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Keywords:

  • Smad3;
  • transforming growth factor β;
  • osteoblast;
  • ALP;
  • mineralization

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Transforming growth factor (TGF) β is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-β signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-β on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-β inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L-azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

BONE MODELING and remodeling are essential for development, maturation, maintenance, and repair of bones. The proliferation and differentiation of osteoblasts are included in these events and are controlled by various local growth factors and cytokines produced in bone as well as by systemic hormones. Among them, transforming growth factor (TGF) β is most abundant in bone matrix compared with other tissues.(1) TGF-β is stored in an inactive form, released from the bone matrix, and activated in the bone microenvironment.(2) It is produced by osteoblasts and appears to regulate bone metabolism in various ways, including skeletal development and bone remodeling.(3) TGF-β modulates the proliferation, differentiation, and production of bone matrix proteins of osteoblasts.(2) Several reports showed that TGF-β induced bone formation when it was locally administered into bone tissues in rats.(4–7) However, it is disputable whether TGF-β would possess bone anabolic effects in vitro,(8–10) and the mechanism by which TGF-β stimulates bone formation in vivo is still unknown.

The Smad family proteins are critical components of the TGF-β signaling pathways.(11) TGF-β exerts growth inhibitory and transcriptional response through the two receptor-regulated Smads: Smad2 and Smad3.(11) Receptor-mediated phosphorylation of Smad2 or Smad3 induces their association with the common partner Smad4, followed by translocation into the nucleus where these complexes activate transcription of specific genes.(12) As for osteoblasts, Li et al.(13) reported that overexpression of Smad2 suppressed Runx2(cbfa1) and osteocalcin (OCN) mRNA expression in primary rat calvaria cells and ROS17/2.8 cells. That study also suggested that Smad2 and Smad3 had independent signaling pathways, which could mediate different aspects of TGF-β actions. Recently, it is reported that integrins regulate osteoblastic differentiation(14) and TGF-β up-regulates the βv-integrin expression via Sp-1 and Smad signaling in MC3T3-E1 cells.(15) Moreover, it is possible that Smad3 plays some role in the actions of calciotropic hormones. Vitamin D receptor potentiates ligand-induced transactivation by TGF-β in MC3T3-E1 cells,(16) and Smad3 mediates cross-talk between vitamin D and TGF-β signaling pathways.(17) However, the roles of Smad3 in the proliferation, expression of bone matrix proteins, and mineralization in osteoblasts remain poorly understood.

Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Materials

MC3T3-E1 cells were kindly provided by Dr. H. Kodama (Ohu Dental College, Japan). Myc-tagged Smad3 was prepared as previously described.(18) Smad3 DNA was derived from rat. A mutant form of Myc-tagged Smad3, in which the MH2 domain corresponding to amino acid residues 278-425 was removed (Smad3ΔC), was kindly provided by Dr. Y. Chen. A type I collagen (COLI) synthesis inhibitor (L-azetidine-2-carboxylic acid), human recombinant TGF-β1, and mouse anti-c-Myc antibody were purchased from Sigma (St. Louis, MO, USA). Anti-Smad3 antibody was purchased from Zymed Laboratories (San Francisco, CA, USA). Neutralizing anti-TGF-β antibody was obtained from Torrey Pines Biolabs, Inc. (San Diego, CA, USA). Anti-COLI antibody and bovine OCN were obtained from Calbiochem-Novabiochem Corp. (San Diego, CA, USA). All other chemicals used were of analytical grade.

Cell culture

MC3T3-E1 cells were cultured in α-minimum essential medium (α-MEM; containing 50 μg/ml of ascorbic acid) supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco BRL, Rockville, MD, USA). The medium was changed twice a week.

Stable transfection

Myc-Smad3, Myc-Smad3ΔC, and empty vector (each 3 μg) were transfected to MC3T3-E1 cells with lipofectamine (Gibco BRL). The ratio between the amounts of empty vector, Myc-Smad3 (or Myc-Smad3ΔC) expression vector was 1:1. Six hours after transfection, the cells were fed with fresh α-MEM containing 10% FBS. After 48 h, cells were passaged and clones were selected in α-MEM supplemented with G418 (0.3 mg/ml; Gibco BRL) and 10% FBS.

Protein extraction and Western blot analysis

Cells were lysed with radioimmunoprecipitation buffer with 0.5 mM of phenylmethylsulfonyl fluoride (PMSF), complete protease inhibitor mixture, 1% Triton X-100, and 1 mM of sodium orthovanadate. Cell lysates were centrifuged at 12,000g for 20 minutes at 4°C, and the supernatants were stored at −80°C. Protein quantitation was performed with BCA protein assay reagent (Pierce, Rockford, IL, USA). Equal amounts of protein were denatured in SDS sample buffer and separated on 10% polyacrylamide-SDS gel. Proteins were transferred in 25 mM of Tris, 192 mM of glycine, and 20% methanol to polyvinylidene difluoride. Blots were blocked with Tris-buffered saline (TBS; 20 mM of Tris-HCl [pH 7.5] and 137 mM of NaCl) plus 0.1% Tween 20 containing 3% dried milk powder. We used anti-Myc antibody and anti-Smad3 antibody to select the most highly expressed clones and anti-COLI antibody to detect the signals of COLI. Anti-Myc antibody was immunized against the sequence of amino acid residues 410-419 in the epitope of human c-Myc. Anti-Smad3 antibody recognizes a center portion of the linker domain of human smad3 and cross-reacts with rat- and mouse-Smad3. The antigen-antibody complexes were visualized using the appropriate secondary antibodies (Sigma) and the enhanced chemiluminescence detection system, as recommended by the manufacturer (Amersham Pharmacia Biotech, Buckinghamshire, UK).

MTT-dye assay

Mitochondrial function was assayed by the ability of viable cells to convert soluble MTT-dye (Sigma) into an insoluble dark blue formazan reaction product, as previously described.(19) MTT was dissolved in PBS at a concentration of 5 mg/ml and sterilized by passage through a 0.22-μm filter. This stock solution was added (1 part to 10 parts medium) to each well of a 96-well tissue culture plate and the plate was incubated at 37°C for 4 h. Acid isopropanol (400 μl of 10 M HCl in 100 ml of isopropanol) was added to each well and mixed thoroughly, to ensure that all the crystals were dissolved. The plates were read on a microplate reader at a wavelength of 595 nm.

[3H]thymidine incorporation assay

MC3T3-E1 cells were seeded at 2 × 104 cells/well in 24-well plates. These cells were maintained in α-MEM with 10% FBS. After 48 h of culture, cells were labeled with 0.5 μCi/ml of [3H]thymidine (Amersham Pharmacia Biotech) for 4 h. The incubation was terminated by removal of the medium, washed with PBS twice, and followed by the addition of 5% trichloroacetic acid (TCA) on ice in 10 minutes. After removal of the TCA, the residue was dissolved in 20 mM of NaOH at 37°C, and scintillation cocktail was added. Each sample was counted in a liquid scintillation counter.

RNA extraction and Northern blot analysis

Total RNA was prepared from MC3T3-E1 cells using the acid guanidinium-thiocyanate-phenol-chloroform extraction method.(20) Twenty micrograms of total RNA was denatured, run on a 1% agarose gel containing 2% formaldehyde, and then transferred to a nitrocellulose membrane and fixed with UV light (FUNA-UV-LINKER; Funakoshi, Tokyo, Japan). The membrane was hybridized to a32P(Amersham Pharmacia Biotech)-labeled DNA probe overnight at 42°C. The hybridization probes were the 2.8-kb fragment of the gene of type I procollagen (COLI; a gift from Dr. T. Kimura, Osaka University, Japan), the 210-kb fragment of mouse OCN, the 495-kb fragment of mouse osteopontin (OPN), and the 395-kb fragment of mouse matrix Gla protein (MGP). After hybridization, the filter was washed twice with 2× SSC containing 0.5% SDS and subsequently washed twice with 0.1× SSC containing 0.5% SDS at 58°C for 1 h. The filter was exposed to X-ray film using intensifying screen at −80°C. All values were normalized for RNA loading by probing blots with human β-actin cDNA (Wako Industries, Ltd., Osaka, Japan).

Assay of ALP activity and DNA content

After reaching confluency, cells in 24-well plates were rinsed three times with PBS and 600 μl of distilled water was added to each well. The DNA assay procedure of Labarca and Paigen(21) was used. ALP activity was assayed at 37°C by a method modified from that of Lowry et al.(22) In brief, the assay mixtures contained 0.1 Mof 2-amino-2-methyl-1-propanol, 1 mM of MgCl2, 8 mM of p-nitrophenyl phosphate disodium (Sigma), and cell homogenates. After 3 minutes of incubation, the reaction was stopped with 0.1N NaOH and the absorbance was read at 405 nm. A standard curve was prepared with p-nitrophenol (Sigma). Each value was normalized with the value in DNA content. ALP staining was performed as previously described by Harlow and Lane.(23) In brief, cultured cells were rinsed in PBS, fixed in 100% methanol, rinsed with PBS, and then overlaid with 1.5 ml of 0.15 mg/ml 5-bromo-4-chloro-3-indolylphosphate (BCIP; Gibco BRL) plus 0.3 mg/mlof nitroblue tetrazolium chloride (NBT) (Gibco BRL) in 0.1 M of Tris-HCl, pH 9.5, 0.01N NaOH, and 0.05 M of MgCl2, followed by incubation at room temperature for 2 h in the dark.

Assay of mineralization

The mineralization of MC3T3-E1 cells was determined in 6-well and 12-well plates using von Kossa staining and Alizarin Red staining, respectively. After confluent cells were grown in α-MEM supplemented with 10% FBS, 1% penicillin-streptomycin and 10 mM of β-glycerophosphate for 2 weeks, the cells were fixed with 95% ethanol and stained with AgNO3 by the von Kossa method to detect phosphate deposits in bone nodules.(14) At the same time, the other plates were fixed with ice-cold 70% ethanol and stained with Alizarin Red (Sigma) to detect calcification. For quantitation, cells stained with Alizarin Red were destained with ethylpyridinium chloride (Wako Industries, Ltd.) and then the extracted stain was transferred to a 96-well plate, and the absorbance at 562 nm was measured using a microplate reader, as previously described.(24)

Statistics

Data were expressed as mean ± SEM. Statistical analysis was performed using an unpaired t-test or ANOVA.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Stable transfection

We picked up 24 clones for each construct after 3 weeks of culture in the presence of G418. Several clones were selected in Western blotting with anti-Myc antibody. As shown in Fig. 1, signals were detected in Myc-Smad3- and Myc-Smad3ΔC-transfected MC3T3-E1 cells, although no signal was observed in empty vector-transfected cells. Similar results were obtained with anti-Smad3 antibody (data not shown).

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Figure FIG. 1. Western blot analysis of empty vector (V)-, Smad3-, and Smad3ΔC-transfected MC3T3-E1 cells. Total cell lysates from empty vector (V)-, Myc-Smad3 (Smad3)-, and Myc-Smad3ΔC (Smad3ΔC)-transfected MC3T3-E1 cells were separated by SDS/PAGE, immunoblotted, and revealed with anti-c-myc antibody. Equivalent amounts (20 μg) of total protein were loaded in each lane. V-transfected cells, clones v1, 2 and3; Smad3-transfected cells, clones s7, 10, and11; Smad3ΔC-transfected cells, clones δ5,10, and 13.

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Smad3 inhibits the proliferation of osteoblastic cells

We examined the effects of TGF-β on osteoblast proliferation. Cell proliferation was analyzed by [3H]thymidine incorporation (TdR) and MTT-dye assay. As shown in Fig. 2A, TGF-β (2.0 ng/ml) inhibited TdR in wild-type MC3T3-E1 cells, which was consistent with the previous evidence.(25) Smad3 transfection significantly inhibited TdR, compared with empty vector-transfected cells, and Smad3ΔC did not affect it (Fig. 2B). We used MTT-dye assay to confirm the results from TdR assay, because TdR can be affected by factors other than cell proliferation. For example, Peterson et al.(26) recently reported that extracellular matrix decreases incorporation of thymidine into DNA in a manner independent of proliferation in MC3T3-E1 cells. However, an MTT-dye assay also showed that Smad3-transfected cells had significantly less fluorescence than cells transfected with empty vector (Table 1). Similar results were obtained in at least three separate cell preparations. These results indicated that Smad3 as well as TGF-β inhibits the proliferation in osteoblastic cells.

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Figure FIG. 2. Smad3 inhibits the proliferation of MC3T3-E1 cells. (A) Effects of TGF-β on TdR in MC3T3-E1 cells. Wild MC3T3-E1 cells were seeded at 2 × 104 cells/well in 24-well plates in α-MEM with 10% FBS. The next day, cells were fed with fresh medium containing 10% FBS with or without TGF-β (2.0 ng/ml) and incubated for 48 h. Then, TdR was measured, as described in the Materials and Methods section. Each bar is expressed as the mean ± SEM (cpm) of four determinations. *Significantly different (p < 0.01) from the control. (B) Effects of Smad3 on TdR in MC3T3-E1 cells. V-, Smad3-, and Smad3ΔC-transfected MC3T3-E1 cells were cultured in 10% FBS-containing medium for 48 h after seeding at 2 × 104 cells/well in 24-well plates. Each bar is expressed as the mean ± SEM (cpm) of four determinations. *Significantly different (p < 0.01) from the value in V-transfected cells.

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Table Table 1.. Effects of Smad3 on the Fluorescent Intensity in MTT-Dye Assay in MC3T3-E1 Cells
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Cell shape

MC3T3-E1 cells change cell shape during their differentiation. In the early stages of differentiation, the MC3T3-E1 cells have a fibroblast-like spindle shape, whereas at the confluent stage, they are cuboidal.(27) However, when treated with TGF-β, cells remain spindle-shaped even at the confluent stage.(28) We found that Smad3 transfection affected the shape of MC3T3-E1 cells; empty vector—transfected cells or Smad3ΔC-transfected cells were cuboidal, whereas Smad3-transfected cells remained spindle-shaped, even at confluence (data not shown).

Smad3 stimulates the expression of COLI, OPN, and MGP in osteoblastic cells

A previous study revealed that TGF-β induces the expression of bone matrix proteins such as COLI, OPN, and MGP in MC3T3-E1 cells.(2) Therefore, we examined whether Smad3 overexpression would affect the expression of COLI, OPN, and MGP mRNA in MC3T3-E1 cells. As shown by a Northern blot analysis (Fig. 3), Smad3-transfected cells had higher levels of COLI, OPN, and MGP mRNA than empty vector-transfected cells and wild-type cells. Moreover, Smad3-transfected cells had a higher level of COLI than empty vector-transfected cells in Western blotting with anti-COLI antibody (data not shown). On the other hand, the levels of the matrix proteins were not affected in Smad3ΔC. These data indicate that Smad3 stimulates the expression of bone matrix proteins such as COLI, OPN, and MGP in osteoblastic cells.

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Figure FIG. 3. Effects of Smad3 on the expression of COLI, OPN, and MGP mRNA in MC3T3-E1 cells. V-, Smad3-, Smad3ΔC-transfected and wild-type MC3T3-E1 cells (W) were grown in 10% FBS-containing medium until reaching confluency for 7 days. Then, total RNA (20 μg) from each cell was analyzed by Northern blotting using cDNA probes of COLI, OPN, MGP, and β-actin, as described in the Materials and Methods section.

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Smad3 enhances ALP activity and mineralization of osteoblastic cells

ALP activity and mineralization also are important factors for bone formation, as well as matrix proteins. Therefore, we examined whether Smad3 would affect ALP activity and mineralization of MC3T3-E1 cells. ALP activity was evaluated biochemically and histochemically. Mineralization was examined by Alizarin Red staining and von Kossa staining as well as by a quantitative assay of mineralization based on Alizarin Red staining. TGF-β significantly inhibited ALP activity (Figs. 4A and 4B) and mineralization (Figs. 5A and 5B) of wild-type MC3T3-E1 cells, in agreement with the previous findings.(9) On the other hand, Smad3-transfected cells had much higher ALP activity (Figs. 4C and 4D) and mineralization (Figs. 5C and 5D) than cells transfected with empty vector. Transfection with Smad3ΔC did not affect either ALP activity and mineralization. These data indicate that Smad3, unlike TGF-β, promotes ALP activity and mineralization of osteoblastic cells.

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Figure FIG. 4. Smad3 increases ALP activity in MC3T3-E1 cells. (A) Effects of TGF-β on ALP activity in MC3T3-E1 cells. Confluent cells were fed with fresh FBS-free medium with or without the indicated concentrations of TGF-β1 for 48 h. ALP activity was measured, as described in the Materials and Methods section. Each bar is expressed as the mean ± SEM (pmol/minute per μg of DNA) of four determinations. *Significantly different (p < 0.01) from the control. (B) ALP staining in MC3T3-E1 cells at the same time as panel A. (C) Effects of Smad3 on ALP activity in MC3T3-E1 cells. Confluent V-, Smad3-, and Smad3ΔC-transfected MC3T3-E1 cells were cultured for 48 h in FBS-free medium. ALP activity was measured, as described in the Materials and Methods section. Each bar is expressed as the mean ± SEM (pmol/minute per μg of DNA) of six determinations. *Significantly different (p < 0.01) from the value in V-transfected cells. (D) ALP staining in MC3T3-E1 cells at the same time as panel C. ALP staining was performed as described in the Materials and Methods section.

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Figure FIG. 5. Smad3 stimulates mineralization in MC3T3-E1 cells. (A) Effects of TGF-β on mineralization in MC3T3-E1 cells. Confluent cells were treated with TGF-β1 (2.0 ng/ml) in the presence of β-glycerophosphate for 2 weeks. The mineralized matrix was stained with the von Kossa method or with Alizarin Red. (B) Quantitation of mineralization using Alizarin Red staining, as described in the Materials and Methods section. Each value is expressed as a ratio of the control value. *Significantly different (p < 0.01) from the control. (C) Effects of V, Smad3, and Smad3ΔC transfection on mineralization in MC3T3-E1 cells. Each confluent cell was cultured in the presence of β-glycerophosphate for 2 weeks. The mineralized matrix was stained with the von Kossa method or with Alizarin Red. (D) Quantitation of mineralization in V-, Smad3-, and Smad3ΔC-transfected MC3T3-E1 cells. Each cell was cultured, as described in panel C. Quantitation of mineralization was performed using Alizarin Red staining as described in the Materials and Methods section. Each value is expressed as a ratio of the value in V-transfected cells. *Significantly different (p < 0.01) from the value in V-transfected cells.

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Role of OCN and COLI in Smad3-stimulated ALP activity and mineralization of osteoblastic cells

The present findings that Smad3 enhanced ALP activity and mineralization are novel. To determine the mechanism by which Smad3 enhanced ALP activity and mineralization, we examined whether TGF-β treatment and Smad3 overexpression would affect the levels of OCN mRNA by using Northern blotting. As shown in Fig. 6A, TGF-β significantly reduced the OCN mRNA level in wild-type MC3T3-E1 cells. The OCN mRNA level was significantly lower in Smad3-transfected cells than in cells transfected with empty vector, but it was not affected in Smad3ΔC-transfected cells (Fig. 6B). Taken together with the findings that Smad3 up-regulated COLI (Fig. 3), we raised hypothesis that Smad3 stimulated ALP activity and mineralization through promotion of COLI synthesis and reduction of OCN expression in MC3T3-E1 cells. To test this hypothesis, we investigated whether ALP activity and mineralization were reduced by either a COLI synthesis inhibitor (L-azetidine-2-carboxylic acid) or bovine-purified OCN. As shown in Fig. 7, 0.3 mM of L-azetidine-2-carboxylic acid significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. COLI synthesis inhibitor did not affect TGF-β-inhibited ALP activity in wild-type MC3T3-E1 cells (vehicle, TGF-β, COLI synthesis inhibitor vs. TGF-β + COLI synthesis inhibitor; 58.6 ± 1.4 pmol/minute per μg of DNA, 23.6 ± 1.1 pmol/minute per μg of DNA, 57.3 ± 2.9 pmol/minute per μg of DNA vs. 21.8 ± 1.3 pmol/minute per μg of DNA). In addition, the levels of COLI in both empty vector-transfected and Smad3-transfected cells were reduced by COLI synthesis inhibitor but not to zero (data not shown) and so that the basal level of COLI required for osteoblastic gene expression would be maintained. OCN also reduced Smad3-stimulated ALP activity (Figs. 8A and 8B) and mineralization (Figs. 8C and 8D). COLI synthesis inhibitor as well as OCN did not affect ALP activity or mineralization of empty vector-transfected cells. These results suggest that reduced OCN as well as increased COLI synthesis would be at least in part involved in Smad3-stimulated ALP activity and mineralization of osteoblastic cells.

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Figure FIG. 6. Effects of Smad3 on OCN mRNA expression in MC3T3-E1 cells. (A) Effects of TGF-β on the expression of OCN mRNA in MC3T3-E1 cells. Total RNA (20 μg) was extracted from MC3T3-E1 cells with 24-h treatment of TGF-β1 (2.0 ng/ml) after reaching confluency. Then, Northern blotting using cDNA probes of mouse OCN and β-actin was performed, as described in the Materials and Methods section. (B) The expression of OCN mRNA in V-, Smad3-, and Smad3ΔC-transfected MC3T3-E1 cells. Total RNA (20 μg) from confluent V-, Smad3-, and Smad3ΔC-transfected MC3T3-E1 cells was analyzed by Northern blotting using cDNA probes of mouse OCN and β-actin, as described in the Materials and Methods section.

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Figure FIG. 7. COLI synthesis inhibitor antagonizes Smad3-stimulated ALP activity and mineralization in MC3T3-E1 cells. (A) Effects of COLI synthesis inhibitor on Smad3-stimulated ALP activity in MC3T3-E1 cells. V- and Smad3-transfected MC3T3-E1 cells were cultured for 7 days with or without L-azetidine-2-carboxylic acid (0.3 mM). ALP activity was measured as described in the Materials and Methods section. Each bar is expressed as the mean ± SEM (pmol/minute per μg of DNA) of four determinations. *Significantly different (p < 0.01) from the value in L-azetidine-2-carboxylic acid-untreated group. (B) ALP staining at the same time as in panel A. ALP staining was performed as described in the Materials and Methods section. (C) The effects of COLI synthesis inhibitor on Smad3-stimulated mineralization in MC3T3-E1 cells. Confluent V- and Myc-Smad3-transfected MC3T3-E1 cells were cultured with or without L-azetidine-2-carboxylic acid (0.3 mM) for 3 weeks. The medium contained β-glycerophosphate in the last 2 weeks. The mineralized matrix was stained with the von Kossa method or with Alizarin Red. (D) The quantitation of mineralization in COLI synthesis inhibitor-treated V- and Smad3-transfected MC3T3-E1 cells, cultured as described in panel C. Quantitation of mineralization was performed using Alizarin Red staining as described in the Materials and Methods section. Each value is expressed as a ratio of the value in untreated V-transfected cells. *Significantly different (p < 0.01) from the value in L-azetidine-2-carboxylic acid-untreated group.

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Figure FIG. 8. OCN antagonizes Smad3-stimulated ALP activity and mineralization in MC3T3-E1 cells. (A) The effects of OCN on Smad3-stimulated ALP activity in MC3T3-E1 cells. Confluent V- and Smad3-transfected MC3T3-E1 cells were cultured for 48 h with or without OCN (100 ng/ml). ALP activity was measured as described in the Materials and Methods section. Each bar is expressed as the mean ± SEM (pmol/minute per μg of DNA) of four determinations. *Significantly different (p < 0.01) from the value in OCN-untreated group. (B) ALP staining at the same time as in panel A. ALP staining was performed as described in the Materials and Methods section. (C) Effects of OCN on Smad3-stimulated mineralization in MC3T3-E1 cells. Confluent V- and Smad3-transfected MC3T3-E1 cells were cultured in the presence of β-glycerophosphate with or without OCN (100 ng/ml) for 2 weeks. The mineralized matrix was stained with the von Kossa method or with Alizarin Red. (D) The quantitation of mineralization in OCN-treated V- and Smad3-transfected MC3T3-E1 cells cultured as described in panel C. Quantitation of mineralization was performed using Alizarin Red staining as described in the Materials and Methods section. Each value is expressed as a ratio of the value in OCN-untreated V-transfected cells. *Significantly different (p < 0.01) from the value in OCN-untreated group.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Previous studies suggested that TGF-β inhibited the proliferation and differentiation of osteoblasts including MC3T3-E1 cells,(8,9,29) although some reports did not confirm these findings.(10,28) This study revealed that both TGF-β treatment and Smad3 overexpression inhibited the proliferation of MC3T3-E1 cells. These data indicate that the effects of Smad3 overexpression on proliferation mimic those of TGF-β.

TGF-β promotes the production of bone matrix proteins in osteoblasts.(2) Several studies have revealed that TGF-β stimulates the production of COLI, OPN, and MGP in MC3T3-E1 cells. Similar findings were obtained (data not shown). This study revealed that Smad3 overexpression increased the expression of COLI as well as OPN and MGP mRNA in MC3T3-E1 cells. These findings indicate that Smad3 mimicked the effects of TGF-β on the expression of bone matrix proteins. Smad3 might be involved in the stimulatory effects of TGF-β on the production of bone matrix proteins such as COLI, OPN, and MGP. A previous study revealed that with TGF-β treatment, the shape of osteoblast-like cells changed from cuboidal to spindle-shaped.(29) The same results were obtained in our preliminary study (date not shown). Our present study revealed that the cell shape with Smad3 overexpression remained spindle-shaped, even after confluency was reached. These data suggest that the effects of Smad3 on the cytoskeleton also mimic those of TGF-β.

Takeuchi et al.(30) reported that the COLI-α2β1-integrin interaction increased ALP activity. This study revealed that Smad3 increased the expression of COLI mRNA and that a COLI synthesis inhibitor significantly antagonized Smad3-stimulated ALP activity and mineralization in MC3T3-E1 cells. Therefore, the present data raise the possibility that Smad3 promotes ALP activity and mineralization through the enhancement of COLI synthesis in osteoblasts. ALP is one of the most important enzymes for osteoblastic mineralization, and β-glycerol phosphate, a substrate for ALP, was shown to stimulate mineralization.(9,31–35) Lee et al.(31) reported that ALP activity was higher in mineralizing cultures than in nonmineralizing cultures, and mRNA level for ALP also was higher during early mineralization. Moreover, osteoblasts in ALP-null mice differentiate normally but are unable to initiate mineralization in vitro.(34) Taken together, our findings suggest that Smad3 enhances the synthesis of COLI, which then increases ALP activity, and, in turn, accelerates the mineralization in osteoblasts.

The role of OCN remains unclear. OCN is highly expressed at the late stage during osteoblastic differentiation.(36) Ducy et al. reported that OCN-deficient mice exhibited increased bone formation and OCN might normally function to limit bone formation.(37) In our present study, Smad3 overexpression suppressed OCN expression and OCN significantly antagonized Smad3-mediated ALP activity and mineralization. These findings suggest that reduced OCN expression would be partly related to Smad3-stimulated ALP activity and mineralization. However, we could not rule out the possibility that COLI synthesis inhibitor and OCN inhibit ALP activity and mineralization in general, and not just Smad3-stimulated ALP activity and mineralization. Further study is necessary to clarify these issues. Moreover, the purity of bovine OCN used in this study was more than 98%, so that should contain very little or no contamination of growth factors, including TGF-β. However, we cannot completely rule out the possibility that a slight amount of contaminated TGF-β affected ALP activity and mineralization in this study, although neutralizing antibody against TGF-β did not affect the effects of OCN in our preliminary experiments.

The increased synthesis of COLI in MC3T3-E1 cells was a common effect of both TGF-β and Smad3. However, Smad3 greatly increased ALP activity as well as mineralization, and TGF-β inhibited them. The fact that Smad3 supports mineralization presumably is caused by the up-regulation of ALP activity. For this reason it can be concluded that the observed change in ALP activity between TGF-β treatment and Smad3 transfection is the “key finding” of this study. We raised three hypotheses to explain this discrepant effect of TGF-β and Smad3 on ALP activity. First, there might be some intracellular signaling pathways by which Smad3 but not TGF-β enhance ALP activity in these cells. Takeuchi et al. reported that the COLI and α2β1-integrin interaction up-regulates ALP activity and down-regulates TGF-β receptor activity, which allows the cells to escape the inhibitory effects of TGF-β in MC3T3-E1 cells.(30) Therefore, α2β1-integrin may play some role in Smad3-stimulated ALP activity. Smad3 overexpression may enhance the interaction between COLI and integrin by up-regulating the expression of the integrin in MC3T3-E1 cells. Second, TGF-β may inhibit ALP activity and mineralization of osteoblasts through a pathway other than the Smad3 pathway. Alternatively, it also is possible that some sort of TGF-β-responsive intracellular signaling that is independent of Smad3 may alter the activity of this cascade. Jun N terminus kinase (JNK) is rapidly and transiently activated by TGF-β receptor type I in a Rho-GTPase-dependent and Smad-independent manner. Coincident activation of the Smad and JNK/AP-1 pathways is necessary for full transcriptional activation in response to TGF-β.(38) Therefore, TGF-β might have inhibitory effects on ALP activity and mineralization via the JNK and/or AP-1 pathways, and the JNK and/or AP-1 pathways might provide an explanation for the divergent effects of Smad3 and TGF-β on ALP activity and mineralization. In preliminary experiments, we found that several MAPK inhibitors antagonize TGF-β-mediated effects in MC3T3-E1 cells but not Smad3-mediated effects (our unpublished data, 2002). Indeed, several studies have shown that JNK as well as c-Jun and JunB represses Smad3-mediated transcriptional activity.(39,40) Taken together, these findings suggest that some MAPK pathways mediate TGF-β-induced negative signals, which antagonized Smad3-stimulated ALP activity and mineralization in osteoblastic cells. Further studies are in progress to clarify these issues in our laboratory. Otherwise, the limited extent of accumulation of Smad3 into the nucleus in response to TGF-β might lead to the inhibition of ALP activity and the mineralization of osteoblasts, and excessive Smad3 might increase ALP activity and mineralization. The third hypothesis is concerned to the transformed state of osteoblasts. TGF-β promotes the production of COLI in ROS 17/2.8(10) and MC3T3-E1 cells.(2,30) However, TGF-β stimulates and inhibits ALP activity in ROS 17/2.8 and MC3T3-E1 cells, respectively.(10,28) Although the effects of Smad3 on ROS 17/2.8 cells are unknown, the intracellular signals that modulate the effects of TGF-β and Smad3 might be different, depending on the cell lines, species, and how the cells have been transformed. Alternatively, Smad3 transfection might change the transformed state, resulting in different phenotype, such as one with a higher ALP activity or a higher degree of mineralization.

Our previous study(41) confirmed that the Smad3 construct that we used is functional. In this study, the actions of Smad3 on osteoblast proliferation, bone matrix protein expression, ALP activity, and mineralization were not observed in Smad3ΔC-transfected cells. These findings were reproducible in at least three clones. These findings indicated that the effects of Smad3 were specific and were not caused by an myc-epitope, and that the MH2 region of Smad3 is indispensable for these effects of Smad3.

Enhancement of bone matrix production, ALP activity, and mineralization are important components of bone formation. Our results suggest that Smad3 is involved in the transcriptional mechanism leading to bone formation. In support of this, Borton et al. recently reported that mice with targeted deletion of Smad3 are osteopenic compared with wild-type littermates, because of a lower rate of bone formation,(42) and Alliston et al. reported that Smad3 decreases Runx2 and OCN expression.(43)

In conclusion, Smad3 inhibited the proliferation of MC3T3-E1 cells and enhanced the levels of bone matrix proteins such as COLI, OPN, and MGP in these cells in a manner similar to that of TGF-β. On the other hand, unlike TGF-β, Smad3 enhanced ALP activity and mineralization. We propose that Smad3 plays an important role in osteoblastic bone formation and that further studies of Smad3 will help to clarify the transcriptional mechanism of bone formation and possibly lead to the development of novel bone-forming drugs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Dr. J.J. Lebrun for providing Smad3 cDNA and acknowledge Y. Higashimaki, C. Ogata, and A. Maeda for excellent technical support.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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