Genetic Rescue of Chondrodysplasia and the Perinatal Lethal Effect of Cartilage Link Protein Deficiency

Abstract

The targeted disruption of cartilage link protein gene (Crtl1) in homozygous mice resulted in a severe chondrodysplasia and perinatal lethality. This raised the question of whether the abnormalities seen in Crtl1 null mice are all caused by the absence of link protein in cartilage or whether the deficiency of the protein in other tissues and organs contributed to the phenotype. To address this question we have generated transgenic mice overexpressing cartilage link protein under the control of a cartilage-specific promoter, and then these transgenic mice were used for a genetic rescue of abnormalities in Crtl1 null mice. While the overexpression of cartilage link protein resulted in no abnormal phenotype, the cartilage-specific transgene expression of link protein could completely prevent the perinatal mortality of link protein-deficient mice and, depending on the level of the link protein expression, rescue skeletal abnormalities. Although link protein was originally isolated from cartilage, we found and determined Crtl1 transcripts and corresponding proteins in every organ tested from mouse embryos to aging animals. We also identified three additional members of the link protein family, all co-localized with hyaluronic acid-binding proteoglycans in the mouse genome. The ubiquitous presence of link protein suggests a general and systemic function of link protein in the organization of extracellular matrix in a number of tissues, possibly interacting with other proteoglycans, such as versican, brevican, and neurocan.A highly specific macromolecular organization of cartilage extracellular matrix (ECM) 1 is required for chondrogenesis, chondrocyte differentiation, endochondral ossification, and for the maintenance of the weight-bearing function of articular cartilage. The major macromolecular components of this ECM are the proteoglycan aggrecan, hyaluronic acid (hyaluronan, HA), link protein (LP) and type II collagen. Hundreds of aggrecan molecules associate with a single HA filament to form large proteoglycan aggregates. The link protein, a glycoprotein, binding simultaneously to both HA and aggrecan, stabilizes the aggregate structure (1), and the aggregates are entrapped within the mesh-like network of type II collagen fibrils (2-4). The negatively charged glycosaminoglycan chains of aggrecan bind large amounts of water and are responsible for the resiliency of cartilage (5). Mutation in a gene encoding components of cartilage ECM may result in skeletal disorders, chondrodysplasias, in mice (6, 7) and humans (8).Cartilage LP is a member of the hyaluronectin superfamily, and shares a high degree of sequence homology in different species, particularly in the HA binding domain (2), also known as "link module" (4, 9). Although LP was originally isolated from cartilage, its presence has been reported in numerous non-cartilaginous tissues, such as aorta (10), mesonephros, and other embryonic tissues (11, 12), brain (13) and sclera (14) in chicken, and the mouse cumulus-oocyte complex, without concomitant expression...

Lp3/Hapln3, a novel link protein that co-localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells

“…The human and mouse genome project revealed that each of the four lectican genes is located pairwise with an LP gene at one of four different chromosome locations (Nomoto et al, 2002;Spicer et al, 2003;Czipri et al, 2003). In the rat genome, the four LP genes and lectican genes were also similarly arranged.…”

“…However, until recently, other members of The LP family had not been reported. The human and mouse genome projects revealed that each of four lectican genes is physically associated with one of the four LP genes on different chromosomes (Nomoto et al, 2002;Spicer et al, 2003;Czipri et al, 2003). Among the new LPs, Bral1 and Bral2 are exclusively expressed in the adult CNS.…”

SOX9-dependent and -independent Transcriptional Regulation of Human Cartilage Link Protein

“…The cartilage in these mice contains significantly reduced aggrecan depositions in the hypertrophic zone and decreased numbers of prehypertrophic and hypertrophic chondrocytes (5). Cartilage-specific transgene expression of CRTL1 can completely prevent perinatal mortality in CRTL1-null mice and rescue skeletal abnormalities at levels dependent upon the amount of CRT-LP expression (6). In addition, CRT-LP may function as a growth factor to up-regulate the synthesis of aggrecan and type II collagen in cartilage (7).…”

Single-nucleotide polymorphism in the hyaluronan and proteoglycan link protein 1 (HAPLN1) gene is associated with spinal osteophyte formation and disc degeneration in Japanese women

“…HAPLN1 is known for its role in cartilage formation, where it stabilizes the hyaluronan/aggrecan and hyaluronan/versican complexes [5,6,14,16,17]. Its importance in cartilage formation has previously been demonstrated in studies on Hapln1-deficient mice [4,29]. These studies have reported that Hapln1-deficient mice exhibit defective cartilage development and delayed bone formation and therefore have short limbs and craniofacial anomalies.…”

“…Chondrocytes of the vertebral bodies are disorganized in Hapln1-null mice [29,30]. Cartilage-specific expression of the Hapln1 transgene can completely prevent perinatal mortality in Hapln1-null mice and can dose dependently correct skeletal abnormalities [4]. In addition, a synthetic peptide of HAPLN1 stimulates the biosynthesis of collagens II, IX, and proteoglycan in the intervertebral disc cells [15].…”