Biomedical subjects

Kevin P Campbell

Publications and source records attributed to Kevin P Campbell.

At least 19 recordsLinked to original sources

PubMed2006

Dystroglycan loss disrupts polarity and beta-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 beta-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 beta-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.

Animals↗

PubMed2006

Interactions of intermediate filament protein synemin with dystrophin and utrophin.

Synemin is a unique, very large intermediate filament (IF) protein present in all types of muscle cells, which forms heteropolymeric intermediate filaments (IFs) with the major IF proteins desmin and/or vimentin. We show herein that tissue-purified avian synemin directly interacts with both dystrophin and utrophin, and that specific expressed regions of both of the mammalian (human) synemin isoforms (alpha-synemin and beta-synemin) directly interact with specific expressed domains/regions of the dystrophin and utrophin molecules. Mammalian synemin is also shown to colocalize with dystrophin within muscle cell cultures. These results indicate that synemin is an important IF protein in muscle cells that helps fortify the linkage between the peripheral layer of cellular myofibrils and the costameric regions located along the sarcolemma and the sarcolemma region located within the neuromuscular and myotendinous junctions (NMJs and MTJs).

Animals↗

PubMed2006

CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus.

Although T-type Ca(2+) channels in the thalamus play a crucial role in determining neuronal excitability and are involved in sensory processing and pathophysiology of epilepsy, little is known about the molecular mechanisms involved in their regulation. Here, we report that reducing agents, including endogenous sulfur-containing amino acid l-cysteine, selectively enhance native T-type currents in reticular thalamic (nRT) neurons and recombinant Ca(V)3.2 (alpha1H) currents, but not native and recombinant Ca(V)3.1 (alpha1G)- and Ca(V)3.3 (alpha1I)-based currents. Consistent with this data, T-type currents of nRT neurons from transgenic mice lacking Ca(V)3.2 channel expression were not modulated by reducing agents. In contrast, oxidizing agents inhibited all native and recombinant T-type currents non-selectively. Thus, our findings directly demonstrate that Ca(V)3.2 channels are the main molecular substrate for redox regulation of neuronal T-type channels. In addition, because thalamic T-type channels generate low-threshold Ca(2+) spikes that directly correlate with burst firing in these neurons, differential redox regulation of these channels may have an important function in controlling cellular excitability in physiological and pathological conditions and fine-tuning of the flow of sensory information into the central nervous system.

Animals↗

PubMed2006

A functional AMPA receptor-calcium channel complex in the postsynaptic membrane.

Ca(2+) channels play critical roles in the regulation of synaptic activity. In contrast to the well established function of voltage-activated Ca(2+) channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the Ca(2+) channels in the postsynaptic membrane. In this study, we demonstrated the functional association of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal Ca(2+) channels. A series of biochemical studies showed the specific association of Ca(v)2.1 (alpha(1A)-class) and Ca(v)2.2 (alpha(1B)-class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and Ca(2+) imaging analyses of recombinant Ca(v)2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of Ca(2+) ([Ca(2+)](i)) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of Ca(2+) channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity.

Animals↗

PubMed2006

Dystroglycan: from biosynthesis to pathogenesis of human disease.

Alpha- and beta-dystroglycan constitute a membrane-spanning complex that connects the extracellular matrix to the cytoskeleton. Although a structural role for dystroglycan had been identified, biochemical and genetic discoveries have recently highlighted the significance of posttranslational processing for dystroglycan function. Glycosylation is the crucial modification that modulates the function of dystroglycan as a receptor for extracellular binding partners. It has become clear that perturbation of dystroglycan glycosylation is the central event in the pathogenesis of several complex disorders, and recent advances suggest that glycosylation could be modulated to ameliorate the pathological features. Our increased understanding of the mechanisms of interaction of dystroglycan with its ligands has become an essential tool in deciphering the biological processes related to the human diseases in which the proteins are implicated.

Animals↗

PubMed2006

Fukutin gene mutations cause dilated cardiomyopathy with minimal muscle weakness.

OBJECTIVE: The fukutin gene (FKTN) is the causative gene for Fukuyama-type congenital muscular dystrophy, characterized by rather homogeneous clinical features of severe muscle wasting and hypotonia from early infancy with mental retardation. In contrast with the severe dystrophic involvement of skeletal muscle, cardiac insufficiency is quite rare. Fukuyama-type congenital muscular dystrophy is one of the disorders associated with glycosylation defects of alpha-dystroglycan, an indispensable molecule for intra-extra cell membrane linkage. METHODS: Protein and functional analyses of alpha-dystroglycan and mutation screening of FKTN and other associated genes were performed. RESULTS: Surprisingly, we identified six patients in four families showing dilated cardiomyopathy with no or minimal limb girdle muscle involvement and normal intelligence, associated with a compound heterozygous FKTN mutation. One patient died by rapid progressive dilated cardiomyopathy at 12 years old, and the other patient received cardiac implantation at 18 years old. Skeletal muscles from the patients showed minimal dystrophic features but have altered glycosylation of alpha-dystroglycan and reduced laminin binding ability. One cardiac muscle that underwent biopsy showed altered glycosylation of alpha-dystroglycan similar to that observed in a Fukuyama-type congenital muscular dystrophy patient. INTERPRETATION: FKTN mutations could cause much wider spectrum of clinical features than previously perceived, including familial dilated cardiomyopathy and mildest limb girdle muscular dystrophy.

Adult↗

PubMed2006

Limb-girdle muscular dystrophy in the United States.

Limb-girdle muscular dystrophy (LGMD) has been linked to 15 chromosomal loci, 7 autosomal-dominant (LGMD1A to E) and 10 autosomal-recessive (LGMD2A to J). To determine the distribution of subtypes among patients in the United States, 6 medical centers evaluated patients with a referral diagnosis of LGMD. Muscle biopsies provided histopathology and immunodiagnostic testing, and their protein abnormalities along with clinical parameters directed mutation screening. The diagnosis in 23 patients was a disorder other than LGMD. Of the remaining 289 unrelated patients, 266 had muscle biopsies sufficient for complete microscopic evaluation; 121 also underwent Western blotting. From this combined evaluation, the distribution of immunophenotypes is 12% calpainopathy, 18% dysferlinopathy, 15% sarcoglycanopathy, 15% dystroglycanopathy, and 1.5% caveolinopathy. Genotypes distributed among 2 dominant and 7 recessive subtypes have been determined for 83 patients. This study of a large racially and ethnically diverse population of patients with LGMD indicates that establishing a putative subtype is possible more than half the time using available diagnostic testing. An efficient approach to genotypic diagnosis is muscle biopsy immunophenotyping followed by directed mutational analysis. The most common LGMDs in the United States are calpainopathies, dysferlinopathies, sarcoglycanopathies, and dystroglycanopathies.

Adolescent↗

PubMed2005

Both laminin and Schwann cell dystroglycan are necessary for proper clustering of sodium channels at nodes of Ranvier.

Nodes of Ranvier are specialized axonal domains, at which voltage-gated sodium channels cluster. How axons cluster molecules in discrete domains is mostly unknown. Both axons and glia probably provide constraining mechanisms that contribute to domain formation. Proper sodium channel clustering in peripheral nerves depends on contact from Schwann cell microvilli, where at least one molecule, gliomedin, binds the sodium channel complex and induces its clustering. Furthermore, mice lacking Schwann cell dystroglycan have aberrant microvilli and poorly clustered sodium channels. Dystroglycan could interact at the basal lamina or at the axonglial surface. Because dystroglycan is a laminin receptor, and laminin 2 mutations [merosin-deficient congenital muscular dystrophy (MDC1A)] cause reduced nerve conduction velocity, we asked whether laminins are involved. Here, we show that the composition of both laminins and the dystroglycan complex at nodes differs from that of internodes. Mice defective in laminin 2 have poorly formed microvilli and abnormal sodium clusters. These abnormalities are similar, albeit less severe, than those of mice lacking dystroglycan. However, mice lacking all Schwann cell laminins show severe nodal abnormalities, suggesting that other laminins compensate for the lack of laminin 2. Thus, although laminins are located at a distance from the axoglial junction, they are required for proper clustering of sodium channels. Laminins, through their specific nodal receptors and cytoskeletal linkages, may participate in the formation of mechanisms that constrain clusters at nodes. Finally, abnormal sodium channel clusters are present in a patient with MDC1A, providing a molecular basis for the reduced nerve conduction velocity in this disorder.

Animals↗

PubMed2005

Centronuclear myopathy in mice lacking a novel muscle-specific protein kinase transcriptionally regulated by MEF2.

Myocyte enhancer factor 2 (MEF2) plays essential roles in transcriptional control of muscle development. However, signaling pathways acting downstream of MEF2 are largely unknown. Here, we performed a microarray analysis using Mef2c-null mouse embryos and identified a novel MEF2-regulated gene encoding a muscle-specific protein kinase, Srpk3, belonging to the serine arginine protein kinase (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins. The Srpk3 gene is specifically expressed in the heart and skeletal muscle from embryogenesis to adulthood and is controlled by a muscle-specific enhancer directly regulated by MEF2. Srpk3-null mice display a new entity of type 2 fiber-specific myopathy with a marked increase in centrally placed nuclei; while transgenic mice overexpressing Srpk3 in skeletal muscle show severe myofiber degeneration and early lethality. We conclude that normal muscle growth and homeostasis require MEF2-dependent signaling by Srpk3.

Amino Acid Sequence↗

PubMed2005

Disruption of perlecan binding and matrix assembly by post-translational or genetic disruption of dystroglycan function.

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. We report reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large(myd) mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystroglycan, laminin and perlecan. These data suggest functional disruption of the trimolecular complex in glycosylation-deficient muscular dystrophy.

Animals↗

PubMed2005

Localization of alpha-dystroglycan on the podocyte: from top to toe.

alpha-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that alpha-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against alpha-DG. Therefore, we studied the exact localization of glomerular alpha-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of alpha-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that alpha-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that alpha-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.

Animals↗

PubMed2005

Muscles of mice deficient in alpha-sarcoglycan maintain large masses and near control force values throughout the life span.

alpha-Sarcoglycan-deficient (Sgca-null) mice provide potential for elucidating the pathogenesis of limb girdle muscular dystrophy type 2D (LGMD 2D) as well as for studying the effectiveness of therapeutic strategies. Skeletal muscles of Sgca-null mice demonstrate an early onset of extensive fiber necrosis, degeneration, and regeneration, but the progression of the pathology and the effects on muscle structure and function throughout the life span are not known. Thus the phenotypic accuracy of the Sgca-null mouse as a model of LGMD 2D has not been fully established. To investigate skeletal muscle structure and function in the absence of alpha-sarcoglycan throughout the life span, we analyzed extensor digitorum longus and soleus muscles of male and female Sgca-null and wild-type mice at 3, 6, 12, and 18 mo of age. Maximum isometric forces and powers were measured in vitro at 25 degrees C. Also determined were individual myofiber cross-sectional areas and numbers, water content, and the proportion of the cross section occupied by connective tissue. Muscle masses were 40-100% larger for Sgca-null compared with age- and gender-matched wild-type mice, with the majority of the increased muscle mass for Sgca-null mice attributable to greater connective tissue and water contents. Although the greater mass of muscles in Sgca-null mice was primarily noncontractile material, absolute forces and powers were maintained near control levels at all ages, indicating a successful adaptation to the deficiency in alpha-sarcoglycan not observed at any age in LGMD 2D patients.

Animals↗

PubMed2005

Aberrant glycosylation of alpha-dystroglycan causes defective binding of laminin in the muscle of chicken muscular dystrophy.

Dystroglycan is a central component of dystrophin-glycoprotein complex that links extracellular matrix and cytoskeleton in skeletal muscle. Although dystrophic chicken is well established as an animal model of human muscular dystrophy, the pathomechanism leading to muscular degeneration remains unknown. We show here that glycosylation and laminin-binding activity of alpha-dystroglycan (alpha-DG) are defective in dystrophic chicken. Extensive glycan structural analysis reveals that Galbeta1-3GalNAc and GalNAc residues are increased while Siaalpha2-3Gal structure is reduced in alpha-DG of dystrophic chicken. These results implicate aberrant glycosylation of alpha-DG in the pathogenesis of muscular degeneration in this model animal of muscular dystrophy.

Animals↗

PubMed2005

Congenital muscular dystrophy with glycosylation defects of alpha-dystroglycan in Japan.

Glycosylation defects of alpha-dystroglycan (alpha-DG) cause various muscular dystrophies. We performed clinical, pathological and genetic analyses of 62 Japanese patients with congenital muscular dystrophy, whose skeletal muscle showed deficiency of glycosylated form of alpha-DG. We found, the first Japanese patient with congenital muscular dystrophy 1C with a novel compound heterozygous mutation in the fukutin-related protein gene. Fukuyama-type congenital muscular dystrophy was genetically confirmed in 54 of 62 patients. Two patients with muscle-eye-brain disease and one Walker-Warburg syndrome were also genetically confirmed. Four patients had no mutation in any known genes associated with glycosylation of alpha-DG. Interestingly, the molecular mass of alpha-DG in the skeletal muscle was similar and was reduced to approximately 90 kDa among these patients, even though the causative gene and the clinico-pathological severity were different. This result suggests that other factors can modify clinical features of the patients with glycosylation defects of alpha-DG.

Blotting, Western↗

PubMed2005

Posttranslational modification of alpha-dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding.

The receptor for lymphocytic choriomeningitis virus (LCMV), the human pathogenic Lassa fever virus (LFV), and clade C New World arenaviruses is alpha-dystroglycan (alpha-DG), a cell surface receptor for proteins of the extracellular matrix (ECM). Specific posttranslational modification of alpha-DG by the glycosyltransferase LARGE is critical for its function as an ECM receptor. In the present study, we show that LARGE-dependent modification is also crucial for alpha-DG's function as a cellular receptor for arenaviruses. Virus binding involves the mucin-type domain of alpha-DG and depends on modification by LARGE. A crucial role of the LARGE-dependent glycosylation of alpha-DG for virus binding is found for several isolates of LCMV, LFV, and the arenaviruses Mobala and Oliveros. Since the posttranslational modification by LARGE is crucial for alpha-DG recognition by both arenaviruses and the host-derived ligand laminin, it also influences competition between virus and laminin for alpha-DG. Hence, LARGE-dependent glycosylation of alpha-DG has important implications for the virus-host cell interaction and the pathogenesis of LFV in humans.

Animals↗

PubMed2004

Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of beta1 integrin-null cells.

The presence of many laminin receptors of the beta1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin alpha6beta4 and dystroglycan. We therefore tested the binding of a beta1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin alpha6Abeta4A variant. GD25 beta1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin alpha6 antibody, but not by a dystroglycan antibody. Hence, integrin alpha6Abeta4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin alpha6Abeta4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin alpha6Abeta4A.

Animals↗

PubMed2004

Cell stiffness and receptors: evidence for cytoskeletal subnetworks.

Viscoelastic models of cells often treat cells as homogeneous objects. However, studies have demonstrated that cellular properties are local and can change dramatically on the basis of the location probed. Because membrane receptors are linked in various ways to the intracellular space, with some receptors linking to the cytoskeleton and others diffusing freely without apparent linkages, the cellular physical response to mechanical stresses is expected to depend on the receptor engaged. In this study, we tested the hypothesis that cellular mechanical stiffness as measured via cytoskeletally linked receptors is greater than stiffness measured via receptors that are not cytoskeletally linked. We used a magnetic micromanipulator to apply linear stresses to magnetic beads attached to living cells via selected receptors. One of the receptor classes probed, the dystroglycan receptors, is linked to the cytoskeleton, while the other, the transferrin receptors, is not. Fibronectin-coated beads were used to test cellular mechanical properties of the cytoskeleton without membrane dependence by allowing the beads to endocytose. For epithelial cells, transferrin-dependent stiffness and endocytosed bead-dependent stiffness were similar, while dystroglycan-dependent stiffness was significantly lower. For smooth muscle cells, dystroglycan-dependent stiffness was similar to the endocytosed bead-dependent stiffness, while the transferrin-dependent stiffness was lower. The conclusion of this study is that the measured cellular stiffness is critically influenced by specific receptor linkage and by cell type and raises the intriguing possibility of the existence of separate cytoskeletal networks with distinct mechanical properties that link different classes of receptors.

Cell Line↗

PubMed2004

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 alpha-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 alpha-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 alpha- or beta-DG. Shedding is dependent on metalloprotease activity and the proprotein convertase furin. Surprisingly, furin is also found to directly process alpha-DG as a proprotein substrate, changing the existing model of DG composition. We also show that the glycosylation of alpha-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.

Amino Acid Sequence↗