PROJECT SUMMARY In the canonical pathway, TGFβs bind to their type I and II receptors TGFβRII and ALK5, respectively, to activate Smads 2 and 3. It has been shown that Smad3 is essential for articular cartilage maintenance, but the function of Smad2 in vivo is unknown. It has been shown that TGFβRII is not required for chondrogenesis during development, but the function of the type I receptor ALK5 is unknown. This is an important gap in knowledge because ALK5 can activate TGFβ pathways independently of TGFβRII. Moreover, ALK5 can interact with the type I BMP receptor ALK1, enabling TGFβs to activate BMP pathways. We found that loss of ALK5 leads to lethal chondrodysplasia, in contrast to the mild viable phenotype in mice lacking TGFβRII, demonstrating that ALK5 acts independently of TGFβRII in cartilage. Furthermore, we found that Smad2/3 mutants are viable, demonstrating that ALK5 does not act primarily as a transducer of canonical TGFβ signaling in cartilage. Unexpectedly, loss of ALK5 led to a significant increase in BMP output. Biochemical studies pointed to ALK1 as the source of elevated BMP signaling. This was confirmed by our finding that loss of Alk1 rescues the severe chondrodysplasia in Alk5 mutants. Our studies therefore uncover a new mechanism of action for ALK5 as a regulator of BMP output and a previously unknown pathological role for ALK1 in cartilage. We will investigate ALK1 and ALK5 action in growth plate cartilage in Aim 1. We also ablated Alk5 in adult articular cartilage (AC). Mutants develop early onset osteoarthritis (OA)-like pathologies. We will address whether ALK5 acts as a transducer of canonical TGFβ pathways and/or as an inhibitor of BMP signaling through ALK1 in AC in Aim 2. Finally, we performed crosslinking, reporter, proximity ligation, and other assays to investigate the basis for the genetic interaction between ALK1 and ALK5. These studies indicate that (a) loss of ALK5 triggers the de novo formation of ALK1/ActRIIB complexes, and (b) provide evidence for ligand- independent BMP signaling through ALK1 in cartilage. Prior in vitro studies have shown that ALK1 is unique among BMP receptors in its ability to activate ligand-independent signaling, but the underlying mechanisms and physiological relevance have never been investigated. We address these unknowns in Aim 3. In summary, these studies demonstrate that the primary role of ALK5 is not to transduce TGFβ signals, but rather is to prevent ligand-independent BMP signaling through ALK1 in cartilage. Completion of these studies could lead to a significant revision of our understanding of the role of ALK5 and its critical importance in regulating TGFβ/BMP crosstalk in cartilage.

Articular cartilage degeneration leads to osteoarthritis, a leading cause of chronic pain and disability in the US. The objective of these studies is to characterize a newly discovered mechanism by which TGFβs and BMPs, two major classes of growth factors that regulate cartilage formation and maintenance, interact with each other. Characterizing this new mechanism should lead to more effective targeting of TGFβ and BMP signaling as therapeutic strategies.