Heparan sulfate 6-O-sulfotransferase 3 is involved in bone marrow mesenchymal stromal cell osteogenic differentiation

Abstract

The roles of sugar chains such as heparan sulfate (HS) in stem cell self-renewal and differentiation are poorly understood. HS is a sugar chain with linear sulfated polyanionic disaccharide repeating structures that interact with many proteins, including structural proteins in the extracellular matrix and growth factors and their receptors. Thus, unraveling the role of HS in stem cell self-renewal and differentiation could provide new insights and technical routes in clinical stem cell applications. Here, we purified rat bone marrow mesenchymal stromal cells (BMMSCs) by density gradient centrifugation, analyzed mesenchymal stromal cell surface stemness marker expression by flow cytometry, and identified the sulfotransferases responsible for sulfation ester modification of HS. An osteogenic differentiation model was established by chemical induction reagents and confirmed via alkaline phosphatase (ALP) activity detection and the expression of the osteogenic differentiation markers Runx2 and Ocn. The expression profiles of HS sulfotransferases in rat BMMSCs before and after osteogenic induction were detected by RT-PCR and Western blot. Cell spheroids were formed in both control and osteogenic culture systems when BMMSCs were grown to high confluence. We determined that this type of cell spheroid was a highly calcified nodule by histochemical staining. Among all the sulfotransferases examined, heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) mRNA and protein were upregulated in these calcified cell spheroids. HS6ST3 knockdown BMMSCs were established with RNA interference, and they had significantly lower ALP activity and decreased expression of the osteogenic differentiation markers Runx2 and Ocn. These findings suggest that HS6ST3 is involved in BMMSC differentiation, and new glycotherapeutic-based technologies could be developed in the future.

Multifarious effects of 17-β-estradiol on apolipoprotein E receptors gene expression during osteoblast differentiation <i>in vitro </i>

“…HSPGs bind to a variety of protein ligands and regulates a wide variety of biological activities, including developmental processes (72,73), such as bone and organ formation (74). A report concluded that there are 15 members in the HSPGs.…”

Heparan Sulfate Proteoglycans: Key Mediators of Stem Cell Function

“…A study from Zhao and colleagues also reported an upregulation of 6OST3 at the mRNA and protein levels as MSCs underwent calcification (Zhao et al, 2015). Collectively, data from these studies indicate that as pre-osteoblasts or MSCs progress through osteogenesis, HS chain initiation may decrease while overall chain sulfation may be increased.…”

“…Collectively, data from these studies indicate that as pre-osteoblasts or MSCs progress through osteogenesis, HS chain initiation may decrease while overall chain sulfation may be increased. Moreover, siRNA knockdown of glypican-3 or 6OST3 was found to decrease differentiation down the osteogenic lineage (Haupt et al, 2009;Zhao et al, 2015), suggesting the necessity of unique HSPG motifs for progression through the lineage commitment process. Further investigations into the functional features of unique HS/heparin signatures in osteogenesis have demonstrated distinct binding properties for proteins that mediate important pro-differentiation signaling cascades.…”

Proteoglycans and Glycosaminoglycans in Stem Cell Homeostasis and Bone Tissue Regeneration

“…Upon osteogenic differentiation, HSPGs including syndecans (syndecan-1,-2,-3, and -4), glypican-1, glypican-3, and glypican-4 showed increased transcript levels, whereas only minimal increases were observed in chondroitin sulfate proteoglycans (CSPGs)/dermatan sulfate proteoglycans (DSPGs) including decorin (DCN), biglycan (BGN) as 2-O-sulfation and 6-O-sulfation of HS under osteogenesis as expression of N-sulfotransferase1/2 (NDST1/2), heparan sulfate 2-O-sulfotransferase (HS2OST) and heparan sulfate 6-O-sulfotransferase (HS6OST) increased under osteogenic induction ( Haupt et al, 2009 , 3). Zhao et al also showed marked upregulation of heparan sulfate 6-O-sulfotransferase-3 (HS6ST3), which encoded enzymes that performed sulfation at the 6-O position in glucosamine in HS ( Zhao et al, 2015 ). These data displayed a distinct profile of proteoglycans/GAGs profile in stem cells.…”

“…The 6-O-sulfation of HS chains was also found to be positively associated with to the osteogenic differentiation of MSCs ( Zhao et al, 2015 ). Knockdown of HS6ST3 in MSCs remarkedly impaired osteogenic differentiation, thus halving ALP activity, and reduced the expression of osteogenic markers such as OCN and RUNX2 by 60 and 75%, respectively.…”