Loss of the Heparan Sulfate Sulfotransferase, Ndst1, in Mammary Epithelial Cells Selectively Blocks Lobuloalveolar Development in Mice

Abstract

BackgroundConsiderable evidence indicates that heparan sulfate is essential for the development of tissues consisting of branching ducts and tubules. However, there are few examples where specific sulfate residues regulate a specific stage in the formation of such tissues.Methodology/Principal FindingsWe examined the role of heparan sulfation in mammary gland branching morphogenesis, lactation and lobuloalveolar development by inactivation of heparan sulfate GlcNAc N-deacetylase/N-sulfotransferase genes (Ndst) in mammary epithelial cells using the Cre-loxP system. Ndst1 deficiency resulted in an overall reduction in glucosamine N-sulfation and decreased binding of FGF to mammary epithelial cells in vitro and in vivo. Mammary epithelia lacking Ndst1 underwent branching morphogenesis, filling the gland with ductal tissue by sexual maturity to the same extent as wildtype epithelia. However, lobuloalveolar expansion did not occur in Ndst1-deficient animals, resulting in insufficient milk production to nurture newly born pups. Lactational differentiation of isolated mammary epithelial cells occurred appropriately via stat5 activation, further supporting the notion that the lack of milk production was due to lack of expansion of the lobuloalveoli.Conclusions/SignificanceThese findings demonstrate a selective, highly penetrant, cell autonomous effect of Ndst1-mediated sulfation on lobuloalveolar development.

Heparan Sulfate Proteoglycans

“…Floxed allele: decreased chemokine transcytosis and presentation and neutrophil infiltration in Tie2Cre mice ; decreased allergen-induced airway hyperresponsiveness and inflammation because of reduction in recruitment of eosinophils, macrophages, neutrophils, and lymphocytes in Tie2Cre mice (Zuberi et al 2009); decreased pathological angiogenesis in Tie2Cre mice (Fuster et al 2007); decreased vascular VEGF-induced hyperpermeability (Xu et al 2010a); decreased vascular smooth muscle cell proliferation, vessel size, and vascular remodeling after arterial injury in SM22aCre mice (Adhikari et al 2010a); mild effect on T-cell response in Tie2Cre;Ndst2 2/2 mice (Garner et al 2008); defective lacrimal gland development and Fgf10-Fgfr2b complex formation and signaling in LeCre mice ; defective lobuloalveolar development in mammary gland (Crawford et al 2010).…”

Glycan-based biomarkers for the mucopolysaccharidoses

“…The high affinity ligand fibroblast growth factor-2 (FGF2; basic FGF) has been used to detect HS on cells, in tissue sections from mice, and in solution [43][44][45]. High sensitivity is achieved by using fluorescent derivatives of FGF2 or biotinylated FGF2 and enzyme-conjugated streptavidin.…”

N-Sulfation of Heparan Sulfate Regulates Early Branching Events in the Developing Mammary Gland

“…Because systemic deletion of many of these enzymes results in either embryonic lethality or a lack of defects in mammary gland development or function (9 -16), we have investigated the role of HS in mammary ductal epithelial branching morphogenesis by utilizing mammary epithelia-specific deletions of various HS biosynthetic enzymes. For example, tissue-specific inactivation of Ndst1, one of only two members of this family expressed in mammary epithelia, had no apparent effect on primary and secondary ductal branching, but selectively perturbed lobuloalveolar development (5). In a subsequent study mammary-specific inactivation of Ext1 (a subunit of the co-polymerase complex that catalyzes the formation of the HS chain) resulted in a highly penetrant and dramatic defect in primary branching (6).…”

“…Whereas specific heparan sulfate proteoglycans have been implicated in mammary development, recent in vivo evidence supports the notion that the heparan sulfate chains (HS) 7 and the pattern of sulfation play a functionally important role in modulating branching morphogenesis of the mammary ductal epithelium (5,6). Biochemical and in vitro cell and organ culture data indicate that the action of HS is dependent upon proper sulfation (7,8) which is established by the action of a series of HS biosynthetic enzymes, including members of the N-deacetylase/N-sulfotransferase (Ndst) family, the uronyl C5-epimerase (Hsglce), and 2-O-sulfotransferase (Hs2st), the glucosaminyl 6-O-sulfotransferases (Hs6st) and 3-O-sulfotransferases (Hs3st) (31).…”

“…In addition, although inactivation of Hs2st (required for 2-O-sulfation of uronic acids in HS) resulted in fewer bifurcated terminal end buds, the ductal network was characterized by a marked reduction in secondary/ductal side branches (6). Thus, whereas assembly of the HS chains (mediated by Ext1) is key to mammary gland development, selective O-sulfation and N-sulfation appear to regulate different stages in branching and lobuloalveolar development (5,6).…”