R.D. Chavez, G. Coricor, J. Perez, H.-S. Seo, R. Serra

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Presentation transcript:

SOX9 protein is stabilized by TGF-β and regulates PAPSS2 mRNA expression in chondrocytes R.D. Chavez, G. Coricor, J. Perez, H.-S. Seo, R. Serra Osteoarthritis and Cartilage Volume 25, Issue 2, Pages 332-340 (February 2017) DOI: 10.1016/j.joca.2016.10.007 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 TGF-β1 regulates PRG4 mRNA and PAPSS2 mRNA in bovine chondrocyte micromass cultures over time. PRG4 mRNA levels increased after 4, 8, and 24 h of TGF-β1 treatment, when compared to mRNA levels of vehicle-treated controls at the same time points (REST, *P < 0.05, n = 5) (A). PAPSS2 mRNA levels increased after 4, 6, 8, and 24 h of TGF-β1 treatment, when compared to mRNA levels of vehicle-treated controls at the same time points (REST, *P < 0.05, n = 5) (B). Osteoarthritis and Cartilage 2017 25, 332-340DOI: (10.1016/j.joca.2016.10.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 SMAD3 and SOX9 adenoviral vectors infect cells, induce expression of FLAG-tagged proteins, and up-regulate SMAD3 and SOX9 function respectively. Bovine chondrocytes that were infected with either Ad-eGFP, Ad-SMAD3, or Ad-SOX9 at an MOI of 75 fluoresced green and showed efficient viral transduction (n = 4) (A). Western blots of extracts from bovine chondrocytes showed expression of FLAG-tagged proteins at approximately 55 kDa and 75 kDa, corresponding to SMAD3 and SOX9 molecular weights respectively (n = 4) (B). Bovine chondrocytes transduced with Ad-SMAD3 exhibited increased PTHrP mRNA levels; Ad-SMAD3 mRNA is relative to Ad-eGFP mRNA (REST, *P < 0.001, n = 5) (C). 293T cells transfected with a Col2a1-luciferase plasmid exhibited increased luciferase activity when infected with Ad-SOX9 (Student's t-test, *P = 0.04, n = 4) (D). Osteoarthritis and Cartilage 2017 25, 332-340DOI: (10.1016/j.joca.2016.10.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 SOX9 regulates PAPSS2 mRNA but not PRG4 mRNA in bovine chondrocytes. In A and B, cells were infected with the indicated viruses at 75 MOI, and relative mRNA levels for PRG4 and PAPSS2 were determined (REST, * = P < 0.05, n.s. = not significant, n = 8). In C and D, cells were infected with the indicated viruses at 75 MOI and treated with either vehicle (−) or TGF-β1 (+), and relative mRNA levels for PRG4 and PAPSS2 were determined (REST, * = P < 0.05, n.s. = not significant, n = 4). In E and F, cells were infected with either 150 MOI of Ad-eGFP (labeled Ad-eGFP), 75 MOI of Ad-eGFP + 75 MOI of Ad-SMAD3 (labeled Ad-SMAD3), 75 MOI of Ad-eGFP + 75 MOI of Ad-SOX9 (labeled Ad-SOX9), or 75 MOI of Ad-SMAD3 + 75 MOI of Ad-SOX9 (labeled Ad-SMAD3 + Ad-SOX9) (REST, * = P < 0.05, n.s. = not significant, n = 4). In all cases, mRNA levels are relative to mRNA levels of cultures treated with only Ad-eGFP (control). Osteoarthritis and Cartilage 2017 25, 332-340DOI: (10.1016/j.joca.2016.10.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Knock-down of Sox9 attenuates TGF-β-mediated regulation of Papss2. ATDC5 cells were transfected with Sox9 siRNA, Smad2/3 siRNA, or control non-specific (NS) siRNA. Sox9 mRNA expression was significantly reduced in cells expressing Sox9 siRNA (A). Acan mRNA was significantly reduced in the presence of Sox9 siRNA (B). Cells containing NS siRNA or Sox9 siRNA were treated with TGF-β1 and expression of Papss2 mRNA was measured by qPCR (C). In A–C, * = P < 0.05 REST, n = 5. Western blots showed Smad2/3 protein levels were reduced in the presence of Smad2/3 siRNA. α-Tubulin was used as a loading control (n = 6) (D). Acan mRNA was significantly down-regulated in the Smad2/3 siRNA-transfected cells compared to cells containing NS siRNA (REST, * = P < 0.05, n = 2) (E). Cells containing NS or Smad2/3 siRNA were treated with TGF-β1 and Papss2 mRNA was, measured by qPCR (REST, * = P < 0.05, n.s = not-significant n = 4) (F). Osteoarthritis and Cartilage 2017 25, 332-340DOI: (10.1016/j.joca.2016.10.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 TGF-β1 stabilizes SOX9 protein in bovine chondrocytes. In A, mRNA was collected from bovine chondrocytes treated with vehicle or TGF-β1 for the indicated amounts of time. SOX9 mRNA levels were determined by qPCR (REST, no statistically significant differences, n = 5). In B, protein was collected from bovine chondrocytes that were treated with either vehicle or TGF-β1 for 4 or 6 h. SOX9, pSMAD3, and SMAD2/3 protein levels were determined by Western blot (n = 3). α-Tubulin was used as a loading control. In C, new protein synthesis was inhibited with CHX. Cells were subsequently treated with either vehicle or TGF-β1 for up to 8 h. Protein was isolated at specified time points, and the levels of SOX9 protein were determined by Western blot (n = 3). Osteoarthritis and Cartilage 2017 25, 332-340DOI: (10.1016/j.joca.2016.10.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions