While it is undisputed that extracellular matrix and soft tissues influence skeletal growth, few specific pathways explaining this effect have been uncovered. In Marfan syndrome, which affects 1-2 in 5000 individuals, skeletal overgrowth, long digits, poorly developed musculature and lax joints result from fibrillin-1 (FBN1) mutations. Strikingly, specific mutations in FBN1 can also cause the ?opposite? of Marfan syndrome, i.e. short stature, disproportionally short digits (brachydactyly), stiff joints, and a ?pseudomuscular? build, which are the hallmarks of acromelic dysplasias, comprising a group of Mendelian disorders. Identical acromelic dysplasias can also be caused by genes encoding ADAMTS proteases, ADAMTS-like (ADAMTSL) proteins, latent transforming growth factor-? (TGF?) binding protein-3 (LTBP3), and SMAD4. The overlapping phenotypes of different gene mutations underlying acromelic dysplasias strongly support my hypothesis that a fibrillin-ADAMTS-TGF? axis constitutes a novel extracellular matrix (ECM) network regulating postnatal limb growth. Mutations in ADAMTSL2 or FBN1 lead to geleophysic dysplasia (GD), a severe, frequently lethal human acromelic dysplasia. ADAMTSL2 is a secreted glycoprotein that binds to FBN1 and FBN2, and is implicated in TGF? signaling. Intriguingly, ADAMTSL2 mRNA is not expressed in growth plate cartilage or bone, but has its strongest expression in tendon. My studies show that FBN2 microfibrils are increased at sites of Adamtsl2 expression in a mouse knockout model of GD, suggesting a role for ADAMTSL2 in switching from prenatal FBN2-dominated microfibrils to postnatal FBN1-dominated microfibrils. Furthermore, skeletal growth is impaired upon limb-specific ADAMTSL2 deletion (Prx1-Cre) or tendon and ligament specific deletion (Scx- Cre). I observed disproportionate distal limb shortening (i.e. acromelic dysplasia) and a reduction in Achilles tendon length in both conditional deletions. A model for skeletal growth in geleophysic dysplasia provides an opportunity to determine how tissue non-autonomous regulation of skeletal growth occurs via mechanical or regulatory input from tendon ECM.
In aim 1, I will test the hypothesis by analyzing postnatal limb growth and ECM alterations in the microfibril system after Adamtsl2 deletion in tendons with Scx-Cre.
In aim 2, I will investigate how ADAMTSL2 interacts with FBN1 and FBN2 and how ADAMTSL2 executes its role in the isoform switch from FBN2 to FBN1. I will analyze the genetic interaction of Adamtsl2 with Fbn1 and Fbn2 in mice and I will use protein-protein interaction studies and cell culture assays to gain mechanistic insights in the function of ADAMTSL2 in regulating the fibrillin isoform switch. Impact: The anticipated results will provide novel insights into the pathophysiology of acromelic dysplasias and other fibrillinopathies. These insights could be translated for targeting tendon ECM in regenerative strategies. The proposal addresses fundamental questions of how functional properties of soft tissues are determined by ECM subsequently might regulate postnatal limb growth.
Short stature is found in human genetic disorders caused by mutations in components of the connective tissue. This proposal uses a novel mouse model for such a disorder, geleophysic dysplasia, to analyze how alterations in the connective tissue of tendon cause bone shortening. The expected results will provide novel insights in the physiological regulation of bone growth by soft tissues and point to novel therapeutic strategies for these disorders.