Osteoarthritis-Related Inflammation Blocks TGF-β’s Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region
Abstract
:1. Introduction
2. Results
2.1. IL-1β and OAS-cm Negate the Anti-Hypertrophic Function of TGF-β in Bovine Cartilage Explants
2.2. IL-1β and OAS-cm Inhibit TGF-β Transcriptional Activity in Different Chondrocyte Cell Lines
2.3. IL-1β and OAS-cm Do Not Inhibit C-Terminal Phosphorylation of SMAD2/3 and Do Not Regulate Receptor Level Expression
2.4. IL-1β and OAS-cm Inhibit TGF-β via(de-)Phosphorylation of the SMAD2/3 Linker Region
3. Discussion
4. Materials and Methods
4.1. Primary Cell Culture
4.2. Chondrocyte Cell Line Culture
4.3. Chondrocyte Stimulation
4.4. Plasmid DNA, Adenoviral Production and Transduction
4.5. SMAD-Luciferase Transcriptional Reporter Assay
4.6. Protein Isolation and Western Blot
4.7. RNA Isolation and Quantitative Real-Time PCR
4.8. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
Appendix A
Appendix B
Appendix C
References
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Gene and Species | Forward Sequence (5′ → 3′) | Reverse Sequence (5′ → 3′) |
---|---|---|
bGAPDH | CACCCACGGCAAGTTCAAC | TCTCGCTCCTGGAAGATGGT |
bRPS14 | CATCACTGCCCTCCACATCA | TTCCAATCCGCCCAATCTTCA |
bRPL22 | GTTCGCTCACCTCCCTTTCTG | GCAGCATCCATGATTCCATCT |
bCOL10A1 | CCATCCAACACCAAGACACAGT | TGCTCTCCTCTCAGTGATACACCTT |
bMMP3 | AAACTCACCTCACGTACAGAATTG | TCCCAGACCGTCAGAGCTTT |
bSMAD7 | GGGCTTTCAGATTCCCAACTT | CTCCCAGTATGCCACCACG |
bTGFBR2 | GGCTGTCTGGAGGAAGAATGA | GTCTCTCCGGACCCCTTTCT |
bALK5 | CAGGACCACTGCAATAAAATAGAACTT | TGCCAGTTCAACAGGACCAA |
hGAPDH | ATCTTCTTTTGCGTCGCCAG | TTCCCCATGGTGTCTGAGC |
hRPL22 | TCGCTCACCTCCCTTTCTAA | TCACGGTGATCTTGCTCTTG |
hRPS27A | TGGCTGTCCTGAAATATTATAAGGT | CCCCAGCACCACATTCATCA |
hSMAD7 | CCTTAGCCGACTCTGCGAACTA | CCAGATAATTCGTTCCCCCTGT |
hTGFBR2 | CTGGTGCTCTGGGAAATGACA | TCGCCCTCGATCTCTCAACA |
hALK5 | CGACGGCGTTACAGTGTTTCT | CCCATCTGTCACACAAGTAAA |
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Thielen, N.; Neefjes, M.; Wiegertjes, R.; van den Akker, G.; Vitters, E.; van Beuningen, H.; Blaney Davidson, E.; Koenders, M.; van Lent, P.; van de Loo, F.; et al. Osteoarthritis-Related Inflammation Blocks TGF-β’s Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region. Int. J. Mol. Sci. 2021, 22, 8124. https://doi.org/10.3390/ijms22158124
Thielen N, Neefjes M, Wiegertjes R, van den Akker G, Vitters E, van Beuningen H, Blaney Davidson E, Koenders M, van Lent P, van de Loo F, et al. Osteoarthritis-Related Inflammation Blocks TGF-β’s Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region. International Journal of Molecular Sciences. 2021; 22(15):8124. https://doi.org/10.3390/ijms22158124
Chicago/Turabian StyleThielen, Nathalie, Margot Neefjes, Renske Wiegertjes, Guus van den Akker, Elly Vitters, Henk van Beuningen, Esmeralda Blaney Davidson, Marije Koenders, Peter van Lent, Fons van de Loo, and et al. 2021. "Osteoarthritis-Related Inflammation Blocks TGF-β’s Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region" International Journal of Molecular Sciences 22, no. 15: 8124. https://doi.org/10.3390/ijms22158124