Katarina Holmborn

Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes.

Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.

Heparan Sulfation–Dependent Fibroblast Growth Factor Signaling Maintains Embryonic Stem Cells Primed for Differentiation in a Heterogeneous State

Embryonic stem (ES) cells continuously decide whether to maintain pluripotency or differentiate. While exogenous leukemia inhibitory factor and BMP4 perpetuate a pluripotent state, less is known about the factors initiating differentiation. We show that heparan sulfate (HS) proteoglycans are critical coreceptors for signals inducing ES cell differentiation. Genetic targeting of NDST1 and NDST2, two enzymes required for N‐sulfation of proteoglycans, blocked differentiation. This phenotype was rescued by HS presented in trans or by soluble heparin. NaClO  3− , which reduces sulfation of proteoglycans, potently blocked differentiation of wild‐type cells. Mechanistically, N‐sulfation was identified to be critical for functional autocrine fibroblast growth factor 4 (FGF4) signaling. Microarray analysis identified the pluripotency maintaining transcription factors Nanog, KLF2/4/8, Tbx3, and Tcf3 to be negatively regulated, whereas markers of differentiation such as Gbx2, Dnmt3b, FGF5, and Brachyury were induced by sulfation‐dependent FGF receptor (FGFR) signaling. We show that several of these genes are heterogeneously expressed in ES cells, and that targeting of heparan sulfation or FGFR‐signaling facilitated a homogenous Nanog/KLF4/Tbx3 positive ES cell state. This finding suggests that the recently discovered heterogeneous state of ES cells is regulated by HS‐dependent FGFR signaling. Similarly, culturing blastocysts with NaClO  3− eliminated GATA6‐positive primitive endoderm progenitors generating a homogenous Nanog‐positive inner cell mass. Functionally, reduction of sulfation robustly improved de novo ES cell derivation efficiency. We conclude that N‐sulfated HS is required for FGF4 signaling to maintain ES cells primed for differentiation in a heterogeneous state. Inhibiting this pathway facilitates a more naïve ground state. STEM CELLS 2010;28:191–200

Heparan Sulfate Synthesized by Mouse Embryonic Stem Cells Deficient in NDST1 and NDST2 Is 6-O-Sulfated but Contains No N-Sulfate Groups

Heparan sulfate structure differs significantly between various cell types and during different developmental stages. The diversity is created during biosynthesis by sulfotransferases, which add sulfate groups to the growing chain, and a C5-epimerase, which converts selected glucuronic acid residues to iduronic acid. All these modifications are believed to depend on initial glucosamine N-sulfation carried out by the enzyme glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here we report that heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 completely lacks N-sulfation but still contains 6-O-sulfate groups, demonstrating that 6-O-sulfation can occur without prior N-sulfation. Reverse transcriptase-PCR analysis indicates that all three identified 6-O-sulfotransferases are expressed by the cells, 6-O-sulfotransferase-1 being the dominating form. The 6-O-sulfated polysaccharide lacking N-sulfate groups also contains N-unsubstituted glucosamine units, raising questions about how these units are generated.

Undersulfation of Heparan Sulfate Restricts Differentiation Potential of Mouse Embryonic Stem Cells

Background: HSPG interacts with growth factors to influence growth and differentiation. Results: ES cells lacking NDST1 and NDST2 show very limited differentiation potential. FGF and heparin rescued formation of neural progenitors. Conclusion: HS-mediated FGF signaling is rate-limiting for commitment of primitive ectoderm to the neural lineage Significance: This study shows the importance of the ratio between HSPG and FGF for neural differentiation. Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.

Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis

Objective—Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis. Methods and Results—Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, transforming growth factor &bgr;, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of transforming growth factor &bgr; and platelet-derived growth factor B signal transduction. Conclusion—The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.

Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 results in increased sulfation of mast cell heparin.

Background: NDST1 and 2 are isoenzymes taking part in heparan sulfate and heparin N-sulfation during biosynthesis. Results: NDST1+/− and NDST1−/− mast cells synthesize heparin with increased degree of sulfation. Conclusion: Tentatively, the Golgi enzyme complex, “the GAGosome,” responsible for heparin biosynthesis, is more efficient when containing only NDST2. Significance: Learning how biosynthesis is regulated is crucial to understanding the biological functions of heparin/heparan sulfate. Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1+/− and NDST1−/− MCs is higher (≈30%) than in control MCs where ≈95% of the 35S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.