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Endocrinology Vol. 141, No. 6 2075-2083
Copyright © 2000 by The Endocrine Society

ARTICLES

Mechanisms of Fibroblast Growth Factor-2 Modulation of Vascular Endothelial Growth Factor Expression by Osteoblastic Cells

Pierre B. Saadeh, Babak J. Mehrara, Douglas S. Steinbrech, Jason A. Spector, Joshua A. Greenwald, Gyu S. Chin, Hikaru Ueno, George K. Gittes and Michael T. Longaker

Laboratory of Developmental Biology and Repair, Department of Surgery, New York University School of Medicine, New York, New York 10016; Department of Surgery, University of Connecticut (P.B.S.), Farmington, Connecticut 06032; Department of Surgery, New York University (B.J.M., D.S.S., J.A.S., J.A.G., G.S.C., G.K.G., M.T.L.), New York, New York 10016; and Department of Cardiology, Kyushu University School of Medicine (H.U.), Fukuoka 812, Japan

Address all correspondence and requests for reprints to: Michael T. Longaker, M.D., Laboratory of Developmental Biology and Repair, Room H-169, New York University Medical Center, 550 First Avenue, New York, New York 10016. E-mail: michael.longaker{at}med.nyu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-ß (TGFß) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFß in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability of VEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFß1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFß2 and TGFß3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFß2 and TGFß3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFß, we disrupted TGFß signal transduction (using adenovirus encoding a truncated form of TGFß receptor II), which attenuated TGFß1 induction of VEGF mRNA, but did not impede FGF-2 induction of VEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFß activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DESPITE THE INTUITIVE importance of angiogenesis to the processes of bone development, growth, and healing, the molecular underpinnings of this assumption remain unclear. Gross and histological evidence of the dependence of bone on an adequate vascular supply includes the findings that vascularized bone grafts maintain more osseous mass than nonvascularized bone grafts (1), the interruption of blood supply to bone results in avascular necrosis (2), osteocyte survival requires a less than 0.1 mm proximity to nutrient vessels (3), and there exists a close correlation between the rate of osteonic bone deposition and the vascular surface area (4). Additionally, a pivotal event during endochondral bone development and repair is the invasion of hypertrophic chondrocytes with new capillaries from existing blood vessels in the developing periosteum (5). Osteogenic cells associate with the invading vasculature and establish the primary spongiosa as calcified cartilage is destroyed. This results in the formation of a scaffold with which osteogenic cells associate and begin to develop bone (6) and the marrow stoma, an important regulator of postnatal skeletal formation.

On a molecular level, cytokines that modulate bone development have been identified and include members of the fibroblast growth factor (FGF) and transforming growth factor-ß (TGFß) families (6, 7, 8, 9). FGF-2, a highly conserved heparin-binding growth factor, has been implicated in the control of skeletal and neural differentiation, angiogenesis, wound healing, and tissue repair (10). FGF-2 is involved in early limb development (11, 12, 13), and in the developing skeleton, FGF-2 and FGF receptor 1 colocalize to proliferating chondrocytes of the epiphyseal growth plate (14). In vitro, FGF-2, which is produced by osteoblastic cells and is stored in the extracellular matrix (15, 16), stimulates osteoblastic proliferation (17) and TGFß1 production (18).

Given the involvement of FGF-2 in differentiation, growth, and repair and the dependence of these related processes on an adequate blood supply, it is not surprising that FGF-2 is a potent modulator of angiogenesis. FGF-2 stimulates endothelial cell migration, proliferation, and angiogenesis in vitro (10) and has been implicated in embryonic vascular development (14, 19). Additionally, FGF-2 induces vascular endothelial growth factor (VEGF) in endothelial cells (20). Finally, in an in vitro angiogenic assay, VEGF-induced angiogenesis is dependent on FGF-2 (21).

VEGF, a dimeric heparin-binding glycoprotein, plays a central role in the development and modulation of angiogenesis. VEGF is expressed in highly vascular tissues and is an endothelial cell-specific mitogen (22). VEGF receptor knockout mice lack adequate blood vessel formation (23), whereas loss of a single VEGF allele is lethal in the mouse embryo (24). With respect to bone, VEGF is expressed in both the normal rat tibia (25) and mandible (26) and by unstimulated osteoblastic cells (27). Additionally, VEGF expression in osteoblastic cells is increased by several cytokines and growth factors, including TGFß1, PGE1, PGE2, insulin-like growth factor, platelet-derived growth factor, and 1,25-dihydroxyvitamin D3 (25, 26, 28, 29).

Although FGF-2 may exert its angiogenic effects both directly and through VEGF, an additional potential angiogenic mechanism may involve alterations in TGFß biological activity, as FGF-2 stimulates both increased TGFß1 production as well as latent TGFß activation. The three defined TGFß isoforms in mammals, TGFß1, TGFß2, and TGFß3, are ubiquitous cytokines with pleiotropic effects and have been implicated in osteoblastic proliferation and differentiation. TGFß1, the largest source of which is bone (30), is expressed in high levels during bone growth and development (27, 31), localized to cells within the developing skeleton (32), and increased in fracture healing (33). In vitro, TGFß1 stimulates osteoblastic migration, modulates osteoblastic proliferation, and increases VEGF expression in osteoblastic cells (26, 34, 35). Despite these findings, the effects of TGFß2 and TGFß3 on VEGF production remain unknown. Given the variable affinity of TGFß receptors for TGFß isoforms, in addition to the finding that individual TGFß subtypes have been implicated in the regulation of palatal and craniofacial development as well as scar formation, analysis of isoform-specific regulation of VEGF expression is warranted. (36, 37, 38, 39) Additionally, the interactions between the TGFß isoforms and FGF-2 in the control of VEGF remain undefined.

Given the importance of FGF-2 and VEGF in the related processes of angiogenesis and bone development, we proposed that FGF-2 may regulate VEGF expression in osteoblastic cells. We found that FGF-2 increased VEGF mRNA in both nonimmortalized and clonal osteoblastic cells. These data suggest a transcriptional mechanism of FGF-2-regulated VEGF expression. Additionally, FGF-2 increased VEGF protein production by osteoblastic cells. Both TGFß2 and TGFß3 increased VEGF mRNA and protein in osteoblastic cells. To dissect the effect of FGF-2 on VEGF from possible FGF-2-induced/activated TGFß (all isoforms of which also increased VEGF), we employed a recombinant adenovirus to mediate the transfer of a dominant negative truncated TGFß receptor II gene, thereby disrupting TGFß signal transduction. Whereas osteoblastic cells transfected with the dominant negative truncated TGFß receptor II adenovirus demonstrated significantly decreased induction of VEGF mRNA by exogenous TGFß1, FGF-2 induction of VEGF mRNA remained similar to control cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture plates and flasks were purchased from Fisher Scientific (Pittsburgh, PA). DMEM, {alpha}MEM, 0.05% trypsin-EDTA, PBS, FBS, and cell culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). Recombinant human FGF-2, TGFß2, and TGFß3 were obtained from Life Technologies, Inc.. Actinomycin D and cycloheximide were purchased from Sigma (St. Louis, MO). Collagenase A and dispase II were obtained from Roche Molecular Biochemicals (Mannheim, Germany).

Animals
Pregnant Sprague Dawley rats were purchased from Taconic Farms, Inc. (Germantown, NY) and housed in separate cages. Animals were kept under a constant 12-h light, 12-h dark schedule and fed Purina rodent chow (Ralston Purina Co., St. Louis, MO) ad libitum. All procedures were approved by the institutional care and use committee at New York University Medical Center. On gestational day 21, the pregnant mothers were killed with carbon dioxide, and the pups were harvested.

Cell culture
Fetal rat calvarial (FRC) cells were cultured from FRC explants according to a modification of the methods described by Centrella et al. (40). Frontal and parietal bones from gestational 21-day-old Sprague Dawley fetal rats were sterilely stripped of all surrounding soft tissue. Calvaria were washed with sterile PBS containing antibiotic/antimycotic and underwent serial digestions (0.1% collagenase A/0.2% dispase II) in a shaking incubator at 180 rpm for 10 min at 37 C. Fractions 2–5 were pooled, centrifuged, and resuspended in medium (DMEM supplemented with 10% FBS, 100 µg/ml penicillin G, 50 µg/ml streptomycin, and 0.25 µg/ml amphotericin). Cells (1.5 x 106 cells/flask) were plated in 75-cm2 flasks and reached confluence (2.5 x 106 cells/flask) in 3 days, after which they were trypsinzed with 0.05% trypsin and replated at a subconfluence (2.0 x 106 cells/flask). The next day, confluent passage 2 cultures were obtained and used for all experiments. Medium was changed every 2 days and after replating. Verification of osteoblastic lineage was performed by mineralized bone nodule formation assay, alkaline phosphatase staining, and Northern blot analysis for osteocalcin (data not shown).

MC3T3-E1 cells, a mouse clonal osteoblastic cell line (gift from Dr. A. Gosain, Medical College of Wisconsin, Milwaukee, WI), were grown in DMEM supplemented with 10% FBS, 100 µg/ml penicillin G, 50 µg/ml streptomycin, and 0.25 µg/ml amphotericin. Medium was changed every 2–3 days. Confluent MC3T3-E1 cell cultures (2.5 x 106 cells/T75 flask) were trypsinized with 0.05% trypsin and replated in a 1:2 ratio. All cultures were maintained in a humidified atmosphere consisting of 95% air-5% CO2 at 37 C.

Probe preparation
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was a 1-kb probe from CLONTECH Laboratories, Inc. (Palo Alto, CA). A 410-bp probe against mouse VEGF was PCR generated from whole mouse embryo cDNA using the following PCR primer sequences: 5'-CGAGACCCTGGTGGACATCT-3' and 5'-CACCGCCTCGGCTTGTCAC-3'. Resultant bands were cloned into PCR.1 plasmids (Invitrogen, Carlsbad, CA) and sequenced to confirm sequence identity (GenBank accession no. NM 009505). The resultant probe was extensively tested using known controls and was found to be capable of recognizing all murine VEGF isoforms (not shown). Probe was generated after EcoRI digestion and gel purification. One hundred nanograms of each probe were labeled with [{alpha}-32P]deoxy-CTP using random oligonucleotide primers and Klenow fragment (Ready To Go labeling beads, Pharmacia Biotech, Cambridge, UK). Unincorporated nucleotides were removed using Sephadex G-50 DNA grade nick columns (Pharmacia Biotech). All probes had a specific activity of more than 105 cpm/ml hybridization solution.

RNA extraction and Northern analysis
Confluent FRC cells (passage 2) and MC3T3-E1 cells in T75 flasks were stimulated with 12.5 ng/ml FGF-2 in antibiotic-containing serum-free medium for 0, 3, 6, or 24 h. Confluent MC3T3-E1 cells in T75 flasks were stimulated with 2.5 ng/ml TGFß2 or 2.5 ng/ml TGFß3 in antibiotic-containing serum-free medium for 0, 3, 6, or 24 h. In experiments designed to investigate the effect of FGF-2 on VEGF mRNA stability, transcription was interrupted with actinomycin D (5 µg/ml) after 5 h of stimulation with FGF-2 (12.5 ng/ml) in serum-free medium. To investigate disruption of protein synthesis, confluent MC3T3-E1 cells in T75 flasks were treated with cyclohexamide (10 µg/ml) with or without FGF-2 (12.5 ng/ml) In addition, to investigate disruption of gene transcription, confluent MC3T3-E1 cells in T75 flasks underwent 1 h of pretreatment, followed by 6 h of exposure to actinomycin D (5 µg/ml) with or without FGF-2 (12.5 ng/ml) (28).

Experiments were designed to disrupt TGFß signal transduction. A dominant negative truncated TGFß receptor II adenovirus and a ß-galactosidase adenovirus have been previously described and characterized in endothelial cells (41) and in MC3T3-E1 clonal osteoblastic cells (42). Transgene expression is regulated via the chicken actin promoter, and at a multiplicity of infection (moi) of 100 (100 plaque-forming units/cell), cells are efficiently transfected and strongly overexpress the truncated TGFß receptor II on their cell surface, which acts in a dominant negative fashion by competing with endogenously expressed receptors. After binding TGFß (all isoforms), the truncated receptor disrupts TGFß biological activity because it fails to phosphorylate TGFß receptor I, thereby interrupting intracellular signaling. An adenovirus containing the Escherichia coli ß-galactosidase gene expressed using the same promoter served as a control for nonspecific viral effects. Confluent MC3T3-E1 cells in T75 flasks were infected with vehicle (PBS with 10% glycerol), the dominant negative truncated TGFß receptor II adenovirus, or the ß-galactosidase adenoviral control (moi = 100). After 60 h of incubation, cells were stimulated with 12.5 ng/ml FGF-2 in antibiotic-containing serum-free medium for 0, 3, 6, or 24 h, after which total cellular RNA was harvested, and VEGF mRNA levels were analyzed using Northern blot analysis. To verify TGFß blockade, identically treated MC3T3-E1 cells were stimulated with 2.5 ng/ml TGFß1 in antibiotic-containing serum-free medium for 0, 3, 6, or 24 h, after which total cellular RNA was harvested, and VEGF mRNA levels were analyzed using Northern blot analysis.

Northern blot analysis
Total cellular RNA was extracted using Trizol solution (Life Technologies, Inc.) according to the manufacturer’s specifications, and were quantified using an Ultraspec2000 spectrophotometer (Pharmacia Biotech). RNA integrity was assessed by ethidium bromide staining of 18S and 28S ribosomal bands. Twenty micrograms of total cellular RNA were loaded onto a 1.0% denaturing formaldehyde gel and resolved using electrophoresis. RNA was transferred to positively charged 0.45-µm pore size nylon membranes (Schleicher & Schuell, Inc., Keene, NH) and UV cross-linked for 2 min (Stratagene, La Jolla, CA) to link the RNA to the membranes. Membranes were prehybridized for 1–2 h at 68 C in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.), followed by hybridization with [{alpha}-P32]deoxy-CTP-labeled cDNA probes against VEGF, TGFß1, or GAPDH in fresh rapid hybridization solution (CLONTECH Laboratories, Inc.) for 2 h at 68 C. Stringency washes were performed twice at room temperature with 2 x SSC-0.1% SDS (1 x SSC = 0.15 M NaCl-15 mM sodium citrate) for 10 min each, followed by two washes in 0.1 x SSC-0.1% SDS at 50 C for 15 min each time. Membrane signal intensity was quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), and the resulting images were analyzed using ImageQuant (Molecular Dynamics, Inc.) image analysis software. All experiments were repeated in triplicate.

VEGF concentration in conditioned medium
A mouse VEGF quantitative sandwich enzyme immunoassay was used to quantify VEGF production (R&D Systems, Minneapolis, MN). This assay measures primarily the quantity of the 165-amino acid isoform of VEGF, because it represents the main soluble isoform of VEGF. Assay and controls were performed according to the manufacturer’s recommendation. Briefly, 2 x 104 MC3T3-E1 cells were plated in each well of 3 24-well plates and allowed to reach confluence over a period of 2–3 days in DMEM supplemented with 10% FBS as described above. Once at confluence, medium was removed, and cells were washed with PBS. Serum-free medium (400 µl) containing antibiotic/antimycotic and recombinant human FGF-2 in concentrations of 0, 1.5, 3.1, 12.5, 25, and 50 ng/ml or either TGFß2 or TGFß3 in concentrations of 0, 0.25, 0.5, 1, 5, and 50 ng/ml was then added to the cultures. Additionally, after stimulating cultures with TGFß1 as previously described (26), costimulatory experiments were performed in which cultures were stimulated with both TGFß1 (0.31 and 0.63 ng/ml) and FGF-2 (12.5 ng/ml). Each cytokine dose was repeated 4 times/experiment. After 24 h, the medium was removed and centrifuged to remove particulate matter. A crystal violet colorimetric assay was used to normalize the data for cell number (see below). All experiments were repeated in triplicate.

Crystal violet staining
To minimize the effect of alterations in cellular proliferation or plating, the number of plated cells was estimated using crystal violet staining as described by Kueng et al. (43). Briefly, cells were washed in PBS and fixed in ice-cold 3.7% paraformaldehyde (Sigma) for 20 min. Cells were washed with PBS, permeabilized with 20% methanol for 20 min, and stained with 0.5% crystal violet (Sigma) in 20% methanol for 30 min. Excess stain was removed after washes in deionized water, followed by elution with 10% acetic acid for 30 min. The OD of the dye was measured at 650 nm using a SPECTRAmax 250 spectrophotometer (Molecular Devices, Menlo Park, CA).

Statistical analysis
All data from quantitative VEGF sandwich enzyme immunoassays are expressed as the mean ± SD. Additionally, the quantitative sandwich enzyme immunoassay and the crystal violet assay underwent statistical significance testing with ANOVA (one-way ANOVA comparing VEGF protein production by each dose of FGF-2, TGFß2, and TGFß3). Post-hoc tests were performed using the Tukey-Kramer multiple comparison test, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FGF-2 increased VEGF mRNA levels in MC3T3-E1 osteoblastic cells and nonimmortalized osteoblastic cells
MC3T3-E1 mouse clonal osteoblastic cells express osteoblastic features such as collagen type I and alkaline phosphatase, and they behave similarly to nonimmortalized osteoblastic cells in response to TGFß1 (30). When osteoblastic cells were stimulated with 12.5 ng/ml FGF-2, VEGF mRNA was increased at all time points compared with unstimulated cells (Fig. 1Go). Initially, FGF-2 stimulation yielded a 3.4-fold increase in VEGF mRNA by 3 h. Increased VEGF mRNA expression was primarily noted in the 3.8-kb isoform of VEGF mRNA. Maximal VEGF mRNA occurred at 6 h, with a 6-fold increase in VEGF mRNA followed by a decline to a 1.7-fold increase in VEGF mRNA by 24 h. Similarly, when FRC cells were stimulated with 12.5 ng/ml FGF-2, VEGF mRNA was increased at all time points compared with unstimulated cells, with a 6-fold peak increase occurring at 6 h (Fig. 2Go). Importantly, the concentration of FGF-2 added to the cell cultures falls within the range of previously reported physiologically relevant levels (~10 ng/ml) (10, 20).



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  		Figure 1. FGF-2 increased VEGF mRNA levels in MC3T3-E1 osteoblastic cells. MC3T3-E1 cells were treated with FGF-2 (12.5 ng/ml) for the indicated times, from 0–24 h. Total cellular RNA (20 µg/lane) was subjected to blot analysis using a mouse VEGF cDNA probe, and the resulting signal intensity was quantified with a PhosphorImager (upper bands). An arrowhead indicates the location of the 28S ribosomal RNAs. Below (lower bands), a GAPDH probe hybridized to the same filter, after stripping, provides a comparison of RNA loading. The figure represents the results from one of three similar experiments. The graph shows quantification of relative VEGF mRNA at the indicated time points. The intensity of VEGF hybridization is given as a value relative to unstimulated MC3T3-E1 cells. The bars represent the means of three experiments, and the brackets represent the SD. VEGF mRNA was increased 6-fold at 6 h with lower, but still elevated, levels at 3 and 24 h.

 


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  		Figure 2. The effect of FGF-2 on VEGF mRNA in FRC cells. FRC cells were treated with FGF-2 (12.5 ng/ml) for the indicated times, from 0–24 h. Total cellular RNA (20 µg/lane) was subjected to blot analysis using a mouse VEGF cDNA probe (upper bands). Below, a GAPDH probe hybridized to the same filter provides a comparison of RNA loading (lower bands). VEGF mRNA was increased at 6 h, with lower, but still elevated, levels at 3 and 24 h. The figure represents the results from one of two similar experiments.

 
Effect of mRNA and protein synthesis inhibitors on FGF-2 stimulation of VEGF mRNA
The short, sharp rise in VEGF mRNA followed by its rapid decline in FGF-2-stimulated nonimmortalized osteoblastic cells and MC3T3-E1 osteoblastic cells were consistent with the pattern shown by other osteogenic cytokines (25, 26, 28) and suggested high turnover and low stability of VEGF mRNA. To further define the mechanisms of action of FGF-2 stimulation of VEGF expression in osteoblastic cells, we employed inhibitors of RNA polymerase and protein synthesis to block transcription and translation, respectively (Fig. 3Go). Blockade of transcription with actinomycin D (5 µg/ml) decreased baseline and FGF-2-stimulated VEGF mRNA expression. In contrast, translational blockade using cycloheximide (10 µg/ml) did not alter baseline or FGF-2-stimulated VEGF mRNA expression. Taken together, these findings suggest that the regulation of VEGF expression by FGF-2 is primarily transcriptionally mediated.



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  		Figure 3. The effects of RNA and protein synthesis inhibitors on FGF-2 stimulation of VEGF mRNA. MC3T3-E1 cells underwent 6-h exposures to 5 µg/ml actinomycin D (A–D) or 10 µg/ml cycloheximide (CHX) with or without 12.5 ng/ml FGF-2. Cells in the actinomycin D group underwent 1 h of pretreatment with actinomycin D before TGFß1 stimulation. Total cellular RNA (20 µg/lane) was subjected to blot analysis using a mouse VEGF cDNA probe. Arrowheads indicate the locations of the 28S and 18S ribosomal RNAs. Below, a GAPDH probe hybridized to the same filter after stripping provides a comparison of RNA loading. +, The presence of the added cytokine or synthesis inhibitor; -, the absence of the respective cytokine or synthesis inhibitor. The figure represents the results from one of two similar experiments.

 
To further elucidate the mechanisms of action of FGF-2 stimulation on VEGF mRNA expression, we compared the stability of VEGF mRNA produced by unstimulated osteoblastic cells to the stability of VEGF mRNA produced by cells stimulated with FGF-2 (Fig. 4Go). Five hours after treatment with either vehicle or FGF-2, transcription was inhibited by the addition of actinomycin D. The VEGF mRNA half-life of cells treated with FGF-2 was not significantly different from the half-life of vehicle-treated cellular mRNA, indicating that the rapid up-regulation of VEGF mRNA by FGF-2 is largely transcriptionally (and not mRNA stabilization) mediated.



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  		Figure 4. The effect of FGF-2 stimulation of MC3T3-E1 osteoblastic cells on VEGF mRNA stability. Five hours after treatment with either vehicle (filled diamonds) or 12.5 ng/ml FGF-2 (filled squares), transcription was inhibited by actinomycin D (A—D; 5 µg/ml). Total cellular RNA was isolated at the indicated time points, and 20 µg RNA/lane were resolved on a denaturing gel followed by transfer to a nylon membrane. The RNA was then subjected to blot analysis using a labeled mouse VEGF cDNA, and the resulting signal intensity was quantified with a PhosphorImager. The membranes were then stripped and rehybridized to a labeled GAPDH probe. Small differences in loading were accounted for by dividing the signal for VEGF intensity by the respective GAPDH signal intensity. The relative amount of VEGF mRNA was expressed as a percentage of 0 h values. The data represent the results of one of two similar experiments. The similar slopes of VEGF mRNA degradation between the vehicle-treated and the FGF-2-treated cells suggested that FGF-2 increases VEGF mRNA through transcriptional mechanisms.

 
Effect of FGF-2 on VEGF concentration in conditioned medium
Having demonstrated FGF-2 stimulation of VEGF mRNA in osteoblastic cells, we proceeded to examine the production of VEGF protein as a result of FGF-2 stimulation. Additionally, we examined the dose-response relationship between FGF-2 and VEGF. The basal level of VEGF production by MC3T3-E1 cells was 300 pg/ml at 24 h (Fig. 5Go). To control for the effect that FGF-2 may have had on cell proliferation, only identically seeded, confluent wells were stimulated with FGF-2. Additionally, a crystal violet assay, performed at the time of medium collection, was used to standardize relative cell number. FGF-2 produced dose-dependent increases in VEGF production, with a maximal increase to 650 pg/ml after stimulation with 50 ng/ml FGF-2. Stimulation with 12.5 ng/ml FGF-2 increased VEGF production to 450 pg/ml. With higher FGF-2 doses, the slope of VEGF production decreased to a plateau, suggesting receptor saturation.



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  		Figure 5. The effect of FGF-2 on VEGF protein production in culture medium of MC3T3-E1 cells. MC3T3-E1 cells were cultured for 24 h in serum-free medium containing the indicated doses of FGF-2. The medium was collected, and VEGF was quantified using a mouse VEGF quantitative sandwich enzyme immunoassay. The bars represent the means of three experiments, and the brackets represent the SD. FGF-2 produced dose-dependent increases in VEGF production beginning at 12.5 ng/ml FGF-2 (P < 0.01).

 
TGFß2 and TGFß3 increased VEGF mRNA levels in MC3T3-E1 osteoblastic cells
MC3T3-E1 cells stimulated with 2.5 ng/ml TGFß2 or TGFß3 demonstrated patterns of increased VEGF mRNA similar to each other, with maximal increases occurring at 6 h (Fig. 6Go). Unlike TGFß1, which in MC3T3-E1 cells induces a peak increase in VEGF mRNA after 3 h (26), neither TGFß2 nor TGFß3 yielded early increases in VEGF mRNA. By 6 h, however, VEGF mRNA was increased 3- and 2-fold in cells stimulated by TGFß2 and TGFß3, respectively. By 24 h, VEGF mRNA levels returned to baseline.



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  		Figure 6. The effects of TGFß2 and TGFß3 on VEGF mRNA in MC3T3-E1 cells. MC3T3-E1 cells were treated with TGFß2 or TGFß3 (2.5 ng/ml) for the indicated times, from 0–24 h. Total cellular RNA (20 µg/lane) was subjected to blot analysis using a mouse VEGF cDNA probe (upper bands). Below, a GAPDH probe hybridized to the same filter provides a comparison of RNA loading (lower bands). VEGF mRNA was increased at 6 h, with baseline levels at 3 and 24 h. The figure represents the results from one of three similar experiments.

 
TGFß2 and TGFß3 increased VEGF protein levels in MC3T3-E1 osteoblastic cells
Having demonstrated an increase in VEGF mRNA in MC3T3-E1 cells as a result of stimulation with TGFß2 and TGFß3, we sought to determine the effects of these cytokines on VEGF protein production. Both TGFß2 and TGFß3 stimulated dose-responsive increases in VEGF protein production (Fig. 7Go). MC3T3-E1 stimulated cells yielded similar patterns of VEGF protein increases, with maximal VEGF stimulation occurring at 5 ng/ml of either cytokine followed by a sharp drop at a supraphysiological dose (50 ng/ml). Consistent with the VEGF mRNA findings, TGFß2-stimulated cells yielded higher absolute increases in VEGF protein than TGFß3-stimulated cells (1900 vs. 1200; P = 0.005).



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  		Figure 7. The effects of TGFß2 and TGFß3 on VEGF protein production in culture medium of MC3T3-E1 cells. MC3T3-E1 cells were cultured for 24 h in serum-free medium containing the indicated doses of TGFß2 or TGFß3. The medium was collected, and VEGF was quantified using a mouse VEGF quantitative sandwich enzyme immunoassay. The bars represent the means of three experiments, and the brackets represent the SD. Both TGFß2 and TGFß3 produced dose-dependent increases in VEGF protein production beginning at 0.25 ng/ml (P < 0.01). Peak VEGF protein stimulation occurred at 5 ng/ml TGFß2 or TGFß3, followed by a sharp decline when supraphysiological doses of either cytokine were used (50 ng/ml)

 
Costimulation of MC3T3-E1 osteoblastic cells with TGFß1 and FGF-2 additively increased VEGF protein levels
To begin to isolate the effects of FGF-2 from those of TGFß, costimulatory experiments were performed. When MC3T3-E1 cells were costimulated with TGFß1 (031 and 0.63 ng/ml) and FGF-2 (12.5 ng/ml), VEGF protein production increased in an additive fashion (Fig. 8Go).



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  		Figure 8. The effect of FGF-2 and TGFß1 costimulation on VEGF protein production. MC3T3-E1 cells were cultured for 24 h in serum-free medium containing the indicated doses of TGFß1 with or without FGF-2 (the doses chosen individually significantly increased VEGF protein production). The medium was collected, and VEGF was quantified using a mouse VEGF quantitative sandwich enzyme immunoassay. The bars represent the means of three experiments, and the brackets represent the SD. When costimulated, VEGF protein production increased in an additive fashion (P < 0.01).

 
TGFß signal blockade did not impede FGF-2 induction of VEGF mRNA
We have shown that FGF-2 stimulation of osteoblastic cells results in the up-regulation of VEGF mRNA and protein expression. The results of studies in which cells were pretreated with actinomycin D or cyclohexamide in addition to VEGF mRNA half-life studies implied that the regulation of VEGF expression occurs primarily at the transcriptional level. These studies, do not, however, directly examine interactions between FGF-2 and endogenously produced TGFß. This point is important, because FGF-2 has been shown to indirectly alter TGFß biological activity by activating latent TGFß secondary to the activation of urokinase-type plasminogen activator (uPA) (21, 44, 45). Thus, to more carefully analyze the potential interactions between FGF-2 and TGFß in the regulation of VEGF expression by osteoblastic cells, we used an adenoviral vector capable of expressing a truncated type II TGFß receptor. Cellular transfection with this adenovirus results in the overexpression of a truncated type II TGFß receptor and a dominant negative effect by binding all TGFß isoforms without causing phosphorylation of the type I TGFß receptor (41). The net effect, therefore, is abrogation of virtually all TGFß biological activity (41).

When control (uninfected or ß-galactosidase adenovirus-infected) or dominant negative TGFß-truncated receptor II-transfected cells were stimulated with FGF-2, VEGF mRNA levels remained similar between the groups, with peak increases in VEGF mRNA at 6 h after stimulation (Fig. 9Go). In contrast, VEGF mRNA levels were significantly curtailed in TGFß1-stimulated cells transfected with dominant negative TGFß-truncated receptor II at all time points (Fig. 9Go). TGFß1-stimulated control cells demonstrated the expected 3 h peak increases in VEGF mRNA (data not shown). These data present evidence that FGF-2 may modulate the up-regulation of VEGF mRNA independently of TGFß.



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  		Figure 9. The effect of TGFß signal blockade on FGF-2 and TGFß1 stimulation of VEGF mRNA. MC3T3-E1 osteoblastic cells were infected with ß-galactosidase adenoviral control or dominant negative truncated TGFß receptor II adenovirus (DN-RII; moi = 100). After 60 h of incubation, cells were stimulated with 12.5 ng/ml FGF-2 or 2.5 ng/ml TGFß1 in antibiotic-containing serum-free medium for 0, 3, 6, or 24 h. Total cellular RNA was isolated, and 20 µg RNA/lane were resolved on a denaturing gel followed by transfer to a nylon membrane. The RNA was subjected to blot analysis using a labeled mouse VEGF cDNA, and the resulting signal intensity was quantified with a PhosphorImager. The membranes were stripped and rehybridized to a labeled GAPDH probe. FGF-2-stimulated control groups (uninfected and ß-galactosidase adenovirus-infected cells) demonstrated peak increases of VEGF mRNA at 6 h and lower (but above baseline) levels at 3 and 24 h. FGF-2-stimulated dominant negative receptor II adenovirus-infected cells demonstrated a similar pattern of VEGF mRNA expression, with a peak increase at 6 h and lower (but above baseline) levels at 3 and 24 h. In contrast, competitive binding of TGFß1 to an overexpressed truncated TGFß receptor II adenovirus substantially interfered with TGFß1 signal transduction, resulting in a very blunted response to TGFß1 stimulation noted particularly at 3 h, the normal peak of stimulated VEGF mRNA expression. TGFß1-stimulated control groups (uninfected or ß-galactosidase adenovirus-infected cells) demonstrated expected peak increases in VEGF mRNA at 3 h and lower (but above baseline) levels at 6 and 24 h, indicating that adenoviral infection did not alter the response of the cells to TGFß1 (data not shown).

 

   Discussion
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 Abstract
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 Materials and Methods
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 Discussion
References
 
The diverse effects and highly conserved nature of FGF-2 suggest its importance in mammalian biological systems. Its actions as a modulator of angiogenesis include its mitogenic and proliferative effects on endothelial cells (46, 47, 48). Additionally, FGF-2 mediates the activity of several modulators of angiogenesis, including uPA, plasminogen activator inhibitor, uPA receptor, and numerous ß-integrin subunits (21, 44, 45). FGF-2 also induces endothelial cell differentiation, which, in turn, leads to the development of capillary-like structures in long-term culture (49). Recently, FGF-2 was identified as an inducer of VEGF in endothelial cells during capillary formation (20). VEGF, which in osteoblastic cells is expressed constitutively and in response to a variety of cytokines and microenvironmental conditions, is a potent angiogenic agent that has been localized to proliferating osteogenic cells and the surrounding callus during fracture healing (26).

Although the roles of FGF-2 in gene patterning and, more specifically, skeletal development are areas of intensive investigation (10, 11, 12, 13, 50, 51), much in vitro research on the effects of FGF-2 on osteoblastic cells has focused on differentiation, proliferation, and extracellular matrix elaboration (17, 52, 53, 54). The significance of angiogenesis during development is well documented and by nature is coincident with skeletal development; however, bone remodeling and repair are life-long processes. Indeed, the fracture milieu contains many of the conditions and factors that have, in other tissues, been found to promote VEGF expression [hypoxia (55, 56), elevated FGF-2 (21), TGFß1 (57), and interleukin 1 (56)]. It is important, therefore, to identify potential mechanisms of FGF-2-induced angiogenesis by osteoblastic cells. We found that physiologically relevant FGF-2 doses promoted VEGF mRNA and protein expression in clonal and nonimmortalized osteoblastic cells. Although static mRNA expression as observed in Northern analysis is difficult to correlate directly with cumulative protein production measured by immunoassay, the finding that both mRNA and protein expression of VEGF are increased by FGF-2 stimulation supports the hypothesis that FGF-2 is a regulator of VEGF synthesis. Similar to the actions of TGFß1, insulin-like growth factor I, PGE1, and PGE2 (25, 26, 28), VEGF mRNA stability was unaffected by FGF-2, whereas actinomycin D, but not cycloheximide, strongly decreased FGF-2 induction of VEGF mRNA, suggesting transcriptional control of FGF-2-mediated increases in VEGF mRNA.

The ability of FGF-2 to induce angiogenesis directly and through VEGF is complicated by the fact that FGF-2 also activates and induces TGFß1, which, itself, modulates angiogenesis and stimulates VEGF production. For example, TGFß1 promotes angiogenesis in both the rabbit corneal micropocket (58) and the chick chorioallantoic membrane (59) models. Furthermore, TGFß1 increases VEGF mRNA and protein in osteoblastic cells, and both growth factors have been colocalized in a healing fracture (26). In contrast, genetically induced TGFß1 overexpression in arteries, liver, epidermis, and respiratory epithelial cells does not result in angiogenesis (60). To account for these diverse effects, a theory has arisen that the microenvironment within which TGFß1 is expressed determines the ultimate biological actions of TGFß1. Specifically, in an inflammatory microenvironment, TGFß1 levels correlate directly with increased angiogenesis (60). This is consistent with the concept that the indirect modulation of angiogenesis by TGFß1 may occur through inflammatory cells temporarily recruited to a site of injury. Thus, given the proper environment and/or effector cells, TGFß1 can be involved in the production of direct angiogenic factors such as VEGF.

To further investigate TGFß, we have demonstrated that its other mammalian isoforms (TGFß2 and TGFß3) also increase VEGF mRNA and protein, again with physiologically relevant doses. Unlike TGFß1, which maximally increases VEGF mRNA at 3 h (26, 28), both TGFß2 and TGFß3 maximally increased VEGF mRNA at 6 h, albeit with lower maximal potency (2- to 3-fold for TGFß3 and TGFß2, respectively, vs. 6-fold for TGFß1). Similar to TGFß1, both TGFß2 and TGFß3 cause dose-dependent increases in VEGF protein production.

As all TGFß isoforms increased VEGF, we investigated the possibility that FGF-2 mediated its induction of VEGF via activation or induction of TGFß. We demonstrated that costimulation with both TGFß1 and FGF-2 yielded additive, and not synergistic, increases in VEGF protein production. Additionally, after blocking TGFß signal transduction with a dominant negative truncated TGFß1 receptor II virus, FGF-2-induced VEGF mRNA was maintained, demonstrating TGFß-independent modulation of VEGF mRNA by FGF-2. Despite these findings, it is not unlikely that FGF-2 induction of VEGF through TGFß occurs physiologically and that this may represent a redundant mechanism of VEGF up-regulation.

The mitogenic and remodeling effects of VEGF on capillary endothelial cells and the ability of FGF-2 to increase VEGF expression by the endothelium suggests that FGF-2 may play an important role in the angiogenic response evidenced by healing bone. Additionally, the increase in VEGF during bone healing (26) may be at least partially an indirect effect of FGF-2, because, as indicated above, FGF-2 increases the levels of several cytokines that have been implicated in VEGF expression. Finally, the effect of FGF-2 on VEGF may by synergistically enhanced by other cytokines and conditions (strain, hypoxia) present in the fracture milieu. A fracture creates the necessary environment for FGF-2 elaboration by osteoblasts, and it is likely that this inflammatory microenvironment sets the stage for the production of VEGF and other direct angiogenic cytokines, without which fracture vascularization, and hence healing, cannot occur. Similarly, significant evidence separately implicates FGF-2 and the TGFß isoforms in skeletal development and VEGF in embryogenesis. These in vivo circumstantial relationships have been explored by investigating possible mechanisms of control of VEGF expression in osteoblastic cells. A better understanding of these mechanisms may lay the groundwork for future manipulations of the developing bone microenvironment and ultimately improve the treatment of bone pathology.

Received May 25, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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