Expression of the Heparan Sulfate Proteoglycan, Perlecan, during Mouse Embryogenesis and Perlecan Chondrogenic Activity In Vitro

Abstract

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.

Mammalian Skeletogenesis and Extracellular Matrix. What can We Learn from Knockout Mice?

“…Initially, it was believed that the expression of this proteoglycan is restricted to basement membranes, but more recent studies showed a high perlecan expression in developing cartilage (Handler et al, 1997;French et al, 1999).…”

Cartilage proteoglycans

“…Perlecan coated onto dishes promoted chondrogenesis and maintains chondrogenic differentiation, perhaps working in concert with SOX9. 45 The perlecan knock-out mouse 46,47 exhibits skeletal abnormalities, defective endochondral ossification, but no early defects in the formation of cartilage anlagen, so in vivo the early events of condensation and chondrocyte differentiation are apparently unaffected by the perlecan deficiency. However, in the knockout animals, there is disorganization of the growth plate, including a disruption of the chondrocyte columnar organization and a reduction of the fibrillar collagen network suggesting the role of perlecan in matrix structure.…”

“…It has also been suggested that perlecan may influence chondrocyte metabolism via signaling receptors or by modulating the expression of FGFs. 44,45 …”

What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?

“…HSPGs can be divided into three major classes: (1) lipid-anchored, e.g., the glypicans; (2) transmembrane, e.g., syndecans; and (3) extracellular or secreted, a group that includes perlecan [42]. HSPGs had been found in cartilage [43], bone stroma [44], uterine decidua [45], and follicular fluid [46]. Perlecan is particularly abundant in the ovary with a level of expression that is stronger than the kidney-a known rich source of this protein.…”