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Several attempts have been made to deliver reactive oxygen and nitrogen species (RONS) produced by non-thermal plasma onto large surfaces in a controllable way compatible with skin treatments. In this paper, the effect of pulse frequency, in the range 0.5–20 kHz, on the discharge behavior was studied on skin tissue models to evaluate potential treatment changes. This has been done through electrical characterization, visualization of the helium flow (by Schlieren technique), produced plasma jet modifications (ICCD imaging) and RONS measurements. The results show that, in addition to its well known important role in the production of the chemical species, the applied discharge frequency plays a very significant role in the size of the treated surface. An enhancement of NO*, OH* and O* production in the gas phase at the higher frequency is reported and assigned to the stronger mixing of the helium flow with ambient air. The efficacy of plasma jet on transporting RONS on/into agarose gel and pig skin has been evaluated. The distribution of the reactive species on the target, or passing through, is strongly dependent on the discharge frequency and consequently induces pH variations. The present study supports a new way for enlarging the treated surface by using a simple jet at high frequency in the 20 kHz range, leading, with appropriate gas flow and distance to target, to conditions of RONS production that are compatible with potential uses for biomedical or cosmetic applications.

International audience; The maintenance of skin integrity is crucial to ensure the physiological barrier against exogenous compounds, microorganisms and dehydration but also to fulfill social and aesthetic purposes. Besides the development of new actives intended to enter a formulation, innovative technologies based on physical principles have been proposed in the last years. Among them, Cold Atmospheric Plasma (CAP) technology, which already showed interesting results in dermatology, is currently being studied for its potential in skin treatments and cares. CAP bio-medical studies gather several different expertise ranging from physics to biology through chemistry and biochemistry, making this topic hard to pin. In this review we provide a broad survey of the interactions between CAP and skin. In the first section, we tried to give some fundamentals on skin structure and physiology, related to its essential functions, together with the main bases on cold plasma and its physicochemical properties. In the following parts we dissected and analyzed each CAP parameter to highlight the already known and the possible effects they can play on skin. This overview aims to get an idea of the potential of cold atmospheric plasma technology in skin biology for the future developments of dermo-cosmetic treatments, for example in aging prevention.

International audience

International audience; Cold atmospheric plasmas (CAPs) have become great challenge due to their vast potentialfor biomedical applications. Theresearch activities in the biological field range from the study of wound healing, cancer treatment, dentistry, sterilization and decontamination,treatment of implants for biocompatibility, dermatology and cosmetics. The biological effects of CAP are strongly linked to generation of reactive oxygen and nitrogen species (RONS). An important issue in application of CAP for tissue treatment concerned the penetration of RONS into biological tissue. RONS distribution and penetration can vary from tissue to tissue and, depending on their concentration, the biological effect can be beneficial or deleterious.In the present work, an agarose gel phantom is used as surrogates of human skin tissue to study its barrier effects on transportation and distribution of RONS after CAP treatment. We showed how the plasma conditions such as the gap distance, the treatment time and thehigh voltage frequency influence the RONS deposition, distribution and diffusion through the target. We also developed a new technique to visualize and quantify the local acidification induced by CAP treatment. These data together with our plasma source characterizationhelp us to determine safe treatment parameters for cosmetic application of CAP on the skin. This work was supportedby Cosmetosciences, a global training and research program dedicated to the cosmetic industry, located in the heart of the Cosmetic Valley, this program led by University of Orléans through the Région Centre-Val de Loire, France

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Skin injury leading to chronic wounds is of high interest due to the increasing number of patients suffering from this symptom. Proliferation, migration, and angiogenesis are key factors in the wound healing processes. For that reason, controlled promotion of these processes is required. In this work, we present the portable helium-dielectric barrier discharge (He-DBD)-based reaction-discharge system of controlled gas temperature for biological activities. To make this He-DBD-based reaction-discharge system safe for biological purposes, a multivariate optimization of the operating parameters was performed. To evaluate the effect of the He-DBD operating parameters on the rotational gas temperature Trot(OH), a design of experiment followed by a Response Surface Methodology was applied. Based on the suggested statistical model, the optimal operating conditions under which the Trot(OH) is less than 37 °C (310 K) were estimated. Then, the resulted model was validated in order to confirm its accuracy. After estimation the optical operating conditions of He-DBD operation, the spectroscopic characteristic of the He-DBD-based reaction-discharge system in relevance to the several optical temperatures in addition to electron number density has been carried out. Additionally, the qualitative and quantitative analyses of the reactive oxygen species and reactive nitrogen species were performed in order to investigate of reactions and processes running in the He-DBD-gaseous phase and in the He-DBD-treated liquid. Next, the developed portable He-DBD-based reaction-discharge system, working under the optimal operating conditions, was used to stimulate the wound healing process. It was found that a 30 s He-DBD treatment significantly increased the proliferation, migration, and angiogenesis of keratinocytes (HaCaT) and fibroblasts (MSU-1.1) cell lines, as well as human skin microvascular endothelial cells (HSkMEC.2). Hence, the application of the cold atmospheric pressure plasma generated in this He-DBD-based reaction-discharge system might be an alternative therapy for patient suffering from chronic wounds.

The biological effects induced by cold atmospheric plasmas (CAPs) on human tissues are mainly due to the production of reactive oxygen and nitrogen species (RONS). Some RONS are also responsible for pH lowering of the treated medium. The CAP-induced acidification has beneficial effect on biological tissues, contributing to the anti-bacterial effect and to the healing improvement observed in treated wound. In this work we investigated the local acidification induced by a helium CAP treatment using tissue models made of agarose gels with adjusted pH around 7.4 to mimic generic organs or around 5.5 to simulate skin surface pH. Using fluorescein as a pH-sensitive fluorescent marker, we developed a useful technique to visualize and quantify the local acidification induced by CAP exposure of tissue surface. The different capillaries used to produce the plasma jet, the treatment time, the initial pH of the surface and the buffer capacity of the tissue model were shown to modulate both the size of the impacted surface and the intensity of the pH decrease. The proposed technique can be advantageous to study the acidifying effect induced by plasma. This method can help to plan safe and controlled plasma treatments in order to avoid hyper-acidification of the tissue, especially when a localized treatment is administered.

International audience

Despite the large number of papers showing the biological effect of cold atmospheric plasmas (CAPs) on skin treatment and although their ability to produce a cocktail of reactive oxygen and nitrogen species (RONS) is largely known, the deep knowledge of these ionized gas sources is far to be uncovered. Many parameters take part in the complex mechanisms induced by plasma, such as voltage, frequency, feeding gas flow rate, temperature, air humidity, and atmospheric gas composition. In this paper, we focus our attention on the influence of oxygen dissolved in the medium in RONS generation during CAP treatment. We evaluate RONS production in Dulbecco’s phosphate-buffered saline equilibrated with two different percentages of oxygen: 18% and 3% O2. The former oxygen concentration is that usually used in most of cell culture and treatment (normoxia) while the latter is close to the physiological level measured inside human skin (physioxia). Intracellular RONS generation upon CAP treatment was also measured in two human skin cell lines maintained either in normoxic or in physioxic conditions. Our data suggest that RONS generation induced by plasma either in the medium or inside cells is influenced by oxygen dissolved in the treated target.

International audience; With the increase of human span life people demand to live and look better. In a world whereappearance is synonymous of health, the demand of new cosmetic has literally exploded. Amongcosmetic treatments, skin care represents a large part of the business of beauty. From 2017 to2023, world-wide non-surgical skin cosmetic treatments business will have a compound annualgrowth rate between 4.7% and 5.3% [1]. The increased demand of non-surgical beautytreatments pushed cosmetic research during the last years. In Europe since 2013 cosmetic testson animal model are definitively banned. This encouraged researchers to develop newapproaches to study skin “in-vitro” using either re-constructed tissue structure or attempting tomimic skin microenvironment parameters. Cell culture has been used since a long time to studyorgan functions in laboratory. In the last decades cell culture has greatly evolved in order tomimic the real tissue structure and microenvironment. Cells are cultivated in three dimensionsand media are adapted to better simulate the extracellular environment. Attempting to get closerto the physiological micro-environment of a cell, scientists underestimated often a keyparameter: the oxygen level [2]. While oxygen represents the 21% of the air gases, in humantissues, its percentage is significantly lower. In skin, it can vary from 7% to 1%. To be as closeas possible to skin microenvironment, in our lab we grow human skin cells either in classicnormoxic condition (18% O2) or in physioxic condition (3% O2). Oxygen has a key role in cellrespiration and in reactive oxygen species (ROS) production. We have already demonstrated thatskin cells raised in physioxia produce lower quantity of reactive oxygen and nitrogen species(RONS) even when exposed to plasma treatment [3]. In this study we evaluated the effect of aCold Atmospheric Plasma (CAP) treatment on human skin cell raised either in normoxia or inphysioxia. In particular, modulating plasma parameters from our helium Plasma Gun, weinvestigated the ability of CAP to improve cell viability and extracellular matrix production suchas collagen, hyaluronic acid and elastin, macromolecules involved in maintaining the health andbeauty of the skin.

Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease- dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the inva- dopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na + /H + exchanger 1 (NHE1)-driven extracellular acidifica - tion, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs pref- erentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1-dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidifi - cation and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.

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Background: FAD synthase is known to catalyze the biosynthesis of FAD in cytosol and mitochondria. Results: The existence of a nuclear FAD synthase and a FAD-hydrolyzing activity is demonstrated. Conclusion: A dynamic pool of FAD exists in the nucleus. Significance: Nuclear, mitochondrial, and cytosolic FAD synthase pools constitute a flavin network involved in the regulation of cellular metabolism and epigenetic events. FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg−1 protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min−1·mg−1 protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

International audience; Nowadays the increases of life expectation in developed world has radically changed people demand and their shopping basket. Since medical research has assuring physical wellness until old age, today people ask not only to stay healthy but also to look young. For this reason in the last decades cosmetic research has literally exploded. New generation cosmetics has appeared on the market and small companies started to develop plasma devices for skin care. Although cold plasma is already used in cosmetics, very little is known about the beneficial mechanism that lead to skin renewing. Cold plasma in fact could produce opposite effects on cells. It could either lead to cell death or stimulate their growth. We have also demonstrated that respecting skin microenvironment and particularly its physiological oxygen level (called physioxia) in in vitro experiments is crucial to be as close as possible to in situ skin [1, 2]. It is especially important for cold plasma treatments as the level of oxygen is closely related to the production of reactive oxygen and nitrogen species (RONS), components of the produced plasma. In this work, we characterized the chemico-physical properties of a He plasma gun [3, 4] (run at 14kV, 2 kHz, He flow of 0.5 sml at 10 mm above target) and its effects on culture media and on skin cells. In particular we analyzed O 2 partial pressure (see Fig. 1), RONS species produced, pH modification, effects on dissolved O 2 levels and electric field generation in order to understand plasma treatment consequences and to define the best settings to achieve a stimulating effect on skin cells. This will be first evaluated in physioxia versus normoxia, by assessing cell viability and motility on human keratinocytes and dermal fibroblasts. These results bring new elements to better understand the physiological response of skin cells to plasma treatment and will help to develop new strategies for cosmetical use. Fig. 1: O 2 pressure and temperature variations versus time in medium (7mm high) in a well of 96 well plate This work was supported by ARD 2020 Cosmetoscience PLASMACOSM project.

International audience; The last decade has seen an impressive development of the research dedicated to the biomedical applications of Low Temperature Non Thermal Plasmas (LTNTP), ionized gases at temperature close to the ambient one. This is especially true with plasma sources working at atmospheric pressure allowing direct applications on human. Medical applications of LTNTP now concern a very wide range of domains from wound healing [1] to cancer treatment [2,3] or transdermal drug delivery [4] . These applications also include dermatology closely linked to cosmetic. At atmospheric pressure, LTNTP are mainly produced by Dielectric Barrier Discharges (DBD) or by single/multi plasma jets, both types based on electrical discharges at very low power. The produced plasmas emit light in wide wavelength range and contain charged species and reactive species which concentrations depend on discharge configuration and electrical parameters. In addition, LTNTP produce external electric field which can, not only, be applied locally in combination with the species mentioned above, but also alone. All of their characteristics render them very attractive for potential applications in cosmetic especially anti-aging through skin structure modifications, hydration, and oxygenation or skin care product delivery. In this presentation, after a very brief reminder on the plasma state, we will first present the different ways to produce atmospheric LTNTP and discuss about their properties (including those related to transient electric field), in particular the ones of clear interest for cosmetic. Then we will give a brief review on the ongoing biomedical plasma research and applications, with a special focus on the ones related to dermatology treatments. We will end by a survey of possible developments and use in cosmetic.AcknowledgmentsThis work is supported by the ARD2020 COSMETSCIENCES Project “PLASMACOSM”References[1] J Heinlin, G Isbary, et a,l JEADV (2011) 25, 1–11[2] H.R. Metelmann et al Clin. Plas Med. doi.org/10.1016/j.cpme.2015.02.001[3] L. Brullé et al, PLOS ONE, 7, DOI: 10.1371/journal.pone.0052653 (2012)[4] S. Khalgathi, ISPC 22nd proceedings, O-22-6 (2015)

L'action des plasmas froids hors équilibre est désormais largement démontrée à la fois in vitro et in vivo (sur des modèles animaux [1,2] et sur l’homme [3,4]) sur de très nombreuses lignées cellulaires, divers types de tumeurs et de maladies. Néanmoins, en raison de la grande variété des composants du plasma capables de jouer un rôle, la compréhension précise de la chaîne de processus conduisant aux effets observés est loin d'être atteinte. Il est difficile d'isoler ou de mesurer l’action individuelle de chaque composante du plasma ou, au contraire, d'évaluer le bénéfice ou la synergie potentielle de leur combinaison. Jusqu'à présent, de nombreuses expériences in vitro ont été réalisées avec cette intention, mais leur transposition in vivo est très compliquée, même, dans de nombreux cas, impossible en raison de l'environnement complexe des cellules in vivo, notamment la vascularisation (impliquant le flux sanguin et l'oxygénation/physioxie) et la réponse du corps (signalisation, recrutement du système immunitaire). En général, la première étape concerne les études consacrées à la viabilité des cellules in vitro ou des études de toxicité dans un milieu de culture avec l'analyse à la fois des changements induits dans les cellules et dans le milieu. Il faut souligner que les conditions expérimentales utilisées, ainsi que le fait de traiter les cellules dans des plaques multipuits en dehors de leur chambre d'incubation peut générer un stress cellulaire qui peut affecter leur comportement et conduire à des résultats biaisés. De plus, même si les jets de plasmas, très largement utilisés dans les applications biomédicales, sont des systèmes relativement simples dans leur conception (principalement à base de réacteur DBD), il n’en reste pas moins que leur étude est complexe, particulièrement dans l’environnement des cibles biologiques. Les interactions entre le plasma lui-même, le débit de gaz et la cible (liquide, tissu, surface inerte) peuvent changer radicalement la production et les interactions des espèces réactives et des espèces chargées pendant les traitements. Enfin, le champ électrique transitoire généré par le jet de plasma peut interagir avec le milieu environnant et, éventuellement, agir en synergie avec les autres composants actifs du plasma délivrés sur la cible.

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na(+)/H(+) exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous DeltaF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent DeltaF508 CFBE41o- and DeltaF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors.

The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0–8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires membrane and released proteases focalized at membrane structures called invadopodia. While extracellular acidification is important in driving tumor invasion, the structure/function mechanisms underlying this regulation are still unknown. Invadopodia are similar in structure and function to osteoclast podosomes responsible for bone degradation, and extracellular acidification is central to podosome action, suggesting that it could also be for invadopodial function. Here, utilizing a novel system for in situ zymography in native matrices, we show that the Na(+)/H(+) exchanger (NHE1) and NHE1-generated extracellular acidification are localized at and necessary for invadopodial-dependent ECM degradation, thereby promoting tumor invasion. Stimulation with EGF increased both NHE1-dependent proton secretion and ECM degradation. Manipulation of the NHE1 expression by RNA interference or activity via either transport-deficient mutation or the specific inhibitor cariporide confirmed that NHE1 expression and activity are required for invadopodia-mediated ECM degradation. Taken together, our data show a concordance among NHE1 localization, the generation of a well-defined acidic extracellular pH in the nanospace surrounding invadopodia, and matrix-degrading activity at invadopodia of human malignant breast carcinoma cells, providing a structural basis for the role of NHE1 in invasion and identifying NHE1 as a strategic target for therapeutic intervention.

BACKGROUND:Neoplastic transformation originates from a large number of different genetic alterations. Despite this genetic variability, a common phenotype to transformed cells is cellular alkalinization. We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 (HPV16), that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na/(+)H(+) exchanger isoform 1 (NHE1) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice. METHODOLOGY:Here, we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1. PRINCIPAL FINDINGS:We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell. Further, using pharmacological agents and transient transfections with dominant interfering, constitutively active, phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1, we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein, RhoA, and its subsequent inhibition. CONCLUSIONS:All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation.

Many physiological and pathophysiological processes, such as embryogenesis, immune defense, wound healing, or metastasis, are based on cell migration and invasion. The activity of the ubiquitously expressed NHE1 isoform of the plasma membrane Na + /H + exchanger is one of the requirements for directed locomotion of migrating cells. The mechanisms by which NHE1 is involved in cell migration are multiple. NHE1 contributes to cell migration by affecting the cell volume, by regulating the intracellular pH and thereby the assembly and activity of cytoskeletal elements, by anchoring the cytoskeleton to the plasma membrane, by the organization of signal transduction and by regulating gene expression. The present review focuses on two additional, extracellular mechanisms by which NHE1 activity contributes to cell migration and invasion. Protons extruded by the NHE1 lead to local, extracellular acidification which, on the one hand, can create pH optima needed for the activity of proteinases at invadopodia/podosomes necessary for extracellular matrix digestion and, on the other hand, facilitates cell/matrix interaction and adhesion at the cell front.

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na+/H+exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1α expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na+/H+exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.

Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.