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.

Osteopontin (OPN) 1 is a non-collagenous, glycosylated phosphoprotein originally found in bone matrix (1, 2), but now known to be expressed in many tissues including kidney, hypertrophic chondrocytes, placenta, T-lymphocytes, macrophages, secretory epithelia and ganglia of the inner ear, and smooth muscle of the vascular system (3, 4). OPN is also found in biological fluids such as milk (5), urine (6), and plasma (7), and it displays elevated expression in many transformed cells (8). OPN is highly acidic with approximately 25% of the amino acid composition aspartate and glutamate and significant numbers of phosphoserine and phosphothreonine (1, 2). The amino acid sequence contains a conserved Gly-Arg-Gly-Asp-Ser (GRGDS) sequence (2, 3). As a result, OPN binds effectively to the ␣ V ␤ 3 integrin (9), as well as to the ␣ V ␤ 5 and ␣ V ␤ 1 integrins (10). OPN also contains a thrombin cleavage site located near the middle of the molecule. Both peptides produced by thrombin cleavage are capable of supporting integrin-mediated cell attachment (11). Potential roles for OPN function through the ␣ V ␤ 3 integrin were proposed (12). OPN can promote attachment of various cell types and can initiate signal transduction through integrin-associated kinases. Other proposed roles for OPN include chemotaxis (13, 14), inhibition of nitric oxide synthase expression (15), activation of pp60 c-src (16), hydroxyapatite binding (17), and Ca 2ϩ binding (18). Hormone regulation plays an important role in OPN production. Our laboratory has examined the effect of the seco-steroid hormone 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) on OPN expression and secretion. After uptake of 1,25-(OH) 2 D 3 into a cell, the hormone binds to the vitamin D receptor (VDR), translocates to the nucleus, dimerizes preferentially with the retinoid X receptor, then binds to the vitamin D response element (VDRE) located in the promoter region of 1,25-(OH) 2 D 3 -responsive genes (19). In the case of OPN, this results in increased transcription as seen by higher OPN mRNA steady state levels at 24 -48 h (20) and, eventually, higher secreted protein levels (21). The OPN gene in rat is regulated by the additive action of two VDREs (3), which respond to both 1,25-(OH) 2 D 3 and to bioactive analogs of 1,25-(OH) 2 D 3 that bind to nuclear receptors (20).1,25-(OH) 2 D 3 regulation also occurs through rapid plasma membrane-initiated responses, which have been well studied in osteoblasts. We previously reported the 1,25-(OH) 2 D 3 regulation of L-type voltage-sensitive calcium channels (VSCC) in ROS 17/2.8 osteosarcoma cells (22,23 activates other osteoblast signaling pathways that are independent of transcription such as a rapid increase in phospholipase C activity (24), activation of protein kinase C (25), and regulation of whole cell chloride currents (26).In this publication, we report the effect of 1,25-(OH) 2 D 3 on osteoblast-like ROS 17/2.8 cells at a time period between the classic genomic response and the rapid membrane-associated responses. Examination of O...

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