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毋庸置疑的，但很少有特定的途径 对这种效应的解释已经被揭开了。在马凡综合征中，每5000人中有1-2人受到影响，骨骼 纤维蛋白-1(FBN1)突变导致过度生长、长指头、肌肉发育不良和关节松弛。 引人注目的是，FBN1的特定突变也会导致马凡氏综合症的“相反”，即身材矮小。 不成比例的短指(短指)、僵硬的关节和“假肌肉”的体型，这些都是特征。 肢端发育不良症，由一组孟德尔病症组成。同样的肢端发育不良也可以是 由编码ADAMTS蛋白酶，ADAMTS样蛋白(ADAMTSL)的基因引起，潜伏转化 生长因子-(转化生长因子)结合蛋白-3和Smad4。不同基因的重叠表型 肢端发育不良的基因突变有力地支持了我的假设，即纤维蛋白-ADAMTS-转化生长因子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。 ADAMTSL2或FBN1基因突变导致遗传性发育不良(GD)，这是一种严重的、经常致命的人类 肢端发育不良。ADAMTSL2是一种分泌型糖蛋白，与FBN1和FBN2结合，并与 转化生长因子信号转导。有趣的是，ADAMTSL2mRNA不在生长板软骨或骨中表达，但有其 肌腱中表达最强。我的研究表明，在Adamtsl2的位置，FBN2微纤维增加 在GD小鼠基因敲除模型中的表达，提示ADAMTSL2在产前转换中的作用 以FB2为主的微纤维到出生后以FBN1为主的微纤维。此外，骨骼生长是 肢体特异性ADAMTSL2缺失(PRX1-CRE)或肌腱和韧带特异性缺失(SCX- Cre)。我观察到肢体远端不成比例的缩短(即肢端发育不良)和跟腱减少。 在两个条件删除中的肌腱长度。骨质疏松症发育不良的骨骼生长模型提供了一个 有机会确定组织对骨骼生长的非自主调节是如何通过机械或 肌腱ECM的调节输入。在目标1中，我将通过分析出生后肢体发育和 伴有SCX-CRE的肌腱Adamtsl2缺失后微原纤维系统的ECM改变。在《目标2》中，我会 调查ADAMTSL2如何与FBN1和FBN2交互，以及ADAMTSL2如何在 从FBN2到FBN1的异构体切换。我将分析Adamtsl2与Fbn1和Fbn2在 老鼠和我将使用蛋白质-蛋白质相互作用研究和细胞培养分析来获得对 ADAMTSL2在调节纤维素异构体开关中的作用 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和 其他纤维蛋白病变。这些见解可以转化为再生策略中的肌腱ECM靶向。 该提案涉及软组织的功能属性如何由以下因素决定的基本问题 细胞外基质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.