Regulation of Skeletal Growth by Soft Tissue Extracellular Matrix

摘要 虽然细胞外基质和软组织影响骨骼生长是无可争议的， 解释了这种效应。在马凡氏综合征中，每5000人中有1-2人受累， 过度生长、长趾、发育不良的肌肉组织和松弛的关节是由BFN-1（FBN 1）突变引起的。 引人注目的是，FBN 1中的特定突变也会导致马凡氏综合征的“相反”，即身材矮小， 手指短小（短指）、关节僵硬和“假肌肉”结构，这些都是特征。 肢端发育不良，包括一组孟德尔疾病。同样的肢端发育不良也可以 由编码ADAMTS蛋白酶、ADAMTS样（ADAMTS SL）蛋白、潜伏转化 生长因子β（TGF β）结合蛋白3（LTBP 3）和SMAD 4。不同基因的重叠表型 肢端发育不良潜在的突变强烈支持我的假设，即原纤维蛋白-ADAMTS-TGF β轴 构成了一个新的细胞外基质（ECM）网络调节出生后肢体的生长。 ADAMTSL 2或FBN 1的突变导致胶质细胞发育不良（GD），这是一种严重的、经常致命的人类疾病， 肢端发育不良ADAMTSL 2是一种分泌型糖蛋白，与FBN 1和FBN 2结合，并参与 TGF β 1信号传导。有趣的是，ADAMTSL 2 mRNA在生长板软骨或骨中不表达，但其在生长板软骨或骨中表达。 在肌腱中表达最强。我的研究表明，FBN 2微纤维在Adamts 12位点增加， 在GD的小鼠敲除模型中表达，表明ADAMTSL 2在从产前基因突变转换为产前基因突变中的作用。 FBN 2主导的微纤维到出生后FBN 1主导的微纤维。此外，骨骼生长是 肢体特异性ADAMTSL 2缺失（Prx 1-Cre）或肌腱和韧带特异性缺失（Scx- Cre）。我观察到不成比例的远端肢体缩短（即肢端发育不良）和跟腱减少 两种条件性缺失中的肌腱长度。一种骨胶性发育不良的骨骼生长模型提供了一种 有机会确定骨骼生长的组织非自主调节是如何通过机械或 来自肌腱ECM的调节输入。在目标1中，我将通过分析出生后肢体的生长来检验这一假设， 在具有Scx-Cre的肌腱中Adamtsl 2缺失后微原纤维系统中的ECM改变。在目标2中，我将 研究ADAMTSL 2如何与FBN 1和FBN 2相互作用，以及ADAMTSL 2如何在 同种型从FBN 2转换为FBN 1。我将分析Adamtsl 2与Fbn 1和Fbn 2的遗传相互作用， 小鼠和我将使用蛋白质-蛋白质相互作用研究和细胞培养试验，以获得机制的见解， ADAMTSL 2在调节β-内酰胺酶同工型开关中的功能。 影响：预期结果将为肢端发育不良的病理生理学提供新的见解， 其他纤维蛋白病。这些见解可以转化为再生策略中针对肌腱ECM的目标。 该提案解决了软组织的功能特性如何由以下因素决定的基本问题： ECM随后可能调节出生后肢体的生长。

ABSTRACT 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.