Expression of TGFβ-family signalling components in ageing cartilage: age-related loss of TGFβ and BMP receptors  A. van Caam, W. Madej, E. Thijssen, A. Garcia de Vinuesa, W. van den Berg, M.-J. Goumans, P. ten Dijke, E. Blaney Davidson, P.M. van der Kraan  Osteoarthritis and Cartilage  Volume 24, Issue 7, Pages 1235-1245 (July 2016) DOI: 10.1016/j.joca.2016.02.008 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Age-related changes in bovine cartilage. (A) Safranin O/Fast green and Hematoxilin/Eosin staining of articular cartilage obtained from a 2 years old (left) or 10 years old cow (right). Pictures are representative of three animals each. 100× Magnification, scale bar = 100 μm. Thickness and cellularity are reduced and a clear tidemark is visible (arrow) in 10 year old cartilage. Note that the surface is intact. (B) 800× Magnification, scale bar = 12.5 μm. Multiple tidemarks (stars) in healthy 10 year old cartilage. No tidemark is visible between non-calcified and calcified cartilage in 2 year old cartilage. (C) Relative gene expression of bovine collagen type 2 alpha 1 (bCol2a1), aggrecan (bAcan) and matrix Gla protein (bMgp) in ageing cartilage as determined by qPCR. Regression analysis (solid line) with 95% CI (dotted line) depicted. Expression of bCol2a1 decreases whereas expression of bMgp increases during ageing. Osteoarthritis and Cartilage 2016 24, 1235-1245DOI: (10.1016/j.joca.2016.02.008) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Age-related drop in TGFβ-family receptor expression in healthy cartilage. (A) Relative gene expression of the Smad1/5 phosphorylating receptors: activin A receptor type IL (bAcvrl1), activin A receptor type I (bAcvr1) and bone morphogenetic protein receptor type IA (bBmpr1a), in ageing cartilage as determined by qPCR. Regression analysis (solid line) with 95% CI (dotted line) depicted. Expression of bBmpr1a clearly decreases with advancing age. (B) Relative gene expression of the Smad2/3 phosphorylating receptors: transforming growth factor beta receptor I (bTgfbr1) and activin A receptor type IB (bAcvr1b) in ageing cartilage as determined by qPCR. Regression analysis (solid line) with 95% CI (dotted line) depicted. Expression of bTgfbr1 and bAcvr1b clearly decreases with age. (C) IHC of bAcvrl1 (left) and bTgfbr1 (right) in 1 year and 5 years old animals in the three zones of articular cartilage. Pictures are representative of four animals each. 1000× Magnification, scale bar = 10 μm. During ageing, bTgfbr1 expression decreases below the detection threshold and unstained cells start to appear (arrows). (D) Scoring of Acvrl1 and Tgfbr1 staining in the superficial, middle and deep zone of 1 and 5 year old bovine cartilage using a 0–3 scale (see Supplementary Fig. 3). Four animals were scored in quadruple for each group for each zone. Error bars represent 95% CI. Statistics were calculated using a Kruskal–Wallis test; * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Osteoarthritis and Cartilage 2016 24, 1235-1245DOI: (10.1016/j.joca.2016.02.008) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Age-related changes in TGFβ-family type II and type III receptors in healthy cartilage. Relative gene expression of bone morphogenetic protein receptor type II (bBmpr2), transforming growth factor beta receptor II (bTgfbr2), endoglin (bEng), and transforming growth factor beta receptor III (bTgfbr3) in ageing cartilage as determined by qPCR. Regression analysis (solid line) with 95% CI (dotted line) depicted. Only expression of bBmpr2 clearly decreases with advancing age. Osteoarthritis and Cartilage 2016 24, 1235-1245DOI: (10.1016/j.joca.2016.02.008) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Age-related drop in TGFβ-family ligands. Relative gene expression of transforming growth factor beta 1 (bTgfb1), transforming growth factor beta 2 (bTgfb2), transforming growth factor beta 3 (bTgfb3), growth differentiation factor 5 (bGdf5), bone morphogenetic protein 2 (bBmp2) and bone morphogenetic protein 7 (bBmp7) in ageing cartilage as determined by qPCR. Regression analysis (solid line) with 95% CI (dotted line) depicted. Expression of both bTgfb1 and bGdf5 clearly decreases with advancing age. Osteoarthritis and Cartilage 2016 24, 1235-1245DOI: (10.1016/j.joca.2016.02.008) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Age-related reduction in Smad3-dependent TGFβ1 responsiveness in cartilage. (A) Relative gene expression of SMAD family member 7 (bSmad7) in ageing cartilage as determined by qPCR. Regression analysis (solid line) with 95% CI (dotted line) depicted. (B) Relative gene expression of serpin peptidase inhibitor, clade E member 1 (bSerpine1) in ageing cartilage as determined by qPCR. Regression analysis (solid line) with 95% CI (dotted line) depicted. (C) Relative gene expression of bSmad7 in ex vivo explants in response to 24 h TGFβ stimulation (1 ng/ml) corrected for unstimulated explants (ΔΔCt). N = 22 animals. Regression analysis (solid line) with 95% CI (dotted line) depicted. (D) Relative gene expression of bSerpine1 in ex vivo explants in response to 24 h TGFβ1 stimulation (1 ng/ml) corrected for unstimulated explants (ΔΔCt). N = 22 animals. Regression analysis (solid line) with 95% CI (dotted line) depicted. (E) Relative gene expression of bId1 in ex vivo explants in response to 24 h TGFβ (1 ng/ml) or BMP9 (5 ng/ml) stimulation corrected for unstimulated explants (ΔΔCt). N = 8 animals. Regression analysis (solid line) depicted. Osteoarthritis and Cartilage 2016 24, 1235-1245DOI: (10.1016/j.joca.2016.02.008) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions