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Bone Morphogenetic Protein-3b (BMP-3b) Gene Expression Is Correlated with Differentiation in Rat Calvarial Osteoblasts
Biochemical and Biophysical Research Communications 256, 419 – 424 (1999) Article ID bbrc.1999.0341, available online at http://www.idealibrary.com on
Bone Morphogenetic Protein-3b (BMP-3b) Gene Expression Is Correlated with Differentiation in Rat Calvarial Osteoblasts Jun Hino, Hisayuki Matsuo, and Kenji Kangawa 1 National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan
Received February 5, 1999
BMP-3b (also called GDF-10) is a novel BMP-3related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor b1 (TGF-b1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-b1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts. © 1999 Academic Press
Bone morphogenetic proteins (BMPs), members of the TGF-b superfamily, originally were identified as proteins that induce endochondral bone formation in adult animals (1). Recent analyses have showed that BMPs have a variety of biological effects on different cell types (2– 8). In mammals, nine forms of BMPs have been identified (8 –12) and subdivided into three groups. BMP-2 and BMP-4 comprise the first group (1, 12), and BMP-5, BMP-6, BMP-7, BMP-8 and BMP-8B 1 To whom correspondence should be addressed. Fax: (181)-66872-7485. E-mail: [email protected]. Abbreviations: BMP, bone morphogenetic protein; GDF-10, growth/differentiation factor 10; ALPase, alkaline phosphatase; TGF-b1, transforming growth factor b1; GAPDH, glyceraldehyde-3phosphate dehydrogenase; RA, retinoic acid; bFGF, basic fibroblast growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline.
the second (9, 11, 12). BMP-3b (GDF-10)/BMP-3, which differs from the members of the other BMP subgroups, comprises the third group (1, 12-15). Recently, we isolated rat and human BMP-3b cDNAs, a novel type of BMP-3, from bone tissues (13, 15), but the physiological and biological functions of BMP-3b are unknown. We showed that the BMP-3b gene primarily is expressed in bone tissues (13, 15), but its regulation has not been studied. To clarify BMP-3b functions and the regulation of its gene transcription in bone tissues, the cells which express the BMP-3b gene must be identified. We here report the gene expression of BMP-3b in primary osteoblastic cells and correlation of its regulation with osteoblast differentiation. We also compared the regulation of BMP-3 and BMP-3b gene expressions in these cells. MATERIALS AND METHODS Cell cultures. Primary cultures of neonatal calvaria were obtained from 1-day-old Sprague-Dawley rats (16). Thirty calvariae were excised, and all adhering soft tissue was removed. The calvariae were cut into pieces then subjected to six sequential 20-min digestions with 2 mg/ml collagenase (Type II, Worthington biochemical corporation), 0.5 mg/ml trypsin (Biken, Osaka, Japan) and 4 mM EDTA at room temperature. Single-cell suspensions were recovered from each digestion and were numbered as populations 1-6 conforming to their order of release. The cells were cultured as a pool of the populations numbered 4-6, which are enriched for cells expressing the osteoblastic phenotype (16, 17). The cells were plated on 24- or 6-well tissue culture plates in a-MEM supplemented with 10% fetal calf serum (FCS), 5 mM b-glycerophosphate and 50 mg/ml ascorbic acid. MC3T3-E1 cells were grown in a-MEM containing 10% FCS, and U2-OS cells in McCoy 5A medium containing 10% FCS. Northern blot analysis. Total RNA was extracted using an RNeasy Kit (QIAGEN). Poly(A) 1 RNA was isolated using Oligo (dT)Latex (Nippon Roche). The total RNA or Poly(A) 1 RNA obtained under each experimental condition was denatured with formaldehyde and formamide then electrophoresed on a 1% agaraose gel containing formaldehyde. The RNA was transferred to a Zeta probe membrane (Bio-Rad) and fixed with ultraviolet irradiation. Hybridization was performed at 42 m in a solution containing 50% formamide (40% formamide for MC3T3-E1 cells), 6 3 SSPE, 5 3 Denhardt’s solution, 0.5% SDS, and 100 mg /ml denatured salmon sperm DNA. The membrane was finally washed at 65°C in 0.1 3 SSC
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containing 0.1% SDS. The same membrane was used for sequential hybridizations with probes, coding region of rat BMP-3b and BMP-3 for bone tissues, primary calvarial osteoblastic cells and MC3T3-E1 cells, coding region of human BMP-3b and BMP-3 for U2-OS cells. The amounts of mRNAs were verified by glyceraldehyde-3phosphate dehydrogenase (GAPDH) or 18S RNA. Signals were detected with a BAS 5000 or FLA 2000 Bioimaging analyzer (Fuji Photo Film). Measurement of cellular alkaline phosphatase activity. The primary osteoblastic cells were washed once with phosphate-buffered saline (PBS) and sonicated in 0.02% Triton X-100. The ALPase activity of the sonicate was measured with p-nitrophenyl phosphate as the substrate, as described elsewhere (18). Alizalin red stain. Cells were washed twice with PBS then fixed with 100% ethanol. The fixed osteoblasts were stained with 1% Alizarin solution. Materials. Recombinant human BMP-2 was purified from Chinese hamster ovary cells transfected with cDNA (1). The following materials were used: recombinant human TGF-b1 purchased from R and D systems (Minneapolis, MN), recombinant human basic fibroblast growth factor (bFGF) from Gibco and all-trans-retinoic acid (RA) from Sigma.
RESULTS Expression of BMP-3b and the BMP-3 gene in primary osteoblastic cells. Several types of osteoblastic cells were subjected to Northern blot analysis to determine whether BMP-3b mRNA is expressed in such cells. The osteoblastic cell lines, MC3T3-E1 and U2-OS first were examined. Although BMP-3b mRNA (2.8 and 4.1kb) was expressed in calvarial tissues (13), it was not detected in MC3T3-E1 and U2-OS cells (Fig. 1). We therefore isolated primary osteoblastic cells from neonatal rat calvariae and examined expression of the BMP-3b gene in these cells. BMP-3b mRNA expression was higher in the primary osteoblastic cells than in the calvaria tissues (Fig. 1). BMP-3 mRNA (6.7, 4.7 and 2.6kb) also was detected only in calvaria tissues and primary osteoblastic cells. Expression of BMP-3b and the BMP-3 gene in prolonged cultures of primary osteoblastic cells. The expression pattern of the BMP-3b gene in prolonged cultures of primary osteoblastic cells and correlation of the expression of BMP-3b with alkaline phosphatase (ALPase) activity, the marker for osteoblast differentiation, were examined. BMP-3b mRNA was increased in these cultures and associated with increased ALPase activity (Fig. 2). Alizarin red staining on day 14 showed mineralized nodules in the cells (data not shown). There was less BMP-3 mRNA than BMP-3b mRNA during prolonged culture, but the gene expression patterns were similar. Effect of BMP-2 on the mRNA levels of BMP-3b and BMP-3 in primary osteoblastic cells. BMP-2 (100 ng/ ml) increased ALPase activity (Fig. 3B) and produced qualitatively more mineralized bone nodule in primary osteoblastic cells than in the controls (Fig. 3C). The time courses of the effects of BMP-2 are shown in Fig.
FIG. 1. RNA blot analysis of BMP-3b and BMP-3 gene transcripts in bone tissues, primary calvarial osteoblastic cells and clonal cell lines. Lanes were loaded with 10 mg of total RNA (bone tissues and primary calvarial osteoblastic cells) and 10 mg of poly(A) 1 RNA (MC3T3-E1 and U2-OS cells). Size markers are given at right.
3A. There was a dramatic, time-dependent increase in BMP-3b gene transcription, which began within 6 h after treatment and lasted for at least 7 days. The maximum increase in BMP-3b mRNA was 6.9-fold after 24 h of stimulation. In contrast, BMP-3 mRNA expression was decreased by BMP-2, especially during the early phase. Effects of various substances on the mRNA levels of BMP-3b and BMP-3 in primary osteoblastic cells. To clarify the regulation of BMP-3b gene expression, the effects of TGF-b1, FCS, basic FGF (bFGF) and retinoic acid (RA) on BMP-3b gene expression were examined (Fig. 4). Of these substances, TGF-b1, FCS and RA inhibited BMP-3b gene expression, but bFGF had scarcely any suppressive effect. On the other hand, BMP-3 gene expression was inhibited by FCS and bFGF but enhanced by RA (Fig. 4). Effect of TGF-b1 on the mRNA levels of BMP-3b and BMP-3 in primary osteoblastic cells. Because TGF-b1 remarkably inhibited BMP-3b gene expression in primary osteoblastic cells (Fig. 4), its effect in these cells was examined in detail. The time course of the TGF-b1 (10 ng/ml) effect on the mRNA levels of BMP-3b and BMP-3 in the osteoblasts is shown in Fig. 5A and B. TGF-b1 caused a dramatic, rapid decrease in BMP-3b
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determine a role of BMP-3b in bone tissues, the regulation of BMP-3b gene transcription in those tissues must be clarified. We therefore examined the regulation of BMP-3b gene expression in primary osteoblastic cells. Interestingly, although the BMP-3b gene is expressed in bone tissues (13, 15), it is not expressed in established osteoblastic cell lines (MC3T3-E1 and U2OS, Fig. 1). Our findings indicate that the BMP-3b gene is expressed only in primary cultured osteoblastic cells. In addition, the BMP-3b mRNA level in primary osteoblastic cells was higher than in calvarial tissues (Fig. 1), indicating that the BMP-3b gene is expressed
FIG. 2. BMP-3b and BMP-3 mRNA expression and ALPase activity in prolonged-cultured primary osteoblastic cells. Day 0: the day the cells reached confluence. The medium (a-MEM containing 10% FCS, 5 mM b-glycerol phosphate and 50 mg/ml ascorbic acid) was replaced every 2 days. (A) Each lane contained 10 mg of total RNA from different stages of culture. The bottom panel shows ethidium bromide-stained gels used as controls. Size markers are given at right. (B) Cellular ALPase activity was measured with p-nitrophenyl phosphate as the substrate. Cells were inoculated to 24-well plates. Means 6 S. D.; n 5 3.
gene transcription; reaching 62% of the control value after 3 h of incubation and almost complete inhibition (14% of control) after 6 h. Inhibition of BMP-3b gene expression by TGF-b1 continued for at least 96 h. The effect of TGF-b1 on the mRNA level of BMP-3 differed. After 12 h of TGF-b1 treatment, BMP-3 mRNA was increased (130% of control), then decreased to less than the control after 24-96 h. Figure 5C shows the time course effect of TGF-b1 on ALPase activity in osteoblasitc cells. TGF-b1 suppressed ALPase activity throughout the 14-day culture period. Figure 5D shows the results of alizarin red staining on day 14, which made visible mineralized nodules in the cells. Treatment with TGF-b1 also suppressed the formation of mineralized nodules. DISCUSSION We recently succeeded in cloning of BMP-3b cDNA from rat femur tissues and showed that the BMP-3b gene is highly expressed in bone tissues (13), but so far nothing is known about the function of BMP-3b. To
FIG. 3. Time courses of BMP-2 effects on BMP-3b and BMP-3 mRNA levels, ALPase activity and formation of mineralized nodules in primary osteoblastic cells. Confluent primary osteoblastic cells were serum deprived for 24 h (day 0) then cultured for the indicated periods in the absence (2) or presence (1) of 100 ng/ml BMP-2. During stimulation, the medium (a-MEM containing 0.1% BSA, 5 mM b-glycerol phosphate, 50 mg/ml ascorbic acid and with or without BMP-2) was replaced every 2 days. (A) Each lane contained 10 mg of total RNA. The bottom panel shows the ethidium bromide-stained gels used as controls. Size markers are given at right. (B) Cellular ALPase activity was measured with p-nitrophenyl phosphate as the substrate. Cells were inoculated to 24-well plates. Means 6 S. D.; n 5 3. (C) Alizarin red staining was done on day 14.
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in osteoblast in bone tissues. In contrast, the BMP-2, 4, 6 and 7 genes are expressed in bone tissues (1, 9, 10, 13, 15), and moreover, all are expressed in U2-OS cells (1, 9, 10, 19) and some in other established osteoblastic cell lines (19, 20), as well as in primary osteoblastic cells. We conclude that the different gene expression pattern of BMP-3b and BMP-2, 4, 6 and 7 reflects that they have different functions. Prolonged culture of primary osteoblastic cells, enriched for cells with the osteoblast phenotype, has proved a useful model for studying the expression of specific genes by osteoblasts during their differentiation into the mature mineralizing phenotype (21– 23). In our study, the increase in BMP-3b mRNA paralleled that of ALPase activity in prolonged cultures (Fig. 2). Using a similar approach, Harris and colleagues showed that BMP-2, 4 and 6 genes were expressed by fetal rat calvarial osteoblasts with the osteoblast phenotype (21). The gene expression patterns of BMP-3b and BMP-2, 4 and 6 differ slightly during prolonged culture (Fig. 2, Ref. 21), probably because they have different functions. These findings indicate that the elevated BMP-3b mRNA level in prolonged cultures is the result of cell differentiation into the mature, mineralizing osteoblast phenotype. To clarify BMP-3b gene regulation in primary osteoblastic cells, we examined the effects of BMP-2, TGFb1, FCS, bFGF and RA on BMP-3b gene transcription. BMP-2 stimulates osteoblast phenotype expression, e.g., increased ALPase activity, in several osteoblastic cell lines and in primary osteoblasts (Fig. 3, Ref. 23, 24). In this study, we showed that BMP-3b gene expression is stimulated by BMP-2 and is associated
FIG. 4. Effects of TGF-b1, FCS, bFGF and RA on BMP-3b and BMP-3 mRNA levels in primary osteoblastic cells. Confluent primary osteoblastic cells were serum deprived for 24 h then cultured in the absence (control) or presence of TGF-b1 (10 ng/ml), FCS (10%), bFGF (100 ng/ml) or RA (5 3 10 26 M) for 24 h. Each lane contained 10 mg of total RNA. Size markers are given at right.
FIG. 5. Time courses of TGF-b1 effects on the levels of BMP-3b and BMP-3 mRNA, ALPase activity and formation of mineralized nodules in primary osteoblastic cells. Confluent primary osteoblastic cells were serum deprived for 24 h (day 0) then cultured for the indicated periods in the absence (control) or presence of 10 ng/ml TGF-b1. During stimulation, the medium (a-MEM containing 0.1% BSA, 5 mM b-glycerol phosphate, 50 mg/ml ascorbic acid and with or without TGF-b1) was replaced every 2-3 days. (A, B) Each lane contained 10 mg of total RNA. Size markers are given at right. (C) Cellular ALPase activity was measured with p-nitrophenyl phosphate as the substrate. Cells were inoculated to 24well plates. Means 6 S. D.; n 5 3. (D) Alizarin red staining was done on day 14.
with increased ALPase activity in primary osteoblastic cells (Fig. 3); whereas, BMP-3 gene expression is decreased by BMP-2 in these cells (Fig. 3). Chen et al. (23) reported that BMP-3 gene expression was enhanced by BMP-2 (5 to 17 days after treatment) but suppressed by it in the early phase (one day after treatment). The different effects of BMP-2 on BMP-3 gene expression may be attribute to the period of analysis. As shown in Fig. 4, TGF-b1 completely inhibited expression of the BMP-3b gene. Surpris-
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ingly, its suppressive effect on gene transcription of BMP-3b in osteoblastic cells was very strong and rapid (Fig. 5). In vivo, TGF-b1 stimulates bone formation (25), but its effects on osteoblastic cells in vitro are numerous and diverse (26 –29). These differences are related to the differentiated state of the osteoblastic cell populations studied. In our study, TGF-b1 suppressed ALPase activity (Fig. 5) and stimulated DNA synthesis (data not shown) in primary osteoblastic cells. A recent report shows that TGF-b1 inhibits BMP-2 gene expression and several osteoblast marker proteins in fetal rat calvarial osteoblasts (22), indicating that TGF-b1 acts as an inhibitor of osteoblastic differentiation in this cell system. FCS and bFGF are mitogenic for bone cells (4, 30), and RA affects bone morphogenesis (31). BMP-3b mRNA synthesis was suppressed by both FCS and RA (Fig. 4), whereas FCS, as well as TGFb1, stimulated DNA synthesis and decreased ALPase activity (data not shown). bFGF had the same effects as TGF-b1 and FCS on the DNA synthesis and ALPase activity in primary osteoblasts, but its effect was very weak. The effect of bFGF on BMP-3b mRNA suppression therefore was lower than that of TGF-b1 and FCS. These findings suggest that transcription of the BMP-3b gene is inhibited when osteoblastic cells are in the dedifferentiated state. RA is reported to affect growth inhibition and certain differentiated functions in malignant osteoblast cells (32, 33). The mechanism of the suppression of BMP-3b mRNA production by RA is thought to differ from that of TGF-b1 and FCS. We also showed that the BMP-3b and BMP-3 mRNA levels in primary osteoblastic cells are differentially regulated by BMP-2, TGF-b1, FCS, bFGF and RA. For example, BMP-3b mRNA synthesis was increased by BMP-2 but rapidly inhibited by TGF-b1; whereas, BMP-3 mRNA was rapidly decreased by BMP-2, and initially increased but slowly suppressed by TGF-b1. These findings suggest that BMP-3b and BMP-3 have different functions in these cells, even though the proteins are closely related (81% amino acid identity in the mature region). In conclusion, the BMP-3b gene expression was closely associated with differentiation in primary osteoblastic cells. These findings provide strong evidence that BMP-3b has a significant role in osteoblasts. ACKNOWLEDGMENTS We thank Dr. M. Iwamoto of Osaka University for valuable discussion, and Ms. H. Iida for excellent technical assistance. This work was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.
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Bone Morphogenetic Protein-3b (BMP-3b) Gene Expression Is Correlated with Differentiation in Rat Calvarial Osteoblasts Jun Hino, Hisayuki Matsuo, and Kenji Kangawa 1 National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan
Received February 5, 1999
BMP-3b (also called GDF-10) is a novel BMP-3related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor b1 (TGF-b1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-b1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts. © 1999 Academic Press
Bone morphogenetic proteins (BMPs), members of the TGF-b superfamily, originally were identified as proteins that induce endochondral bone formation in adult animals (1). Recent analyses have showed that BMPs have a variety of biological effects on different cell types (2– 8). In mammals, nine forms of BMPs have been identified (8 –12) and subdivided into three groups. BMP-2 and BMP-4 comprise the first group (1, 12), and BMP-5, BMP-6, BMP-7, BMP-8 and BMP-8B 1 To whom correspondence should be addressed. Fax: (181)-66872-7485. E-mail: [email protected]. Abbreviations: BMP, bone morphogenetic protein; GDF-10, growth/differentiation factor 10; ALPase, alkaline phosphatase; TGF-b1, transforming growth factor b1; GAPDH, glyceraldehyde-3phosphate dehydrogenase; RA, retinoic acid; bFGF, basic fibroblast growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline.
the second (9, 11, 12). BMP-3b (GDF-10)/BMP-3, which differs from the members of the other BMP subgroups, comprises the third group (1, 12-15). Recently, we isolated rat and human BMP-3b cDNAs, a novel type of BMP-3, from bone tissues (13, 15), but the physiological and biological functions of BMP-3b are unknown. We showed that the BMP-3b gene primarily is expressed in bone tissues (13, 15), but its regulation has not been studied. To clarify BMP-3b functions and the regulation of its gene transcription in bone tissues, the cells which express the BMP-3b gene must be identified. We here report the gene expression of BMP-3b in primary osteoblastic cells and correlation of its regulation with osteoblast differentiation. We also compared the regulation of BMP-3 and BMP-3b gene expressions in these cells. MATERIALS AND METHODS Cell cultures. Primary cultures of neonatal calvaria were obtained from 1-day-old Sprague-Dawley rats (16). Thirty calvariae were excised, and all adhering soft tissue was removed. The calvariae were cut into pieces then subjected to six sequential 20-min digestions with 2 mg/ml collagenase (Type II, Worthington biochemical corporation), 0.5 mg/ml trypsin (Biken, Osaka, Japan) and 4 mM EDTA at room temperature. Single-cell suspensions were recovered from each digestion and were numbered as populations 1-6 conforming to their order of release. The cells were cultured as a pool of the populations numbered 4-6, which are enriched for cells expressing the osteoblastic phenotype (16, 17). The cells were plated on 24- or 6-well tissue culture plates in a-MEM supplemented with 10% fetal calf serum (FCS), 5 mM b-glycerophosphate and 50 mg/ml ascorbic acid. MC3T3-E1 cells were grown in a-MEM containing 10% FCS, and U2-OS cells in McCoy 5A medium containing 10% FCS. Northern blot analysis. Total RNA was extracted using an RNeasy Kit (QIAGEN). Poly(A) 1 RNA was isolated using Oligo (dT)Latex (Nippon Roche). The total RNA or Poly(A) 1 RNA obtained under each experimental condition was denatured with formaldehyde and formamide then electrophoresed on a 1% agaraose gel containing formaldehyde. The RNA was transferred to a Zeta probe membrane (Bio-Rad) and fixed with ultraviolet irradiation. Hybridization was performed at 42 m in a solution containing 50% formamide (40% formamide for MC3T3-E1 cells), 6 3 SSPE, 5 3 Denhardt’s solution, 0.5% SDS, and 100 mg /ml denatured salmon sperm DNA. The membrane was finally washed at 65°C in 0.1 3 SSC
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containing 0.1% SDS. The same membrane was used for sequential hybridizations with probes, coding region of rat BMP-3b and BMP-3 for bone tissues, primary calvarial osteoblastic cells and MC3T3-E1 cells, coding region of human BMP-3b and BMP-3 for U2-OS cells. The amounts of mRNAs were verified by glyceraldehyde-3phosphate dehydrogenase (GAPDH) or 18S RNA. Signals were detected with a BAS 5000 or FLA 2000 Bioimaging analyzer (Fuji Photo Film). Measurement of cellular alkaline phosphatase activity. The primary osteoblastic cells were washed once with phosphate-buffered saline (PBS) and sonicated in 0.02% Triton X-100. The ALPase activity of the sonicate was measured with p-nitrophenyl phosphate as the substrate, as described elsewhere (18). Alizalin red stain. Cells were washed twice with PBS then fixed with 100% ethanol. The fixed osteoblasts were stained with 1% Alizarin solution. Materials. Recombinant human BMP-2 was purified from Chinese hamster ovary cells transfected with cDNA (1). The following materials were used: recombinant human TGF-b1 purchased from R and D systems (Minneapolis, MN), recombinant human basic fibroblast growth factor (bFGF) from Gibco and all-trans-retinoic acid (RA) from Sigma.
RESULTS Expression of BMP-3b and the BMP-3 gene in primary osteoblastic cells. Several types of osteoblastic cells were subjected to Northern blot analysis to determine whether BMP-3b mRNA is expressed in such cells. The osteoblastic cell lines, MC3T3-E1 and U2-OS first were examined. Although BMP-3b mRNA (2.8 and 4.1kb) was expressed in calvarial tissues (13), it was not detected in MC3T3-E1 and U2-OS cells (Fig. 1). We therefore isolated primary osteoblastic cells from neonatal rat calvariae and examined expression of the BMP-3b gene in these cells. BMP-3b mRNA expression was higher in the primary osteoblastic cells than in the calvaria tissues (Fig. 1). BMP-3 mRNA (6.7, 4.7 and 2.6kb) also was detected only in calvaria tissues and primary osteoblastic cells. Expression of BMP-3b and the BMP-3 gene in prolonged cultures of primary osteoblastic cells. The expression pattern of the BMP-3b gene in prolonged cultures of primary osteoblastic cells and correlation of the expression of BMP-3b with alkaline phosphatase (ALPase) activity, the marker for osteoblast differentiation, were examined. BMP-3b mRNA was increased in these cultures and associated with increased ALPase activity (Fig. 2). Alizarin red staining on day 14 showed mineralized nodules in the cells (data not shown). There was less BMP-3 mRNA than BMP-3b mRNA during prolonged culture, but the gene expression patterns were similar. Effect of BMP-2 on the mRNA levels of BMP-3b and BMP-3 in primary osteoblastic cells. BMP-2 (100 ng/ ml) increased ALPase activity (Fig. 3B) and produced qualitatively more mineralized bone nodule in primary osteoblastic cells than in the controls (Fig. 3C). The time courses of the effects of BMP-2 are shown in Fig.
FIG. 1. RNA blot analysis of BMP-3b and BMP-3 gene transcripts in bone tissues, primary calvarial osteoblastic cells and clonal cell lines. Lanes were loaded with 10 mg of total RNA (bone tissues and primary calvarial osteoblastic cells) and 10 mg of poly(A) 1 RNA (MC3T3-E1 and U2-OS cells). Size markers are given at right.
3A. There was a dramatic, time-dependent increase in BMP-3b gene transcription, which began within 6 h after treatment and lasted for at least 7 days. The maximum increase in BMP-3b mRNA was 6.9-fold after 24 h of stimulation. In contrast, BMP-3 mRNA expression was decreased by BMP-2, especially during the early phase. Effects of various substances on the mRNA levels of BMP-3b and BMP-3 in primary osteoblastic cells. To clarify the regulation of BMP-3b gene expression, the effects of TGF-b1, FCS, basic FGF (bFGF) and retinoic acid (RA) on BMP-3b gene expression were examined (Fig. 4). Of these substances, TGF-b1, FCS and RA inhibited BMP-3b gene expression, but bFGF had scarcely any suppressive effect. On the other hand, BMP-3 gene expression was inhibited by FCS and bFGF but enhanced by RA (Fig. 4). Effect of TGF-b1 on the mRNA levels of BMP-3b and BMP-3 in primary osteoblastic cells. Because TGF-b1 remarkably inhibited BMP-3b gene expression in primary osteoblastic cells (Fig. 4), its effect in these cells was examined in detail. The time course of the TGF-b1 (10 ng/ml) effect on the mRNA levels of BMP-3b and BMP-3 in the osteoblasts is shown in Fig. 5A and B. TGF-b1 caused a dramatic, rapid decrease in BMP-3b
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determine a role of BMP-3b in bone tissues, the regulation of BMP-3b gene transcription in those tissues must be clarified. We therefore examined the regulation of BMP-3b gene expression in primary osteoblastic cells. Interestingly, although the BMP-3b gene is expressed in bone tissues (13, 15), it is not expressed in established osteoblastic cell lines (MC3T3-E1 and U2OS, Fig. 1). Our findings indicate that the BMP-3b gene is expressed only in primary cultured osteoblastic cells. In addition, the BMP-3b mRNA level in primary osteoblastic cells was higher than in calvarial tissues (Fig. 1), indicating that the BMP-3b gene is expressed
FIG. 2. BMP-3b and BMP-3 mRNA expression and ALPase activity in prolonged-cultured primary osteoblastic cells. Day 0: the day the cells reached confluence. The medium (a-MEM containing 10% FCS, 5 mM b-glycerol phosphate and 50 mg/ml ascorbic acid) was replaced every 2 days. (A) Each lane contained 10 mg of total RNA from different stages of culture. The bottom panel shows ethidium bromide-stained gels used as controls. Size markers are given at right. (B) Cellular ALPase activity was measured with p-nitrophenyl phosphate as the substrate. Cells were inoculated to 24-well plates. Means 6 S. D.; n 5 3.
gene transcription; reaching 62% of the control value after 3 h of incubation and almost complete inhibition (14% of control) after 6 h. Inhibition of BMP-3b gene expression by TGF-b1 continued for at least 96 h. The effect of TGF-b1 on the mRNA level of BMP-3 differed. After 12 h of TGF-b1 treatment, BMP-3 mRNA was increased (130% of control), then decreased to less than the control after 24-96 h. Figure 5C shows the time course effect of TGF-b1 on ALPase activity in osteoblasitc cells. TGF-b1 suppressed ALPase activity throughout the 14-day culture period. Figure 5D shows the results of alizarin red staining on day 14, which made visible mineralized nodules in the cells. Treatment with TGF-b1 also suppressed the formation of mineralized nodules. DISCUSSION We recently succeeded in cloning of BMP-3b cDNA from rat femur tissues and showed that the BMP-3b gene is highly expressed in bone tissues (13), but so far nothing is known about the function of BMP-3b. To
FIG. 3. Time courses of BMP-2 effects on BMP-3b and BMP-3 mRNA levels, ALPase activity and formation of mineralized nodules in primary osteoblastic cells. Confluent primary osteoblastic cells were serum deprived for 24 h (day 0) then cultured for the indicated periods in the absence (2) or presence (1) of 100 ng/ml BMP-2. During stimulation, the medium (a-MEM containing 0.1% BSA, 5 mM b-glycerol phosphate, 50 mg/ml ascorbic acid and with or without BMP-2) was replaced every 2 days. (A) Each lane contained 10 mg of total RNA. The bottom panel shows the ethidium bromide-stained gels used as controls. Size markers are given at right. (B) Cellular ALPase activity was measured with p-nitrophenyl phosphate as the substrate. Cells were inoculated to 24-well plates. Means 6 S. D.; n 5 3. (C) Alizarin red staining was done on day 14.
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in osteoblast in bone tissues. In contrast, the BMP-2, 4, 6 and 7 genes are expressed in bone tissues (1, 9, 10, 13, 15), and moreover, all are expressed in U2-OS cells (1, 9, 10, 19) and some in other established osteoblastic cell lines (19, 20), as well as in primary osteoblastic cells. We conclude that the different gene expression pattern of BMP-3b and BMP-2, 4, 6 and 7 reflects that they have different functions. Prolonged culture of primary osteoblastic cells, enriched for cells with the osteoblast phenotype, has proved a useful model for studying the expression of specific genes by osteoblasts during their differentiation into the mature mineralizing phenotype (21– 23). In our study, the increase in BMP-3b mRNA paralleled that of ALPase activity in prolonged cultures (Fig. 2). Using a similar approach, Harris and colleagues showed that BMP-2, 4 and 6 genes were expressed by fetal rat calvarial osteoblasts with the osteoblast phenotype (21). The gene expression patterns of BMP-3b and BMP-2, 4 and 6 differ slightly during prolonged culture (Fig. 2, Ref. 21), probably because they have different functions. These findings indicate that the elevated BMP-3b mRNA level in prolonged cultures is the result of cell differentiation into the mature, mineralizing osteoblast phenotype. To clarify BMP-3b gene regulation in primary osteoblastic cells, we examined the effects of BMP-2, TGFb1, FCS, bFGF and RA on BMP-3b gene transcription. BMP-2 stimulates osteoblast phenotype expression, e.g., increased ALPase activity, in several osteoblastic cell lines and in primary osteoblasts (Fig. 3, Ref. 23, 24). In this study, we showed that BMP-3b gene expression is stimulated by BMP-2 and is associated
FIG. 4. Effects of TGF-b1, FCS, bFGF and RA on BMP-3b and BMP-3 mRNA levels in primary osteoblastic cells. Confluent primary osteoblastic cells were serum deprived for 24 h then cultured in the absence (control) or presence of TGF-b1 (10 ng/ml), FCS (10%), bFGF (100 ng/ml) or RA (5 3 10 26 M) for 24 h. Each lane contained 10 mg of total RNA. Size markers are given at right.
FIG. 5. Time courses of TGF-b1 effects on the levels of BMP-3b and BMP-3 mRNA, ALPase activity and formation of mineralized nodules in primary osteoblastic cells. Confluent primary osteoblastic cells were serum deprived for 24 h (day 0) then cultured for the indicated periods in the absence (control) or presence of 10 ng/ml TGF-b1. During stimulation, the medium (a-MEM containing 0.1% BSA, 5 mM b-glycerol phosphate, 50 mg/ml ascorbic acid and with or without TGF-b1) was replaced every 2-3 days. (A, B) Each lane contained 10 mg of total RNA. Size markers are given at right. (C) Cellular ALPase activity was measured with p-nitrophenyl phosphate as the substrate. Cells were inoculated to 24well plates. Means 6 S. D.; n 5 3. (D) Alizarin red staining was done on day 14.
with increased ALPase activity in primary osteoblastic cells (Fig. 3); whereas, BMP-3 gene expression is decreased by BMP-2 in these cells (Fig. 3). Chen et al. (23) reported that BMP-3 gene expression was enhanced by BMP-2 (5 to 17 days after treatment) but suppressed by it in the early phase (one day after treatment). The different effects of BMP-2 on BMP-3 gene expression may be attribute to the period of analysis. As shown in Fig. 4, TGF-b1 completely inhibited expression of the BMP-3b gene. Surpris-
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ingly, its suppressive effect on gene transcription of BMP-3b in osteoblastic cells was very strong and rapid (Fig. 5). In vivo, TGF-b1 stimulates bone formation (25), but its effects on osteoblastic cells in vitro are numerous and diverse (26 –29). These differences are related to the differentiated state of the osteoblastic cell populations studied. In our study, TGF-b1 suppressed ALPase activity (Fig. 5) and stimulated DNA synthesis (data not shown) in primary osteoblastic cells. A recent report shows that TGF-b1 inhibits BMP-2 gene expression and several osteoblast marker proteins in fetal rat calvarial osteoblasts (22), indicating that TGF-b1 acts as an inhibitor of osteoblastic differentiation in this cell system. FCS and bFGF are mitogenic for bone cells (4, 30), and RA affects bone morphogenesis (31). BMP-3b mRNA synthesis was suppressed by both FCS and RA (Fig. 4), whereas FCS, as well as TGFb1, stimulated DNA synthesis and decreased ALPase activity (data not shown). bFGF had the same effects as TGF-b1 and FCS on the DNA synthesis and ALPase activity in primary osteoblasts, but its effect was very weak. The effect of bFGF on BMP-3b mRNA suppression therefore was lower than that of TGF-b1 and FCS. These findings suggest that transcription of the BMP-3b gene is inhibited when osteoblastic cells are in the dedifferentiated state. RA is reported to affect growth inhibition and certain differentiated functions in malignant osteoblast cells (32, 33). The mechanism of the suppression of BMP-3b mRNA production by RA is thought to differ from that of TGF-b1 and FCS. We also showed that the BMP-3b and BMP-3 mRNA levels in primary osteoblastic cells are differentially regulated by BMP-2, TGF-b1, FCS, bFGF and RA. For example, BMP-3b mRNA synthesis was increased by BMP-2 but rapidly inhibited by TGF-b1; whereas, BMP-3 mRNA was rapidly decreased by BMP-2, and initially increased but slowly suppressed by TGF-b1. These findings suggest that BMP-3b and BMP-3 have different functions in these cells, even though the proteins are closely related (81% amino acid identity in the mature region). In conclusion, the BMP-3b gene expression was closely associated with differentiation in primary osteoblastic cells. These findings provide strong evidence that BMP-3b has a significant role in osteoblasts. ACKNOWLEDGMENTS We thank Dr. M. Iwamoto of Osaka University for valuable discussion, and Ms. H. Iida for excellent technical assistance. This work was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.
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