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Expression of bone protein mRNA at physiological fluoride concentrations in rat osteoblast culture
Bone and Mineral, 22 (1993) 187-196
187
9 1993 Elsevier Scientific Publishers Ireland Ltd. all rights reserved. 0169-6009/93/$06.00 BAM 0600
Expression of bone protein mRNA at physiological fluoride concentrations in rat osteoblast culture R o n g s h a n Li and P a m e l a K. D e n B e s t e n Department of Pediatric Dentistry, Eastman Dental Center, 625 Elmwood Ave, Rochester, N Y 14620, USA (Received 10 November 1992) (Revision received 22 March 1993) (Accepted 4 May 1993)
Summary Fluoride causes an increase in the amount of unmineralized osteoid. To determine whether the increase in osteoid is due to greater protein expression in the presence of fluoride, we measured the relative amount of mRNA expressed by fetal rat calvaria cells maintained in culture for either 18 or 26 days in the presence of 0, 5, 20 or 300 #M fluoride. There were no differences in the level of expression ofmRNA for collagenous or non-collagenous proteins in fluoride-treated cells as compared with control cells at 18 days in culture. Expression of mRN A for osteocalcin and al-type I collagen was decreased at 300 #M fluoride after 26 days culture. The amount of [3H]thymidine incorporation in cells exposed to the different amounts of fluoride was measured at various time points. Fluoride did not alter the time at which rapid cell proliferation ended. These studies indicate that at physiological serum levels, fluoride does not increase expression ofmRNA by osteoblasts. The relative increase in osteoid in bone may be related to other mechanisms such as altered matrix mineralization.
Key words: Osteoblast; Bone cell; Fluoride; mRNA; Gene expression
Introduction Fluoride has been shown to both accumulate in mineralizing tissues and to result in altered development of bones and teeth. In the treatment of osteoporosis, fluoride levels which are sufficient to maintain fasting serum fluoride at 5-10 t~M (0.095-0.19 ppm F) result in a net gain in bone mass. New bone formation is most pronounced in the trabecular bone of the axial skeleton. In cortical bone, there is no net gain in bone formation and some reports have indicated a decrease in cortical Correspondence to: P.K. DenBesten, Department of Pediatric Dentistry, Eastman Dental Center, 625 Elmwood Ave, Rochester, NY 14620, USA.
188
bone mass with fluoride treatment [1,2,3]. Chronic fluoride exposure in humans has been shown to increase serum osteocalcin [4,5] which may indicate increased osteoblastic activity. Prolonged exposure to high levels of fluoride (120 ppm) in rats, results in the formation of bone with widened, unmineralized osteoid seams on the periosteal surface of the femur [6,7]. This level of fluoride in the drinking water of a rat would result in serum fluoride levels between 10 and 20 t~M fluoride [8]. Cheng and Bader [9] gave sodium fluoride in drinking water to rats, and found that levels resulting in serum concentrations from 5 to 13 /~M fluoride caused an increase in the rat cancellous bone volume through trabecular thickening. They suggested that this change was due to reduced bone resorption. Direct cellular effects of physiological (micromolar) levels of fluoride include stimulation of osteogenesis in embryonic mesenchyme [10]. A concentration of 10 #M (0.18 ppm) fluoride induced osteogenesis under culture conditions which would otherwise not result in bone formation. Several reports show that fluoride at concentrations of 2-25/~M (0.038-0.475 ppm) increases the rate of cell proliferation and alkaline phosphatase activity of isolated chick [11], and human osteoblasts [12]. However, in human fetal bone cell culture, fluoride exposure of up to 6 days was not found to affect cell proliferation or protein secretion [131. These studies indicate that alterations in bone formation by fluoride may be a result of a direct effect of fluoride on the osteoblasts, affecting cell proliferation, differentiation and matrix production. Expression of certain matrix proteins, such as osteocalcin, is related to the onset of mineralization [14,15]. Changes in the matrix (such as in the type and amount of mineral formed) may indirectly affect matrix expression, and could further result in the delayed mineralization found in fluorideexposed bone. In the present study, we have used the rat calvaria cell culture system to determine whether long-term fluoride exposure to bone cells in vitro, alters expression of matrix proteins. Materials and methods
Cell culture Calvaria from 21-day gestation fetal rats were isolated and sequentially digested with collagenase trypsin as described by Aronow et al. [16]. Cells were plated in minimal essential medium (MEM; Gibco) supplemented with 1.5% penicillin, 0.05% streptomycin, 10% fetal bovine serum (FBS) in 100 mm dishes (for mRNA isolation) and 6-well dishes (for thymidine incorporation) at 5 x 105 or 3.6 x 105 per dish, respectively. The culture dishes were divided into four groups and fluoride was added to a final concentration in the media of either 0, 5, 20, or 300 #M from day 1 of culture. These levels of fluoride were maintained in the medium throughout the time of culture. At day 7, the cultures were supplemented with 50/~g/ml ascorbic acid and 10 mM/3-glycerol phosphate, the supplements were added every feeding day thereafter. The cells were fed every 2-3 days, and maintained in a 5% CO 2 atmosphere. DNA synthesis To determine whether the cessation of cell proliferation occurred at the same time under the various fluoride conditions, DNA synthesis was determined according to
189
the method of Owen et al. [14]. Cells were plated into 6-well dishes with a final concentration of 0, 5, 20 or 300 t~M fluoride in the medium containing 10% FBS. At days 5, 8, 11, 14 and 18, [3H]thymidine (20 Ci/mmol) was added to the medium of 2 wells from each group to a final concentration of 10/zCi/ml for 1 h. Cells were rinsed with phosphate-buffered saline (PBS), incubated with 5% (w/v) trichloracetic acid (TCA) for 5 min, rinsed and the incubation repeated. The cell layer was solubilized in 0.5 ml 10% SDS, and 4 ml of scintillation fluid was added prior to counting on a scintillation counter.
RNA &olation and analys& In one experiment, message levels of osteocalcin, osteonectin, osteopontin, transin (rat stromelysin), type I collagen, fl-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPD) were determined at day 18 of culture. Day 18 was chosen as a time point in osteoblast culture where matrix maturation occurs, and matrix mineralization has begun [14,16]. Cells exposed to the different levels of fluoride were cultured in triplicate, and on day 18, they were washed with PBS, immersed in SDS buffer containing RNAase/protein degrader provided in a Micro-Fast-track mRNA isolation kit (Invitrogen, San Diego, CA). The cells were scraped from the dishes, and polyadenylated RNA was then isolated from the cells according to the manufacturer's directions. The mRNA isolated from each group was quantitated by absorbance at 260 nm and 1 t~g of each sample was separated on a 1% agarose gel containing 2 M formalde-
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Fig. 1. Northern blot analysis of mRNA from RC cells, probed with 32p-labeled cDNA for transin (group B) and for GAPD (group A). The blot is visualizied, the area to be counted is marked, and an output of counts/min for each marked area is generated. The counts/min for each matrix protein was divided by the counts/min for either GAPD or/3-actin, to correct for variations in the amount of mRNA initially loaded on the agarose gel.
190
hyde using standard methodology [17]. Following separation, the RNA was transferred overnight with 20 x SSC by capillary diffusion to a Duralon nylon membrane (Stratagene, La Jolla, CA). The mRNA was cross-linked to the membrane using a Stratalinker (Stratagene) ultraviolet cross-linker. DNA probes used for hybridization were: osteocalcin, a gift from Genetics Institute, Cambridge MA, [18]; osteonectin and osteopontin, a gift from Dr. Marion Young, National Institutes of Health, [19]; transin, a gift from Dr. Lynn Matrision, Vanderbilt University [20]; rat c~-I collagen, a gift from Dr. Barbara Kream, University of Connecticut, [21]; and B-actin and GAPD, gifts of Dr. David Wong, Harvard School of Dental Medicine. DNA probes were labeled with [32p]dCTP by random priming, and the membranes were hybridized using standard methodology [22]. The membranes were washed 4 times, with a final wash of 0.1 x SSC and 0.5% SDS at 68~ for 30 min, and the relative radioactivity emitted from a hybridized band was quantitated on a Betascope Blot Analyzer (Betagen, Waltham, MA). The Betascope allows direct counting of radioactive emissions from the hybridized band (see Fig. 1). The hybridization signal for each of the matrix proteins was quantitated relative to the signal for either B-actin or GAPD, and the average was obtained for each level of fluorid6 exposure. In one set of experiments, the time of culture was extended to 26 days. The mRNA was isolated from duplicate samples from each group and analyzed for expression of osteocalcin and type I collagen. The mineral contained in the matrix of 2 wells from each group of cells cultured for 18 days was analyzed by chemical analysis, infrared spectroscopy [23] and X-ray diffraction (XRD). For X-ray diffraction, a sample from each group was mounted on a low background holder and analyzed in a Philips powder difractometer using copper K,, radiation. Fluoride in the matrix was measured by the diffusion method of Taves [24]. The calcium and phosphate contents of the mineral were determined from the acid solution (remaining following fluoride diffusion) by atomic absorption spectrophotometry, and phosphate was determined by the method of Chen et al. [251. Statistical evaluation
The statistical significance of the differences between the means of the control and experimental groups was evaluated by analysis of variance (ANOVA). Values indicating a probability of < 0.05 were regarded as statistically significant. Results
There were no gross changes in the morphology of cells cultured in the presence of fluoride, as compared with controls. Mineralizing nodules similar to those described in previous studies using rat calvaria cells [16,26,27] were identified by light microscopy within 24 h after supplementation of ~GPO 4. Cell proliferation, as measured by incorporation of [3H]thymidine, decreased sharply at day 11 in all groups (Fig. 2), when cell differentiation and matrix nodule formation began. Longterm exposure to fluoride in vitro did not cause a change in the time at which cell proliferation ceased, as compared with the controls. The expression of cd-type I collagen (Fig. 3) and the non-collagenous proteins (osteocalcin, osteonectin, osteopontin, and transin) (Fig. 4) were measured relative
191
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to the level of expression of either fl-actin or GAPD. There were no significant differences in the levels of mRNA expression for these proteins when the fluoride-exposed groups were compared with controls. There was a significant increase in the levels of osteopontin in the 20/~M and 300 #M fluoride-exposed groups compared with
mRNA Expression for Type I Collagen 5
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Fig. 3. Expression of mRNA for collagen in RC cells exposed to various levels of fluoride for 18 days. Cells were cultured in triplicate to obtain a mean value for each fluoride level. These results were repeated in a duplicate experiment. No significant differences were found between any of the groups. Error bars represent standard deviations for each group.
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Fig. 4. Expression ofmRNA for non-collagenous proteins in RC cells exposed to various levels of fluoride for 18 days. Cells were cultured in triplicate, to obtain a mean value for each fluoride level. Error bars represent standard deviations for each group. *Significantly different from the group exposed to 5#M F.
the 5/~M fluoride group relative to expression o f ~-actin. These differences were not found to be significant when compared with G A P D . In the 26 day cultures, the message for osteocalcin and type I collagen was greatly reduced in the cells exposed to 300 #M fluoride (Fig. 5) relative to both/3-actin and G A P D . However, the message levels were similar in the lower fluoride groups, as compared with the control group. The time at which message levels decreased appeared to be somewhat dependent on culture condition. Some experiments performed with more densely plated cultures showed a drop in m R N A expression earlier than 26 days (results not shown).
3
ooa osteocalcin 26d collagen 26d
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pmolar F Fig. 5. Expression of mRNA for osteocalcin and collagen in RC cells exposed to fluoride for 26 days. Values are the average of duplicate analyses.
193 Although sample amounts were very small, all matrices from 18-day-old cell culture produced infrared spectra characteristic of poorly-formed apatite-like or brushite-like calcium phosphate, comparable with that found by us for mouse calvaria (unpublished data). Phosphate, carbonate and structural water bands were present. The sample amounts were too small to allow definitive characterization by XRD. The infrared spectra coupled with the chemical analyses of the material, which showed calcium (20-45 ~g per sample) and phosphate (6-50 ~g, as P, per sample) to be present in the particles analyzed, indicated the presence of poorly crystalline calcium phosphate mineral. The samples could not be accurately weighed, therefore, the amount of fluoride present was related to the amount of calcium and phosphate present, assumed to be in the form of hydroxyapatite for purposes of calculation. Measurements were done as an average of 2 samples from each group and showed increasing amounts of fluoride in the matrix with increased amounts of fluoride in the media (control, 633 ppm; 5 #M F, 590 ppm; 20 #M F, 1056 ppm; 300 #M F, 4305 ppm). Discussion Studies of the rat osteoblast culture system have shown a developmental sequence of gene expression associated with bone cell differentiation [14,16]. In the present study, this culture system was used to study the long-term effects of fluoride on osteoblast-enriched rat calvaria cells. Fluoride levels of 5 t~M and 20 ~M were chosen as similar to the serum levels of humans ingesting from 5 to 15 ppm F in drinking water. Long-term exposure to fluoride in drinking water resulting in these serum levels causes moderate to severe fluorosis in both humans [4,28,29] and rats [6,7,8]. Fasting serum levels maintained in the treatment of osteoporosis are between 10 and 15 #M [30,31]. Cells exposed to 300 #M fluoride were included to determine whether the effects of fluoride were altered at higher than physiologic serum levels. Message levels for bone proteins were determined at day 18 which allowed accurate measurements for both type I collagen, and the non-collagenous proteins associated with mineralization [16]. Studies by Farley et al. [11] and Wergedal et al. [12] of osteoblast-enriched populations cultured from 18 h to 3 days showed that fluoride (2-25 #M) increased the rate of [3H]thymidine incorporation, compared with controls. However, Kopp and Robey [13] found that fluoride in doses ranging from 10 -6 to 10 -3 M did not alter thymidine incorporation of human bone cells cultured for 16 h, and then exposed to fluoride for 30 h. Wergedal et al. [12] suggested that fluoride response appears to be sensitive to changes in culture conditions, which may further explain the differences between the various in vitro studies. In the present study, the timing of cell proliferation and differentiation, as measured by [3H]thymidine incorporation, was not altered by long-term fluoride exposure. Bone exposed to fluoride is characterized by an increased amount of osteoid in the trabecular bone. Possible reasons for the increase in osteoid following fluoride exposure include an increase in osteoblast proliferation, increased expression of matrix proteins in the fluoride-exposed bone, or a delay in mineralization of the osteoid matrix. Fluoride-exposed bone has been reported to have both increased osteoblast surface area [32,33] and a decrease in osteoblast surface area [34,351.
194
Therefore, although an increase in osteoid may be related to increased numbers of osteoblasts, it is not clear that this occurs in vivo. The findings by several investigators that serum osteocalcin levels were elevated in humans chronically exposed to high levels of fluoride [4,5] suggested that fluoride may increase the expression of bone matrix protein. However, we found no difference in the level of expression of either osteocalcin, cd-type I collagen, or transin in 18-day cultures exposed to different levels of fluoride. The amount of osteopontin expressed by cells exposed to 5 ~M F was significantly less than that expressed by cells exposed to 20 and 300 ~M F, when compared with f3-actin. However, because of a greater standard deviation in the control groups, there was no significant difference between the control and the three groups of fluoride-exposed cells. These results suggest that fluoride does not have a major effect on protein expression during the proliferative (days 5-11) or differentiation stages of matrix formation. Rather, the increases in serum osteocalcin in humans exposed to high levels of fluoride may be due to effects of fluoride on other aspects of bone metabolism, which may result in an increased number or activity of osteoblasts. In a similar study by Bellows et al. [36], cells were cultured for 18 days in the presence of varying amounts of fluoride. At fluoride concentrations ~ 100 #M F, there were no significant differences between control and fluoride-exposed cells in the numbers of mineralizing nodules. In addition, concentrations of < 500 ~M F in culture showed no significant effect on cell growth rate (as measured by cell number at various time points) or colony formation. The results of that study, as well the present study, suggest that physiological levels of fluoride do not have an effect on cell growth, differentiation, or matrix protein expression. In one experiment, cells were cultured for 26 days, with a resulting large reduction in the expression of both osteocalcin and cd-type I collagen in the high (300 ~M) fluoride cultures. The difference in protein expression in 26-day culture, compared with the 18-day culture, may indicate alterations by fluoride in the mineralizing matrix which may indirectly affect cell function and protein expression. Exposure of cells to 300 ~M fluoride does not appear to be physiologically relevant when compared with serum fluoride levels. However, fluoride levels of > 1000 ppm or higher accumulate in the bone mineral of adult humans. It is possible that the fluoridated mineral establishes an equilibrium with the surrounding fluid, resulting in local fluoride concentrations which are higher than serum levels. Fluoride at higher concentrations (millimolar) in culture has been shown to have effects on many aspects of cell metabolism. These effects include an inhibition of cell proliferation [37], delayed cell differentiation [38], and increased concentrations of cAMP due to fluoride interactions with G proteins [39-41]. If higher concentrations exist in the extracellular matrix fluid surrounding the osteoblasts than in the serum, then these effects may be relevant to our understanding of the effects of fluoride on bone. Further experiments to measure fluoride levels in the bone fluid which surround the cells are needed to establish the relevance of these and other studies of the effects of fluoride on cells in vitro. The results of these in vitro studies suggest that direct effects of fluoride on osteoblast proliferation or protein expression are not major effects of fluoride which result in the formation of fluorosed bone. In tooth enamel, a major effect of fluoride on forming enamel, is a delay in mineralization. Similar effects of fluoride on forming bone may result from mechanisms such as an inhibition of matrix enzymes which
195
remove crystal growth inhibitors prior to the onset of mineralization [36], changes in the composition of bone glycosaminoglycans [42,43], or post-translational effects of fluoride on the bone matrix proteins. It is likely that more than one mechanism is responsible for the formation of fluorosed bone.
Acknowledgements The authors gratefully acknowledge technical assistance of Carol P. Shields for the chemical analyses and of Richard E. Glena in the XDR and IR analyses. Helpful discussions with Dr. John Featherstone regarding these studies are also acknowledged.
References 1 Thiebaud M, Zender R, Courvoisier B, Baud CA, Jacot C. The action of fluoride on diffuse bone atrophies. In: Visher TL, ed. Fluoride in medicine. Bern: Haber, 1971;136-142. 2 Riggs BL. Treatment of osteoporosis with sodium fluoride: an appraisal. In: Peck WA, ed. Bone and mineral research, Annual 1. Amsterdam: Elsevier, 1983;366-393. 3 Dur-Smith BA, Kraenzlin ME, Farley SM, Libanati CR, Schulz EE, Baylink DJ. Fluoride therapy for osteoporosis: a review of dose response, duration of treatment, and skeletal sites of action. Calcif Tissue lnt 1991;49($5):64-72. 4 Strivastava RN, Gill DS, Moudgel A, Menon RK, Thomas M, Dandona P. Normal ionised calcium, parathyroid hypersecretion and elevated osteocalcin in a family with fluorosis. Metabolism 1989;2:120-124. 5 Dondona P, Coumar A, Gill DS, Bell J, Thomas M. Sodium fluoride stimulates osteocalcin in normal subjects. Clin Endocrinol 1988;29:437-441. 6 Bernick S, Zipkin I. Histochemical study of bone in hydrocortisone- and fluoride-treated rats. J Dent Res 1967;46:1404-14.'??.. 7 Ream L. The effects of short-term fluoride ingestion on bone formation and resorption in the rat femur. Cell Tissue Res 1981;221:421-430. 8 DenBesten PK, Crenshaw MA. The effects of chronic high fluoride levels on forming enamel in the rat. Arch Oral Biol 1984;29:675-679. 9 Cheng PT, Bader SM. Effects of fluoride on rat cancellous bone. Bone Miner 1990;11:153-161. l0 Hall BK. Sodium fluoride as an initiator of osteogenesis from embryonic mesenchyme in vitro, Bone 1987;8:111-116. 11 Farley JR, Wergedal JE, Baylink DJ. Fluoride directly stimulated proliferation and alkaline phosphatase activity of bone-forming cells. Science 1983;222:330-332. 12 Wergedal JE, Lau KHW, Baylink DJ. Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures. Clin Orthop Relat Res 1988;233:274-282. 13 Kopp JB, Robey PG. Sodium fluoride does not increase human bone cell proliferation or protein synthesis in vitro. Calcif Tissue Int 1990;47:221-229. 14 Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS. Progressive development of the rat osteoblast phenotype in vitro: peeiprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990;143:420-430. 15 Collin P, Nefussi JR, Wetterwald A, Nicolas V, Boy-Lefevre M, Fleisch H, Forest N. Expression of collagen, osteocalein, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture. Calcif Tissue lnt 1992;51:175-183. 16 Aronow MA, Gerstenfeld LC, Owen TA, Tassinari MS, Stein GS, Lian JB. Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells. J Cell Physiol 1990;143:213-221. 17 Ogedn RC, Adams DA. Electrophoresis in agarose and acrylamide gels. In: Berger SL, ed. Methods in enzymology: guide to molecular cloning techniques. New York: Academic Press, 1987;61-87. 18 Celeste AJ, Rosen V, Buecker JL, Kriz R, Wang EA, Wozney JM. Isolation of the human gene for bone gla protein utilizing mouse and rat eDNA clones. EMBO J 1986;5:1885-1890.
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22 23 24 25 26 27 28 29 30
31 32 33 34 35 36 37 38 39 40 41 42 43 44
Bolander ME, Young MF, Fisher LW, Yamada Y, Termine JD. Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc Natl Acad Sci USA 1988;85:2919-2923. Matrisian LM, Leroy P, Ruhlmann M, Gesnell C, Breathnach R. Isolation of oncogene and epidermal growth factor-induced transin gene: complex control in rat fibroblasts. Mol Cell Biol 1986;6:1679-1686. Genovese C, Rowe D and Kream B. Construction of DNA sequences complementary to rat a I and a 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25dihydroxyvitamin D. Biochemistry 1984;23:6210-6215. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1989, pp. 9.52-9.55. Featherstone JDB, Pearson S, LeGeros RZ. An infrared method for quantitfication of carbonate in carbonated apatites. Caries Res 1984;18:63-66. Taves DR. Determinations of submicromotar concentrations of fluoride in biological samples. Talanta 1968;1015-1023. Chen PS, Toribara TY, Warner H. Micro-determinations of phosphorous. Anal Chem 1956;28:1756-1758. Bellows CG, Aubin JE, Heersche JNM, Antosz ME. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue int 1986;38:143-154. Bhargava U, Bar-Lev M, Bellows CG, Aubin JE. Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells. Bone 1988;9:155-163. Guy WS, Taves DR. Relation between F in drinking water and human plasma. J Dent Res 1973;52:238. Singer L, Ophaug R. Ionic and nonionic fluoride in plasma (or serum). CRC Crit Rev Clin Lab Sci 1982;18:111-140. Van Kesteren R J, Duursma SA, Visser W J, van der Sluys Veer J, Backer Dirks O. Fluoride in serum and bone during treatment of osteoporosis with sodium fluoride, calcium and vitamin D. Metab Bone Dis Relat Res 1982;4:31-37. Mohan S, Stauffer M, Baylink DJ. Clinical use of fluoride in osteoporosis, In: Kanis JA, ed. Calcium metabolism. Basel: Karger, 1990;4:137-164. Marie PJ, Hott M. Short-term effects of fluoride and strontium on bone formation and resorption in the mouse. Metabolism 1986;7:547-551. Mosekilde L, Kragstrup J, Richards A. Compressive strength, ash weight, and volume of vertebral trabecular bone in experimental fluorosis in pigs. Calcif Tissue Int 1987;40:318-322. Snow GR, Anderson C. Short-term chronic fluoride administration and trabecular bone remodeling in beagles: a pilot study. Calcif Tissue lnt 1986;38:217-221. Cheng P-T, Bader SM. Effects of fluoride on rat cancellous bone. Bone Miner 1990~11:153-161. Bellows CG, Heersche JNM, Aubin JE. The effects of fluoride on osteoblast progenitors in vitro. J Bone Miner Res 1990;5:S101-S105. Holland RI, Hongslo JK. The effect of fluoride on the cellular uptake and pool of amino acids. Acta Pharmacol Toxicol 1979;44:354-358. Kerley MA, Kolar EJ. Regeneration of tooth development in vitro following fluoride treatment. Am J Anat 1977;149:181-196. Stadel JM, Crooke ST. Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding. Biochem J 1988;254:15-20. Hall A. The cellular functions of small GTP-binding proteins. Science 1990;249:635-640. Habara Y, Satoh T, Saito T, Kanno T. A G-protein activator, NaF induced [Ca 2+] oscillation and secretory response in rat pancreatic acini. Biomed Res 1990;11:389-398. Robey PG. The biochemistry of bone. Endocrin Metab Clin North Am 1989:18:859-902. Jha M, Susheela AK. Characterization of glycosaminoglycans from normal and fluoride treated rabbit iliac crest. Biochem Biophys Res Commun 1982;105:711-716. Prince CW, Navia JM. Glycosaminoglycan alterations in rat bone due to growth and fluorosis. J Nutr 1983;113:1576-1582.
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9 1993 Elsevier Scientific Publishers Ireland Ltd. all rights reserved. 0169-6009/93/$06.00 BAM 0600
Expression of bone protein mRNA at physiological fluoride concentrations in rat osteoblast culture R o n g s h a n Li and P a m e l a K. D e n B e s t e n Department of Pediatric Dentistry, Eastman Dental Center, 625 Elmwood Ave, Rochester, N Y 14620, USA (Received 10 November 1992) (Revision received 22 March 1993) (Accepted 4 May 1993)
Summary Fluoride causes an increase in the amount of unmineralized osteoid. To determine whether the increase in osteoid is due to greater protein expression in the presence of fluoride, we measured the relative amount of mRNA expressed by fetal rat calvaria cells maintained in culture for either 18 or 26 days in the presence of 0, 5, 20 or 300 #M fluoride. There were no differences in the level of expression ofmRNA for collagenous or non-collagenous proteins in fluoride-treated cells as compared with control cells at 18 days in culture. Expression of mRN A for osteocalcin and al-type I collagen was decreased at 300 #M fluoride after 26 days culture. The amount of [3H]thymidine incorporation in cells exposed to the different amounts of fluoride was measured at various time points. Fluoride did not alter the time at which rapid cell proliferation ended. These studies indicate that at physiological serum levels, fluoride does not increase expression ofmRNA by osteoblasts. The relative increase in osteoid in bone may be related to other mechanisms such as altered matrix mineralization.
Key words: Osteoblast; Bone cell; Fluoride; mRNA; Gene expression
Introduction Fluoride has been shown to both accumulate in mineralizing tissues and to result in altered development of bones and teeth. In the treatment of osteoporosis, fluoride levels which are sufficient to maintain fasting serum fluoride at 5-10 t~M (0.095-0.19 ppm F) result in a net gain in bone mass. New bone formation is most pronounced in the trabecular bone of the axial skeleton. In cortical bone, there is no net gain in bone formation and some reports have indicated a decrease in cortical Correspondence to: P.K. DenBesten, Department of Pediatric Dentistry, Eastman Dental Center, 625 Elmwood Ave, Rochester, NY 14620, USA.
188
bone mass with fluoride treatment [1,2,3]. Chronic fluoride exposure in humans has been shown to increase serum osteocalcin [4,5] which may indicate increased osteoblastic activity. Prolonged exposure to high levels of fluoride (120 ppm) in rats, results in the formation of bone with widened, unmineralized osteoid seams on the periosteal surface of the femur [6,7]. This level of fluoride in the drinking water of a rat would result in serum fluoride levels between 10 and 20 t~M fluoride [8]. Cheng and Bader [9] gave sodium fluoride in drinking water to rats, and found that levels resulting in serum concentrations from 5 to 13 /~M fluoride caused an increase in the rat cancellous bone volume through trabecular thickening. They suggested that this change was due to reduced bone resorption. Direct cellular effects of physiological (micromolar) levels of fluoride include stimulation of osteogenesis in embryonic mesenchyme [10]. A concentration of 10 #M (0.18 ppm) fluoride induced osteogenesis under culture conditions which would otherwise not result in bone formation. Several reports show that fluoride at concentrations of 2-25/~M (0.038-0.475 ppm) increases the rate of cell proliferation and alkaline phosphatase activity of isolated chick [11], and human osteoblasts [12]. However, in human fetal bone cell culture, fluoride exposure of up to 6 days was not found to affect cell proliferation or protein secretion [131. These studies indicate that alterations in bone formation by fluoride may be a result of a direct effect of fluoride on the osteoblasts, affecting cell proliferation, differentiation and matrix production. Expression of certain matrix proteins, such as osteocalcin, is related to the onset of mineralization [14,15]. Changes in the matrix (such as in the type and amount of mineral formed) may indirectly affect matrix expression, and could further result in the delayed mineralization found in fluorideexposed bone. In the present study, we have used the rat calvaria cell culture system to determine whether long-term fluoride exposure to bone cells in vitro, alters expression of matrix proteins. Materials and methods
Cell culture Calvaria from 21-day gestation fetal rats were isolated and sequentially digested with collagenase trypsin as described by Aronow et al. [16]. Cells were plated in minimal essential medium (MEM; Gibco) supplemented with 1.5% penicillin, 0.05% streptomycin, 10% fetal bovine serum (FBS) in 100 mm dishes (for mRNA isolation) and 6-well dishes (for thymidine incorporation) at 5 x 105 or 3.6 x 105 per dish, respectively. The culture dishes were divided into four groups and fluoride was added to a final concentration in the media of either 0, 5, 20, or 300 #M from day 1 of culture. These levels of fluoride were maintained in the medium throughout the time of culture. At day 7, the cultures were supplemented with 50/~g/ml ascorbic acid and 10 mM/3-glycerol phosphate, the supplements were added every feeding day thereafter. The cells were fed every 2-3 days, and maintained in a 5% CO 2 atmosphere. DNA synthesis To determine whether the cessation of cell proliferation occurred at the same time under the various fluoride conditions, DNA synthesis was determined according to
189
the method of Owen et al. [14]. Cells were plated into 6-well dishes with a final concentration of 0, 5, 20 or 300 t~M fluoride in the medium containing 10% FBS. At days 5, 8, 11, 14 and 18, [3H]thymidine (20 Ci/mmol) was added to the medium of 2 wells from each group to a final concentration of 10/zCi/ml for 1 h. Cells were rinsed with phosphate-buffered saline (PBS), incubated with 5% (w/v) trichloracetic acid (TCA) for 5 min, rinsed and the incubation repeated. The cell layer was solubilized in 0.5 ml 10% SDS, and 4 ml of scintillation fluid was added prior to counting on a scintillation counter.
RNA &olation and analys& In one experiment, message levels of osteocalcin, osteonectin, osteopontin, transin (rat stromelysin), type I collagen, fl-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPD) were determined at day 18 of culture. Day 18 was chosen as a time point in osteoblast culture where matrix maturation occurs, and matrix mineralization has begun [14,16]. Cells exposed to the different levels of fluoride were cultured in triplicate, and on day 18, they were washed with PBS, immersed in SDS buffer containing RNAase/protein degrader provided in a Micro-Fast-track mRNA isolation kit (Invitrogen, San Diego, CA). The cells were scraped from the dishes, and polyadenylated RNA was then isolated from the cells according to the manufacturer's directions. The mRNA isolated from each group was quantitated by absorbance at 260 nm and 1 t~g of each sample was separated on a 1% agarose gel containing 2 M formalde-
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Fig. 1. Northern blot analysis of mRNA from RC cells, probed with 32p-labeled cDNA for transin (group B) and for GAPD (group A). The blot is visualizied, the area to be counted is marked, and an output of counts/min for each marked area is generated. The counts/min for each matrix protein was divided by the counts/min for either GAPD or/3-actin, to correct for variations in the amount of mRNA initially loaded on the agarose gel.
190
hyde using standard methodology [17]. Following separation, the RNA was transferred overnight with 20 x SSC by capillary diffusion to a Duralon nylon membrane (Stratagene, La Jolla, CA). The mRNA was cross-linked to the membrane using a Stratalinker (Stratagene) ultraviolet cross-linker. DNA probes used for hybridization were: osteocalcin, a gift from Genetics Institute, Cambridge MA, [18]; osteonectin and osteopontin, a gift from Dr. Marion Young, National Institutes of Health, [19]; transin, a gift from Dr. Lynn Matrision, Vanderbilt University [20]; rat c~-I collagen, a gift from Dr. Barbara Kream, University of Connecticut, [21]; and B-actin and GAPD, gifts of Dr. David Wong, Harvard School of Dental Medicine. DNA probes were labeled with [32p]dCTP by random priming, and the membranes were hybridized using standard methodology [22]. The membranes were washed 4 times, with a final wash of 0.1 x SSC and 0.5% SDS at 68~ for 30 min, and the relative radioactivity emitted from a hybridized band was quantitated on a Betascope Blot Analyzer (Betagen, Waltham, MA). The Betascope allows direct counting of radioactive emissions from the hybridized band (see Fig. 1). The hybridization signal for each of the matrix proteins was quantitated relative to the signal for either B-actin or GAPD, and the average was obtained for each level of fluorid6 exposure. In one set of experiments, the time of culture was extended to 26 days. The mRNA was isolated from duplicate samples from each group and analyzed for expression of osteocalcin and type I collagen. The mineral contained in the matrix of 2 wells from each group of cells cultured for 18 days was analyzed by chemical analysis, infrared spectroscopy [23] and X-ray diffraction (XRD). For X-ray diffraction, a sample from each group was mounted on a low background holder and analyzed in a Philips powder difractometer using copper K,, radiation. Fluoride in the matrix was measured by the diffusion method of Taves [24]. The calcium and phosphate contents of the mineral were determined from the acid solution (remaining following fluoride diffusion) by atomic absorption spectrophotometry, and phosphate was determined by the method of Chen et al. [251. Statistical evaluation
The statistical significance of the differences between the means of the control and experimental groups was evaluated by analysis of variance (ANOVA). Values indicating a probability of < 0.05 were regarded as statistically significant. Results
There were no gross changes in the morphology of cells cultured in the presence of fluoride, as compared with controls. Mineralizing nodules similar to those described in previous studies using rat calvaria cells [16,26,27] were identified by light microscopy within 24 h after supplementation of ~GPO 4. Cell proliferation, as measured by incorporation of [3H]thymidine, decreased sharply at day 11 in all groups (Fig. 2), when cell differentiation and matrix nodule formation began. Longterm exposure to fluoride in vitro did not cause a change in the time at which cell proliferation ceased, as compared with the controls. The expression of cd-type I collagen (Fig. 3) and the non-collagenous proteins (osteocalcin, osteonectin, osteopontin, and transin) (Fig. 4) were measured relative
191
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to the level of expression of either fl-actin or GAPD. There were no significant differences in the levels of mRNA expression for these proteins when the fluoride-exposed groups were compared with controls. There was a significant increase in the levels of osteopontin in the 20/~M and 300 #M fluoride-exposed groups compared with
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the 5/~M fluoride group relative to expression o f ~-actin. These differences were not found to be significant when compared with G A P D . In the 26 day cultures, the message for osteocalcin and type I collagen was greatly reduced in the cells exposed to 300 #M fluoride (Fig. 5) relative to both/3-actin and G A P D . However, the message levels were similar in the lower fluoride groups, as compared with the control group. The time at which message levels decreased appeared to be somewhat dependent on culture condition. Some experiments performed with more densely plated cultures showed a drop in m R N A expression earlier than 26 days (results not shown).
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193 Although sample amounts were very small, all matrices from 18-day-old cell culture produced infrared spectra characteristic of poorly-formed apatite-like or brushite-like calcium phosphate, comparable with that found by us for mouse calvaria (unpublished data). Phosphate, carbonate and structural water bands were present. The sample amounts were too small to allow definitive characterization by XRD. The infrared spectra coupled with the chemical analyses of the material, which showed calcium (20-45 ~g per sample) and phosphate (6-50 ~g, as P, per sample) to be present in the particles analyzed, indicated the presence of poorly crystalline calcium phosphate mineral. The samples could not be accurately weighed, therefore, the amount of fluoride present was related to the amount of calcium and phosphate present, assumed to be in the form of hydroxyapatite for purposes of calculation. Measurements were done as an average of 2 samples from each group and showed increasing amounts of fluoride in the matrix with increased amounts of fluoride in the media (control, 633 ppm; 5 #M F, 590 ppm; 20 #M F, 1056 ppm; 300 #M F, 4305 ppm). Discussion Studies of the rat osteoblast culture system have shown a developmental sequence of gene expression associated with bone cell differentiation [14,16]. In the present study, this culture system was used to study the long-term effects of fluoride on osteoblast-enriched rat calvaria cells. Fluoride levels of 5 t~M and 20 ~M were chosen as similar to the serum levels of humans ingesting from 5 to 15 ppm F in drinking water. Long-term exposure to fluoride in drinking water resulting in these serum levels causes moderate to severe fluorosis in both humans [4,28,29] and rats [6,7,8]. Fasting serum levels maintained in the treatment of osteoporosis are between 10 and 15 #M [30,31]. Cells exposed to 300 #M fluoride were included to determine whether the effects of fluoride were altered at higher than physiologic serum levels. Message levels for bone proteins were determined at day 18 which allowed accurate measurements for both type I collagen, and the non-collagenous proteins associated with mineralization [16]. Studies by Farley et al. [11] and Wergedal et al. [12] of osteoblast-enriched populations cultured from 18 h to 3 days showed that fluoride (2-25 #M) increased the rate of [3H]thymidine incorporation, compared with controls. However, Kopp and Robey [13] found that fluoride in doses ranging from 10 -6 to 10 -3 M did not alter thymidine incorporation of human bone cells cultured for 16 h, and then exposed to fluoride for 30 h. Wergedal et al. [12] suggested that fluoride response appears to be sensitive to changes in culture conditions, which may further explain the differences between the various in vitro studies. In the present study, the timing of cell proliferation and differentiation, as measured by [3H]thymidine incorporation, was not altered by long-term fluoride exposure. Bone exposed to fluoride is characterized by an increased amount of osteoid in the trabecular bone. Possible reasons for the increase in osteoid following fluoride exposure include an increase in osteoblast proliferation, increased expression of matrix proteins in the fluoride-exposed bone, or a delay in mineralization of the osteoid matrix. Fluoride-exposed bone has been reported to have both increased osteoblast surface area [32,33] and a decrease in osteoblast surface area [34,351.
194
Therefore, although an increase in osteoid may be related to increased numbers of osteoblasts, it is not clear that this occurs in vivo. The findings by several investigators that serum osteocalcin levels were elevated in humans chronically exposed to high levels of fluoride [4,5] suggested that fluoride may increase the expression of bone matrix protein. However, we found no difference in the level of expression of either osteocalcin, cd-type I collagen, or transin in 18-day cultures exposed to different levels of fluoride. The amount of osteopontin expressed by cells exposed to 5 ~M F was significantly less than that expressed by cells exposed to 20 and 300 ~M F, when compared with f3-actin. However, because of a greater standard deviation in the control groups, there was no significant difference between the control and the three groups of fluoride-exposed cells. These results suggest that fluoride does not have a major effect on protein expression during the proliferative (days 5-11) or differentiation stages of matrix formation. Rather, the increases in serum osteocalcin in humans exposed to high levels of fluoride may be due to effects of fluoride on other aspects of bone metabolism, which may result in an increased number or activity of osteoblasts. In a similar study by Bellows et al. [36], cells were cultured for 18 days in the presence of varying amounts of fluoride. At fluoride concentrations ~ 100 #M F, there were no significant differences between control and fluoride-exposed cells in the numbers of mineralizing nodules. In addition, concentrations of < 500 ~M F in culture showed no significant effect on cell growth rate (as measured by cell number at various time points) or colony formation. The results of that study, as well the present study, suggest that physiological levels of fluoride do not have an effect on cell growth, differentiation, or matrix protein expression. In one experiment, cells were cultured for 26 days, with a resulting large reduction in the expression of both osteocalcin and cd-type I collagen in the high (300 ~M) fluoride cultures. The difference in protein expression in 26-day culture, compared with the 18-day culture, may indicate alterations by fluoride in the mineralizing matrix which may indirectly affect cell function and protein expression. Exposure of cells to 300 ~M fluoride does not appear to be physiologically relevant when compared with serum fluoride levels. However, fluoride levels of > 1000 ppm or higher accumulate in the bone mineral of adult humans. It is possible that the fluoridated mineral establishes an equilibrium with the surrounding fluid, resulting in local fluoride concentrations which are higher than serum levels. Fluoride at higher concentrations (millimolar) in culture has been shown to have effects on many aspects of cell metabolism. These effects include an inhibition of cell proliferation [37], delayed cell differentiation [38], and increased concentrations of cAMP due to fluoride interactions with G proteins [39-41]. If higher concentrations exist in the extracellular matrix fluid surrounding the osteoblasts than in the serum, then these effects may be relevant to our understanding of the effects of fluoride on bone. Further experiments to measure fluoride levels in the bone fluid which surround the cells are needed to establish the relevance of these and other studies of the effects of fluoride on cells in vitro. The results of these in vitro studies suggest that direct effects of fluoride on osteoblast proliferation or protein expression are not major effects of fluoride which result in the formation of fluorosed bone. In tooth enamel, a major effect of fluoride on forming enamel, is a delay in mineralization. Similar effects of fluoride on forming bone may result from mechanisms such as an inhibition of matrix enzymes which
195
remove crystal growth inhibitors prior to the onset of mineralization [36], changes in the composition of bone glycosaminoglycans [42,43], or post-translational effects of fluoride on the bone matrix proteins. It is likely that more than one mechanism is responsible for the formation of fluorosed bone.
Acknowledgements The authors gratefully acknowledge technical assistance of Carol P. Shields for the chemical analyses and of Richard E. Glena in the XDR and IR analyses. Helpful discussions with Dr. John Featherstone regarding these studies are also acknowledged.
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22 23 24 25 26 27 28 29 30
31 32 33 34 35 36 37 38 39 40 41 42 43 44
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