Abstract
The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional importance in human disease. In contrast, very little is known about the roles of chondroitin sulfate molecules and sulfation patterns during mammalian development and cell lineage specification. Here, we report a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through modulation of Wnt/beta-catenin signaling. Lineage marker analysis demonstrates that enzymatic elimination of endogenous chondroitin sulfates leads to defects specifically in cardiac differentiation. This is accompanied by a reduction in the number of beating cardiac foci. Mechanistically, we show that endogenous chondroitin sulfate controls cardiac differentiation in a temporal biphasic manner through inhibition of the Wnt/beta-catenin pathway, a known regulatory pathway for the cardiac lineage. Treatment with a specific exogenous chondroitin sulfate, CS-E, could mimic these biphasic effects on cardiac differentiation and Wnt/beta-catenin signaling. These results establish chondroitin sulfate and its sulfation balance as important regulators of cardiac cell lineage decisions through control of the Wnt/beta-catenin pathway. Our work suggests that targeting the chondroitin biosynthesis and sulfation machinery is a novel promising avenue in regenerative strategies after heart injury.
Highlights
The glycosaminoglycan chondroitin sulfate (CS) consists of linear chains of repeating disaccharide units covalently linked to cell surface and secreted proteins to form chondroitin sulfate proteoglycans [1,2], which have been shown to control multiple aspects of cellular behavior and communication [2]
Cultures were treated with the bacterial enzyme Chondroitinase ABC (ChABC) at 10 mU/ml, previously demonstrated to efficiently to eliminate chondroitin sulfate side chains of chondroitin sulfate proteoglycans (CSPGs) [48]
Cells were not treated with ChABC; no immunofluorescence signal was detected in embryonic stem (ES) cells cultured in the presence or absence of Leukemia Inhibitory factor (LIF), or in embryoid body (EB) at d6 or d12 of differentiation (Figure 1C)
Summary
The glycosaminoglycan chondroitin sulfate (CS) consists of linear chains of repeating disaccharide units covalently linked to cell surface and secreted proteins to form chondroitin sulfate proteoglycans [1,2], which have been shown to control multiple aspects of cellular behavior and communication [2]. We have previously described severe embryonic skeletal abnormalities and perinatal lethality in mice carrying a loss-of-function mutation in the Chondroitin-4-sulfotransferase-1 (C4st-1) gene [7], demonstrating the critical importance of a proper balance of chondroitin sulfation in cartilage development. These conclusions were supported by other studies, which identified skeletal abnormalities in loss-of-function mutations of a number of CS biosynthesis enzymes [22,23]. Distinct expression domains for CS and CS biosynthesis enzymes have been described in the developing and mature mammalian heart [4,28,29]; the functional roles of CS in heart development or cardiac lineage development are not understood
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