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Endocrinology Vol. 140, No. 1 434-444
Copyright © 1999 by The Endocrine Society

ARTICLES

Prostaglandins Regulate the Expression of Fibroblast Growth Factor-2 in Bone

M. G. Sabbieti, L. Marchetti, C. Abreu, A. Montero, A. R. Hand, L. G. Raisz and M. M. Hurley

Universita degli Studi di Camerino, Departimento Di Scienze (M.G.S., L.M.), Morphologiche E Biochimiche Comparate, Camerino (MC) Italy; University of Connecticut School of Medicine, Division of Endocrinology and Metabolism (C.A., A.M., L.G.R., M.M.H.), Farmington, Connecticut 06030-1850; and University of Connecticut School of Dental Medicine, Department of Pediatric Dentistry (A.R.H.), Farmington, Connecticut 06030-1610

Address all correspondence and requests for reprints to: Marja M. Hurley, M.D., The University of Connecticut Health Center, Department of Medicine, Division of Endocrinology and Metabolism, Farmington, Connecticut 06030-1850.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the effect of PGs, particularly PGF2{alpha}, on basic fibroblast growth factor-2 (FGF-2) messenger RNA (mRNA) and protein in the rat osteoblastic cell line Py1a and in fetal rat calvariae. Py1a cells expressed multiple FGF-2 mRNA transcripts. PGF2{alpha} dose-dependently increased the 6-kb transcript at 6 h. The selective PGF2{alpha} agonist, fluprostenol (Flup), was more potent than PGF2{alpha}. Phorbol myristate acetate (10-6 M) also increased a 6-kb mRNA at 6 h. By immunofluorescence microscopy, Flup increased perinuclear staining for FGF-2 protein at 6 h and nuclear labeling at 24 h. Immunogold labeling of calvariae revealed that treatment with Flup for 3 h caused a transition of FGF expression from matrix to cells and an increase in cytoplasmic labeling for FGF-2 protein in periosteal cells and in osteoblasts. After treatment with Flup for 24 h, nuclear labeling was marked in periosteal cells and in osteoblasts, and a further increase in cytoplasmic labeling for FGF-2 was noted in osteocytes, periosteal cells, and osteoblasts. We conclude that PGs can increase FGF-2 mRNA and protein in bone cells. Because the effect of Flup was mimicked by phorbol myristate acetate, we hypothesize that PGs’ regulation of FGF-2 is mediated by a PGF2{alpha}-selective receptor acting through protein kinase C. Hence, effects of PGs on bone remodeling may be mediated, in part, by endogenous FGF-2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PGs HAVE both stimulatory and inhibitory effects on bone formation in cell and organ culture (1, 2). PGE2 has been shown to stimulate new bone formation in vivo either at low concentrations or in the presence of cortisol (1, 3, 4). In contrast, high concentrations of PGE2 inhibit collagen synthesis in organ cultures and in clonal osteoblastic cell lines. Data on structure-activity relations and signal transduction mechanisms for the anabolic effects of PGs are conflicting. Stimulation of cAMP production is probably important in the ability of these compounds to increase bone formation (5, 6, 7), whereas activation of protein kinase C (PKC) has been implicated in their mitogenic effect on osteoblasts and inhibitory effect on collagen synthesis (8, 9).

Basic fibroblast growth factor-2 (FGF-2) is a member of a protein family that influences the proliferation and differentiation of cell types of epithelial, neuroectodermal, and mesenchymal origin. These proteins play an important role in autocrine and paracrine regulation of cell proliferation and differentiation (9). FGF-2 is a potent regulator of both bone formation and bone resorption. FGF-2 is produced by cells of the osteoblast lineage and can act as a local regulator of bone remodeling (10, 11). In vitro studies have shown that continuous treatment with FGF-2 stimulated bone cell replication (12) and reduced differentiation-related markers, such as alkaline phosphatase (13) and PTH-responsive adenylate cyclase activity (14). In vitro studies have also shown that continuous FGF-2 treatment inhibited collagen synthesis in osteoblasts (15, 16). However, experiments using brief administration, followed by withdrawal in vitro (17), as well as in vivo (18), show that FGF-2 can stimulate bone formation. This is probably caused by an increase in the number of preosteoblasts, which can then differentiate into mature osteoblasts capable of forming new bone.

The effects of FGF-2 are similar to those of PGs; FGF-2 increases bone resorption (13) and is a potent inhibitor of type 1 collagen synthesis in osteoblasts (16). In addition, both FGF-2 and PGs have anabolic effects in vivo (18, 4, 5). Like PGs, FGF-2 is mitogenic for osteoblasts (16, 19). The local factors that mediate the effects of PGs in bone have not been clearly defined. Although factors such as IGF-1 and IGF-BP have been implicated (20, 21), they do not fully explain the effects of PGs in bone.

In this study, we examined the effect of PGs, particularly PGF2{alpha} and the selective PGF2{alpha} agonist, fluprostenol (Flup), on FGF-2 messenger RNA (mRNA) and protein synthesis in Py1a cells and 21-day fetal rat calvariae. In this report, we show that PGs, important regulators of bone remodeling, modulated FGF-2 gene expression in osteoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Immortalized rat osteoblastic Py1a cells were plated in 6-well dishes (Costar Corp, Cambridge, MA), at a density of 5000 cells/cm2 unless stated otherwise, and grown for 6–7 days in F-12 medium containing 5% nonheat inactivated FCS with penicillin and streptomycin. They were precultured for another 24 h in serum-free DMEM with antibiotics, 1 mg/ml BSA (RIA grade, Sigma Chemical Co., St. Louis, MO), and (50 µg/ml) phosphoascorbic acid (L-ascorbic acid phosphate magnesium salt, Wako Pure Chemical Industries Ltd., Osaka, Japan) before treatment. Cycloheximide (CHX, 3 µg/ml) was added 1 h before addition of effectors. Control cultures were treated with appropriate vehicles. PG F2{alpha} (PGF2{alpha}) was obtained from Sigma Chemical Co.. Flup was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).

Twenty-one-day fetal rat calvarial cultures
Time pregnant Sprague Dawley rats (Charles River Farms, Wilmington, MA) were killed by CO2 narcosis, followed by cervical dislocation on the 21st day of gestation. The fetuses were removed, and calvariae were dissected. Parietal bones were precultured for 2 h in a chemically defined medium, BGJb (1 mM proline, 3 mM phosphate, 0.4 mM L-glutamine, 100 µg/ml L-ascorbic acid phosphate, and 1 mg/ml BSA) (Gibco BRL, Gaithersburg, MD). After preculture, bones were cultured in the experimental medium in 6-well dishes (Costar Corp.). Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All animal protocols were approved by the Animal Care and Use Committee of the University of Connecticut Health Center.

Complementary DNA (cDNA) probes
A 1.4-kb rat FGF-2 cDNA (22) (a gift from Dr. Andrew Baird, Prizm Pharmaceuticals, La Jolla, CA) was used in these studies. A rat glyceraldehyde 3-phosphate dehydrogenase cDNA probe was used as a control.

Measurement of mRNA levels
Total RNA was extracted from cells by the method of Chomczynski and Sacchi (23). Cells were scraped in 4 M guanidinium thiocyanate and extracted with phenol/chloroform isoamyl alcohol (24:1) and total RNA precipitated with isopropanol. For Northern analysis, 12.5 µg total RNA was denatured and fractionated on a 1% agarose/2.2 Mq formaldehyde gel, transferred to nylon membrane by positive pressure, and fixed to the filter by UV irradiation (Stratalinker, Stratagene, La Jolla, CA) (24, 25). After a 4-h prehybridization, filters were hybridized overnight with a random primer deoxycycidine triphosphate-labeled [32P]cDNA probe for the mRNA of interest overnight. Bands were normalized to glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Northern blots were quantitated by autoradiography and densitometry.

Immunofluorescence
Py1a cells were plated at 1500 cells/well on NUNC coverslips and were grown for 3 days. When approximately 80% confluent, cells were serum-deprived for 24 h and treated with effectors for 6 or 24 h (26). Cells were washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 25 min at room temperature. After three washes in PBS, cells were permeabilized with 0.3% Triton X-100 for 30 min. Cells were washed in PBS and incubated with 0.5% BSA dissolved in PBS at room temperature for 20 min. The cells were then incubated with a 1:100 dilution of polyclonal anti-FGF-2 antibody raised in rabbit (Sigma Chemical Co.) for 90 min. A 1:70 dilution of goat antirabbit IgG, conjugated with fluorescein isothiocyanate, was added to the cells for 1 h at room temperature. Reaction controls were performed using a nonimmune rabbit IgG. After 3 washes (10 min each) the coverslips were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA) to prevent quenching of the fluorescence.

Immunogold labeling of bones
Twenty-one-day fetal rat calvariae (parietal bones) were cultured for 2 h in BJGb medium. The cultures were treated for 3 or 24 h with Flup (10-8 M) and then fixed in 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. After fixation, tissues were dehydrated in graded methanol solutions (30%, 50%, and 70%, 15 min each on ice; and 90%, 3 x 15 min at -20 C), infiltrated, and embedded in Lowicryl K4M (Polysciences, Warrington, PA) at -20 C, and polymerized under UV light (365 nm) for 5 days.

For immunogold localization of FGF-2 protein, ultrathin sections were collected on nickel grids coated with formvar. The sections were treated for 30 min at room temperature with 1% BSA-1% instant milk in PBS (pH 7.4) to block nonspecific binding. They were then incubated with monoclonal anti-FGF-2 (125 µg/ml, Transduction Laboratories, Inc., Lexington, KY) in 1% BSA in PBS (pH 7.4) overnight at 4 C. After rinsing with PBS, sections were incubated with goat antimouse gold-labeled secondary antibody (Amersham, Arlington Heights, IL) diluted 1:10 in PBS (pH 7.4) for 60 min at room temperature. After thorough rinses with PBS and distilled water, sections were counterstained with 2% aqueous uranyl acetate (1 min) and lead citrate (1 min).

Immunogold-labeled sections were observed in a transmission electron microscope. Representative periosteal cells, osteoblasts, and osteocytes were identified at low magnification (so that gold particles were not visible), then photographed at higher magnification of 11,500 x. Gold particle counts were done on prints at a final magnification of 31,000 x, using a digitizing tablet and SigmaScan software (Jandel, Corte Madera, CA). From three to ten areas of cytoplasm and nuclei, and three to five areas of bone matrix were counted for each condition.

Statistical analysis
Means of groups were compared using ANOVA, and the Bonferroni post hoc test when ANOVA demonstrated significant differences. Immunogold labeling densities (gold particles/µm2) of control and treated calvarial cells were compared using a t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Py1a cells constitutively expressed FGF-2 mRNA transcripts of approximately 4, 2, and 1.5 kb with low levels of a 6-kb transcript. Treatment of cells with PGF2{alpha} (10-5–10-8 M) for 6 h caused a concentration-dependent 3- to 4-fold increase of the 6-kb mRNA transcript (Fig. 1Go). The selective agonist of PGF2{alpha}, Flup, was more potent, with a dose-related response from 10-8 M–10-10 M (Figs. 1Go and 2Go).



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  		Figure 1. Dose response effect of PGF2{alpha} on FGF-2 mRNA in Py1a cells treated for 6 h. Cells were serum deprived for 24 h and then treated with various concentrations of PGF2{alpha}. RNA was extracted for Northern blot analysis, as described in Materials and Methods.

 


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  		Figure 2. Dose response effect of Flup on FGF-2 mRNA in Py1a cells treated for 6 h. Cells were serum deprived for 24 h and then treated with various concentrations of Flup. RNA was extracted for Northern blot analysis, as described in Materials and Methods.

 
We determined the time course of PGF2{alpha} and Flup-mediated increase in FGF-2 mRNA in Py1a cells. Cells were serum-deprived for 24 h and treated with PGF2{alpha} (10-6 M) for 2, 4, 6, and 24 h. The increase by PGF2{alpha} of the 6-kb mRNA transcript was maximal between 4 and 6 h, with a small residual effect after 24 h (Fig. 3Go). As shown in Fig. 4Go, the time course of the Flup (10-8 M) effect was similar to that of PGF2{alpha}.



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  		Figure 3. Time course of the effects of PGF2{alpha} (10-6 M) on FGF-2 mRNA in rat osteoblastic Py1a cells. Cells were serum deprived for 24 h and treated with or without Flup. RNA was extracted at each time point for Northern blot analysis, as described in Materials and Methods.

 


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  		Figure 4. Time course of the effects of Flup (10-8 M) on FGF-2 mRNA in rat osteoblastic Py1a cells. Cells were serum deprived for 24 h and treated with or without Flup. RNA was extracted at each time point for Northern analysis.

 
We compared the effect of 4 h treatment with PGF2{alpha} (10-6 M) and PGE2 (10-6 M) on FGF-2 mRNA expression in serum-deprived cultures. Pooled data from two to three experiments showed that, at similar concentrations of 10-6 M, PGF2{alpha} increased the 6-kb FGF-2 mRNA 5-fold (5.3 ± 1.2), whereas 10-6 M PGE2 increased the 6-kb FGF-2 mRNA only 2-fold (2.1 ± 0.5).

In some experiments, cultures were pretreated for 1 h with the protein synthesis inhibitor CHX (3 µg/ml), and then Flup was added for an additional 6 h. CHX abrogated the effect of Flup on FGF-2 mRNA (Fig. 5Go), indicating that new protein synthesis was required for the stimulatory effect of Flup on FGF-2 mRNA.



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  		Figure 5. Effect of Flup on FGF-2 mRNA in the absence or presence of CHX (3 µg/ml). Cells were serum deprived for 24 h and then were pretreated with CHX for 1 h. Flup was then added for an additional 6 h. RNA was extracted for Northern blot analysis.

 
We determined whether serum alone stimulated FGF-2 expression in Py1a cells. We also assessed whether phorbol myristate 12–13 acetate (PMA), an activator of PKC, or stimulators of cAMP/PKA, such as forskolin (FSK) and 8 Bromo-cAMP (8 Br-cAMP) regulated FGF-2 mRNA expression in Py1a cells. The results are shown in Fig. 6Go. In these experiments, 24 h before treatment with effectors, culture media was changed to either 5% nonheat inactivated FCS (lane 1) or serum-free media containing 1 mg/ml BSA (lanes 2–6). Serum-free cultures were pulsed for an additional 6 h with either PGF2{alpha} (10-6 M), FSK (10-5 M), PMA (10-6 M), or 8 Br-cAMP (3 mM). As shown in Fig. 6Go, the 6-kb transcript was visualized in serum-replete (lane 1) (but not in serum-deprived) cultures (lane 2). PGF2{alpha} and PMA increased FGF-2 mRNA (lanes 3 and 5). In contrast, neither FSK nor 8 Br-cAMP induced the 6-kb FGF-2 mRNA transcript (lanes 4 and 6).



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  		Figure 6. Effects of serum, or activators of the PKC or cAMP/PKA signaling pathways, on FGF-2 mRNA levels in Py1a cells. Cells were either treated with 5% serum (lane 1) or were serum deprived in the presence of BSA (1 mg/ml) for 24 h (lanes 2–6). After 24 h, cultures were treated for an additional 6 h with PGF2{alpha} (10-6 M, lane 3), FSK (10-6 M, lane 4), PMA (10-6 M, lane 5), or 8 Br-cAMP (3 mM, lanes 6). Total RNA was extracted for Northern blot analysis. Con, Control.

 
To further determine whether the PKC pathway was involved in PG-mediated FGF-2 regulation in rat osteoblastic Py1a cells, we examined the ability of Flup (10-8 M) and PMA (10-6 M) alone, or in combination, to increase FGF-2 mRNA after pretreatment with PMA (10-6 M) for 24 h. As shown in Fig. 7Go, a 6-h treatment with Flup alone, PMA alone, or the combination increased the 6-kb transcript (lanes 2–4). In contrast, 24-h PMA pretreatment blocked the stimulatory effect of both Flup and further PMA treatment on FGF-2 mRNA expression (lanes 6–8).



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  		Figure 7. Effect of down-regulation of the PKC pathway with 24 h PMA pretreatment on Flup (10-8 M) and PMA (10-6 M)-mediated increase in FGF-2 mRNA expression in Py1a cells. Confluent cultures were serum deprived in the absence (Lanes 1–4) or presence of PMA (10-6 M). After 24 h, media was changed, and effectors were added for an additional 6 h. Total RNA was extracted for Northern blot analysis.

 
We determined whether the increase in FGF-2 mRNA reflected the synthesis of FGF-2 protein. Immunofluorescence microscopy was performed on cells that were approximately 80% confluent. Py1a cells serum deprived for 24 h were treated with Flup (10-8 M) for 6 and 24 h. The untreated control cultures showed low levels of uniform cytoplasmic and nuclear staining for FGF-2 protein (Fig. 8Go). Treatment with Flup for 6 h increased staining, particularly in the perinuclear and nuclear regions (Fig. 8Go). This increased staining was more evident after 24 h of incubation; and at this time point, FGF-2 seemed to have accumulated in the nucleus of 50–60% of the cells (Fig. 8Go). There was no staining in cultures incubated with nonimmune rabbit IgG.



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  		Figure 8. Time course of Flup (10-8 M) effects on FGF-2 protein accumulation and localization in Py1a cells. Cells were grown on Nunc coverslips for 3 days, at which time they were precultured in serum-free media for 24 h. After preculture, cells were treated with vehicle or Flup (10-8 M) for an additional 6 or 24 h. Staining for FGF-2 protein was performed as described in Materials and Methods. Slight cytoplasmic staining for FGF-2 is seen at 6 and 24 h in cells treated with BSA alone (Control). Treatment with Flup 10-8 M for 6 h resulted in increased FGF-2 staining in the perinuclear region. This increased staining was more evident after 24 h of incubation, at which time the FGF-2 in 50–60% of cells seemed localized in the nuclei (magnification, x300; bar, 20 µm). There was no staining in cultures incubated with nonimmune IgG (NONI).

 
The cellular localization and accumulation of FGF-2 protein in a bone organ culture model was examined by electron microscopy. In control cultures, low levels of FGF-2 protein were present in the cytoplasm and nuclei of osteoblasts, osteocytes, and periosteal cells, as well as in bone matrix. Treatment with Flup (10-8 M) for 3 h did not alter nuclear labeling but caused increased cytoplasmic labeling for FGF-2 protein in periosteal cells and in osteoblasts (Figs. 9Go and 10Go). A quantitative analysis revealed that Flup caused a 2-fold increase in cytoplasmic labeling in periosteal cells and osteoblasts using either the monoclonal (Fig. 10Go) (P < 0.05) or a polyclonal FGF-2 antibody (data not shown). In contrast to increased labeling of bone cells, in two independent experiments, 3 h treatment with Flup significantly decreased labeling of the matrix, relative to control (Figs. 9Go and 10Go). Treatment with Flup (10-8 M) for 24 h resulted in significant increases in cytoplasmic and nuclear labeling for FGF-2 protein in osteoblasts (Figs. 11Go and 12Go) and periosteal cells (Fig. 12Go). Cytoplasmic labeling of osteocytes was also increased (Fig. 12Go), but no significant changes occurred in nuclear labeling. In contrast to the results of 3 h Flup treatment, no difference in matrix labeling was observed between control and treated bones in 24-h cultures (data not shown).





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  		Figure 9. Effect of 3 h treatment with Flup (10-8 M) on FGF-2 protein localization and accumulation in 21-day fetal rat calvariae. Fetal rat parietal bones were precultured in serum-free media for 2 h. After preculture, bones were placed in culture with or without Flup and cultured for an additional 3 h. Electron microscopic immunogold labeling for FGF-2 protein was performed as described in Materials and Methods. Gold particles are present over the cytoplasm and the nuclei (Nu) of periosteal cells, osteoblasts, and osteocytes. Increased cytoplasmic labeling is seen after treatment with Flup. Arrowheads indicate particles over the nuclei. A, Periosteal cells, control; B, periosteal cells, 3 h Flup; C, osteoblasts, control; D, osteoblasts, 3 h Flup; E, osteocytes, control; F, osteocytes, 3 h Flup; G, bone matrix, control; H, bone matrix, 3 h Flup.

 


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  		Figure 10. Quantitation of immunogold labeling for FGF-2 protein in 21-day fetal rat calvariae treated with Flup (10-8 M) for 3 h. Culture of fetal rat parietal bones, electron microscopic immunogold labeling, and quantitative analysis were performed as described in Materials and Methods. Cytoplasmic labeling was increased 2-fold in periosteal cells and osteoblasts and in osteocytes, 1.3-fold after treatment. Nuclear labeling was unchanged after 3 h treatment with Flup. Labeling for FGF-2 protein in bone matrix was markedly reduced at 3 h in Flup-treated bones. *, Significantly different from control at P < 0.05.

 


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  		Figure 11. Effect of 24 h Flup (10-8 M) treatment on FGF-2 protein localization in 21-day fetal rat calvariae treated and prepared as in Fig. 9Go. A, Osteoblast, control; B, osteoblasts, 24 h Flup. The control cells show low levels of cytoplasmic and nuclear (Nu) labeling; increased labeling is seen in osteoblasts after treatment with Flup. Arrowheads indicate gold particles over the nucleus.

 


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  		Figure 12. Quantitation of immunogold labeling for FGF-2 protein in 21-day fetal rat calvariae treated with Flup (10-8 M) for 24 h. Gold particle counts from Flup-treated cultures were normalized to untreated controls. Cytoplasmic labeling in periosteal cells, osteoblasts, and osteocytes was increased 3-fold at 24 h; and nuclear labeling of periosteal cells and osteoblasts was increased 3- to 11-fold after 24 h treatment. Matrix labeling in treated cultures was no different from controls (data not shown). *, Significantly different from control at P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we determined the dose response and time course of the effects of PGs on FGF-2 mRNA in the immortalized rat osteoblastic clonal cell line Py1a. In the absence of serum, Py1a osteoblasts constitutively expressed FGF-2 mRNA transcripts of 4.5 and 1.5 kb. The present study demonstrated that, in serum-free cultures, a high-molecular-weight 6-kb transcript was transiently increased, between 4 and 6 h, by both PGF2{alpha} and Flup. Comparison of the dose response of PGF2{alpha} and Flup on FGF-2 mRNA showed that Flup was more potent, suggesting that the effect is mediated by a PGF-selective receptor.

We used PGF2{alpha}, instead of PGE2, in these studies because, in previous structure activity studies, we showed that PGF2{alpha} was approximately 100x more potent than PGE2 in inhibiting collagen synthesis in Py1a cells (9). In addition, our data show that, at similar concentrations of 10-6 M, PGF2{alpha} caused a greater increase in the 6-kb FGF-2 mRNA than PGE2.

The increase in FGF-2 mRNA by Flup was blocked by CHX, suggesting that de novo protein synthesis was required for this effect of Flup on the FGF-2 gene.

Because previous studies demonstrated that a serum factor altered FGF-2 mRNA expression in nonosteoblastic cells (27), we examined FGF-2 expression in serum-replete and serum-free cultures. Both the 6- and 4-kb FGF-2 mRNA transcripts were expressed in Py1a cells in the presence of serum (Fig. 6Go, lane 1). In cultures that are serum-deprived for 24 h, the 6-kb transcript is poorly visualized in unstimulated Py1a cells. This suggests that either the 6-kb mRNA is unstable and degraded immediately after translation or that factors that maintain its expression are present in serum. In the absence of serum, our studies show that stimulation of Pyla cells with PGs or PMA increased mainly the 6-kb transcripts, suggesting that this mRNA could mediate the increase in FGF-2 production in response to PGs in these cells.

We have previously published that cAMP is increased in response to activators of the PKA pathway in Py1a cells (9). We have also shown, in studies in mouse osteoblastic cell line MC3T3-E1, that both PMA and FSK stimulated FGF-2 mRNA expression, indicating that activation of both the PKC and PKA may be involved in FGF-2 expression (28). In other studies, Stachowiak et al. (26) have shown that neurotransmitters, as well as PMA and FSK, regulate FGF-2 mRNA and protein in bovine adrenal medullary cells. We therefore examined the ability of activators of the PKA and PKC to increase FGF-2 mRNA expression in rat osteoblastic Py1a cells. Our results show that activators of the cAMP/PKA pathway, such as FSK and 8 Br-cAMP, did not mimic the stimulatory effect of PGs on FGF-2 mRNA, suggesting that the PG effect is not mediated via cAMP/PKA. It is possible that the PKA pathway is down-regulated in these cells or that there are species differences in the response to FSK in rat and mouse osteoblastic cell lines.

In contrast, the PKC activator PMA significantly increased FGF-2 mRNA. Furthermore, down-regulation of the PKC pathway by PMA pretreatment blocked the PG-induced increase in FGF-2 mRNA expression. These experiments suggest that PGs may increase FGF-2 mRNA levels by a mechanism that involves PKC activation in rat osteoblastic cells. The stimulatory effect of PMA on FGF-2 mRNA has been described in nonosteoblast cell models. Weich et al. (29) reported that FGF-2 mRNA in endothelial cells was increased in response to thrombin and phorbol ester treatment.

Our studies in Py1a cells and 21-day fetal rat calvariae revealed that short-term treatment with Flup (6 and 3 h, respectively) increased cytoplasmic (but not nuclear) labeling for FGF-2. Flup treatment of calvariae also significantly decreased matrix labeling for FGF-2. This pattern of increased cellular staining with decreased matrix labeling suggests an additional action of PGs to mobilize FGF-2 from matrix stores. However, we were unable to measure FGF-2 in the medium of cultured cells. Similar to our observations, studies in nonosteoblastic cells showed that the accumulation of FGF-2 protein in the cytoplasm or the nucleus of adrenal medullary cells, stimulated with either FSK or PMA, did not result in increased FGF-2 proteins in the culture medium (26). A number of studies have shown that FGF-2, which lacks a signal sequence necessary for secreted proteins, is ordinarily not expressed in culture medium but remains primarily cell associated (26, 30, 31, 32).

The data from 21-day fetal rat calvariae immunolabeling showed increased labeling of FGF-2 protein in the cytoplasm of periosteal cells, osteoblasts, and osteocytes after 24 h of treatment with Flup. Moreover, the immunolabeling performed in calvariae treated with Flup for 24 h showed significantly increased labeling for FGF-2 in the nuclei of bone cells, confirming the results obtained in Py1a cells.

Although FGF-2 lacks a signal sequence that would direct release via the classic secretory pathway (33, 34), it functions extracellularly, because multiple forms of FGF plasma membrane receptors are expressed by a variety of cell types (35, 36, 37, 38, 39), and it is localized in the extracellular matrix (11). Moreover, neutralizing antibodies to FGF-2, which presumably do not enter the cell, altered properties of FGF-producing cells, including morphology, growth in soft agar, plasminogen activator synthesis, and cell migration (40, 41, 42, 43). The nuclear accumulation of FGF-2 in Flup-treated osteoblasts may be relevant, because a number of studies have suggested that there is a pathway of FGF-2 action, involving intracrine regulation at the nucleus (44, 45). By this mechanism, transport/trafficking of FGF-2 into the nucleus might be sufficient to initiate an effect on growth or differentiation (46, 47, 48, 49). Several laboratories have reported on nuclear localization of peptide growth factors, suggesting that some of their biologic effects are mediated through direct interaction with nuclear factors (50). In support of intracellular localization and function of FGF, studies have shown that FGF could induce stimulation and proliferation of fibroblast (51) and differentiation of schawnn cells (52) without being secreted. Extracellular-added FGF-2 accumulates in the nucleus in a cell-cycle-dependent manner (48). The addition of FGF-2 to isolated nuclei stimulates ribosomal RNA synthesis (49) and modulates in vitro gene transcription in nuclear extracts (53). Although the biologic effects of extracellular FGF-2 in-volves activation of high-affinity tyrosine kinase receptors (54), other studies suggest that intracellular FGF-2 may stimulate cell proliferation via cell-surface-receptor-independent mechanisms (51).

Based on our data, we hypothesize that PGs are able to regulate endogenous FGF-2 production. This regulation may reflect a translocation of cytoplasmic FGF-2 into the nuclei to modulate gene transcription, such as Type (I) procollagen.

Previous studies found that the inhibitory effect of PGs on collagen synthesis in the Py1a osteoblastic cells seems to be mediated by the F-type receptors and could be mimicked by PMA (but not by FSK), suggesting that PKC (and not PKA) is the mediator of the inhibition (9). Furthermore, activation of PKC has been implicated in the mitogenic effect of PGs on osteoblasts (8). We have shown that activation of PKC pathway mediates the mitogenic response to FGF-2 in Py1a cells (19)

In this study, we have demonstrated that PMA (but not FSK) mimicked the effect induced by Flup on the 6-kb FGF-2 transcript, suggesting that the effect of PGs on FGF-2 is mediated by PKC. Moreover, we hypothesize that the effect of PGs on FGF-2 is mediated by a PGF2{alpha}-selective receptor, acting through PKC. These data suggest that some effects of PGs on bone remodeling may be mediated by the regulation of endogenous FGF-2 production.


   Acknowledgments
 
The authors would like to thank Ms. Jan Figueroa for her clerical assistance.

Received February 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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