Session Type: Abstract Submissions (ACR)
Osteoarthritis is characterized by degradation of articular cartilage. TGFβ-superfamily signaling via Smad phosphorylation (pSmad) plays a crucial role in cartilage maintenance. Two distinct pSmad pathways exist: pSmad1/5/8 and pSmad2/3. pSmad2/3 induces matrix formation and protects cartilage against deleterious processes like chondrocyte hypertrophy and IL1-signalling. In contrast, pSmad1/5/8 is linked to chondrocyte hypertrophy and expression of the main cartilage degrading enzyme: MMP13. Recently a very potent pSmad1/5/8 inducing ligand has been identified: BMP9. BMP9 is produced by the liver and circulates in high levels. Over 60% of all BMP activity in serum can be attributed to BMP9, showing the abundance of this BMP. In this study we investigated the effect of this potent pSmad1/5/8 inducing ligand, BMP9, on chondrocyte phenotype. Furthermore, because of pSmad2/3’s importance in chondrocyte homeostasis, we studied the interaction between BMP9-signaling and TGFβ1-induced pSmad2/3 signaling.
In this study, primary bovine chondrocytes were used. BMP9-induced Smad phosphorylation was detected after 1 h and 2 h using Western Blot . Subsequently, BMP9-induced gene expression was measured up to 24 h using real time qPCR. Biological activity of pSmad2/3 was measured using the (CAGA)12-luc reporter assay, which produces luciferase in response to pSmad3. Cellular hypertrophy was investigated by culturing chondrocytes 1 week in the presence of growth factors and analyzing gene expression of hypertrophy markers.
In primary bovine chondrocytes, BMP9 stimulation in doses upwards of 50 pg/ml induced pSmad1/5/8, which was reflected in expression of the Smad1/5/8 response gene bId1. Remarkably, BMP9 doses of 1 ng/ml and higher also induced pSmad2, but, expression of the pSmad3 response gene bSerpine1 was not induced. Co-stimulation of chondrocytes with BMP9 and a low dose TGFβ1 (0.1 ng/ml) reduced BMP9-induced pSmad1/5/8 and, surprisingly, enhanced TGFβ1-induced pSmad2. After 24 hours, this interaction was reflected in mRNA levels, as co-stimulation increased expression of the pSmad2/3 responsive genes: bSerpine1, bTgfb1 and bSmad7 and decreased expression of bId1. Furthermore, BMP9 also synergistically enhanced biological activity of TGFβ1 in the CAGA12Luc reporter assay. The uniqueness of this synergy between BMP9 and TGFβ1 was illustrated by co-stimulation of chondrocytes with TGFβ1 and two other BMPs important in chondrocyte biology: BMP2 and BMP7, which showed that both these BMPs do not synergize with TGFβ1 on Smad2/3p. As a more long term effect, BMP9 induced chondrocyte hypertrophy after one week of stimulation, as indicated by upregulation of Alkaline phosphatase and Col10a1, however addition of 0.1 ng/ml of TGFβ1 inhibited this BMP9-induced hypertrophy.
Our results show that BMP9 potently induces pSmad1/5/8 and its downstream gene expression in chondrocytes. In long term culture this results in induction of chondrocyte hypertrophy. However TGFβ1 can potently inhibit this hypertrophy. Possibly, this runs via the observed but yet unexplained pSmad2/3 enhancing interaction between both growth factors, which is unique for BMP9 compared to BMP2 or BMP7.
A. van Caam,
E. Blaney Davidson,
E. W. van Geffen,
W. B. van den Berg,
P. M. van der Kraan,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bmp9-induced-psmad158-signaling-and-chondrocyte-hypertrophy-are-effectively-inhibited-by-tgf%ce%b21/