Session Title: Biology and Pathology of Bone and Joint
Session Type: Abstract Submissions (ACR)
Background/Purpose: Osteoarthritis (OA) is a multifactorial disease characterized by loss of articular cartilage. TGFβ is considered as a protective factor against cartilage loss in young cartilage by inducing Smad2/3 phosphorylation via the TGFβ receptor ALK5. Smad2/3 phosphorylation (Smad2/3P) lowers MMP13 expression, and prevents deleterious terminal differentiation of chondrocytes. In contrast to Smad2/3P, Smad1/5 phosphorylation (Smad1/5P), induced by BMPs but also by TGFβ, is linked to terminal differentiation and MMP13 production. Type I receptors that phosphorylate Smad1/5/8 include ALK1 and ALK3. BMP-9 signals via ALK1 and BMP-2 via ALK3, and both ligands induce enhanced glycosaminoglycan synthesis in young cartilage. BMP2 is induced in damaged cartilage while BMP9 is constitutively present in high concentrations in body fluids. If and how these factors modulate chondroprotective TGFβ signaling is still unclear. Therefore, we analyzed BMP-2 and BMP-9 induced Smad signaling and gene expression in chondrocytes, and studied their interaction with TGFβ.
Methods: Primary bovine chondrocytes, isolated from the metacarpophalangeal joint of 2 year old animals, or the human G6 chondrocyte cell line were cultured to near confluency and incubated with TGFβ1, BMP-2, BMP-9 or a combination thereof. Smad phosphorylation kinetics were analyzed by specific Smad2P and Smad1/5P staining of Western blots. Expression patterns of Smad specific response genes: PAI-1 (Smad3 signaling), and ID-1, (Smad1/5 signaling) were analyzed from 0-48h by quantitative real time PCR (qPCR). Biological activity was tested with a CAGA12-luc transcriptional reporter construct that produces luciferase in response to Smad3 signaling. Adenovirally transduced cells (MOI = 5) were serum starved for 8h, stimulated with growth factors for 20h, and luciferase activity was measured.
Results: In primary chondrocytes, both BMP-2 and BMP-9 potently induced Smad1/5 phosphorylation, which peeked after 1 h and lasted up to 3 h. BMP-9 was more potent than BMP2. Remarkably BMP-9 (5 ng/ml or more) was also capable of inducing Smad2 phosphorylation, whereas BMP-2 (50 ng/ml) was not. BMP-9-induced Smad2 phosphorylation lasted from 1 up to 3 hours. Moreover, in contrast to BMP-2, BMP-9 was also able to rapidly (from 2 up to 24h) induce PAI-1 transcription. Both growth factors potently induced ID1 expression. Gene transcription was reflected in biological activity, as BMP-9 induced luciferase production in the CAGA12-luc assay. Interestingly, co-stimulation of TGFβ with these BMPs revealed an even more remarkable difference between both factors. BMP-2 was able to dose dependently inhibit Smad2 phosphorylation. On the contrary, BMP-9 synergized with TGFβ on Smad2/3 phosphorylation, resulting in a 50% increase in luciferase production after co-stimulation compared to TGFβ alone (p<0.01).
Conclusion: Both BMPs show a distinct difference in Smad phosphorylation and interaction with TGFβ. Although both BMPs promote matrix synthesis (Glycosaminoglycans production), long term effects of both factors on cartilage will most likely differ due to their different effects on chondroprotective Smad2/3 signaling.
A. P. van Caam,
E. N. Blaney Davidson,
E. L. Vitters,
L. de Kroon,
E. W. van Geffen,
P. ten Dijke,
W. B. van den Berg,
P. M. van der Kraan,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/differential-effects-of-bone-morphogenetic-protein-2-and-9-on-chondroprotective-transforming-growth-factor-%ce%b2-signaling/