Activin A Promotes Osteoblastic Differentiation of Human Preosteoblasts through the ALK1-Smad1/5/9 Pathway
Abstract
:1. Introduction
2. Results
2.1. Expression of Activin A in Rat PDL Tissue and Saos2 Cells
2.2. Effects of Activin A on Osteoblastic Differentiation of Saos2 Cells
2.3. Effects of Activin Type I Receptors on Activin A-Induced Osteoblastic Differentiation in Saos2 Cells
2.4. Expression of ALK1 in Rat PDL Tissue
2.5. Expression of ALK1 in Surgically Damaged PDL Tissue
2.6. Effects of ALK1 Knockdown on Phosphorylation of Activin A-Related Intracellular Signaling Molecules in Saos2 Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Animal Model
4.3. Western Blotting Analysis
4.4. Immunofluorescence and Immunochemical Analyses
4.5. Treatment of Saos2 Cells with IL-1β or TNF-α
4.6. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR)
4.7. Osteoblastic Differentiation of Saos2 Cells
4.8. Small Interfering RNA Transfection
4.9. Proliferation Assay
4.10. ALP Activity Assay
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Target Gene (Abbreviation) | Forward (Top) and Reverse (Bottom) Primer Sequences | Size of Amplified Products (bp) | Annealing Temperature (°C) | Sequence ID |
---|---|---|---|---|
β-actin | 5’-ATTGCCGACAGGATGCAGA-3’ 5’-GAGTACTTGCGCTCAGGAGGA-3’ | 89 | 60 | NM_001101.3 |
ALK1 | 5’-ATCCTAGGCTTCATCGCCTC-3’ 5’-TAGCCTCAGAGCCAGATGGG-3’ | 141 | 60 | NM_000020.3 |
ALK2 | 5’-AGGCTGCTTCCAGGTTTATGAG-3’ 5’-TGGCAGCACTCCACAGCTT-3’ | 81 | 60 | NM_001105.5 |
ALK3 | 5’-AGTGTCTCCAGTCAAGCTCTGGGTA-3’ 5’-CCATCTCTGCTGCGCTCATTTA-3’ | 97 | 60 | NM_004329.3 |
ALK4 | 5’-TGCTGCGCCATGAAAACATC-3’ 5’-GGGACCCGTGCTCATGATAG-3’ | 105 | 60 | NM_020327.3 |
ALK5 | 5’-CCTTCTGACCCATCAGTTGAAGA-3’ 5’-CCTAGCTGCTCCATTGGCAT-3’ | 150 | 60 | NM_004612.4 |
ALK6 | 5’-GTTACGCCCCTCATTCCCAA-3’ 5’-GTTTTCTTAACCCGCAGGGC-3’ | 126 | 60 | NM_001256793.2 |
Inhibinβa | 5’-TGTGCCCACCAAGCTGAGAC-3’ 5’-CTGGGCTGGGCAACTCTATGA-3’ | 124 | 60 | NM_002192.2 |
OCN | 5’-GTGCAGAGTCCAGCAAAGGT-3’ 5’-TCAGCCAACTCGTCACAGTC-3’ | 110 | 60 | NM_199173.4 |
OPN | 5’-ACACATATGATGGCCGAGGTGA-3’ 5’-TGTGAGGTGATGTCCTCGTCTGT-3’ | 115 | 60 | NM_000582.3 |
RUNX2 | 5’-AACCCTTAATTTGCACTGGGTCA-3’ 5’-CAAATTCCAGCAATGTTTGTGCTAC-3’ | 145 | 60 | NM_001024630.3 |
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Sugii, H.; Albougha, M.S.; Adachi, O.; Tomita, H.; Tomokiyo, A.; Hamano, S.; Hasegawa, D.; Yoshida, S.; Itoyama, T.; Maeda, H. Activin A Promotes Osteoblastic Differentiation of Human Preosteoblasts through the ALK1-Smad1/5/9 Pathway. Int. J. Mol. Sci. 2021, 22, 13491. https://doi.org/10.3390/ijms222413491
Sugii H, Albougha MS, Adachi O, Tomita H, Tomokiyo A, Hamano S, Hasegawa D, Yoshida S, Itoyama T, Maeda H. Activin A Promotes Osteoblastic Differentiation of Human Preosteoblasts through the ALK1-Smad1/5/9 Pathway. International Journal of Molecular Sciences. 2021; 22(24):13491. https://doi.org/10.3390/ijms222413491
Chicago/Turabian StyleSugii, Hideki, Mhd Safwan Albougha, Orie Adachi, Hiroka Tomita, Atsushi Tomokiyo, Sayuri Hamano, Daigaku Hasegawa, Shinichiro Yoshida, Tomohiro Itoyama, and Hidefumi Maeda. 2021. "Activin A Promotes Osteoblastic Differentiation of Human Preosteoblasts through the ALK1-Smad1/5/9 Pathway" International Journal of Molecular Sciences 22, no. 24: 13491. https://doi.org/10.3390/ijms222413491