A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation.

Perlecan/HSPG2 is a large, multi-domain, multifunctional heparan sulfate proteoglycan with a wide tissue distribution. With the exception of its unique domain I, each of perlecan's other four domains shares sequence similarity to other protein families including low density lipoprotein (LDL) receptor, laminin alpha chain, neural cell adhesion molecule (NCAM), immunoglobulin (Ig) superfamily members, and epidermal growth factor (EGF). Previous studies demonstrated that glycosaminoglycan-bearing perlecan domain I supports early chondrogenesis and growth factor delivery. Other sites in the core protein interact with other matrix molecules and support cell adhesion, although the peptide sequences involved remain unidentified. To identify novel functional motifs within perlecan, we used a bioinformatics approach to predict regions likely to be on the exterior of the folded protein. Unique hydrophilic sequences of about 18 amino acids were selected for testing in cell adhesion assays. A novel peptide sequence (TWSKVGGHLRPGIVQSG) from an immunoglobulin (Ig) repeat in domain IV supported rapid cell adhesion, spreading and focal adhesion kinase (FAK) activation when compared to other peptides, a randomly scrambled sequence of the domain IV peptide or a negative control protein. MG-63 human osteosarcoma cells, epithelial cells and multipotent C(3)H10T1/2 cells, but not bone marrow cells, rapidly, i.e., within 30 min, formed focal adhesions and assembled an actin cytoskeleton on domain IV peptide. Cell lines differentially adhered to the domain IV peptide, suggesting adhesion is receptor specific. Adhesion was divalent cation independent and heparin sensitive, a finding that may explain some previously poorly understood observations obtained with intact perlecan. Collectively, these studies demonstrate the feasibility of using bioinformatics-based strategies to identify novel functional motifs in matrix proteins such as perlecan.

Chondrogenic differentiation on perlecan domain I, collagen II, and bone morphogenetic protein-2-based matrices.

Extracellular matrix (ECM) molecules in cartilage cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). We hypothesized that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on 2-dimensional P-C fibrils or 3-dimensional P-C/BMP-2-coated (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II, and aggrecan. Immunostaining for aggrecan, perlecan, tenascin, and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We conclude that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenic differentiation.

Heparan sulfate proteoglycans: coordinators of multiple signaling pathways during chondrogenesis.

Heparan sulfate proteoglycans are abundantly expressed in the pericellular matrix of both developing and mature cartilage. Increasing evidence indicates that the action of numerous chondroregulatory molecules depends on these proteoglycans. This review summarizes the current understanding of the interactions of heparan sulfate chains of cartilage proteoglycans with both soluble and nonsoluble ligands during the process of chondrogenesis. In addition, the consequences of mutating genes encoding heparan sulfate biosynthetic enzymes or heparan sulfate proteoglycan core proteins on cartilage development are discussed.

HIP/RPL29 down-regulation accompanies terminal chondrocyte differentiation.

HIP is a heparin/heparan sulfate (Hp/HS) binding protein identical to ribosomal protein L29 that displays diverse biological functions. There is strong evidence that abnormal expression and quantitative deficiencies of essential molecules such as extracellular matrix (ECM) proteins, transcription factors, and ribosomal proteins can seriously impair embryonic development. As observed for HS-bearing molecules, high levels of HIP/RPL29 are found in proliferating chondrocytic precursors and chondrocytes of developing growth plate. Here, we demonstrate both in vitro and in developing mouse embryos that HIP/RPL29 is down-regulated in terminally differentiated chondrocytes corresponding to the late hypertrophic zone of the growth plate. Because cartilage serves as a template for endochondral bone formation, we hypothesize that the presence of HIP/RPL29 during early chondrogenesis is essential for normal skeletal growth and patterning. In particular, we believe that HIP/RPL29 expression is required to maintain proliferation of chondrocytes and avoid skeletal shortening. Increasing evidence suggests that multifunctional ribosomal proteins of eukaryotic cells are important regulators of cell growth and differentiation, not simply structural parts of translational machinery. To investigate the role of HIP/RPL29 normal expression during cartilage formation, we designed a ribozyme-mediated knock-down approach to partially down-regulate HIP/RPL29 expression in the multipotent mouse embryonic skin fibroblast cell line C3H/10T (1/2). This technology permitted us to avoid the insufficient expression associated with more severe consequences, such as lethality, and provided advantages similar to those obtained with mutations generating hypomorphic phenotypes. Our results show that partial reduction of HIP/RPL29 levels accelerates differentiation of C3H/10T(1/2) into cartilage-like cells. In conclusion, our data indicate that HIP/RPL29 constitutes an important novel regulator of chondrocytic growth and differentiation.