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. 2018 Jun 20;77(9):1372–1380. doi: 10.1136/annrheumdis-2018-212964

Figure 4.

Figure 4

Connective tissue growth factor (CTGF) is required for release and activation of latent TGFβ in a TGFβR3-dependent manner. Conditional, inducible deletion of CTGF (CtgfcKO) was achieved by crossing Ctgffl/fl (wt) mice with an inducible Cre recombinase driven by the ubiquitin promoter (UbiCreER T2). Mice were treated with tamoxifen at 4 weeks to induce deletion. Hip avulsion, a model of murine cartilage injury, was performed in 6-week-old CtgfcKO or wt mice. Injury medium (serum free) was conditioned for either 1 hour (A–C) or 24 hours (D–G). Some medium was conditioned from hips that had been rested for 48 hours then re-cut to obtain control (ctrl) and re-cut injury medium. (A) Injury CM or explant lysates were immunoblotted for CTGF and TGFβ (run under reducing conditions). (B) Protein levels of injury CM CTGF and TGFβ were quantified (wt, n=7; null, n=6) and their correlation examined. (C) Control and injury CM was used to stimulate porcine chondrocytes (45 min) and lysates immunoblotted for pSMAD2. (D) Injury CM (24 hours) was generated from CtgfcKO and wt hips and were immunoblotted for CTGF and TGFβ (run under reducing conditions). Injury CM was also used to stimulate monolayer chondrocytes for 45 min and the lysates were immunoblotted for pSMAD2 (lower panels). Released CTGF (E) and TGFβ (F) from the injury CM were quantified (wt, n=17; null, n=16) and expressed relative to wt levels. (G) Injury CM was used to stimulate porcine chondrocytes (45 min) and pSMAD2 was quantified from Western blots. (H–J) To determine the mechanism of activation of latent injury CM, porcine chondrocytes were stimulated with either injury CM or TGFβ with the following pre-treatments: (H) 10 mU/mL heparitinase or chondroitinase for 4 hours prior to stimulation. (I) 50 ng/mL or 500 ng/mL soluble TGFβR3, 1 hour prior to stimulation. (J) TGFβR3 was knocked down by siRNA for 72 hours and the cells were rested in serum-free dulbecco modified eagles medium (DMEM) for 18 hours prior to stimulation. Error bars represent SE. **p<0.01, ***p<0.001 by a two-sided Student’s t-test; ns, not significant. Pearson’s coefficients of linear correlation and p values are shown.