Josette Noël

pH Nanoenvironment at the Surface of Single Melanoma Cells

Extracellular pH and the Na+/H+ exchanger (NHE1) modulate tumor cell migration. Yet, the pH nanoenvironment at the outer surface of the cell membrane (pHem) where cell/matrix interaction occurs and matrix metalloproteinases work was never measured. We present a method to measure this pH nanoenvironment using proton-sensitive dyes to label the outer leaflet of the plasma membrane or the glycocalyx of human melanoma cells. Polarized cells generate an extracellular proton gradient at their surface that increases from the rear end to the leading edge of the lamellipodium along the direction of movement. This gradient collapses upon NHE1 inhibition by HOE642. NHE1 stimulation by intracellular acidification increases the difference in pHem between the tips of lamellipodia and the cell body in a Na+ dependent way. Thus, cells create a pH nanoenvironment that promotes cell migration by facilitating cell adhesion at their front and the release of cell/matrix contacts at their rear part.

EGF and K+ channel activity control normal and cystic fibrosis bronchial epithelia repair

Severe lesions of airway epithelia are observed in cystic fibrosis (CF) patients. The regulatory mechanisms of cell migration and proliferation processes, involved in the repair of injured epithelia, then need to be better understood. A model of mechanical wounding of non-CF (NuLi) and CF (CuFi) bronchial monolayers was employed to study the repair mechanisms. We first observed that wound repair, under paracrine and autocrine EGF control, was slower (up to 33%) in CuFi than in NuLi. Furthermore, EGF receptor (EGFR) activation, following wounding, was lower in CuFi than in NuLi monolayers. Cell proliferation and migration assays indicated a similar rate of proliferation in both cell lines but with reduced (by 25%) CuFi cell migration. In addition, cell migration experiments performed in the presence of conditioned medium, collected from NuLi and CuFi wounded bronchial monolayers, suggested a defect in EGF/EGFR signaling in CF cells. We (49) recently demonstrated coupling between the EGF response and K(+) channel function, which is crucial for EGF-stimulated alveolar repair. In CuFi cells, lower EGF/EGFR signaling was accompanied by a 40-70% reduction in K(+) currents and KvLQT1, ATP-sensitive potassium (K(ATP)), and Ca(2+)-activated K(+) (KCa3.1) channel expression. In addition, EGF-stimulated bronchial wound healing, cell migration, and proliferation were severely decreased by K(+) channel inhibitors. Finally, acute CFTR inhibition failed to reduce wound healing, EGF secretion, and K(+) channel expression in NuLi. In summary, the delay in CuFi wound healing could be due to diminished EGFR signaling coupled with lower K(+) channel function, which play a crucial role in bronchial repair.

Insulin-sensitizing and Anti-proliferative Effects of Argania spinosa Seed Extracts

Argania spinosa is an evergreen tree endemic of southwestern Morocco. Many preparations have been used in traditional Moroccan medicine for centuries to treat several illnesses including diabetes. However, scientific evidence supporting these actions is lacking. Therefore, we prepared various extracts of the argan fruit, namely keel, cake and argan oil extracts, which we tested in the HTC hepatoma cell line for their potential to affect cellular insulin responses. Cell viability was measured by Trypan Blue exclusion and the response to insulin evaluated by the activation of the extracellular regulated kinase (ERK1/2), ERK kinase (MEK1/2) and protein kinase B (PKB/Akt) signaling components. None of the extracts demonstrated significant cytotoxic activity. Certain extracts demonstrated a bi-phasic effect on ERK1/2 activation; low doses of the extract slightly increased ERK1/2 activation in response to insulin, whereas higher doses completely abolished the response. In contrast, none of the extracts had any significant effect on MEK whereas only a cake saponin subfraction enhanced insulin-induced PKB/Akt activation. The specific action of argan oil extracts on ERK1/2 activation made us consider an anti-proliferative action. We have thus tested other transformed cell lines (HT-1080 and MSV-MDCK-INV cells) and found similar results. Inhibition of ERK1/2 activation was also associated with decreased DNA synthesis as evidenced by [3H]thymidine incorporation experiments. These results suggest that the products of Argania spinosa may provide a new therapeutic avenue against proliferative diseases.

Functional expression of tagged human Na+—glucose cotransporter in Xenopus laevis oocytes

1 High‐affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLT1 (sodium glucose cotransporter). Though basic properties of the transporter are now defined, many questions regarding the structure‐ function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to better address these questions, we produced a functional hSGLT1 protein (from human) containing a reporter tag. 2 Six constructs, made from three tags (myc, haemaglutinin and poly‐His) inserted at both the C‐ and N‐terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLT1 construct with the myc tag inserted at the N‐terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc‐tagged and the untagged hSGLT1 proteins. 3 On the basis of both steady‐state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady‐state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4 The myc‐tagged hSGLT1 was also successfully expressed in polarized MDCK cells. α‐Methylglucose uptake studies on transfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5 These comparative studies demonstrate that the myc epitope inserted at the N‐terminus of hSGLT1 produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.

The glycocalyx maintains a cell surface pH nanoenvironment crucial for integrin-mediated migration of human melanoma cells

The glycocalyx consists of proteoglycans, glycoproteins, glycosaminoglycans, associated plasma proteins, and soluble glycosaminoglycans and covers the surface of all eukaryotic cells. It mediates specific recognition events, modulates biological processes such as ligand–receptor interactions, and has been proposed to affect tumor metastasis. Here, we studied whether the glycocalyx is required for melanoma cell migration. We diminished the glycocalyx of human melanoma cells by inhibiting posttranslational N-glycosylation or by enzymatic digestion of the N-glycosides. This partial destruction of the glycocalyx reduced melanoma cell migration by up to 60%. It was accompanied by the disintegration of a characteristic pH nanoenvironment typically surrounding migrating cells. Restoring this pH profile by stimulating the activity of the Na+/H+ exchanger NHE1 rescued cell migration even in the absence of an intact glycocalyx. The effects of partially removing the glycocalyx compared to those of knocking down β1-integrin expression points to a close functional correlation between glycocalyx, integrins, and cell surface pH nanoenvironment. We conclude that the glycocalyx is required for tumor cell migration. It stabilizes the cell surface pH nanoenvironment allowing a concerted pH-dependent interaction of adhesion receptors and extracellular matrix.

Molecular characterization of a human cation-Cl- cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport.

Cation‐Cl− cotransporters (CCCs) belong to a large family of proteins that includes 9 isoforms, two of which have still not been ascribed a transport function (CCC8 and CCC9) while the others are all known to promote Cl−‐coupled Na+ and/or K+ movement at the cell surface. The CCCs are also included in a larger family termed amino acid‐polyamine‐organocation carriers (APCs). In contrast to the CCCs, however, polyamine (PA) transporters have thus far been isolated from unicellular species exclusively and do not all belong to the APC family. In this work, we have found that a splice variant of CCC9 (CCC9a) promotes PA‐amino acid transport at the surface of HEK‐293 cells. We have also found that the influx of PAs in CCC9a‐expressing cells is inhibited by pentamidine as well as furosemide, and that it increases further in the presence of specific amino acids but not of Na+, K+, or Cl−. Hence, a group of substrates that are directly transported by CCC9 and the molecular identity of a PA transport system in animal cells may have been uncovered for the first time. These findings are of special interest given that intracellular PAs play a key role in cell proliferation. J. Cell. Physiol. 220: 680–689, 2009.

Regulation of Na+/H+ exchanger 1 allosteric balance by its localization in cholesterol‐ and caveolin‐rich membrane microdomains

The Na+/H+ exchanger 1, which plays an essential role in intracellular pH regulation in most tissues, is also known to be a key actor in both proliferative and apoptotic processes. Its activation by H+ is best described by the Monod–Wyman–Changeux model: the dimeric NHE‐1 oscillates between a low and a high affinity conformation, the balance between the two forms being defined by the allosteric constant L0. In this study, influence of cholesterol‐ and caveolin‐rich microdomains on NHE‐1 activity was examined by using cholesterol depleting agents, including methyl‐β‐cyclodextrin (MBCD). These agents activated NHE‐1 by modulating its L0 parameter, which was reverted by cholesterol repletion. This activation was associated with NHE‐1 relocation outside microdomains, and was distinct from NHE‐1 mitogenic and hormonal stimulation; indeed MBCD and serum treatments were additive, and serum alone did not change NHE‐1 localization. Besides, MBCD activated a serum‐insensitive, constitutively active mutated NHE‐1 (625KDKEEEIRK635 into KNKQQQIRK). Finally, the membrane‐dependent NHE‐1 regulation occurred independently of Mitogen Activated Protein Kinases, especially Extracellular Regulated Kinase activation, although this kinase was activated by MBCD. In conclusion, localization of NHE‐1 in membrane cholesterol‐ and caveolin‐rich microdomains constitutes a novel physiological negative regulator of NHE‐1 activity. J. Cell. Physiol. 216: 207–220, 2008.

Endothelin-1 promotes migration of endothelial cells through the activation of ARF6 and the regulation of FAK activity

Several proteins act in concert to promote remodeling of the actin cytoskeleton during migration. This process is highly regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family of proteins. Here, we show that endothelin-1 (ET-1) can promote the activation of ARF6 and migration of endothelial cells through the activation of ET(B) receptors. Inhibition of ARF6 expression using RNA interference markedly impairs basal and ET-1 stimulated cell migration. In contrast, depletion of ARF1 has no significant effect. In order to delineate the underlying mechanism, we examined the signaling events activated in endothelial cells following ET-1 stimulation. Here, we show that this hormone promotes the phosphorylation of focal adhesion kinase (FAK), Erk1/2, and the association of FAK to Src, as well as of FAK to GIT1. These have been shown to be important for the formation and turnover of focal adhesions. In non-stimulated cells, depletion of ARF6 leads to increased FAK and Erk1/2 phosphorylation, similar to what is observed in ET-1 treated cells. In these conditions, FAK is found constitutively associated with the soluble tyrosine kinase, Src. In contrast, depletion of ARF6 impairs the ability of GIT1 to form an agonist promoted complex with FAK, thereby preventing disassembly of focal adhesions. As a consequence, ARF6 depleted endothelial cells are impaired in their ability to form capillary tubes. Taken together, our data suggest that ARF6 is central in regulating focal adhesion turnover in endothelial cells. Our study provides a molecular mechanism by which, this small GTPase regulates cell motility, and ultimately angiogenesis.

Rho/ROCK-dependent pseudopodial protrusion and cellular blebbing are regulated by p38 MAPK in tumour cells exhibiting autocrine c-Met activation

Background information. The c‐Met‐dependent, β‐actin‐rich, blebbed pseudopodia of MSV‐MDCK‐INV (invasive Moloney‐sarcoma‐virus‐transformed Madin—Darby canine kidney) cells are induced by Rho/ROCK (Rho kinase) activation, and are morphologically distinct from flat extended lamellipodia.

Autocrine Activation of the Hepatocyte Growth Factor Receptor/Met Tyrosine Kinase Induces Tumor Cell Motility by Regulating Pseudopodial Protrusion

The multiple β-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFα antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFα antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line.