John L. Muschler

Molecular Recognition by LARGE Is Essential for Expression of Functional Dystroglycan

Reduced ligand binding activity of alpha-dystroglycan is associated with muscle and central nervous system pathogenesis in a growing number of muscular dystrophies. Posttranslational processing of alpha-dystroglycan is generally accepted to be critical for the expression of functional dystroglycan. Here we show that both the N-terminal domain and a portion of the mucin-like domain of alpha-dystroglycan are essential for high-affinity laminin-receptor function. Posttranslational modification of alpha-dystroglycan by glycosyltransferase, LARGE, occurs within the mucin-like domain, but the N-terminal domain interacts with LARGE, defining an intracellular enzyme-substrate recognition motif necessary to initiate functional glycosylation. Gene replacement in dystroglycan-deficient muscle demonstrates that the dystroglycan C-terminal domain is sufficient only for dystrophin-glycoprotein complex assembly, but to prevent muscle degeneration the expression of a functional dystroglycan through LARGE recognition and glycosylation is required. Therefore, molecular recognition of dystroglycan by LARGE is a key determinant in the biosynthetic pathway to produce mature and functional dystroglycan.

Cell–Matrix Interactions in Mammary Gland Development and Breast Cancer

The mammary gland is an organ that at once gives life to the young, but at the same time poses one of the greatest threats to the mother. Understanding how the tissue develops and functions is of pressing importance in determining how its control mechanisms break down in breast cancer. Here we argue that the interactions between mammary epithelial cells and their extracellular matrix (ECM) are crucial in the development and function of the tissue. Current strategies for treating breast cancer take advantage of our knowledge of the endocrine regulation of breast development, and the emerging role of stromal-epithelial interactions (Fig. 1). Focusing, in addition, on the microenvironmental influences that arise from cell-matrix interactions will open new opportunities for therapeutic intervention. We suggest that ultimately a three-pronged approach targeting endocrine, growth factor, and cell-matrix interactions will provide the best chance of curing the disease.

Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and β-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

Proteolytic Enzymes and Altered Glycosylation Modulate Dystroglycan Function in Carcinoma Cells

Alterations in the basement membrane receptor dystroglycan (DG) are evident in muscular dystrophies and carcinoma cells and characterized by a selective loss or modification of the extracellular α-DG subunit. Defects in posttranslational modifications of DG have been identified in some muscular dystrophies, but the underlying modifications in carcinoma cells have not yet been defined. We reveal here multiple posttranslational modifications that modulate the composition and function of DG in normal epithelial cells and carcinoma cells. We show that α-DG is shed from the cell surface of normal and tumorigenic epithelial cells through a proteolytic mechanism that does not require direct cleavage of either α- or β-DG. Shedding is dependent on metalloprotease activity and the proprotein convertase furin. Surprisingly, furin is also found to directly process α-DG as a proprotein substrate, changing the existing model of DG composition. We also show that the glycosylation of α-DG is altered in invasive carcinoma cells, and this modification causes complete loss of laminin binding properties. Together, these data elucidate several novel events regulating the functional composition of DG and reveal defects that arise during cancer progression, providing direction for efforts to restore this link with the basement membrane in carcinoma cells.

Dystroglycan loss disrupts polarity and β-casein induction in mammary epithelial cells by perturbing laminin anchoring

Precise contact between epithelial cells and their underlying basement membrane is crucial to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG gene (Dag1) expression in cultured mammary epithelial cells. Strikingly, DG loss disrupted laminin-111-induced polarity and β-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. Dystroglycan re-expression restored these deficiencies. Investigations of the mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in mammary epithelial cells that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity and β-casein induction. The observed loss of laminin-111 assembly and signaling in Dag1-/- mammary epithelial cells provides insights into the signaling changes occurring in breast carcinomas and other cancers, where the binding function of DG to laminin is frequently defective.

SEA domain proteolysis determines the functional composition of dystroglycan

Post‐translational modifications of the extracellular matrix receptor dystroglycan (DG) determine its functional state, and defects in these modifications are linked to muscular dystrophies and cancers. A prominent feature of DG biosynthesis is a precursor cleavage that segregates the ligand‐binding and transmembrane domains into the noncovalently attached α‐and β‐subunits. We investigate here the structural determinants and functional significance of this cleavage. We show that cleavage of DG elicits a conspicuous change in its ligand‐binding activity. Mutations that obstruct this cleavage result in increased capacity to bind laminin, in part, due to enhanced glycosylation of α‐DG. Reconstitution of DG cleavage in a cell‐free expression system demonstrates that cleavage takes place in the endoplasmic reticulum, providing a suitable regulatory point for later processing events. Sequence and mutational analyses reveal that the cleavage occurs within a full SEA (sea urchin, enterokinase, agrin) module with traits matching those ascribed to autoproteolysis. Thus, cleavage of DG constitutes a control point for the modulation of its ligand‐binding properties, with therapeutic implications for muscular dystrophies. We provide a structural model for the cleavage domain that is validated by experimental analysis and discuss this cleavage in the context of mucin protein and SEA domain evolution. Akhavan, A., Crivelli, S. N., Singh, M., Lingappa, V. R., Muschler, J. L. SEA domain proteolysis determines the functional composition of dystroglycan. FASEB J. 22, 612–621 (2008)

Loss of Cell-Surface Laminin Anchoring Promotes Tumor Growth and Is Associated with Poor Clinical Outcomes

Perturbations in the composition and assembly of extracellular matrices (ECM) contribute to progression of numerous diseases, including cancers. Anchoring of laminins at the cell surface enables assembly and signaling of many ECMs, but the possible contributions of altered laminin anchoring to cancer progression remain undetermined. In this study, we investigated the prominence and origins of defective laminin anchoring in cancer cells and its association with cancer subtypes and clinical outcomes. We found loss of laminin anchoring to be widespread in cancer cells. Perturbation of laminin anchoring originated from several distinct defects, which all led to dysfunctional glycosylation of the ECM receptor dystroglycan. In aggressive breast and brain cancers, defective laminin anchoring was often due to suppressed expression of the glycosyltransferase LARGE. Reduced expression of LARGE characterized a broad array of human tumors in which it was associated with aggressive cancer subtypes and poor clinical outcomes. Notably, this defect robustly predicted poor survival in patients with brain cancers. Restoring LARGE expression repaired anchoring of exogenous and endogenous laminin and modulated cell proliferation and tumor growth. Together, our findings suggest that defects in laminin anchoring occur commonly in cancer cells, are characteristic of aggressive cancer subtypes, and are important drivers of disease progression.

Nuclear translocation of β-dystroglycan reveals a distinctive trafficking pattern of autoproteolyzed mucins

Dystroglycan (DG) is an extracellular matrix receptor implicated in muscular dystrophies and cancers. DG belongs to the membrane‐tethered mucin family and is composed of extracellular (α‐DG) and transmembrane (β‐DG) subunits stably coupled at the cell surface. These two subunits are generated by autoproteolysis of a monomeric precursor within a distinctive protein motif called sea urchin–enterokinase–agrin (SEA) domain, yet the purpose of this cleavage and heterodimer creation is uncertain. In this study, we identify a functional nuclear localization signal within β‐DG and show that, in addition to associating with α‐DG at the cell surface, the full‐length and glycosylated β‐DG autonomously traffics to the cytoplasm and nucleoplasm in a process that occurs independent of α‐DG ligand binding. The trafficking pattern of β‐DG mirrors that of MUC1‐C, the transmembrane subunit of the related MUC1 oncoprotein, also a heterodimeric membrane‐tethered mucin created by SEA autoproteolysis. We show that the transmembrane subunits of both MUC1 and DG transit the secretory pathway prior to nuclear targeting and that their monomeric precursors maintain the capacity for nuclear trafficking. A screen of breast carcinoma cell lines of distinct pathophysiological origins revealed considerable variability in the nuclear partitioning of β‐DG, indicating that nuclear localization of β‐DG is regulated, albeit independent of extracellular ligand binding. These findings point to novel intracellular functions for β‐DG, with possible disease implications. They also reveal an evolutionarily conserved role for SEA autoproteolysis, serving to enable independent functions of mucin transmembrane subunits, enacted by a shared and poorly understood pathway of segregated subunit trafficking.

Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

Receptors for basement membrane (BM) proteins, including dystroglycan (DG), coordinate tissue development and function by mechanisms that are only partially defined. To further elucidate these mechanisms, we generated a conditional knockout of DG in the epithelial compartment of the mouse mammary gland. Deletion of DG caused an inhibition of mammary epithelial outgrowth and a failure of lactation. Surprisingly, loss of DG in vivo did not disrupt normal tissue architecture or BM formation, even though cultured Dag1-null epithelial cells failed to assemble laminin-111 at the cell surface. The absence of DG was, however, associated with a marked loss in activity of signal transducer and activator of transcription 5 (STAT5). Loss of DG perturbed STAT5 signaling induced by either prolactin or growth hormone. We found that DG regulates signaling by both hormones in a manner that is dependent on laminin-111 binding, but independent of the DG cytoplasmic domain, suggesting that it acts via a co-receptor mechanism reliant on DG-mediated laminin assembly. These results demonstrate a requirement for DG in the growth and function of a mammalian epithelial tissue in vivo. Moreover, we reveal a selective role for DG in the control of multiple STAT5-dependent hormone signaling pathways, with implications for numerous diseases in which DG function is compromised.

Dystroglycan: emerging roles in mammary gland function.

Dystroglycan (DG) is a single receptor that binds to multiple basement membrane proteins and forms a transmembrane link to the actin cytoskeleton. It was first isolated as a component of the dystrophin–glycoprotein complex, which plays a role in the maintenance of muscle cell integrity and is defective in many muscular dystrophies. Although studied most extensively in muscle tissues, DG is present at most cell–basement membrane interfaces, and only recently has investigation of DG functions in nonmuscle cells gained momentum. Information emerging from recent studies in epithelial cells is implicating DG in a wide range of critical cell responses to the basement membrane, ranging from organization of tissue architecture to cell survival. Moreover, DG functions appear to be frequently absent in carcinoma cells, implicating its loss in cancer progression. Although many questions remain as to its precise role in mammary tissue, DG is emerging as a potentially important player in mammary gland function.