Oversulfated chondroitin sulfate‐E binds to BMP‐4 and enhances osteoblast differentiation

Abstract

Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N-acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization.

Glycosaminoglycan chains of biglycan promote bone morphogenetic protein-4-induced osteoblast differentiation

“…In the present report, we studied the effects of GAG chains of BGN on the BMP-4-induced osteoblast differentiation. Previous reports support our findings; Miyazaki et al (23) reported that hypersulfated chondroitin sulfate (CS)-E binds to BMP-4 and enhances osteoblast differentiation by increasing the level of exogenous sulfated GAGs to MC3T3-E1 osteoblastic cells. It has also been reported that collagen chondroitin sulfate promotes the in vitro mineralization of 3-dimensional collagen matrices seeded with bone-derived cells (24).…”

Sulfation patterns of exogenous chondroitin sulfate affect chondrogenic differentiation of ATDC5 cells

“…These two CS variants also show strong affinity to some growth factors, such as fibroblast growth factors and bone morphogenetic proteins (BMPs) [24,25]. CS-E enhances osteoblast differentiation by binding to BMP-4, which is an essential growth factor for chondrogenic differentiation [26]. ATDC5 cells express the BMP-4 gene and autocrine BMP-4 is required for the differentiation of ATDC5 [27].…”

Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells

“…It is known that osteoblastic MC3T3-E1 cells express oversulfated CS structures and the differentiation was inhibited by CHase digestion 8) . MC3T3-E1 cells were inoculated into 48-well plates in α-MEM containing 5% FBS and 10 µg/mL gentamicin (growth medium) at 37°C in a humidified atmosphere of 5% CO2.…”

“…Recently, it was reported that oversulfated CS structures existed in the osteoblast cell surface and induced its own mineralization by binding with bone morphogenetic protein (BMP) and upregulating the activity 8) . As sulfated GAGs have high affinity for calcium phosphate (CaP), such as hydroxyapatite (HA) 9) , it is thought that sulfated GAGs would also exist in the HA layer of the bone matrix.…”

“…Special attention was given to the CS-E because it enhanced the growth and differentiation of osteoblasts 8) . …”