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Non-alcoholic fatty liver disease (NAFLD) is the fastest-growing cause of liver-associated death globally. Whole-body knockout (KO) of Na+/H+ exchanger 1 (NHE1, SLC9A1) was previously proposed to protect against high-fat diet-induced liver damage; however, mechanistic insight was lacking. The aim of the present work was to address this question in vitro to determine how NHE1, specifically in hepatocytes, impacts lipid overload-induced inflammation, fibrosis, and hepatocyte--hepatic stellate cell (HSC) crosstalk. We induced palmitate (PA)-based steatosis in AML12 and HepG2 hepatocytes; manipulated NHE1 activity pharmacologically and by CRISPR/Cas9-mediated KO and overexpression; and measured intracellular pH (pHi), steatosis-associated inflammatory and fibrotic mediators, and cell death. PA treatment increased NHE1 mRNA levels but modestly reduced NHE1 protein expression and hepatocyte pHi. NHE1 KO in hepatocytes did not alter lipid droplet accumulation but reduced inflammatory signaling (p38 MAPK activity), lipotoxicity (4-HNE accumulation), and apoptosis (poly-ADP-ribose-polymerase-1 (PARP) cleavage). Conditioned medium from PA-treated hepatocytes increased the expression of NHE1 and of the fibrosis regulator tissue inhibitor of matrix metalloproteinases-2 in LX-2 HSCs, in a manner abolished by NHE1 KO in hepatocytes. We conclude that NHE1 is regulated in NAFLD in vitro and contributes to the ensuing damage by aggravating hepatocyte injury and stimulating hepatocyte--HSC crosstalk. [ABSTRACT FROM AUTHOR]

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal β-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression. [ABSTRACT FROM AUTHOR]

Solid tumors are characterized by a microenvironment that is highly acidic, while intracellular pH (pH,) is normal or even elevated. This is the result of elevated metabolic rates in the highly proliferative cancer cells, in conjunction with often greatly increased rates of net cellular acid extrusion. Studies in various cancers have suggested that while the acid extrusion mechanisms employed are generally the same as those in healthy cells, the specific transporters upregulated vary with the cancer type. The main such transporters include [Na.sup.+]/[H.sup.+] exchangers, various HC[O.sub.3.sup.-]transporters, [H.sup.+] pumps, and lactate-[H.sup.+] cotransporters. The mechanisms leading to their dysregulation in cancer are incompletely understood but include changes in transporter expression levels, trafficking and membrane localization, and posttranslational modifications. In turn, accumulating evidence has revealed that in addition to supporting their elevated metabolic rate, their increased acid efflux capacity endows the cancer cells with increased capacity for invasiveness, proliferation, and chemotherapy resistance. The pancreatic duct exhibits an enormous capacity for acid-base transport, rendering pHi dysregulation a potentially very important topic in pancreatic ductal adenocarcinoma (PDAC). PDAC--accounting for about 90% of all pancreatic cancers--has one of the highest cancer mortality rates known, and new diagnostic and treatment options are highly needed. However, very little is known about whether pH regulation is altered in PDAC and, if so, the possible role of this in cancer development. Here, we review current models for pancreatic acid-base transport and pH homeostasis and summarize current views on acid-base dysregulation in cancer, focusing where possible on the few studies to date in PDAC. Finally, we present new data-mining analyses of acid-base transporter expression changes in PDAC and discuss essential directions for future work. Key words: PDAC, NHE, MCT, [H.sup.+]/[K.sup.+]-ATPase, V-ATPase, NBCs, [H.sup.+], proton, bicarbonate, metabolism. Les tumeurs solides se caracterisent par un microenvironnement tres acide, alors que le pH intracellulaire est normal, voire eleve. Ceci est le resultat de taux metaboliques eleves dans les cellules cancereuses hautement proliferatives, en conjonction avec des taux d'extrusion acide souvent fortement accrus. Des etudes portant sur differents cancers ont suggere qu'alors que les mecanismes d'extrusion acide utilises sont generalement les memes que chez les cellules saines, les transporteurs specifiques regules a la hausse varient en fonction du type de cancer. Les principaux transporteurs incluent les echangeurs [Na.sup.+]/[H.sup.+], differents transporteurs de HC[O.sub.3.sup.-], les pompes a [H.sup.+] et les cotransporteurs lactate-[H.sup.+]. Les mecanismes qui conduisent a leur deregulation dans le cancer sont incompletement compris, mais ils incluent des changements des niveaux d'expression, du trafic et de la localisation membranaire des transporteurs, et les modifications post-traductionnelles. Par ailleurs, de plus en plus de donnees revelent qu'en plus de soutenir leur taux metabolique eleve, leur capacite accrue d'efflux acide dote les cellules cancereuses d'une capacite accrue d'invasion, de proliferation et de chimioresistance. Le canal pancreatique possede une enorme capacite de transport acide-base, faisant de la deregulation du pHi un element potentiellement tres important des adenocarcinomes canalaires pancreatiques (ACCP). L'ACCP--qui constitue environ 90% de tous les cancers pancreatiques--possede un des taux de mortalite connus les plus eleves, et de nouvelles options de diagnostic et de traitement sont absolument requises. Cependant, on ignore si la regulation du pH est modifiee chez les ACCP, et, le cas echeant, si elle joue un role dans le developpement du cancer. Les auteurs passent ici en revue les modeles actuels de transport acide-base et d'homeostasie du pH dans le pancreas, et ils resument les connaissances actuelles de la deregulation acide-base dans le cancer, en se concentrant lorsque possible sur les quelques etudes realisees a ce jour sur l'ACCP. Finalement, ils presentent de nouvelles analyses en forage de donnees des changements de l'expression du transporteur acide-base chez les ACCP, et discutent des directions qu'il sera essentiel de suivre dans les travaux a venir. [Traduit par la Redaction] Mots-cles: ACCP, NHE, MCT, [H.sup.+]/[K.sup.+]-ATPase, V-ATPase, NBC, [H.sup.+], proton, bicarbonate, metabolisme., Introduction Recent years have seen a surge of interest in how dysregulation of intra- and extracellular pH ([pH.sub.i], [pH.sub.e]) occurs early in cellular transformation and contributes, via multiple different mechanisms, [...]

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with minimal treatment options and a global rise in prevalence. PDAC is characterized by frequent driver mutations including KRAS and TP53 (p53), and a dense, acidic tumor microenvironment (TME). The relation between genotype and TME in PDAC development is unknown. Strikingly, when wild type (WT) Panc02 PDAC cells were adapted to growth in an acidic TME and returned to normal pH to mimic invasive cells escaping acidic regions, they displayed a strong increase of aggressive traits such as increased growth in 3‐dimensional (3D) culture, adhesion‐independent colony formation and invasive outgrowth. This pattern of acidosis‐induced aggressiveness was observed in 3D spheroid culture as well as upon organotypic growth in matrigel, collagen‐I and combination thereof, mimicking early and later stages of PDAC development. Acid‐adaptation‐induced gain of cancerous traits was further increased by p53 knockout (KO), but only in specific extracellular matrix (ECM) compositions. Akt‐ and Transforming growth factor‐β (TGFβ) signaling, as well as expression of the Na+/H+ exchanger NHE1, were increased by acid adaptation. Whereas Akt inhibition decreased spheroid growth regardless of treatment and genotype, stimulation with TGFβI increased growth of WT control spheroids, and inhibition of TGFβ signaling tended to limit growth under acidic conditions only. Our results indicate that a complex crosstalk between tumor acidosis, ECM composition and genotype contributes to PDAC development. The findings may guide future strategies for acidosis‐targeted therapies. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers globally, with a 5-year overall survival of less than 10%. The development and progression of PDAC are linked to its fluctuating acidic tumor microenvironment. Ion channels act as important sensors of this acidic tumor microenvironment. They transduce extracellular signals and regulate signaling pathways involved in all hallmarks of cancer. In this study, we evaluated the interplay between a pH-sensitive ion channel, the calcium (Ca2+) channel transient receptor potential C1 (TRPC1), and three different stages of the tumor microenvironment, normal pH, acid adaptation, and acid recovery, and its impact on PDAC cell migration, proliferation, and cell cycle progression. In acid adaptation and recovery conditions, TRPC1 localizes to the plasma membrane, where it interacts with PI3K and calmodulin, and permits Ca2+ entry, which results in downstream signaling, leading to proliferation and migration. Thus, TRPC1 exerts a more aggressive role after adaptation to the acidic tumor microenvironment. Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a low overall survival rate of less than 10% and limited therapeutic options. Fluctuations in tumor microenvironment pH are a hallmark of PDAC development and progression. Many ion channels are bona fide cellular sensors of changes in pH. Yet, the interplay between the acidic tumor microenvironment and ion channel regulation in PDAC is poorly understood. In this study, we show that acid adaption increases PANC-1 cell migration but attenuates proliferation and spheroid growth, which are restored upon recovery. Moreover, acid adaptation and recovery conditions favor the plasma membrane localization of the pH-sensitive calcium (Ca2+) channel transient receptor potential C1 (TRPC1), TRPC1-mediated Ca2+ influx, channel interaction with the PI3K p85α subunit and calmodulin (CaM), and AKT and ERK1/2 activation. Knockdown (KD) of TRPC1 suppresses cell migration, proliferation, and spheroid growth, notably in acid-recovered cells. KD of TRPC1 causes the accumulation of cells in G0/G1 and G2/M phases, along with reduced expression of CDK6, −2, and −1, and cyclin A, and increased expression of p21CIP1. TRPC1 silencing decreases the basal Ca2+ influx in acid-adapted and -recovered cells, but not in normal pH conditions, and Ca2+ chelation reduces cell migration and proliferation solely in acid adaptation and recovery conditions. In conclusion, acid adaptation and recovery reinforce the involvement of TRPC1 in migration, proliferation, and cell cycle progression by permitting Ca2+ entry and forming a complex with the PI3K p85α subunit and CaM. [ABSTRACT FROM AUTHOR]

Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of [K.sup.+], [Na.sup.+], and [Cl.sup.-] ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in [G.sub.0], [G.sub.1], and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of [Na.sup.+] and [Cl.sup.-] were reduced in the [G.sub.0]-[G.sub.1] phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after [Na.sup.+] and [Cl.sup.-] substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of [Na.sup.+] was observed to upregulate the activity of the [Na.sup.+]/[H.sup.+] exchanger NHE1 as well as of [Na.sup.+]-independent acid extrusion mechanisms, facilitating intracellular pH ([pH.sub.i]) recovery after acid loading and increasing [pH.sub.i]. Results using the potential sensitive dye [DiBaC.sub.4](3) showed a reduced [Cl.sup.-] conductance in S compared with [G.sub.1] followed by transmembrane potential ([E.sub.m]) hyperpolarization in S. [Cl.sup.-] substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with [G.sub.1]. Our results suggest that [Na.sup.+] regulates ELA cell proliferation by regulating intracellular pH while [Cl.sup.-] may regulate proliferation by fine-tuning of [E.sub.m] in S phase and altered [Ca.sup.2+] signaling. cell cycle; chloride channel; volume regulatory anion channel; [Na.sup.+]/[H.sup.+] exchanger 1 doi: 10.1152/ajpcell.00445.2009.

The [Ca.sup.2+]-independent phospholipase [A.sub.2] VI ([iPLA.sub.2]-VI) and the [Na.sup.+][H.sup.+] exchanger isoform l (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH ([pH.sub.o]) in ischemia-reperfusion injury and death and in regulation and function of [iPLA.sub.2]-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% [O.sub.2], 8 mM [K.sup.+], and 20 mM lactate) at [pH.sub.o] 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of [iPLA.sub.2]-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of [iPLA.sub.2]-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content, [iPLA.sub.2]-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a [pH.sub.o]-dependent manner during SI/R. This also affected the subcellular localization of [iPLA.sub.2]-VIA. Thus, the mode of cell death and the roles and regulation of [iPLA.sub.2]-VI and NHE1 are at least in part determined by the [pH.sub.o] during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of [iPLA.sub.2]-VIA and NHE1 during cardiac I/R. cytoskeleton; cell death; extracellular pH; NHE1; [iPLA.sub.2]; EIPA; necrosis

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) [alpha]--mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-[alpha] and the [Na.sup.+]/[H.sup.+] exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in [Tg737.sup.orpk] MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-[alpha] ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5'-(N-ethyl-N-isopropyl)amiloride potently reduces PDGF-AA-mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in [Tg737.sup.orpk] MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-[alpha] signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-[alpha] stimulation.

Dysregulation of the transient receptor canonical ion channel (TRPC1) has been found in several cancer types, yet the underlying molecular mechanisms through which TRPC1 impacts pancreatic ductal adenocarcinoma (PDAC) cell proliferation are incompletely understood. Here, we found that TRPC1 is upregulated in human PDAC tissue compared to adjacent pancreatic tissue and this higher expression correlates with low overall survival. TRPC1 is, as well, upregulated in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, and its knockdown (KD) reduced cell proliferation along with PANC-1 3D spheroid growth by arresting cells in the G1/S phase whilst decreasing cyclin A, CDK2, CDK6, and increasing p21CIP1 expression. In addition, the KD of TRPC1 neither affected Ca2+ influx nor store-operated Ca2+ entry (SOCE) and reduced cell proliferation independently of extracellular calcium. Interestingly, TRPC1 interacted with the PI3K-p85α subunit and calmodulin (CaM); both the CaM protein level and AKT phosphorylation were reduced upon TRPC1 KD. In conclusion, our results show that TRPC1 regulates PDAC cell proliferation and cell cycle progression by interacting with PI3K-p85α and CaM through a Ca2+-independent pathway. [ABSTRACT FROM AUTHOR]