The authors have declared that no competing interests exist.
This study aimed to preliminary investigate the role of activin receptor-like kinase (ALK) 5 as one of TGF-βR1 subtypes in bone turnover and osteoblastic differentiation induced by fluoride. We analyzed bone mineral density and the expression of genes related with transforming growth factor-β1(TGF-β1) signaling and bone turnover in rats treated by different concentrations of fluoride with or without SB431542
Fluoride is an important element for human in maintaining bone strength and stimulates bone growth [
Previous study concluded that fluoride exerted influence on bone turnover by regulating certain factors such as runt-related transcription factor 2 (Runx2) and receptor activator for nuclear factor-κ B ligand (RANKL), which was considered as key factors for osteoblast and osteoclast differentiation. Besides, transforming growth factor-β1 (TGF-β1) is known to be essential for osteoblast and osteoclast differentiation [
Numerous studies concluded that many factors affected TGF-β1 signaling pathway [
The male Wistar rats (6 weeks old, 150g) used in the study were provided by the Experimental Animal Center of Bethune Medical College, Jilin University. The study protocol was subject to approval by the Ethics Committee on the Use and Care of Animals of Jilin University (Changchun, China). All experimental animals implemented anesthesia before they were euthanized by cervical dislocation. Each rat was kept in an individual cage with a standard environment. The rats were randomly divided into control group, low fluoride group and high fluoride group (n = 20 for each group). One third of rats were treated with sodium fluoride (NaF, Sigma–Aldrich Co., USA) by gavage at a dose of 10 mg fluoride/kg.bw as low fluoride, and one third of rats were treated with 20 mg fluoride/kg.bw as high fluoride and the remaining were kept as the control group. The doses of fluoride were selected based on past reports [
After treatment, a sample of fresh bone tissue was obtained from each rat. Bone was immersed and grinded in liquid nitrogen, then total RNA was extracted by TRIzol reagent (Invitrogen; Carlsbad, CA, USA) and quantified by scanning spectrophotomer. Next, cDNA was prepared by using a Transcriptor First Strand cDNA Synthesis Kit (Applied Biosystems, USA), and genes expression of the bone samples was examined by real-time PCR using SYBR green detection method (Applied Biosystems, USA). All the primers were validated before PCR reaction. Fold change was calculated using ddCT method (2∧-ddCt). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference. All experiments were repeated three times. The primers used in the study were synthesized by Sangon Biotech (Shanghai, China) and were shown in
Genens | Number | Sequences(5′-3′) |
---|---|---|
Runx2 | NM_053470 | Forward: Reverse: |
RANKL | Forward: Reverse: |
|
Smad3 | Forward: Reverse: |
|
ALK5 | Forward: Reverse: |
|
GAPDH | Forward: Reverse: |
Bone marrow stem cells (BMSC) were isolated from the femurs of young Kunming mice (3 weeks old, 30g) in a sterile environment. Cells were washed out by phosphate-buffered saline (PBS, pH = 7.4) from marrow cavity at 4°C, then, they were collected and centrifuged for 10 min at 1800rpm. After that, cells were cultured in DMEM/F12 medium (Hyclone Co, USA) containing mineralization induction agents (0.05 g/L vitamin C, 40 ng/mL dexamethasone and 10 mmol/L β-sodium glycerol phosphate) and 10% fetal bovine serum at 37°C with 5% CO2. After two weeks of induced culturing, according to our previous study [
MTT assay was performed to detect cells viability at 4-day and 7-dayafter exposure to fluoride and SB431542. Cells were treated with MTT reagent (5mg/ml), then incubated for 4 h at 37°C with 5% CO2. Following incubation, dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) was added to allow color development, and the optical density (OD) of each well at 490nm was measured with a spectrophotometer. Cell viability was calculated as follows:
Cells were cultured by fluoride at concentrations of 1 mg/L, 4 mg/L and 16 mg/L with or without 10 μmol/L SB431542 and 10 μg/L TGF-β1 for 7 days as previously described [
Cells were exposed to fluoride at concentrations of 1 mg/L, 4 mg/L and 16 mg/L with or without 10μmol/L SB431542 for 7 days. Following treatment, total RNA was extracted by using TRIzol reagent (Invitrogen; Carlsbad, CA, USA) and quantified by a scanning spectrophotometer. First-strand cDNA was synthesized from 1 μg of total RNA using an Oligo(dT) 18 primers and reverse transcriptase (Applied Biosystems, USA). The real-time PCR were performed under the same procedures stated above. All experiments were repeated three times. The primers used were synthesized by Sangon Biotech (Shanghai, China) and were shown in
Genens | Number | Sequences(5′-3′) |
---|---|---|
Runx2 | Forward: Reverse: |
|
Smad3 | Forward: Reverse: |
|
ALK5 | Forward: Reverse: |
|
GAPDH | Forward: Reverse: |
The immunocytochemical analysis was utilized to detect the expression of osteocalcin (OCN) in BMSC. Cells were exposed to different concentrations of fluoride with or without 10 μmol/L SB431542 for 28 days. At the end of incubation, the cells were washed in PBS, fixed with 4% paraformaldehyde for 20 min at 4°C, and treated with 0.5% TritonX-100 for 15 min. Next, they were blocked with 10% H2O2 at room temperature for 10 min, then treated with 3% bovine serum albumin (BSA) at room temperature for 30 min. The cells were then incubated overnight at 4°C with OCN rabbit polyclonal antibody (OCN, 1:100; Santa Cruz, USA) which was diluted in 2% BSA/0.1M PBS, and cells were further incubated with a goat anti-rabbit IgG at room temperature for 1h. The cell coverslips sections were treated with an avidin-biotin-peroxidase (Maixin Biotech Co, China) complex for 30min. Then, they were immersed in diaminobenzidine (Maixin Biotech Co, China) for 1 min. Hematoxylin was used for nuclear counterstaining.
Smad2/3 and phosphorylation of Smad2/3 expression were measured by western bolt analysis. Cells were lysed with the lysis buffer (Beyotime, China), and the protein concentration was quantified using the bicinchoninic acid (BCA) method. Samples of protein (20 μg) were separated on 10% SDS-PAGE gels, and transferred onto PVDF membranes (PALL, USA). The transfer membranes were then blocked with 5% milk-TBST (20 mmol/L Tris-HCl (pH 8.0), 8 g/L NaCl, and 0.1% Tween 20) for 1h at room temperature. After blocking, the membranes were incubated with primary antibodies (GAPDH, 1:5000; Smad2/3, 1:800; P-Smad2/3, 1:500; Santa Cruz Co, USA) at 4°C overnight, then incubated with a secondary antibody (goat anti-rabbit IgG-HRP, 1:3000; Santa Cruz Co, USA) for 2h. Finally, immunoreactive bands were detected by EasySee Western Blot Kit (Transgen, China). The staining results were analyzed by Gel-Pro Analyzer 4.0 software. The expression levels of individual proteins were indicated as a ratio relative to GAPDH expression.
All data were expressed as the mean ± SD. Statistical differences between the groups were analyzed using LSD and Duncan’s test, Statistical analysis was performed by SPSS 13.0 software (SPSS Inc, Chicago). A P-value of less than 0.05was considered statistically significant.
As an indicator of bone mass bone mineral density (BMD) implies the bone turnover state [
Rats was treated with sodium fluoride by gavage at 10 mgF-/kg.bw and 20 mgF-/kg.bw for 2 months, and half of rats in each group were injected with an ALK5 inhibitor (SB431542, 2.1 mg/kg.bw). The tibias were collected and bone mineral density was measured by using a Hologic Discovery WA scanner. Results are expressed as mean±SD (n = 10). Results indicated significant changes (*P < 0.05, compare with control group; aa P < 0.01, compare with two groups).
Gene expression ofRunx2, RANKL, ALK5 and Smad3 was detected in bone tissue. Results showed that gene expression ofRunx2 slightly enhanced after low-dose fluoride treatment, but decreased in high-dose fluoride group compared with control group. Moreover, fluoride treatment increased RANKL expression and significantly higher expression was observed in high-dose fluoride treatment group. The expressions of Runx2 and RANKL were inhibited in co-treatment with fluoride and SB431542 compared to the fluoride treatment alone. The gene expression of intracellular signaling factors of TGF-β1 signaling, such as ALK5 and Smad3, were also measured. Results indicated that fluoride treatment stimulated expression of Smad3 compared to the control, but SB431542 administration with fluoride markedly inhibited Smad3comparedwith fluoride treatment alone. The expression of ALK5 was slightly lower than that in the control group, and SB431542 co-treated with fluoride further decreased ALK5 expression, which might attribute to the activity decreases of osteoblastic cells mentioned in above results(
Rats was treated with sodium fluoride by gavage at 10 mgF-/kg.bw and 20 mgF-/kg.bw for 2 months, and half of rats in each group were injected with an ALK5 inhibitor (SB431542, 2.1 mg/kg.bw). The femurs were collected and extracted mRNA by Trizol reagent. Realtime PCR was used to analyze Runx2, RANKL, Smad3 and ALK5 expression. Results are expressed as mean± SD(n = 3). (**P < 0.01 compare with control group; aa P < 0.01, compare with two groups).
Results showed that 1, 4 mg/L of fluoride treatment enhanced the viability of BMSC at 4-day and7-day, while the viability significantly decreased in cells exposed to16 mg/L of fluoride compared with the control. SB431542 co-treated with 1 mg/L fluoride inhibited cells viability compared with fluoride treatment alone after 4 days of culture. SB431542 co-treated with 1 mg/L and 4 mg/L fluoride inhibited cells viability compared with fluoride treatment alone after 7 days of culture (
Cells were treated with 1 mg/L, 4 mg/L, and 16 mg/L of fluoride with or without 10μmol/L SB431542 for 4 and 7 days. MTT assay was used to detect cell viability. Absorbance was measured at 490nm in aspectrophotometer. Average optical density (OD) value of cells viability was represented as mean±SD (n = 8) (*P < 0.05, **P < 0.01 compare with control group; aa P< 0.05, compare with SB431542 group).
The ALP regulated calcium deposition and was considered as a key factor in osteoblast early differentiation process. Results showed that treatment with fluoride at concentrations of 1 mg/L and 4 mg/L increased the levels of ALP positive staining in cells, while dark staining area was reduced after 16 mg/L fluoride treatment compared with control (
Cells were treated with 1 mg/L, 4 mg/L, and 16 mg/L of fluoride with or without 10μmol/L SB431542 for 7 days. The deposition of Cobalt sulfide particles was stained into light black granules in cytoplasm. The positive staining for deposition of Cobalt sulfide particles was shown in control group (a), 1 mg F−/L group (b), 4 mg F−/L (c), 16 mg F−/L group (d), SB431542 control group (e), SB431542 +1 mg F−/L group (f), SB431542 +4 mg F−/L (g), SB431542 + 16 mg F−/L group (h), TGF-β1control group (i), TGF-β1 + 1 mg F−/L group (j), TGF-β1 +4 mg F−/L (k), TGF-β1 + 16 mg F−/L group (l) under microscopy (red arrowheads; scale bar, 50 μm).
We detected the expression of Runx2, ALK5 and Smad3 in BMSCs. Results showed that compared with control, Runx2 gene expression increased by stimulation with 1 mg/L and 4 mg/L fluoride, but it was inhibited by 16 mg/L fluoride treatment. The level of ALK5 gene expression increased by 1 mg/L fluoride treatment while it was inhibited by 16 mg/L fluoride treatment compared with control. The expression of Smad3 markedly decreased in cells treated with 4 mg/L and 16 mg/L of fluoride compared with control. With SB431542 treatment in cells, Runx2 expression significantly decreased compared with fluoride treatment alone. The ALK5 expression was inhibited by 1 mg/L fluoride and SB431542 co-treatment compared with fluoride treatment alone. While the expression of Smad3 increased with 16 mg/L fluoride and SB431542 co-treatment (
The BMSCs were collected and extracted mRNA by Trizol reagent. Realtime PCR was used to analyze Runx2, Smad3 and ALK5 expression. The GAPDH was used as inner control. Results are expressed as mean± SD (n = 3). (*P < 0.05, **P < 0.01 compare with control group; aa P < 0.01, compare with two groups).
The OCN is commonly used as a marker for osteoblast differentiation. As it was shown in
Cells were treated with 1 mg/L, 4 mg/L, and 16 mg/L of fluoride with or without 10μmol/L SB431542 for 28 days. Immunocytochemistry analysis was used to test osteocalcin expression in situ. Positive staining showed brown granules in cytoplasm as that in control group (a), 1 mg F−/L group (b), 4 mg F−/L (c),16 mg F−/L group (d), SB431542 control group (e), SB431542 + 1 mg F−/L group (f), SB431542 +4 mg F−/L (g), SB431542+ 16 mg F−/L group (h) under microscopy (red arrowheads; scale bar, 50 μm). Immunocytochemistry was analyzed by using Image-Pro Plus 6.0 software to measure integrated optical density. Results are expressed as mean±SD (n = 3). Results indicated the significant changes(*P < 0.05, **P < 0.01 compare with control group; a P< 0.05, compare with two groups).
P-Smad2/3 protein is an important downstream mediator of TGF-β1/ALK5 signaling in regulating osteoblast differentiation. We measured p-Smad2/3 and Smad2/3 expressions in BMSCs treated by fluoride and ALK5 inhibitor (SB431542). As shown in
Cells were lysed with lysis buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membrane, which were incubated with primary and secondary antibodies. Immunostained proteins were detected by EasySee Western Blot Kit. Result was shown in control group (a), 1 mg F−/L group (b), 4 mg F−/L (c), 16 mg F−/L group (d), SB431542 control group (e), SB431542 + 1 mg F−/L group (f), SB431542 +4 mg F−/L (g), SB431542+ 16 mg F−/L group (h). The gels were analyzed by using Gel-Pro Analyzer 4.0 software to measure integrated optical density. Results are expressed as mean±SD (n = 3). Results indicated the significant changes (*P < 0.05, **P < 0.01 Vs control group; a P< 0.05, compare with two groups, aa P< 0.01, compare with two groups).
Up to date, role of TGF-β1 signaling on the skeletal fluorosis is was rarely studied. Aberrant change of Runx2 mediated signaling cascade is one of the decisive steps during the pathogenesis of fluorosis. The present study showed that bone mineral density decreased after treated with fluoride for two months, which was similar as previous study [
TGF-β1 is known to regulate majority stages involved in the osteoblast and osteoclast differentiation pathways [
Protein analysis showed that SB431542 treatment significantly inhibited phosphorylation of Smad2/3, which implied that ALK5 inhibitor effectively impeded the couple between ALK5 and Smad2/3. Co-treated with fluoride and SB431542 not only reduced the cell viability, ALP activity and OCN expression, but also inhibited expression of Runx2 and ALK5. The low-dose of fluoride and SB431542 treatment greatly reduced the expression of Smad3. However, the reason why high dose of fluoride and SB431542 mildly enhanced Smad3 expression was still unclear. We speculated that less active cells collected in the high-dose fluoride group influenced gene expression analysis. Research indicated that TGF-β1 enhanced ALP activity in MC3T3-E1 cells [
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This work was supported by grant for skeletal fluorosis research from National Natural Science Foundation of China [81673111].