The effects of altering N-cadherin N-glycosylation on several cadherin-mediated cellular behaviors were investigated using small interfering RNA and site-directed mutagenesis. In HT1080 fibrosarcoma cells, small interfering RNA-directed knockdown of N-acetylglucosaminyltransferase V (GnT-V), a glycosyltransferase up-regulated by oncogene signaling, caused decreased expression of N-linked ␤(1,6)-branched glycans expressed on N-cadherin, resulting in enhanced N-cadherin-mediated cell-cell adhesion, but had no effect on N-cadherin expression on the cell surface. This effect on adhesion was accompanied by decreased cell migration and invasion, opposite of the effects observed when GnT-V was overexpressed in these cells (Guo, H. B., Lee, I., Kamar, M., and Pierce, M. (2003) J. Biol. Chem. 278, 52412-52424). A detailed study using site-directed mutagenesis demonstrated that three of the eight putative N-glycosylation sites in the N-cadherin sequence showed N-glycan expression. Moreover, all three of these sites, located in the extracellular domains EC2 and EC3, were shown by leucoagglutinating phytohemagglutinin binding to express at least some ␤(1,6)-branched glycans, products of GnT-V activity. Deletion of these sites had no effect on cadherin levels on the cell surface but led to increased stabilization of cell-cell contacts, cell-cell adhesion-mediated intracellular signaling, and reduced cell migration. We show for the first time that these deletions had little effect on formation of the N-cadherincatenin complex but instead resulted in increased N-cadherin cis-dimerization. Branched N-glycan expression at three sites in the EC2 and -3 domains regulates N-cadherin-mediated cell-cell contact formation, outside-in signaling, and cell migration and is probably a significant contributor to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling.Cadherins are single-pass transmembrane receptors that mediate calcium-dependent cell-cell adhesion at adherens junctions and play an essential role in regulating major cellular behaviors, including cell growth, motility, and differentiation (1, 2). Several cadherins, including E-cadherin and N-cadherin, have in common an extracellular domain with five segments of repeated sequences and regulate cell-cell adhesion in a homotypic manner through their association of amino-terminal extracellular domains, such as EC1 (3, 4). Calcium binding to the extracellular domain triggers a conformation that initiates the homotypic binding of cadherin between cells. The conserved cytoplasmic domain of cadherin interacts with various proteins, collectively termed catenins, that link cadherins to the actin-based cytoskeleton and promote strong cell-cell adhesion (5). Evidence indicates that the formation and tyrosine phosphorylation of the cadherin-catenin complex are critical for the maintenance of the stabilization of cell-cell adhesion (6, 7). It has been well documented that cadherins are implicated in the regul...

Changes in the expression of glycosyltransferases that branch N-linked glycans can alter the function of several types of cell surface receptors and a glucose transporter. To study in detail the mechanisms by which aberrant N-glycosylation caused by altered N-acetylglucosaminyltransferase V(GnT-V, GnT-Va, and Mgat5a) expression can regulate the invasiveness-related phenotypes found in some carcinomas, we utilized specific small interfering RNA (siRNA) to selectively knock down GnT-V expression in the highly metastatic and invasive human breast carcinoma cell line, MDA-MB231. Knockdown of GnT-V by siRNA expression had no effect on epidermal growth factor receptor expression levels but lowered expression of N-linked ␤(1,6)-branching on epidermal growth factor receptor, as expected. Compared with control cells, knockdown of GnT-V caused significant inhibition of the morphological changes and cell detachment from matrix that is normally seen after stimulation with epidermal growth factor (EGF). Decreased expression of GnT-V caused a marked inhibition of EGF-induced dephosphorylation of focal adhesion kinase (FAK), consistent with the lack of cell morphology changes in the cells expressing GnT-V siRNA. The attenuation of EGF-mediated phosphorylation and activation of the tyrosine phosphatase SHP-2 was dramatically observed in GnT-V knockdown cells, and these effects could be rescued by reintroduction of GnT-V into these cells, indicating that reduced EGF-mediated activation of SHP-2 was GnT-V related. Concomitantly, knockdown of GnT-V caused reduced EGF-mediated ERK signaling and tumor cell invasiveness-related phenotypes, including effects on actin rearrangement and cell motility. No changes in EGF binding were observed, however, after knockdown of GnT-V. Our results demonstrate that decreased GnT-V activity due to siRNA expression in human breast carcinoma cells resulted in an inhibition of EGF-stimulated SHP-2 activation and, consequently, caused attenuation of the dephosphorylation of FAK induced by EGF. These effects suppressed EGF-mediated downstream signaling and invasiveness-related phenotypes and suggest GnT-V as a potential therapeutic target.The expression of specific glycan structures on several cell surface adhesion molecules, growth factor receptors, and the GLUT2 transporter is associated with an alteration of their functions (1-5). For example, studies have shown that aberrant N-glycosylation on several cell surface receptors, including integrins and cadherins, is associated with changes in carcinoma progression and metastasis (6 -11). A glycan whose expression is often up-regulated during malignant transformation contains the ␤(1,6)-linked N-acetylglucosamine found on some N-glycans (12), synthesized by N-acetylglucosaminyltransferase V (GnT-V 2 or Mgat5, also called GnT-Va; EC 2.4.1.155), a key enzyme in the processing of multiantennary N-glycans during glycoprotein biosynthesis whose expression is regulated by oncogene expression (13)(14)(15)(16)(17)(18). Studies have demonstrated the association o...

The human body contains approximately 640 individual skeletal muscles. Despite the fact that all of these muscles are composed of striated muscle tissue, the biology of these muscles and their associated muscle stem cell populations are quite diverse. Skeletal muscles are affected differentially by various muscular dystrophies (MDs), such that certain genetic mutations specifically alter muscle function in only a subset of muscles. Additionally, defective muscle stem cells have been implicated in the pathology of some MDs. The biology of muscle stem cells varies depending on the muscles with which they are associated. Here we review the biology of skeletal muscle stem cell populations of eight different muscle groups. Understanding the biological variation of skeletal muscles and their resident stem cells could provide valuable insight into mechanisms underlying the susceptibility of certain muscles to myopathic disease.

The expression of an enzyme, GnT-V, that catalyzes a specific posttranslational modification of a family of glycoproteins, namely a branched N-glycan, is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V, we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation, an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover, her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors, evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumorinitiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls, and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/ SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that, in turn, regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset. T he amplification and overexpression of her-2/erbB2, a member of the epidermal growth factor (EGF) receptor family, play a pivotal role in the development of several different types of cancers, including breast carcinoma (1, 2). Oncogenesis observed in mouse mammary epithelia induced by her-2 expression shares similarities with that of human breast carcinoma (3). Her-2 signaling is activated through its interactions with other EGF family receptors after they bind ligands, including epidermal growth factor (EGF) and neuregulin (NRG) (4). Ligand-induced phosphorylation of this family of receptors recruits various docking proteins and signaling molecules that convey proliferative and survival signals via MAPK and PI3K/PKB pathways (5). In a reconstituted basement membrane culture system (3D culture), activation or overexpression of the her-2 receptor in a nontransformed mammary epithelial cell line (MCF-10A) elicits a multiacinar phenotype characterized by excessive cell proliferation and filling of the acinar luminal space that results from inhibited apoptosis and altered apicobasal polarization (6, 7). These in vitro alterations caused by her-2 overexpression are linked to the phenotypes observed for human breast ductal carcinoma in situ (DCIS) with erbB2 amplification (8). Recent studies have shown that her-2 regulates the mammary epithelial stem/ progenitor cell population that drives tumorigenesis and progression (9, 10), and th...

The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging.