Publications by authors named "Karen K McKee"

20 Publications

  • Page 1 of 1

A deletion in the N-terminal polymerizing domain of laminin β2 is a new mouse model of chronic nephrotic syndrome.

Kidney Int 2020 07 20;98(1):133-146. Epub 2020 Feb 20.

Department of Internal Medicine, Division of Nephrology, Washington University, St. Louis, Missouri, USA. Electronic address:

The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin β2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2 mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.
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http://dx.doi.org/10.1016/j.kint.2020.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311232PMC
July 2020

A mutation affecting laminin alpha 5 polymerisation gives rise to a syndromic developmental disorder.

Development 2020 06 22;147(21). Epub 2020 Jun 22.

Department of Anatomy and Developmental Biology, Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Melbourne 3800, Australia

Laminin alpha 5 (LAMA5) is a member of a large family of proteins that trimerise and then polymerise to form a central component of all basement membranes. Consequently, the protein plays an instrumental role in shaping the normal development of the kidney, skin, neural tube, lung and limb, and many other organs and tissues. Pathogenic mutations in some laminins have been shown to cause a range of largely syndromic conditions affecting the competency of the basement membranes to which they contribute. We report the identification of a mutation in the polymerisation domain of LAMA5 in a patient with a complex syndromic disease characterised by defects in kidney, craniofacial and limb development, and by a range of other congenital defects. Using CRISPR-generated mouse models and biochemical assays, we demonstrate the pathogenicity of this variant, showing that the change results in a failure of the polymerisation of α/β/γ laminin trimers. Comparing these phenotypes with those apparent upon gene deletion in mice provides insights into the specific functional importance of laminin polymerisation during development and tissue homeostasis.
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http://dx.doi.org/10.1242/dev.189183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540250PMC
June 2020

Linker Protein Repair of LAMA2 Dystrophic Neuromuscular Basement Membranes.

Front Mol Neurosci 2019 13;12:305. Epub 2019 Dec 13.

Department of Pathology and Laboratory Medicine, Rutgers Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, United States.

An understanding of basement membrane (BM) assembly at a molecular level provides a foundation with which to develop repair strategies for diseases with defects of BM structure. As currently understood, laminins become anchored to cell surfaces through receptor-mediated interactions and polymerize. This provisional matrix binds to proteoglycans, nidogens and type IV collagen to form a mature BM. Identification of BM binding domains and their binding targets has enabled investigators to engineer proteins that link BM components to modify and improve their functions. This approach is illustrated by the development of two linker proteins to repair the LAMA2-deficient muscular dystrophy (LAMA2-MD). Dystrophy-causing mutations of the gene product (Lmα2) disrupt the BM molecular architecture, destabilizing it. In a mild ambulatory type of the dystrophy, α2LN mutations in laminin-211 prevents polymerization. In the more common and severe non-ambulatory type (MDC1A), an absent Lmα2 subunit is replaced by the naturally occurring Lmα4 subunit that is normally largely confined to the microvasculature. The compensatory laminin, however, is a poor substitute because it neither polymerizes nor binds adequately to the anchoring receptor α-dystroglycan. A chimeric laminin-binding protein called αLNNd enables laminins with defective or absent αLN domains to polymerize while another engineered protein, miniagrin (), promotes efficient α-dystroglycan receptor-binding in otherwise weakly adhesive laminins. Alone, αLNNd enables Lm211 with a self-assembly defect to polymerize and was used to ameliorate a mouse model of the ambulatory dystrophy. Together, these linker proteins alter Lm411 such that it both polymerizes and binds αDG such that it properly assembles. This combination was used to ameliorate a mouse model of the non-ambulatory dystrophy in which Lm411 replaced Lm211 as seen in the human disease. Collectively, these studies pave the way for the development of somatic gene delivery of repair proteins for treatment of LAMA2-MD. The studies further suggest a more general approach of linker-protein mediated repair in which a variety of existing BM protein domains can be combined together to stabilize BMs in other diseases.
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http://dx.doi.org/10.3389/fnmol.2019.00305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923227PMC
December 2019

Chimeric protein identification of dystrophic, Pierson and other laminin polymerization residues.

Matrix Biol 2018 04 3;67:32-46. Epub 2018 Mar 3.

Department of Pathology and Laboratory Medicine, Rutgers University - Robert Wood Johnson Medical School, Piscataway, NJ 08854, United States. Electronic address:

Laminin polymerization is a key step of basement membrane self-assembly that depends on the binding of the three different N-terminal globular LN domains. Several mutations in the LN domains cause LAMA2-deficient muscular dystrophy and LAMB2-deficient Pierson syndrome. These mutations may affect polymerization. A novel approach to identify the amino acid residues required for polymerization has been applied to an analysis of these and other laminin LN mutations. The approach utilizes laminin-nidogen chimeric fusion proteins that bind to recombinant non-polymerizing laminins to provide a missing functional LN domain. Single amino acid substitutions introduced into these chimeras were tested to determine if polymerization activity and the ability to assemble on cell surfaces were lost. Several laminin-deficient muscular dystrophy mutations, renal Pierson syndrome mutations, and Drosophila mutations causing defects of heart development were identified as ones causing loss of laminin polymerization. In addition, two novel residues required for polymerization were identified in the laminin γ1 LN domain.
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http://dx.doi.org/10.1016/j.matbio.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910262PMC
April 2018

Pathogenicity of a Human Laminin 2 Mutation Revealed in Models of Alport Syndrome.

J Am Soc Nephrol 2018 03 20;29(3):949-960. Epub 2017 Dec 20.

Division of Nephrology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri and

Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin 2 (), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a mouse model of autosomal recessive Alport syndrome and increased proteinuria in females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen 345(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.
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http://dx.doi.org/10.1681/ASN.2017090997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827610PMC
March 2018

Laminin-deficient muscular dystrophy: Molecular pathogenesis and structural repair strategies.

Matrix Biol 2018 10 27;71-72:174-187. Epub 2017 Nov 27.

Biozentrum, University of Basel, 4056 Basel, Switzerland. Electronic address:

Laminins are large heterotrimers composed of the α, β and γ subunits with distinct tissue-specific and developmentally regulated expression patterns. The laminin-α2 subunit, encoded by the LAMA2 gene, is expressed in skeletal muscle, Schwann cells of the peripheral nerve and astrocytes and pericytes of the capillaries in the brain. Mutations in LAMA2 cause the most common type of congenital muscular dystrophies, called LAMA2 MD or MDC1A. The disorder manifests mostly as a muscular dystrophy but slowing of nerve conduction contributes to the disease. There are severe, non-ambulatory or milder, ambulatory variants, the latter resulting from reduced laminin-α2 expression and/or deficient laminin-α2 function. Lm-211 (α2β1γ1) is responsible for initiating basement membrane assembly. This is primarily accomplished by anchorage of Lm-211 to dystroglycan and α7β1 integrin receptors, polymerization, and binding to nidogen and other structural components. In LAMA2 MD, Lm-411 replaces Lm-211; however, Lm-411 lacks the ability to polymerize and bind to receptors. This results in a weakened basement membrane leading to the disease. The possibility of introducing structural repair proteins that correct the underlying abnormality is an attractive therapeutic goal. Recent studies in mouse models for LAMA2 MD reveal that introduction of laminin-binding linker proteins that restore lost functional activities can substantially ameliorate the disease. This review discusses the underlying mechanism of this repair and compares this approach to other developing therapies employing pharmacological treatments.
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http://dx.doi.org/10.1016/j.matbio.2017.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971131PMC
October 2018

Linker proteins restore basement membrane and correct -related muscular dystrophy in mice.

Sci Transl Med 2017 06;9(396)

Biozentrum, University of Basel, 4056 Basel, Switzerland.

-related muscular dystrophy ( MD or MDC1A) is the most frequent form of early-onset, fatal congenital muscular dystrophies. It is caused by mutations in , the gene encoding laminin-α2, the long arm of the heterotrimeric (α2, β1, and γ1) basement membrane protein laminin-211 (Lm-211). We establish that despite compensatory expression of laminin-α4, giving rise to Lm-411 (α4, β1, and γ1), muscle basement membrane is labile in MD biopsies. Consistent with this deficit, recombinant Lm-411 polymerized and bound to cultured myotubes only weakly. Polymerization and cell binding of Lm-411 were enhanced by addition of two specifically designed linker proteins. One, called αLNNd, consists of the N-terminal part of laminin-α1 and the laminin-binding site of nidogen-1. The second, called mini-agrin (mag), contains binding sites for laminins and α-dystroglycan. Transgenic expression of mag and αLNNd in a mouse model for MD fully restored basement membrane stability, recovered muscle force and size, increased overall body weight, and extended life span more than five times to a maximum survival beyond 2 years. These findings provide a mechanistic understanding of MD and establish a strong basis for a potential treatment.
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http://dx.doi.org/10.1126/scitranslmed.aal4649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744687PMC
June 2017

Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype.

J Clin Invest 2017 Mar 20;127(3):1075-1089. Epub 2017 Feb 20.

Mutations in laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.
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http://dx.doi.org/10.1172/JCI90854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330723PMC
March 2017

Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion.

mBio 2017 01 10;8(1). Epub 2017 Jan 10.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA

To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2β1γ1/laminin-α2β2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens.

Importance: Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin α2 subunit. This finding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs.
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http://dx.doi.org/10.1128/mBio.02128-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225314PMC
January 2017

Integrin alpha6 maintains the structural integrity of the kidney collecting system.

Matrix Biol 2017 01 30;57-58:244-257. Epub 2016 Dec 30.

Division of Nephrology and Hypertension and Vanderbilt Center for Kidney Disease, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Veterans Affairs Hospital, Nashville, TN 37232, USA. Electronic address:

Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and β subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3β1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6β1 and α6β4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or β4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.
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http://dx.doi.org/10.1016/j.matbio.2016.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330664PMC
January 2017

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes.

Nat Commun 2016 11 30;7:13515. Epub 2016 Nov 30.

Institute for Dental Research and Oral Musculoskeletal Biology, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, Cologne 50931, Germany.

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.
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http://dx.doi.org/10.1038/ncomms13515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141367PMC
November 2016

Integrin α3β1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt.

Mol Biol Cell 2015 May 25;26(10):1857-74. Epub 2015 Mar 25.

Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 Veterans Affairs Hospital, Nashville, TN 37232

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin α3β1 is crucial for this developmental program; however, the LM types and LM/integrin α3β1-dependent signaling pathways are poorly defined. We show that α3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3β1-dependent collecting duct cell functions. We demonstrate that integrin α3β1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin β1 subunit and regulator of integrin α3β1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3β1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways.
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http://dx.doi.org/10.1091/mbc.E14-07-1203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436831PMC
May 2015

Perlecan is recruited by dystroglycan to nodes of Ranvier and binds the clustering molecule gliomedin.

J Cell Biol 2015 Feb;208(3):313-29

Division of Genetics and Cell Biology, San Raffaele Hospital, 20132 Milan, Italy Department of Biochemistry and Department of Neurology, Hunter James Kelly Research Institute, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203 Department of Biochemistry and Department of Neurology, Hunter James Kelly Research Institute, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203

Fast neural conduction requires accumulation of Na(+) channels at nodes of Ranvier. Dedicated adhesion molecules on myelinating cells and axons govern node organization. Among those, specific laminins and dystroglycan complexes contribute to Na(+) channel clustering at peripheral nodes by unknown mechanisms. We show that in addition to facing the basal lamina, dystroglycan is found near the nodal matrix around axons, binds matrix components, and participates in initial events of nodogenesis. We identify the dystroglycan-ligand perlecan as a novel nodal component and show that dystroglycan is required for the selective accumulation of perlecan at nodes. Perlecan binds the clustering molecule gliomedin and enhances clustering of node of Ranvier components. These data show that proteoglycans have specific roles in peripheral nodes and indicate that peripheral and central axons use similar strategies but different molecules to form nodes of Ranvier. Further, our data indicate that dystroglycan binds free matrix that is not organized in a basal lamina.
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http://dx.doi.org/10.1083/jcb.201403111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315246PMC
February 2015

α6β1 and α7β1 integrins are required in Schwann cells to sort axons.

J Neurosci 2013 Nov;33(46):17995-8007

San Raffaele Scientific Institute, Milano 20132, Italy, Hunter James Kelly Research Institute, University at Buffalo, State University of New York, New York 14203, Development and Stem Cells Program, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, Institut National de la Santé et de la Recherche Médicale U964, Université de Strasbourg, Illkirch 67404, France, Department of Medicine, Children's Hospital Boston and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, and Robert Wood Johnson Medical School, Piscataway, New Jersey, New Jersey 08854.

During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the β1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with β1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding β1 integrins in Schwann cells and show that only α6β1 and α7β1 integrins are required and that α7β1 compensates for the absence of α6β1 during development. The absence of either α7β1 or α6β1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all β1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6β1 and α7β1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell β1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.
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http://dx.doi.org/10.1523/JNEUROSCI.3179-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893357PMC
November 2013

Schwann cell myelination requires integration of laminin activities.

J Cell Sci 2012 Oct 5;125(Pt 19):4609-19. Epub 2012 Jul 5.

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of β1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for laminin γ1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to α6β1 and/or α7β1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional α6β1/α7β1-laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination.
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http://dx.doi.org/10.1242/jcs.107995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500866PMC
October 2012

Renal collecting system growth and function depend upon embryonic γ1 laminin expression.

Development 2011 Oct 8;138(20):4535-44. Epub 2011 Sep 8.

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of α6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and β1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of γ1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.
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http://dx.doi.org/10.1242/dev.071266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177319PMC
October 2011

Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

J Biol Chem 2009 Mar 2;284(13):8984-94. Epub 2009 Feb 2.

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states.
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http://dx.doi.org/10.1074/jbc.M809719200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659255PMC
March 2009

Role of laminin terminal globular domains in basement membrane assembly.

J Biol Chem 2007 Jul 21;282(29):21437-47. Epub 2007 May 21.

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin.
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http://dx.doi.org/10.1074/jbc.M702963200DOI Listing
July 2007

Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering.

J Biol Chem 2005 Dec 11;280(50):41449-57. Epub 2005 Oct 11.

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.
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http://dx.doi.org/10.1074/jbc.M508939200DOI Listing
December 2005

Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.

J Cell Biol 2005 Apr;169(1):179-89

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.
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http://dx.doi.org/10.1083/jcb.200501098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171891PMC
April 2005