The sodium channel ?1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis.
ABSTRACT: Voltage-gated Na(+) channels (VGSCs) are heteromeric proteins composed of pore-forming ? subunits and smaller ? subunits. The ? subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). ?1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. ?1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/?1 protein were up-regulated in BCa specimens, compared with normal breast tissue. ?1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. ?1 over-expression also increased vascularization and reduced apoptosis in the primary tumours, and ?1 over-expressing tumour cells had an elongate morphology. In vitro, ?1 potentiated outgrowth of processes from BCa cells co-cultured with fibroblasts, via trans-homophilic adhesion. ?1-mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na(+) current, thus replicating the mechanism by which ?1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, ?1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for ?1 in tumour growth and metastasis in vivo. We propose that ?1 warrants further study as a potential biomarker and targeting ?1-mediated adhesion interactions may have value as a novel anti-cancer therapy.
Project description:Voltage-gated Na(+) channel (VGSC) beta1 subunits regulate cell-cell adhesion and channel activity in vitro. We previously showed that beta1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that beta1-mediated neurite outgrowth requires Na(+) current (I(Na)) mediated by Na(v)1.6. In addition, beta1 is required for high-frequency action potential firing. Transient I(Na) is unchanged in Scn1b (beta1) null CGNs; however, the resurgent I(Na), thought to underlie high-frequency firing in Na(v)1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Na(v)1.6 is reduced in Scn1b null cerebellar neurons. In place of Na(v)1.6 at the AIS, we observed an increase in Na(v)1.1, whereas Na(v)1.2 was unchanged. This indicates that beta1 is required for normal localization of Na(v)1.6 at the AIS during the postnatal developmental switch to Na(v)1.6-mediated high-frequency firing. In agreement with this, beta1 is normally expressed with alpha subunits at the AIS of P14 CGNs. We propose reciprocity of function between beta1 and Na(v)1.6 such that beta1-mediated neurite outgrowth requires Na(v)1.6-mediated I(Na), and Na(v)1.6 localization and consequent high-frequency firing require beta1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.
Project description:Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming ? subunits together with non-pore-forming ? subunits. There are nine ? subunits, Nav1.1-Nav1.9, and four ? subunits, ?1-?4. The ? subunits are multifunctional, modulating channel activity, cell surface expression, and are members of the immunoglobulin superfamily of cell adhesion molecules. VGSCs are classically responsible for action potential initiation and conduction in electrically excitable cells, including neurons and muscle cells. In addition, through the ?1 subunit, VGSCs regulate neurite outgrowth and pathfinding in the developing central nervous system. Reciprocal signalling through Nav1.6 and ?1 collectively regulates Na(+) current, electrical excitability and neurite outgrowth in cerebellar granule neurons. Thus, ? and ? subunits may have diverse interacting roles dependent on cell/tissue type. VGSCs are also expressed in non-excitable cells, including cells derived from a number of types of cancer. In cancer cells, VGSC ? and ? subunits regulate cellular morphology, migration, invasion and metastasis. VGSC expression associates with poor prognosis in several studies. It is hypothesised that VGSCs are up-regulated in metastatic tumours, favouring an invasive phenotype. Thus, VGSCs may have utility as prognostic markers, and/or as novel therapeutic targets for reducing/preventing metastatic disease burden. VGSCs appear to regulate a number of key cellular processes, both during normal postnatal development of the CNS and during cancer metastasis, by a combination of conducting (i.e. via Na(+) current) and non-conducting mechanisms.
Project description:Voltage-gated sodium channels are protein complexes comprised of one pore forming ? subunit and two, non-pore forming, ? subunits. The voltage-gated sodium channel ? subunits were originally identified to function as auxiliary subunits, which modulate the gating, kinetics, and localization of the ion channel pore. Since that time, the five ? subunits have been shown to play crucial roles as multifunctional signaling molecules involved in cell adhesion, cell migration, neuronal pathfinding, fasciculation, and neurite outgrowth. Here, we provide an overview of the evidence implicating the ? subunits in their conducting and non-conducting roles. Mutations in the ? subunit genes (SCN1B-SCN4B) have been linked to a variety of diseases. These include cancer, epilepsy, cardiac arrhythmias, sudden infant death syndrome/sudden unexpected death in epilepsy, neuropathic pain, and multiple neurodegenerative disorders. ? subunits thus provide novel therapeutic targets for future drug discovery.
Project description:SALMs are a family of five adhesion molecules whose expression is largely restricted to the CNS. Initial reports showed that SALM1 functions in neurite outgrowth while SALM2 is involved in synapse formation. To investigate the function of SALMs in detail, we asked if all five are involved in neurite outgrowth. Expression of epitope-tagged proteins in cultured hippocampal neurons showed that SALMs are distributed throughout neurons, including axons, dendrites, and growth cones. Over-expression of each SALM resulted in enhanced neurite outgrowth, but with different phenotypes. Neurite outgrowth could be reduced by applying antibodies targeting the extracellular leucine rich regions of SALMs and with RNAi. Through over-expression of deletion constructs, we found that the C-terminal PDZ binding domains of SALMs 1-3 are required for most aspects of neurite outgrowth. In addition, by using a chimera of SALMs 2 and 4, we found that the N-terminus is also involved in neurite outgrowth.
Project description:Modifier of cell adhesion (MOCA) is a member of the dedicator of cytokinesis 180 family of proteins and is highly expressed in CNS neurons. MOCA is associated with Alzheimer's disease tangles and regulates the accumulation of amyloid precursor protein and beta-amyloid. Here, we report that MOCA modulates cell-cell adhesion and morphology. MOCA increases the accumulation of adherens junction proteins, including N-cadherin and beta-catenin, whereas reducing endogenous MOCA expression lowers cell-cell aggregation and N-cadherin expression. MOCA colocalizes with N-cadherin and actin in areas of cell-cell and cell substratum contact and is expressed in neuronal processes. MOCA accumulates during neuronal differentiation, and its expression enhances NGF-induced neurite outgrowth and morphological complexity. We conclude that MOCA regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth.
Project description:Voltage-gated Na+ channel beta1 (Scn1b) subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that beta1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel alpha subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of beta1 subunits in vivo we cloned two beta1-like subunit cDNAs from D. rerio.Two beta1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian beta1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian beta1 subunits. In contrast to mammalian beta1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation.These data add to our understanding of structure-function relationships in Na+ channel beta1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.
Project description:We previously identified a human monoclonal antibody, termed HIgM12 that stimulates spontaneous locomotor activity in a chronically demyelinating mouse model of multiple sclerosis. When tested as a molecular substrate, HIgM12 stimulated neurite outgrowth in vitro. We recently reported that polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) is one of the cellular antigens for HIgM12. Fluorescent double-labeling of astrocytes using HIgM12 and commercially available anti-PSA antibody showed dramatic co-localization. Neural tissue homogenates and primary CNS cultures from mice lacking the three major NCAM splice variants NCAM180, NCAM140 and NCAM120 (NCAM KO) were no longer able to bind HIgM12. Furthermore, enzymatic digestion of PSA on wild type (WT) glia abolished HIgM12-binding. Moreover, neurons and glia from NCAM KO animals did not attach to HIgM12-coated nitrocellulose in neurite outgrowth assays. We conclude that HIgM12 targets PSA attached to NCAM, and that the PSA moiety mediates neuronal and glial adhesion and subsequent neurite outgrowth in our in vitro assay. Therefore, this anti-PSA antibody may serve as a future therapeutic to stimulate functional improvement in multiple sclerosis patients and other neurodegenerative diseases.
Project description:Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in the CNS that inhibit axonal regeneration after CNS injury. Signaling pathways in neurons triggered by CSPGs are still largely unknown. In this study, using well-characterized in vitro assays for neurite outgrowth and neurite guidance, we demonstrate a major role for myosin II in the response of neurons to CSPGs. We found that the phosphorylation of myosin II regulatory light chains is increased by CSPGs. Specific inhibition of myosin II activity with blebbistatin allows growing neurites to cross onto CSPG-rich areas and increases the length of neurites of neurons growing on CSPGs. Using specific gene knockdown, we demonstrate selective roles for myosin IIA and IIB in these processes. Time lapse microscopy and immunocytochemistry demonstrated that CSPGs also inhibit cell adhesion and cell spreading. Inhibition of myosin II selectively accelerated neurite initiation without altering cell adhesion and spreading on CSPGs.
Project description:Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSCα subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independently of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.
Project description:Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, ?1 and ?1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of ?1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to ?1B. We show that wild-type ?1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of ?1B with voltage-gated Na+ channels Na(v)1.1 or Na(v)1.3 is not detectable by immunoprecipitation and ?1B does not affect Na(v)1.3 cell surface expression as measured by [(3)H]saxitoxin binding. However, ?1B coexpression results in subtle alteration of Na(v)1.3 currents in transfected cells, suggesting that ?1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of ?1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that ?1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.