1. TGF-β is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5- Smad2/3 pathway. Potential role in OA related pain? 
E.N. Blaney Davidson, A.P.M. van Caam, E.L. Vitters, M.B. Bennink, E. Thijssen, W.B. van den Berg, M.I. Koenders, P.L.E.M. van Lent, F.A.J. van de Loo, P.M. van der Kraan 
Osteoarthritis and Cartilage 
Volume 23, Issue 3, Pages (March 2015)
DOI: /j.joca
Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
TGF-β1 induces NGF in murine articular chondrocytes in an ALK5-Smad2/3 and TAK1 dependent manner. Murine H4 chondrocytes were stimulated with 10 ng/ml TGF-β1 for 24 h in the presence or absence of ALK5-Smad2/3 signaling inhibitor SB and compared to controls. RNA was isolated and QPCR was performed for NGF mRNA expression. TGF-β1 stimulation upregulated NGF expression compared to controls, which could be prevented by inhibiting ALK5-Smad2/3 signaling. Statistical analysis was performed using Krusskall–Wallis and Dunn's comparison (A). Murine H2 chondrocytes were stimulated with 10 ng/ml of TGF-β1 for 24 h in the presence or absence of 0.5 μM or 1 μM oxozeaenol to prevent TAK1 signaling. TGF-β1 exposure resulted in an upregulation of NGF, which could be prevented by blocking TAK1 (B).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
TGF-β1 induces NGF expression in human chondrocytes in higher levels than IL-1β; TGF-β1 and IL-1β do not have an additive effect on NGF expression. Human G6 chondrocytes were stimulated with TGF-β1 or IL-1β for 24 h, followed by RNA isolation and QPCR for NGF expression. Clearly, TGF-β1 induced higher levels of NGF compared to IL-1β already at a dose of 1 ng/ml TGF-β1 (A). When TGF-β1 and IL-1β were combined, NGF levels were not enhanced showing a lack of additive effect of TGF-β1 and IL-1 β (B).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
TGF-β1 induces higher levels of NGF than IL-1β in bovine chondrocytes and bovine articular cartilage in a ALK5-Smad2/3 and partially TAK1 dependent manner. Primary bovine chondrocytes isolated from the bovine metacarpophelangeal joint were stimulated with TGF-β1 or IL-1β or both with or without oxozeaenol. Both TGF-β1 and IL-1β induced NGF, but TGF-β1 induced NGF levels were much higher. Co-incubation with oxozeaenol, thereby preventing TAK1 signaling could reduce TGF-β1-induced NGF expression, and fully reverse IL-1β induced NGF expression (A). Bovine articular cartilage explants were stimulated with TGF-β1 or IL-1β or a combination of both for 24 h after which mRNA was isolated to measure NGF expression. This shows that TGF-β1 induced NGF expression is higher than IL-1β induced NGF expression (B). Bovine articular cartilage was stimulated with TGF-β1 with or without inhibitors for ALK5-Smad2/3 signaling (SB ) 5 μM, Smad 1/5/8 signaling (LDN) 0.05 μM or TAK1 signaling (oxozeaenol) 0.5 μM for 24 h after which RNA was isolated to measure NGF expression. This showed that TGF-β1 induced NGF expression could only be fully blocked in the presence of SB , could not be blocked by LDN and slightly reduced by oxozeaenol (C). Statistical analysis was performed using Krusskall–Wallis and Dunn's comparison.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
TGF-β1 induces higher NGF expression than IL-1βin human primary OA chondrocytes, but basal NGF levels show very high variation. Human primary OA chondrocytes were isolated from patients undergoing total joint replacement surgery after informed consent. After isolation, chondrocytes were stimulated with TGF-β1 or IL-1β for 24 h followed by RNA isolation and Q-PCR for NGF. Clearly, TGF-β1 stimulated samples reached higher levels of NGF than IL-1β stimulated samples. However, a striking observation was that the basal levels of NGF in human OA chondrocytes showed a very large variation (A). In these samples MMP13 was measured to ensure IL-1βresponsiveness which showed that IL-1β indeed induced MMP13 whereas TGF-β1 inhibited MMP13 Statistical analysis was performed using Krusskall–Wallis and Dunn's comparison (B). Immunohistochemistry for NGF was performed on articular cartilage of three random OA patients showing diversity in NGF protein expression (C). Human OA cartilage explants were stimulated with TGF-β1 with or without an inhibitor for ALK5-Smad2/3 signaling (SB ) 5 μMfor 24 h after which RNA was isolated to measure NGF expression. Co-incubation with SB resulted in a clear blockage of TGF-β1 induced NGF expression. After normality test, analysis was performed with a one-way ANOVA and Bonferroni for comparison (D).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions