ORIGINAL PAPER Temporal and functional changes in glycosaminoglycan expression during osteogenesis Victor Nurcombe Æ Fuqi Jack Goh Æ Larisa M. Haupt Æ Sadasivam Murali Æ Simon M. Cool Received: 11 June 2007 / Accepted: 12 July 2007 / Published online: 3 August 2007 Ó Springer Science+Business Media B.V. 2007 Abstract Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the sur- faces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of hepa- ran sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts. Keywords Heparan sulfate Á Differentiation Á Phenotype Á Mineralization Á Proteoglycans Á Extracellular matrix Á Growth factors Introduction Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix (ECM) macromolecules that are composed of a core protein with one or more covalently linked, unbranched glycosaminoglycan (GAG) sugar chains (Bernfield et al. 1999; Esko and Lindahl 2001). They are virtually ubiquitous, as well as abundant, on animal cell surfaces. The HS polysaccharide chain, which usually carries the bulk of the bioactivity of the macro- molecular complex (Herndon et al. 1999), consists of alternating hexuronic acid and D-glucosamine units that are substituted with sulfate groups at various points. The sul- fated saccharides within the chain tend to be grouped into domains, which provide numerous docking sites for protein ligands (Gallagher 2006). To date, a plethora of extracel- lular proteins have been found to rely on HS sugars to drive their interactions with other proteins, including the Wnt, Hedgehog, transforming growth factor-b, and fibroblast growth factor pathways; thus HS has been hypothesized to catalyze molecular interactions (Lander and Selleck 2000). Two main types of cell surface-bound PG core protein have been identified: the glycosylphosphatidyl inositol- linked glypicans and the transmembrane syndecans. As well as these, several forms, most prominently the perlecans, are secreted into the extracellular matrix (ECM) (Perrimon and Bernfield 2001). Among the variety of ligands dependent on their capacities apart from growth factors are morphogens, as well as their receptors, enzymes, enzyme inhibitors, cell adhesion molecules, chemokines and various ECM proteins V. Nurcombe (&) Á F. J. Goh Á L. M. Haupt Á S. Murali Á S. M. Cool Stem Cell and Tissue Repair Group, Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Singapore e-mail: vnurcombe@imcb.a-star.edu.sg V. Nurcombe Á S. M. Cool Department of Orthopaedic Surgery, Yong Loo School of Medicine, National University of Singapore, Singapore 117597, Singapore 123 J Mol Hist (2007) 38:469–481 DOI 10.1007/s10735-007-9123-4