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Originally published In Press as doi:10.1074/jbc.M412938200 on April 14, 2005

J. Biol. Chem., Vol. 280, Issue 24, 23251-23261, June 17, 2005
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Presenilin/{gamma}-Secretase-mediated Cleavage of the Voltage-gated Sodium Channel {beta}2-Subunit Regulates Cell Adhesion and Migration*{boxs}

Doo Yeon Kim{ddagger}, Laura A. MacKenzie Ingano{ddagger}, Bryce W. Carey, Warren H. Pettingell, and Dora M. Kovacs§

From the Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

Received for publication, November 16, 2004 , and in revised form, March 29, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The voltage-gated sodium channel {beta}2-subunit ({beta}2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that {beta}2 undergoes ectodomain shedding followed by presenilin (PS)-dependent {gamma}-secretase-mediated cleavage. 12-O-Tetradecanoylphorbol-13-acetate treatment or expression of an {alpha}-secretase enzyme, ADAM10, resulted in ectodomain cleavage of {beta}2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa C-terminal fragment ({beta}2-CTF) was independently inhibited by three specific {gamma}-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. {gamma}-Secretase inhibitor treatment also increased endogenous {beta}2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free {gamma}-secretase assay, we detected {gamma}-secretase activity-dependent generation of a 12 kDa {beta}2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of {beta}2 processing by {gamma}-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific {gamma}-secretase inhibitor, would alter {beta}2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing {beta}2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased {beta}2-CTF levels, we also tested whether {beta}2-CTFs or {beta}2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of {beta}2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that {beta}2 is a PS/{gamma}-secretase substrate and {gamma}-secretase mediated cleavage of {beta}2-CTF is required for cell-cell adhesion and migration of {beta}2-expressing cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major pathologic hallmark of Alzheimer disease is the deposition of amyloid {beta}-peptide (A{beta})1 into senile plaques. A{beta} is produced from {beta}-amyloid precursor protein (APP) by sequential {beta}- and {gamma}-secretase-mediated cleavages. An alternative to the {beta}-secretase pathway is a cleavage mediated by {alpha}-secretase, which cuts in the middle of the A{beta} region of APP (1). APP has been shown to serially undergo either {beta}-{gamma} and {alpha}-{gamma} cleavages. Both A{beta} and the {beta}-secretase-mediated C-terminal cleavage product of APP, C99 or CTF{beta}, have been shown to contribute to neurodegeneration in Alzheimer disease (2, 3).

{gamma}-Secretase is a multisubunit aspartyl protease complex, harboring presenilin 1 (PS1) or 2 (PS2), nicastrin, aph-1, pen2, and likely additional factors (4). The presenilins are polytopic membrane proteins that constitute either catalytic subunits or necessary cofactors of the {gamma}-secretase complex (5, 6). Besides APP, PS/{gamma}-secretase is required for the intramembraneous cleavages of at least 15 additional proteins, including Notch, nectin-1{alpha}, and recently NRADD (4, 7–11). All the known substrates, with the exception of GluR3, are type I membrane proteins and require extracellular domain cleavage prior to subsequent {gamma}-cleavage. Interestingly, many {gamma}-secretase substrates (E-, N-cadherin, nectin-1, CD44, APP, ErbB4, p75NTR, APOER2, LRP, syndecan-3, and DCC) share functions in cell adhesion and/or migration, implicating a possible role for PS/{gamma}-secretase in these processes (11–16). For example, {gamma}-secretase-dependent cleavage of E-cadherin, a well characterized cell adhesion molecule, promotes disassembly of the E-cadherin-catenin adhesion complex (17). This results in the release of {beta}-catenin, a key mediator of the Wnt signaling pathway that controls cell migration and cell adhesion (18, 19). Studies on central nervous system morphogenesis in PS1 knock-out mice also strongly support a function for PS/{gamma}-secretase in neuronal cell migration and/or cell adhesion during brain development (20, 21).

{alpha}-Secretase is a generic name for several ectodomain-shedding proteases, primarily of the metalloprotease type, based on their sensitivity to generic metalloprotease inhibitors and inducible activation by phorbol ester treatment (22). ADAM-10 and ADAM-17 are among the best characterized {alpha}-secretases of APP (23, 24). Ectodomain shedding of currently reported PS/{gamma}-secretase substrates is likely to be mediated by {alpha}-secretase-like metalloproteases. However, the identity of the metalloproteases responsible for each {gamma}-secretase substrate remains to be elucidated.

Voltage-gated sodium channels consist of a single pore-forming {alpha} subunit and one or two {beta}-subunits (25). All {beta}-subunits, {beta}1, 1A, 2, 3, and recently 4, serve as auxiliary subunits of the voltage-gated sodium channel and have been known to be involved both in channel gating and cell surface expression of {alpha} subunits (25). Similarly to nectin-1 and DCC, both substrates for PS/{gamma}-secretase-mediated cleavages, the {beta}-subunits belong to the immunoglobulin superfamily of cell adhesion molecules (CAMs) (26). The {beta}2-subunit, in particular, is enriched in the central nervous system, covalently linked to an {alpha} subunit, and well characterized as a cell-cell adhesion protein (25, 27, 28). {beta}2-Subunits interact homophilically, and also heterophilically with tenascin-R and {beta}1-subunits via their N-CAM-like extracellular domains, mediating their function as CAMs (27, 29, 30).

Here we report the identification of {beta}2 as a substrate for PS/{gamma}-secretase mediated cleavage. TPA treatment or ADAM10 expression induce ectodomain cleavage of {beta}2. The remaining 15-kDa membrane-anchored {beta}2-CTF undergoes PS/{gamma}-secretase-mediated cleavage to generate a 12-kDa {beta}2 intracellular domain ({beta}2-ICD). Lack of {gamma}-secretase cleavage of {beta}2, resulting in accumulation of {beta}2-CTFs, inhibits cell-cell adhesion and migration in CHO cells. These data suggest a functional role for PS/{gamma}-secretase in cell adhesion and migration via processing of {beta}2-CTFs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids, Transfection, and Primary Neuronal Cultures—An expression construct encoding full-length human voltage-gated sodium channel {beta}2 subunit ({beta}2, GeneBankTM accession number gi:21361089) containing a C-terminal V5/His tag (in pcDNA3.1/GS) was purchased from Invitrogen. An expression vector containing human ADAM10 was a kind gift of Dr. Lichtenthaler (Ludwig-Maximilians-Universität). Effectene (Qiagen) was routinely used for transfecting cell lines. We produced stably transfected CHO cells with {beta}2, {beta}2-CTF, {beta}2-ICD, nectin, NE-CTF, NE-ICD, wild-type PS1, and PS1(D385A) expression constructs. The sequences of primers used to generate these constructs are available by request. Individual clones with similar expression levels were maintained in selection medium. The PS1/PS2 knock-out ES cell line (BD1) and control wild-type ES cells (BD6) were a generous gift of Dr. Sisodia (University of Chicago). SH-SY5Y cells were routinely grown in DMEM medium (Cambrex) with 10% fetal bovine serum and switched to RPMI 1640 medium (Cambrex) during migration assays. Mouse primary cortical neuronal cultures (DIV16) were prepared as described (8). Briefly, cortices were dissected from E16 mouse fetal brains and dissociated by repeated passages through a fire-polished Pasteur pipette. The dissociated cells were plated on L-polylysine/laminine coated 6-well dishes and maintained in growth media consisting of neurobasal medium (Invitrogen) medium supplemented with B27 and 0.5 mM L-glutamine. Cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere for 16 days (DIV16).

Western Blot Analysis, Antibodies, and Inhibitors—For immunoblotting, cell extracts were prepared by directly extracting cells in a buffer containing 10 mM Tris-HCl, pH 6.8, 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40, 1% Triton X-100, and a protease inhibitor mixture (Roche Applied Science), followed by a spin at 16,000 x g. Protein levels were quantitated using the BCA protein assay kit (Pierce). 20–100 µg of protein were resolved on 4–12% gradient BisTris gels or 12% BisTris gels (Invitrogen). Primary antibodies were used at the following dilutions: anti-V5 antibody (1:5000 dilution (Invitrogen)), anti-sodium channel {beta}2 subunit antibodies (Nav{beta}2, 1:500 dilution (Chemicon) and 1:500 (USBiological)), anti-nectin-1 (1:1000 dilution (a kind gift from Dr. Federoff at the University of Rochester)), anti-transferrin receptor (1:1000 (BD Bioscience)), anti-poly-(D-ribose) polymerase (1:1000 (Pharmingen)), and anti-cleaved caspase 3 (1:2000 (Cell Signaling)). The blots were visualized by ECL using Lumiglo (KPL) or SuperSignal CL-HRP substrates (Pierce) according to the manufacturer's instructions. The {gamma}-secretase inhibitors DAPT and L-685,458 were obtained from Calbiochem, and WPE31C was a kind gift of Dr. M. Wolfe (Brigham and Women's Hospital). The {alpha}-secretase inhibitor TAPI-1 (tumor necrosis factor-{alpha} processing inhibitor-1, IC-2) was purchased from Biomol.

In Vitro Generation of {beta}2-ICD—Membrane preparation and in vitro generation of {beta}2-ICD were performed as described (8, 31). The P2-P3 fractions were resuspended in Buffer H (20 mM HEPES, 150 mM NaCl, 10% glycerol, 5 mM EDTA, pH 7.4) with protease inhibitors. In vitro cleavage experiments were performed by incubating the membrane fractions at 37 °C for 1 h in the presence or absence of the indicated amounts of DAPT and L-685,458. After incubation, the soluble and membrane-associated fragments were separated by centrifugation of the reaction mixture at 120,000 x g for 45 min. To dissociate ICD fragments from the membranes, 1 mM NaOH solution was added to the membrane fraction and centrifuged at 120,000 x g for 45 min.

Fluorescence Confocal Analysis—For immunostaining, cells were fixed with 4% paraformaldehyde, rinsed, permeabilized, and blocked by using 0.1% Triton X-100 and 1.5% goat IgG (Molecular Probes), respectively. Cells were then washed three times with PBS and incubated for 3 h with an anti-V5 antibody (1:200, Invitrogen) in PBS containing 1.5% goat IgG. After washing three times with PBS, cells were incubated with secondary antibodies conjugated with Alexa Fluor 488. Confocal fluorescence images were obtained using a LSM 5 Pascal laser scanning microscope (Zeiss).

Cell-Cell Aggregation Assay—In vitro cell-cell aggregation assays were performed as described by Miura et al. (32) with slight modifications. To induce {beta}2-CTF accumulation, cells were pretreated with DAPT for 12 h. Cells were then incubated for 15 min at room temperature with 1 mM EDTA/Hanks' balanced salt solution containing 500 nM DAPT and dispersed by gentle pipetting. After washing three times, cells were resuspended in Ca2+/Mg2+-free Hanks' balanced salt solution containing DAPT (500 nM), transferred into 35-mm polysterene dishes precoated with bovine serum albumin, and agitated (75 rpm) at room temperature for the indicated time intervals. The number of cell aggregates was counted three times in a hemocytometer. Aggregation was quantified by the index N(t)/N(0), where N(t) and N(0) are the total number of particles at incubation time at t and 0, respectively.

Wound Healing Assay—CHO cells expressing {beta}2 were grown in monolayer, scraped with a P200 pipette tip, washed with PBS, and incubated for 18–21 h in the presence and absence of 500 nM DAPT (33). For {beta}2-CTF accumulation, cells were pretreated with DAPT for 12 h. Migration of cells into the wound was examined by phase contrast microscopy. Photographic images were obtained immediately after scraping and after 21 h in the same locations. Wound healing activity was quantified by measuring the total area of the wound in each 10x field and the area covered by the migrating cells within the wound by using public image analysis software, NIH image software, Version 1.61. At least three different fields were chosen randomly, and the mean percentage of wound area covered by cells was calculated.

Cell Migration Assay with Cell Culture Inserts—SH-SY5Y cell migration assays were performed as described by Pola et al. (34) with slight modifications. Briefly, cell culture inserts with polyethylene terephthalate membranes (8 µm pore, USA Scientific, Inc.) were coated with collagen type IV (Sigma) and placed in 6-well dishes. Confluent SH-SY5Y cells were preincubated with serum-free RPMI 1640 medium for 24 h in the presence or absence of 500 nM DAPT. Cells were then dispersed with 0.02% EDTA solution in PBS. 4 x 105 cells were replated into cell culture inserts placed in 6-well plates and incubated for 24 h to allow for cell migration. PDGF-BB (20 ng/ml, Sigma) was added to the lower chamber as an attractant of SH-SY5Y cells. Cells on the upper surface of the inserts were removed by sterile cell scraper (Falcon), to leave the cells that had migrated across the membrane. Cells that had migrated and attached to the lower surface of the inserts were stained with Hoechst33342 (Molecular Probes) and quantitated by a Victor3 fluorescence plate reader (PerkinElmer Life Sciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
{beta}2 Is Sequentially Cleaved by {alpha}-Secretase and PS/{gamma}-Secretase Activities—Common characteristics among currently reported PS/{gamma}-secretase substrates include: 1) sequence homology with other PS/{gamma}-secretase substrates at the membrane-cytosol interface (8, 9); 2) ectodomain cleavage by {alpha}-secretase alone or both {alpha}- and {beta}-secretase-like activities, which generate CTF derivatives; 3) accumulation of CTF derivatives with extracellular domains shorter than 300 amino acids in response to PS/{gamma}-secretase inhibition (35); 4) detection of a PS/{gamma}-secretase-dependent cleavage product, termed ICD; 5) in many cases, modulation of nuclear transcriptional activity by the ICD that is released into the cytosol (36–38).

Although A{beta} is released by {gamma}-secretase cleavage of APP-CTF{beta}, a second intramembraneous clip ({epsilon}-cleavage) in APP is also PS-mediated, at a site homologous to the S3 cleavage of Notch (31, 39–41). Similarly, all other PS/{gamma}-secretase substrates harbor a loose consensus sequence at the membrane-cytosol interface, corresponding to the {epsilon}/S3-cleavage sites in APP/Notch (8, 9, 17). Using a BLAST search based on the homology between the {epsilon}/S3-cleavage sites, we found a large number of proteins containing homologous amino acid sequences, including {beta}2. We chose to characterize {beta}2 because, similarly to nectin-1 and DCC, {beta}2 is a cell adhesion protein belonging to the wide family of IgCAMs (25). Of the numerous members of the voltage-gated sodium channel proteins, including {alpha} and {beta} subunits, only {beta}2 harbors strong sequence homology to the APP-{epsilon} and Notch-S3 cleavage sites (Fig. 1A). Interestingly, this putative cleavage site in {beta}2 also contains a lysine residue for potential monoubiquitination, which has recently been reported to control Notch-S3 cleavage (42).

Previously reported PS/{gamma}-secretase substrates, such as APP, erbB4, LRP, and nectin-1, undergo phorbol ester-inducible ectodomain shedding to produce short CTFs, which are subsequently cleaved in a PS-dependent manner (8, 43–45) (Fig. 1B). To assess whether {beta}2 also yields shorter cleavage products in response to phorbol esters, we treated CHO cells expressing {beta}2 with 12-O-tetradecanoylphorbol-13-acetate (TPA). Employing an antibody against the C-terminal V5 epitope tag, Western blot analysis showed that full-length {beta}2 migrated at ~47 kDa (Fig. 1C). Unlike nectin-1{alpha}, but similarly to APP, untreated cells already processed {beta}2 to yield a CTF of ~15 kDa. This indicates that {beta}2 undergoes constitutive ectodomain shedding similar to APP, in addition to a phorbol ester inducible activity. The size of this fragment was consistent with the predicted molecular mass of the {beta}2-CTF-V5/His cleavage product, ~14–15 kDa, if ectodomain shedding occurred near the plasma membrane. TPA treatment only slightly increased levels of the 15-kDa fragment, presumably because of immediate PS/{gamma}-secretase processing of the protein. However, DAPT, a {gamma}-secretase inhibitor, alone or in conjunction with TPA, further elevated cellular levels of the 15-kDa CTF (Fig. 1C). These data suggest that {beta}2 undergoes TPA-inducible ectodomain shedding followed by {gamma}-secretase activity-dependent processing of its CTF.

TPA-inducible cleavage as shown in Fig. 1C is characteristic of metalloprotease or {alpha}-secretase-mediated protease activities (46). To test whether an {alpha}-secretase activity is involved in ectodomain shedding of {beta}2, CHO cells expressing {beta}2 were treated with 10 µM TAPI-1, a hydroxamic acid-based {alpha}-secretase inhibitor (47). DAPT treatment alone induced accumulation of the 15 kDa {beta}2-CTF, and this was blocked by the {alpha}-secretase inhibitor TAPI-1 (Fig. 1D). To further assess whether {alpha}-secretase was partially responsible for ectodomain shedding of {beta}2, we directly tested a well known {alpha}-secretase in our cells. For these experiments, we obtained a hemagglutinin-tagged ADAM10 construct from Dr. Lichtenthaler (Ludwig-Maximilians-Universität). Overexpression of ADAM10 alone was sufficient to increase {beta}2-CTF levels in CHO cells expressing {beta}2 (Fig. 1E). Taken together, these data demonstrate that {alpha}-secretase-like proteases including ADAM10 partially mediate ectodomain cleavage of {beta}2.

In Fig. 1, we have shown that DAPT increased the levels of the 15 kDa {beta}2-CTF in CHO cells overexpressing {beta}2. This suggests that {gamma}-secretase activity is responsible for the degradation of the 15-kDa {beta}2-CTF, generated from ectodomain shedding of {beta}2. To confirm that this 15-kDa {beta}2-CTF is a substrate for PS/{gamma}-secretase activity, we treated {beta}2-transfected CHO cells with two additional {gamma}-secretase inhibitors, L-685,458 and WPE31C. These inhibitors independently elevated {beta}2-CTF levels similarly to DAPT, suggesting that specific inhibition of {gamma}-secretase activity is responsible for the increased {beta}2-CTF levels (Fig. 2A). We also found that {beta}2-CTF levels increased with increasing concentrations of DAPT (Fig. 2B). Moreover, {beta}2-CTFs specifically accumulated in CHO cells stably transfected with a construct expressing the dominant negative PS1(D385A), and this was further enhanced by TPA treatment (Fig. 2C). Similarly, {beta}2-CTF levels largely increased in PS1/PS2 knock-out ES cells transfected with full-length {beta}2 (Fig. 2D). These data show that the proteolytic processing of the 15-kDa {beta}2-CTF is PS/{gamma}-secretase-mediated.

In neuronal cells, {beta}2 proteins are predominantly associated with voltage-gated sodium channel {alpha} subunits (48). To test whether {beta}2 undergoes {gamma}-secretase-mediated cleavage in neuronal cells, we generated rat neuroblastoma cell lines (B104) stably expressing {beta}2-V5/His. We chose to use B104 cells because they have been reported to express functionally active voltage-gated sodium channels, consisting of {alpha} and {beta}1 subunits (49, 50). 8 h of DAPT treatment, in the absence of TPA, induced the accumulation of the 15-kDa {beta}2-CTF in B104 cells stably expressing {beta}2, suggesting that {beta}2 undergoes constitutive ectodomain shedding followed by {gamma}-secretase cleavage in these cells (Fig. 2E). To test whether endogenous {beta}2 in neurons also undergoes {gamma}-secretase-mediated cleavage, cultured cortical neurons from E16 mouse embryos were also treated with DAPT for 8 h. Western blot analysis using an antibody against the C-terminal sequence of {beta}2 showed that the putative endogenous 10-kDa {beta}2-CTF (lacking the V5/His tag) largely increased following DAPT treatment (Fig. 2F). This 10-kDa {beta}2-CTF band was absent when the antibodies were preincubated with the same antigenic peptide used to generate the {beta}2 antibody (data not shown). Full-length {beta}2 was also detected in mouse cortical neuronal cultures (Fig. 2F). These data demonstrate that {beta}2 is an endogenous substrate for PS/{gamma}-secretase-mediated cleavage in neuronal cells.

The PS/{gamma}-secretase-derived cleavage products (intracellular domains or ICDs) of previously identified substrates are rapidly degraded in intact cells (8, 9, 38, 51). Therefore, we used a cell-free {gamma}-secretase assay to detect {beta}2-ICD (8). Membranes from {beta}2-overexpressing CHO cells were incubated for 1 h in the presence and absence of the {gamma}-secretase inhibitor DAPT (8). Incubation at 37 °C resulted in the generation of a band at ~12 kDa, the expected molecular mass of the {beta}2-ICD containing a V5/His tag (Fig. 3A). DAPT and another {gamma}-secretase inhibitor, L-685,458, blocked the generation of this band (Fig. 3B). The 12-kDa fragment, as opposed to the 15-kDa {beta}2-CTF, could be dissociated from the membranes when washed with 0.1 M sodium hydroxide (Fig. 3B). Therefore, the 12-kDa {beta}2 band likely represents the PS-dependent ICD fragment of {beta}2 because it is not membrane-tethered, it is undetectable in intact cells, and its generation is inhibited by PS/{gamma}-secretase inhibitors. Similar only to nectin-1{alpha}-ICD, {beta}2-ICD is peripherally associated with membranes presumably via protein-protein interactions (8). However, while 0.1 M sodium carbonate was sufficient to dissociate nectin-1{alpha}-ICD, the same treatment did not dissociate {beta}2-ICD from the membrane fraction, suggesting that the latter is more tightly associated with membranes than nectin-1{alpha}-ICD (8). Since {beta}2- and nectin-ICDs remain associated with the membrane pool, it is unlikely that they would directly function in modulating transcription in the nucleus. However, it cannot be excluded that a fraction of these ICDs and/or selected adaptor proteins are released and enter the nucleus in intact cells, similar to {beta}-catenin following PS/{gamma}-secretase-like cleavage of cadherin (17).

PS/{gamma}-Secretase Activity Is Required for {beta}2-Mediated Cell-Cell Adhesion and Cell Migration—Since {beta}2 is involved in cell adhesion, we tested whether PS/{gamma}-secretase activity modulates the cell adhesion function of {beta}2. For these experiments, we used an in vitro cell aggregation model system in Ca2+/Mg2+-free conditions (32). CHO cells were pretreated with DAPT for 12 h, dispersed into single cells by mechanical agitation in the presence of 1 mM EDTA, and incubated in Ca2+/Mg2+-free Hanks' balanced salt solution. 60-min incubation at room temperature induced cell-cell aggregation, enhanced by stable overexpression of {beta}2 (Fig. 4A, bottom left panel). These data reconfirmed previous reports that {beta}2 functions as a cell-cell adhesion molecule in non-neuronal cells (27). Quantification showed a 2-fold increase in cell-cell aggregation in {beta}2-expressing CHO cells as compared with parental CHO cells (Fig. 4B). Interestingly, {beta}2-induced cell-cell aggregation was completely blocked by the {gamma}-secretase inhibitor DAPT, while DAPT did not change cell-cell aggregation in CHO parental cell lines (Fig. 4B). We then tested whether inhibition of {gamma}-secretase modulates cell migration, a complex cellular event that involves cytoskeletal reorganization, detachment from the extracellular matrix, and generation of new cell-cell junctions. Using a wound healing assay system (33), we found that 21 h of DAPT treatment inhibited cell migration of CHO cells stably expressing {beta}2 by ~40% as compared with control Me2SO-treated cells (Fig. 4C, D). These data demonstrate that {gamma}-secretase activity is required for cell-cell adhesion and cell migration in CHO cells stably expressing {beta}2.



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  		FIG. 1.
The ectodomain of {beta}2 is first cleaved by an {alpha}-secretase activity. A, sequence comparison of the APP-{epsilon}/Notch-S3 domains in nine human PS1/{gamma}-secretase substrates and in {beta}2. B, expected proteolytic processing of {beta}2, based on previously identified PS/{gamma}-secretase substrates. C, Western blot analysis of CHO cells expressing {beta}2-V5/His. {beta}2-CTF levels are increased by TPA treatment, further enhanced by treatment with a {gamma}-secretase inhibitor, DAPT, which is expected to block further processing of {beta}2-CTFs by {gamma}-secretase. D, DAPT treatment elevates {beta}2-CTF levels in CHO cells, while co-treatment with the {alpha}-secretase inhibitor TAPI-1 (10 µM) blocks this increase. E, co-expression of {beta}2 with the {alpha}-secretase ADAM10 increases levels of the 15-kDa {beta}2-CTF. F.L., full length; NTF, N-terminal fragment.

 



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  		FIG. 2.
Following ectodomain shedding, {beta}2-CTF is further processed by PS/{gamma}-secretase. A, Western blot analysis of CHO cells expressing {beta}2-V5/His. {beta}2-CTF levels are increased by three different {gamma}-secretase inhibitors: DAPT, L-685,458, and WPE31C; this increase is further potentiated by TPA. B, DAPT increases {beta}2-CTF levels in a dose-dependent manner. C, expression of the dominant negative form of PS1, PS1(D385A), also results in the accumulation of the 15-kDa {beta}2-CTF (arrowhead), only slightly intensified by TPA treatment. D, {beta}2-CTF levels are elevated by the absence of functional presenilins in PS1/PS2 double knock-out ES cells, slightly increased by TPA. E, DAPT treatment also increases {beta}2-CTF levels in a rat neuroblastoma cell line (B104) stably expressing human {beta}2-V5/His. F, mouse cortical neurons (DIV16) produce detectable levels of endogenous {beta}2-CTF following DAPT treatment. F.L., full length.

 



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  		FIG. 3.
Cell-free generation of {beta}2-ICD is PS/{gamma}-secretase activity-dependent. A, a 12-kDa {beta}2-ICD is only detectable when membrane fractions from {beta}2-transfected CHO cells are incubated at 37 °C and in the absence of the {gamma}-secretase inhibitor DAPT. B, Western blot analysis of membrane and soluble fractions from a cell-free {gamma}-secretase assay. In addition to DAPT, the {gamma}-secretase inhibitor L-685,458 also blocks {beta}2-ICD generation. The {beta}2-ICD does not appear in the soluble fraction, because it remains loosely associated with membranes. A sodium hydroxide (NaOH) wash, which also removes actin associated with the membranes but not the control transferrin receptor, is needed for dissociation of {beta}2-ICD from membranes. F.L., full length.

 
{beta}2-CTF and NE-CTF Independently Inhibit Cell Migration—In cells transfected with {beta}2, {gamma}-secretase inhibition elevates {beta}2-CTF levels. To directly assess whether accumulation of {beta}2-CTFs inhibits cell migration, we generated N-terminally truncated deletion mutant constructs of {beta}2-V5/His, and as a control, of nectin-V5/His (Fig. 5A). Following a murine Ig {kappa}-chain signal peptide (pSecTag, Invitrogen) and a short linker, the {beta}2 and nectin-1 sequences of these CTFs started two amino acids from the putative transmembrane domains of the proteins. When transiently overexpressed in CHO cells, already stably expressing dominant negative PS1(D385A), both CTFs were predominantly localized to cell surface membranes (Fig. 5B). Stable expression of {beta}2-CTF and nectin-CTF in CHO cells did not cause detectable levels of apoptotic cell death, as assessed by two markers of apoptosis, poly-(D-ribose) polymerase cleavage (supplemental Figs. S1 and S2) and generation of active caspase 3 (data not shown). Given that {beta}2-expressing cells treated with {gamma}-secretase inhibitors contain both full-length {beta}2 and {beta}2-CTFs, we decided to replicate similar conditions by stably co-expressing both forms of the protein in CHO cells (Fig. 5C). As a control, CHO cell lines stably co-expressing full-length nectin-1 and NE-CTF were also generated for these experiments (Fig. 5C). Stable expression of full-length {beta}2 and {beta}2-CTF decreased cell migration in wound healing assays by 81–93%, while full-length nectin-1 and NE-CTF reduced cell migration by 84% (Fig. 5, D and E). These inhibitory effects on cell migration were not due to decreased rates of cell division, because we could not detect any growth rate difference among the different cell lines (data not shown). To test whether reduced ICD signaling could account for the observed decrease in cell migration, we generated cells co-expressing {beta}2-ICD or NE-ICD together with full-length {beta}2 (supplemental Fig. S3). Interestingly, overexpression of {beta}2 or nectin-ICD does not induce statistically meaningful changes in cell migration (Fig. 5F). These data indicate that the inhibitory effects of CTFs on cell migration derive from increased CTF itself. Taken together, our data suggest that CTFs generated by ectodomain shedding of {beta}2 and nectin are processed by {gamma}-secretase activity to prevent adverse effects of the CTFs on cell migration.



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  		FIG. 4.
DAPT inhibits {beta}2-mediated cell-cell adhesion and cell migration in a wound-healing assay. A, representative photomicrographs showing cell-cell aggregation after 60 min of incubation. Cell clumps were generated specifically in CHO cells stably expressing {beta}2 (CHO-{beta}2, see arrowhead in bottom left panel), blocked by 500 nM DAPT (bottom right panel). CHO(-), wild-type CHO cells without the expression of {beta}2. B, quantitation of the extent of cell-cell aggregation shows that DAPT inhibits cell-cell aggregation of cells stably expressing {beta}2. N(t), total particle number at time t of incubation; N(0), initial particle number. C, representative photomicrographs showing that 500 nM DAPT treatment reduces wound healing in CHO-{beta}2 cell lines. Dotted lines delimit the initially wounded regions, as determined by superimposing the picture taken immediately after the wound was made. D, the extent of wound healing was quantified by counting the number of migrated cells after 21 h. Statistical analysis was performed using analysis of variance followed by a post hoc Tukey's analysis. *, p < 0.05 compared with dimethyl sulfoxide (DMSO)-treated cells (n = 3 per each condition).

 
DAPT Treatment Inhibits PDGF-induced Cell Migration of SH-SY5Y Neuroblastoma Cells—To show that {gamma}-secretase cleavage of endogenous substrates, including {beta}2, is required for cell migration of neuronal cells, we used SH-SY5Y neuroblastoma cells as a model system. SH-SY5Y cells have been reported to express {beta}2 mRNA in a previous study (52). In preliminary studies, we reconfirmed that this cell line expresses {beta}2 mRNA by using reverse transcription-PCR (data not shown). To show that endogenous {beta}2 and nectin-1 in SY5Y cells undergo {alpha}- and {gamma}-secretase-mediated cleavages, we performed Western blot analysis using antibodies against the C termini of endogenous {beta}2 and nectin-1. As expected, endogenous {beta}2- and nectin-CTFs specifically increased when cells were co-treated with DAPT and TPA (Fig. 6A). To test whether {gamma}-secretase cleavage modulates migration SH-SY5Y cells, we employed a modified Boyden chamber assay using PDGF as a chemoattractant (34) (Fig. 6B). We found that PDGF induced cell migration by 4.6-fold as compared with the bovine serum albumin control in the lower chambers. However, DAPT decreased PDGF-induced cell migration by 43% (Fig. 6C). These data support the notion that {gamma}-secretase cleavage of endogenous {beta}2 and/or nectin-1 promotes cell migration of neuronal cells.



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  		FIG. 5.
Overexpressed recombinant {beta}2- or NE-CTFs inhibit cell migration in a wound-healing assay. A, schematic representation of recombinant {beta}2-CTF, NE-CTF, {beta}2-ICD, and NE-ICD proteins used in the assay (cloned into pSecTag/FRT/V5-His TOPO). B, subcellular localization of {beta}2- and NE-CTF expressed in CHO cells stably expressing dominant negative PS1(D385A). C, Western blot analysis by using an anti-V5 antibody of two different stable clones co-expressing full-length {beta}2 and {beta}2-CTF (CHO-{beta}2 F.L.+{beta}2-CTF) and one clone with nectin and NE-CTF (CHO-nectin F.L.+NE-CTF). CTF levels in these cell lines increased by 2–3-fold compared with parental cell lines (CHO-{beta}2 F.L. and CHO-nectin F.L.) expressing only full-length proteins, respectively. D, representative photomicrographs showing that wound healing activity of CHO cell lines co-expressing full-length and CTF of {beta}2(CHO-{beta}2 F.L.+{beta}2-CTF) or nectin-1 (CHO-nectin F.L.+NE-CTF) was decreased as compared with CHO cells expressing only full-length {beta}2 (CHO-{beta}2 F.L.) or nectin-1 (CHO-nectin F.L.), respectively. Dotted lines delimit the initially woundedregions, as determined by superimposing the picture taken immediately after the wound was made. E, quantitative analysis of the experiment shown in D. Statistical analyses used were a Student's t test (left panel) and analysis of variance followed by a post hoc Tukey's analysis (right panel). The control for the right panel is nectin F.L. and {beta}2 F.L. for the left panel (***, p < 0.001 compared with control; n = 3 per each condition). F, cell cultures co-expressing {beta}2 F.L. ({beta}2 F.L) and {beta}2-ICD ({beta}2 F.L.+{beta}2-ICD) or NE-ICD ({beta}2 F.L.+ NE-ICD) were generated and their wound-healing activity was measured. Co-expression of {beta}2-ICD or NE-ICD in CHO-{beta}2 F.L cells does not show any statistically meaningful effect on cell migration (n = 6 per each condition). F.L., full length.

 



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  		FIG. 6.
Inhibition of {gamma}-secretase activity decreases PDGF-induced cell migration of SH-SY5Y cells in a modified Boyden chamber assay. A, Western blot analysis of SH-SY5Y cells treated with 500 nM DAPT alone or together with 100 nM TPA. The antibodies against endogeous human {beta}2 or nectin-1 detected both full-length proteins and CTFs. {beta}2- and nectin-CTFs were specifically increased by co-treatment with TPA and DAPT. B, schematic representation of the PDGF-induced cell migration assay. SH-SY5Y cells migrated through 8-µm membrane pores, attracted by PDGF added to the lower chamber (upper panel). bovine serum albumin in the lower chambers served as a control for cell migration. The migrated cells were stained with Hoechst33342 and visualized under fluorescence microscope (lower panel). DAPT treatment reduced the number of cells migrating to the lower chamber (right panel). C, the extent of cell migration was quantitated by using Victor3 fluorescence plate reader (PerkinElmer Life Sciences). A Student's t test was used for statistical analysis (*, p < 0.05 compared with dimethyl sulfoxide (DMSO)-treated cells; n = 3 per each condition). F.L., full length.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we show that {beta}2, an auxiliary subunit of the voltage-gated sodium channel and a cell adhesion protein, is a substrate for both {alpha}- and PS/{gamma}-secretase-mediated cleavages. We also found that increased levels of {beta}2-CTFs, either by inhibiting {gamma}-secretase activity or by expressing recombinant {beta}2-CTF, block cell aggregation and migration in CHO cells. Interestingly, similar results were found when the CTF, but not ICD, of another {gamma}-secretase substrate and cell adhesion protein, nectin, was overexpressed. These data indicate that PS/{gamma}-secretase-mediated processing of {gamma}-secretase substrates, such as {beta}2-CTF and NE-CTF, is required for cell-cell adhesion and migration, supporting a role for PS/{gamma}-secretase in cellular functions requiring these processes such as neurite outgrowth and axon guidance.

{beta}2-CTF and NE-CTF are likely to inhibit cell migration via a complex interplay among different cellular events, including direct interference with cell adhesion or altered intracellular signaling. Considering that only two extracellular amino acids are preserved in our CTF constructs, CTF effects on cell migration are probably mediated by the transmembrane or cytoplasmic domains. However, direct overexpression of ICDs did not inhibit cell migration. The cytoplasmic domain of the {beta}2 subunit recruits ankyrin in response to cell adhesion, thereby anchoring the extracellular space to the spectrin cytoskeletal system (27, 53). Likewise, nectin-1, which is also a PS/{gamma}-secretase substrate, is anchored to the cytoskeleton via several adaptor proteins including afadin and {alpha}- and {beta}-catenin (8, 54). E-cadherin, another binding partner of {alpha}- and {beta}-catenin, undergoes {gamma}-secretase-mediated cleavage, which releases a cytoplasmic E-cadherin fragment together with {beta}- and {alpha}-catenin (17). Fe65, an adaptor protein of APP, is also released and translocated to the nucleus in a {gamma}-secretase-dependent manner (37, 55, 56). These studies support a function for PS/{gamma}-secretase in modulating the release of adaptor signaling proteins, which are attached to the cytoplasmic domains of {gamma}-secretase substrate proteins. Thus, signaling through interactions among CTFs, adaptor molecules, and the cytoskeletal system might be relevant to the CTF-mediated inhibition of cell migration.

{beta}2 is involved in both cell adhesion and sodium channel function, thus, its misprocessing may interfere with neuronal function at multiple levels. Additionally, a recent study suggests that {beta}-subunits are involved in neurite outgrowth (57). Moreover, another recent study demonstrated that the accumulation of membrane-tethered DCC-CTF (one of the reported {gamma}-secretase substrate proteins) increased neurite outgrowth in in vitro and in vivo model systems (58). Here we show that {beta}2-CTF, which is similar to membrane-tethered DCC-CTF, directly inhibits cell aggregation and migration in cells that do not express the {alpha} subunit of the voltage-gated sodium channel. It will be interesting to see whether elevated levels of {beta}2-CTFs also affect neurite outgrowth, which is mechanistically close to cell migration. Considering the function of {beta}2 in controlling cell surface sodium channel density, elevated {beta}2-CTFs may also alter sodium channel function in neuronal systems. Several studies have previously shown that co-expression of {alpha} and {beta} subunits results in stabilization of cell surface sodium channel levels (59, 60). Conversely, absence of the {beta}2 subunit in {beta}2 knock-out mice leads to a general decrease in active voltage-gated sodium channels on the cell surface (61). Therefore, it will be interesting to study whether PS/{gamma}-secretase-mediated proteolytic cleavage of {beta}2 modulates sodium channel activity.

The identification of additional PS/{gamma}-secretase substrates may raise questions about the predicted safety of {gamma}-secretase inhibitors in anti-{beta}-amyloid therapy. Complete blocking of {gamma}-secretase activity should elevate CTF levels of currently known {gamma}-secretase substrate proteins, such as {beta}2 and nectin-1. Studies show strong developmental defects including abnormal neuronal migration in PS1-/- mice and neurodegeneration in 6-month-old conditional PS1/2 knock-out mice (21, 62). However, partial inhibition of {gamma}-secretase activity may reduce A{beta} generation without significantly affect cleavage of other substrates. Development of {gamma}-secretase inhibitors designed to specifically block A{beta} generation such as nonsteroidal anti-inflammatory drugs would be desirable for treatment of Alzheimer disease patients (63, 64).


   FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health/NIA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. Back

{ddagger} These authors contributed equally to this work. Back

§ To whom correspondence should be addressed. E-mail: dora_kovacs{at}hms.harvard.edu.

1 The abbreviations used are: A{beta}, amyloid {beta}-peptide; APP, {beta}-amyloid precursor protein; PS, presenilin; PS1, presenilin 1; PS2, presenilin 2; TPA, 12-O-tetradecanoylphorbol-13-acetate; CAM, cell adhesion molecule; {beta}2, voltage-gated sodium channel {beta}2-subunit; {beta}2-CTF, voltage-gated sodium channel {beta}2-subunit C-terminal fragment; {beta}2-ICD, voltage-gated sodium channel {beta}2-subunit intracellular domain; NE-CTF, nectin-1 C-terminal fragment; NE-ICD, nectin-1 intracellular domain; CHO, Chinese hamster ovary; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; TAPI-1, tumor necrosis factor-{alpha} processing inhibitor-1; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor. Back


   ACKNOWLEDGMENTS
 
We thank Dr. S. S. Sisodia (University of Chicago) for the BD1 and BD6 ES cells, Dr. M. Wolfe (Brigham and Women's Hospital) for WPE31C, Dr. Lichtenthaler (Ludwig-Maximilians-Universität) for the ADAM10 construct, Dr. L. L. Isom (University of Michigan) for helpful discussions, and Igor Bagayer (Massachusetts General Hospital) for his technical assistance in confocal imaging.



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M. Zhang, A. Haapasalo, D. Y. Kim, L. A. M. Ingano, W. H. Pettingell, and D. M. Kovacs
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C. Bohm, N. M. Seibel, B. Henkel, H. Steiner, C. Haass, and W. Hampe
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D. G. Cook, X. Li, S. D. Cherry, and A. R. Cantrell
Presenilin 1 Deficiency Alters the Activity of Voltage-Gated Ca2+ Channels in Cultured Cortical Neurons
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