In cultured fetal calvarial cells, the expression of OP and OC is stimulated at the onset of mineralization but not during active cell proliferation and extracellular matrix maturation (Owen et al., 1990). In rat femur, the expression of OP and OC is elevated during both bone formation and resorption following marrow injury (Liang et al., 1992). Although OP was first isolated from mineralized bone matrix (Franzen and Heinegard, 1984), it is also produced in many other tissues including skin, kidney, uterus, neural cells and others (Nomura et al., 1988; Young et al., 1990; Tanaka et al., 1996), suggesting that it may play a wider role than previously realized. OP enhances cell attachment (Oldberg et al., 1986), and has been postulated to act as the anchorage of osteoclasts to bone matrix (Reinholt et al., 1990). A recent study has shown that potent bone-resorbing agents such as IL-1, TNF-a, LPS and 1,25(OH)2D3 increase the expression of OP mRNA in both the clonal osteoblastic cells and the primary osteoblast-like cells, suggesting that OP is a possible candidate involved in the interaction between osteoblasts and osteoclasts (Jin et al., 1990). bFGF has been shown to stimulate the resorption of cultured fetal rat long bone (Simmons and Raisz, 1991). It is conceivable that bFGF may stimulate bone resorption via its effect on OP expression. In bone, osteoprogenitor cells and osteoblasts are exposed to multiple local growth factors constantly and the effects of one of these multifunctional factors are likely modulated by the presence of others. In addition to bFGF, IGF-I and PDGF are known to regulate the proliferation and the expression of osteoblast-related genes in stromal cells (Tanaka et al., 1996; Tanaka and Liang, 1996). Here we demonstrate that there is an augmentative stimulatory effect on DNA synthesis and OP gene expression by IGF-I and bFGF. Interaction between IGF-I and bFGF on mitogenic activity has been reported in osteoblasts (Slootweg et al., 1990), chondrocytes (Hiraki et al., 1987), heart mesenchymal cells (Balk et al., 1984) and adipocytes (Butterwith et al., 1992). This type of interaction between growth factors is not limited to bFGF and IGF-I. PDGF and IGF-I are known to augment each other on cell proliferation and OP expression in marrow progenitor cells (Tanaka and Liang, 1995). Synergistic interaction can be caused by activation of different signal transduction mechanisms or altering the number or the affinity of the receptor toward one growth factor by the other growth factor. A previous study has shown that bFGF increases the number of IGF-I receptors in muscle cells (Rosenthal et al., 1991). Failure of bFGF and PDGF to elicit an additional increase in activity over that obtained with PDGF alone suggests that these two growth factors may share the same pathway. However, synergistic effect of the same combination has been demonstrated in adipocytes (Butterwith et al., 1992).

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The effect of bFGF on bone formation has been examined in several studies. bFGF stimulate the ingrowth rate of new bone in bone induction chambers implanted in rat tibiae (Wang and Aspenberg, 1996). However, bFGF at high dose is not effective in stimulating the ingrowth rate. Systemic administration of bFGF on bone formation has been examined in growing rats (Nagai et al., 1995). At low dose, bFGF stimulates endosteal and endochondral bone formation and depresses periosteal bone formation. At high dose of bFGF, calcification at the growth plate metaphyseal junction and the endocortical surface are retarded. The longitudinal growth rate, endocortical labeled surface and bone formation rate are also decreased with high dose of bFGF. The causes of the dose dependent variation are not discussed in these studies. In transgenic mice with overexpression of bFGF, skeletal malformations including shortening and flattening of long bones were observed (Coffin et al., 1995). In the present study using cultured progenitor cells, we showed that bFGF can stimulate the proliferation of these osteoprogenitor cells in the range of 0.1 – 10 ng/ml of bFGF. However, the expression of osteogenic markers such as COL I and AP was inhibited at higher dose of bFGF ( \ 50 ng/ml). Perhaps the dose dependent effects of bFGF on bone metabolism observed in in vivo are the result of the differential effects of bFGF on cell proliferation and osteogenic function as shown in our studies using cultured cells. In the current study we also compared the bFGF induced changes in the mitogenic activity and osteogenic lineage expression in osteoprogenitor cells derived from marrow of adult and old bones. The effective dose of bFGF to stimulate cell proliferation was not different between cells from two age groups. However, the maximal mitogenic activity was reduced about half for cells from old animals. In contrast, the expression of osteoblast-related genes in response to bFGF was, by and large, not altered with age. Similar findings of age-associated differences in the response of bone progenitor cells to growth factors have also been observed in studies with PDGF (Tanaka and Liang, 1995). Even though the mitogenic activity of progenitor cells from old animals in response to bFGF was reduced, positive increase in the proliferation of progenitor cells by bFGF was still possible in these animals. Therefore, it can not exclude the possible application of bFGF in treatment of age-associated osteoporosis. One useful strategy would be using the combination of IGF-I and a loss of bFGF which should maximize the mitogenic activity of progenitor cells. Previously, we showed that IGF-I infusion can increase bone formation activity in old rats and substantially negate the decrease in trabecular bone (Wakisaka et al., 1998). It would be interesting to test the combined effect of bFGF and IGF-I in the same model.

Previously, we and other laboratories have shown that the number of osteoprogenitor cells reduces with age. This age-related abnormality may be due in part to the deficient mitogenic response of progenitor cells from old animals to growth factors as described above. It should be noted that despite the consistent results from numerous animal studies which show that the number of osteoprogenitor cells reduces with age, similar findings have not been reported in limited studies using human cells. It is unclear whether this is due to differences in species, experimental protocol or other unknown factors. In conclusion, we have demonstrated that bFGF modulates proliferation and the expression of osteoblast-related genes in adult bone marrow osteoprogenitor cells. Our findings suggest that bFGF may play a dual role in the complex regulation of bone remodeling by local growth factors. We have also shown that the interaction between growth factors may augment the effect of individual growth factor on bone cell metabolism. Perhaps, a better strategy can be developed for possible use of growth factors in the treatment of impaired bone activity associated with osteoporosis.

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