Thomas Harnois

Semaphorin SEMA3F and VEGF have opposing effects on cell attachment and spreading.

SEMA3F, isolated from a 3p21.3 deletion, has antitumor activity in transfected cells, and protein expression correlates with tumor stage and histology. In primary tumors, SEMA3F and VEGF surface staining is inversely correlated. Coupled with SEMA3F at the leading edge of motile cells, we previously suggested that both proteins competitively regulate cell motility and adhesion. We have investigated this using the breast cancer cell line, MCF7. SEMA3F inhibited cell attachment and spreading as evidenced by loss of lamellipodia extensions, membrane ruffling, and cell-cell contacts, with cells eventually rounding-up and detaching. In contrast, VEGF had opposite effects. Although SEMA3F binds NRP2 with 10-fold greater affinity than NRP1, the effects in MCF7 were mediated by NRP1. This was determined by receptor expression and blocking of anti-NRP1 antibodies. Similar effects, but through NRP2, were observed in the C100 breast cancer cell line. Although we were unable to demonstrate changes in total GTP-bound Rac1 or RhoA, we did observe changes in the localization of Rac1-GFP using time lapse microscopy. Following SEMA3F, Rac1 moved to the base of lamellipodia and - with their collapse - to the membrane. These results support the concept that SEMA3F and VEGF have antagonistic actions affecting motility in primary tumor cell.

Differential interaction and activation of Rho family GTPases by p210bcr-abl and p190bcr-abl.

The p210bcr-abl and p190bcr-abl fusion proteins, respectively responsible for chronic myelogenous leukemia and acute lymphoblastic leukemia, present deregulated tyrosine kinase activity and abnormal localization. The Dbl homology domain of Bcr, activating Rho GTPases, is present in p210bcr-abl, but absent in p190bcr-abl. We investigated the interaction of Bcr-Abl chimeras and Rho proteins by coimmunoprecipitation, pull-down experiments and GEF activity measurement. RhoA, Rac1 and Cdc42 interact in vivo with p210bcr-abl only. Moreover, the three types of GTPases are activated in vitro and in vivo by p210bcr-abl. Nevertheless, Rac1 and Cdc42, but not RhoA, are activated by p190bcr-abl in vitro and in vivo. Part of this GEF activity of p190bcr-abl is probably attributable to p95vav, which is complexed with both p190bcr-abl and p210bcr-abl in an activated form. p160bcr, also in complex with Bcr-Abl, presents no GEF activity in p190bcr-abl-expressing cells. These results suggest that differential activation of Rho proteins should play a major role in Bcr-Abl-induced leukemogenesis.

The β1-Subunit of NaV1.5 Cardiac Sodium Channel is required for a Dominant Negative Effect through α-α Interaction

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Nav1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Nav1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Nav1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β1-subunit was essential for this dominant negative effect. Indeed, the absence of the β1-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β1-subunit was present. Our findings reveal a new role for β1-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.

Nav1.5 channels can reach the plasma membrane through distinct N-glycosylation states.

BACKGROUND Like many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors. METHODS Patch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation. RESULTS The present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current. CONCLUSIONS Our findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route. GENERAL SIGNIFICANCE This work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.

An Endogenous Vitamin K-Dependent Mechanism Regulates Cell Proliferation in the Brain Subventricular Stem Cell Niche†‡§

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K‐dependent proteins (VKDPs) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin ((S(−)‐3‐acetonylbenzyl)‐4‐hydroxycoumarin)), a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDPs production, in vitro, by exposure of SVZ cell cultures to warfarin or, in vivo, by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anticoagulant factors, protein S and its structural homolog Gas6, as the two only VKDPs produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, and Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDPs in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem‐like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDPs, and anticoagulants in NSC biology in health and disease. STEM CELLS 2012; 30:719–731

SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline

Background Barely 10-20% of patients with metastatic colorectal cancer (mCRC) receive a clinical benefit from the use of anti-EGFR monoclonal antibodies (mAbs). We hypothesized that this could depends on their efficiency to reduce Store Operated Calcium Entry (SOCE) that are known to enhance cancer cells. Results In the present study, we demonstrate that SOCE promotes migration of colon cancer cell following the formation of a lipid raft ion channel complex composed of TRPC1/Orai1 and SK3 channels. Formation of this complex is stimulated by the phosphorylation of the reticular protein STIM1 by EGF and activation of the Akt pathway. Our data show that, in a positive feedback loop SOCE activates both Akt pathway and SK3 channel activity which lead to SOCE amplification. This amplification occurs through the activation of Rac1/Calpain mediated by Akt. We also show that Anti-EGFR mAbs can modulate SOCE and cancer cell migration through the Akt pathway. Interestingly, the alkyl-lipid Ohmline, which we previously showed to be an inhibitor of SK3 channel, can dissociated the lipid raft ion channel complex through decreased phosphorylation of Akt and modulation of mAbs action. Conclusions This study demonstrates that the inhibition of the SOCE-dependent colon cancer cell migration trough SK3/TRPC1/Orai1 channel complex by the alkyl-lipid Ohmline may be a novel strategy to modulate Anti-EGFR mAb action in mCRC.

Galanin promotes neuronal differentiation in murine subventricular zone cell cultures.

Neural stem cells of the subventricular zone (SVZ) represent a potentially important source of surrogate cells for the treatment of brain damage. Proper use of these cells for neuronal replacement depends on the ability to drive neuronal differentiation. Several neuromodulators stimulate neurogenesis. Here we examined the effects of the neuropeptide galanin, on neuronal differentiation in murine SVZ cultures. SVZ neurospheres obtained from early postnatal mice were treated with 10 nM to 2 μM galanin. Galanin promoted neuronal differentiation, increasing numbers of NeuN-, vesicular GABA transporter- and tyrosine hydroxylase-expressing neurons. In contrast, galanin neither affected cell proliferation assessed by BrdU incorporation nor cell death evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Neuronal differentiation was further confirmed at the functional level by measuring [Ca(2+)]i variations in single SVZ cells after KCl and histamine stimulations to distinguish neurons from immature cells. Galanin treatment increased the numbers of neuronal-like responding cells compared to immature cells. Using selective agonists (M617, AR-M1896) and antagonists (galantide, M871) for galanin receptors 1 and 2, we showed that both galanin receptors mediated neuronal differentiation. Early proneuronal effects of galanin included positive regulation of the transcription factor neurogenin-1 (Ngn1). In addition, galanin promoted axonogenesis and dendritogenesis, increasing both the length of phosphorylated stress-activated protein kinase- and Tau-positive axons and the numbers of microtubule associated protein-2 (MAP-2)-labelled dendrites. Moreover, galanin inhibited SVZ cell migration in the transwell assay. Our results show a proneurogenic effect of galanin and open new perspectives for future applications in stem cell-based therapies for neuronal replacement.

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates.

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.

Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes

In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.

p210bcr-abl induces amoeboid motility by recruiting ADF/destrin through RhoA/ROCK1

We previously demonstrated that the Bcr‐Abl oncogene, p210bcr‐abl, through its unique GEF domain, specifically activates RhoA and induces spontaneous amoeboid motility. We intend to study the pathways downstream RhoA controlling amoeboid motility. Mouse prolymphoblastic cells (Ba/F3 cell line) expressing different forms of Bcr‐Abl were embedded in 3‐dimensional (3D) Matrigel to study motility and explore the effects of inhibiting Rho pathway (inhibitors and siRNAs). The phosphorylation levels of cofilin‐1 and destrin were analyzed by 2‐dimensional electrophoresis. Composition of Bcr‐Abl signalplex in different conditions was determined by coimmunoprecipitation. Ba/F3p190 and Ba/F3 expressing a mutant form of p210bcr‐abl (unable to activate RhoA) cells presented a spontaneous motility, but not an amoeboid type. p210bcr‐abl‐induced amoeboid motility in a 3D matrix requires isoform‐specific RhoA/ROCK‐1/destrin signaling. Next to the conventional Rho/ROCK/MLC/myosin pathway, this pathway is a crucial determinant for amoeboid motility, specific for the destrin isoform (and not its coexpressed homologue cofilin‐1). Also, the presence of destrin (and not cofilin‐1) in the p210bcr‐abl complex is dependent on ROCK1, and this signalplex is required for amoeboid motility. This underscores isoform‐specific function within the ADF/cofilin family and provides new insight into Bcr‐Abl signaling to amoeboid motility and possible impact on understanding chronic myeloid leukemia progression.—Rochelle, T., Daubon, T., Van Troys, M., Harnois, T., Waterschoot, D., Ampe, C., Roy, L., Bourmeyster, N., Constantin, B. p210bcr‐abl induces amoeboid motility by recruiting ADF/destrin through RhoA/ROCK1. FASEB J. 27, 123–134 (2013). www.fasebj.org