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A, B Hyposmolarity-induced epidermal growth factor receptor (EGFR) phosphorylation in Swiss 3T3 fibroblast cell line cultures. Serum-starved cells (24 h) were exposed for 3 min to the conditions indicated, fixed, incubated overnight with phosphoEGFR (Tyr 845) antibody, incubated with a secondary fluorescent antibody and visualized by confocal microscopy in sections collected at 0.5-µm intervals. The images shown are from the second section in the series. A Isosmotic medium (a); 35% hyposmotic (H35%) medium (b); isosmotic medium plus EGF (200 ng/ml) (c); 35% hyposmotic medium plus EGF (d) and 35% hyposmotic medium plus 50 µM AG213 (e). Bar 10 µm. B Quantitative summary of fluorescence intensity. Means±SE from analysis of five fields, containing 10-15 cells each, from at least three independent experiments. *P<0.001 vs. isosmotic; § P<0.001 vs. H35%
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Exposure of cultured Swiss 3T3 fibroblasts to 35% hyposmotic solution activated epidermal growth factor receptor (EGFR) phosphorylation to a greater extent than the ligand, EGF. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) had the same effect. EGFR phosphorylation seems to be involved in the transduction signalling for hyposmotically indu...
Citations
... Previous results obtained from immunocytochemistry studies suggested that certain lectins activate the EGFR in the absence of a cognate ligand. 56 However, detailed investigations of lectin-promoted EGFR activation have not yet been performed. Thus, we utilized EGFR128-AZDye 488 to determine if lectin binding to glycans of the EGFR induces EGFR activation. ...
The epidermal growth factor receptor (EGFR) is a cell-surface glycoprotein that is involved mainly in cell proliferation. Overexpression of this receptor is intimately related to the development of a broad spectrum of tumors. In addition, glycans linked to the EGFR are known to affect its EGF-induced activation. Because of the pathophysiological significance of the EGFR, we prepared a fluorescently labeled EGFR (EGFR128-AZDye 488) on the cell surface by employing the genetic code expansion technique and bioorthogonal chemistry. EGFR128-AZDye 488 was initially utilized to investigate time-dependent endocytosis of the EGFR in live cells. The results showed that an EGFR inhibitor and antibody suppress endocytosis of the EGFR promoted by the EGF, and that lectins recognizing glycans of the EGFR do not enhance EGFR internalization into cells. Observations made in studies of the effects of appended glycans on the entry of the EGFR into cells indicate that a de-sialylated or de-fucosylated EGFR is internalized into cells more efficiently than a wild-type EGFR. Furthermore, by using the FRET-based imaging method of cells which contain an EGFR linked to AZDye 488 (a FRET donor) and cellular glycans labeled with rhodamine (a FRET acceptor), sialic acid residues attached to the EGFR were specifically detected on the live cell surface. Taken together, the results suggest that a fluorescently labeled EGFR will be a valuable tool in studies aimed at gaining an understanding of cellular functions of the EGFR.
... Downstream of RTKs are phosphoinositide 3 kinase (PI3K), 3-phosphoinositide-dependent protein kinase-1 (PDK1) and protein kinase B (PKB, also known as Akt) [130]. In Swiss 3T3 fibroblasts, hyposmolarity activates the EGFR/PI3K/Akt axis, resulting in taurine efflux [131]. The muscle isoform of myosin II isolated from rabbit skeletal muscle binds to the pleckstrin homology (PH) domain of PKB [132]. ...
Changes in plasma membrane curvature and intracellular ionic strength are two key features of cell volume perturbations. In this hypothesis we present a model of the responsible molecular apparatus which is assembled of two molecular motors [non-muscle myosin II (NMMII) and protrusive actin polymerization], a spring [a complex between the plasma membrane (PM) and the submembrane actin-based cytoskeleton (smACSK) which behaves like a viscoelastic solid] and the associated signaling proteins. We hypothesize that this apparatus senses changes in both the plasma membrane curvature and the ionic strength and in turn activates signaling pathways responsible for regulatory volume increase (RVI) and regulatory volume decrease (RVD). During cell volume changes hydrostatic pressure (HP) changes drive alterations in the cell membrane curvature. HP difference has opposite directions in swelling versus shrinkage, thus allowing distinction between them. By analogy with actomyosin contractility that appears to sense stiffness of the extracellular matrix we propose that NMMII and actin polymerization can actively probe the transmembrane gradient in HP. Furthermore, NMMII and protein-protein interactions in the actin cortex are sensitive to ionic strength. Emerging data on direct binding to and regulating activities of transmembrane mechanosensors by NMMII and actin cortex provide routes for signal transduction from transmembrane mechanosensors to cell volume regulatory mechanisms.
... It has been suggested that the decrease in tension during cell shrinkage causes the clustering and activation of epidermal growth factor (EGF) receptors in some cell types [112,113]. In addition, EGF receptors on Swiss 3T3 fibroblasts and the ErbB4 EGF receptor on cerebellar granular neurons are activated by membrane stretching [114,115], which regulates taurine efflux via the PI3K-PKB and MEK1/2-ERK1/2 pathways [114]. ...
... It has been suggested that the decrease in tension during cell shrinkage causes the clustering and activation of epidermal growth factor (EGF) receptors in some cell types [112,113]. In addition, EGF receptors on Swiss 3T3 fibroblasts and the ErbB4 EGF receptor on cerebellar granular neurons are activated by membrane stretching [114,115], which regulates taurine efflux via the PI3K-PKB and MEK1/2-ERK1/2 pathways [114]. ...
Cells are constantly exposed to the risk of volume perturbation under physiological conditions. The increase or decrease in cell volume accompanies intracellular changes in cell membrane tension, ionic strength/concentration and macromolecular crowding. To avoid deleterious consequences caused by cell volume perturbation, cells have volume recovery systems that regulate osmotic water flow by transporting ions and organic osmolytes across the cell membrane. Thus far, a number of biomolecules have been reported to regulate cell volume. However, the question of how cells sense volume change and modulate volume regulatory systems is not fully understood. Recently, the existence and significance of phaseseparated biomolecular condensates have been revealed in numerous physiological events, including cell volume perturbation. In this review, we summarize the current understanding of cell volume-sensing mechanisms, introduce recent studies on biomolecular condensates induced by cell volume change and discuss how biomolecular condensates contribute to cell volume sensing and cell volume maintenance. In addition to previous studies of biochemistry, molecular biology and cell biology, a phase separation perspective will allow us to understand the complicated volume regulatory systems of cells.
... These results indicate that integrin β 1 plays an important role in RVD in adherent ELA cells, whereas it has no effect on RVD in nonadherent EATC-WT. Pharmacological evidence has indicated that release of the organic osmolyte taurine, which is known to contribute to RVD, is regulated by RTKs, FAK, RhoA, and members of the Src family, which are all kinases known to be regulated by integrin clustering and activation [53][54][55][56]. We therefore investigated the effect of integrin β 1 knockdown on swelling-induced taurine efflux via the volume sensitive organic anion channel (VSOAC). ...
Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin β1, in cell volume regulation and drug-induced apoptosis in adherent and non-adherent Ehrlich ascites cell lines.
Adhesion phenotypes were verified by colorimetric cell-adhesion-assay. Quantitative real-time PCR and western blot were used to compare expression levels of integrin subunits. Small interfering RNA was used to silence integrin β1 expression. Regulatory volume decrease (RVD) after cell swelling was studied with calcein-fluorescence-self-quenching and Coulter counter analysis. Taurine efflux was estimated with tracer technique. Caspase assay was used to determine apoptosis.
We show that adherent cells have stronger fibronectin binding and a significantly increased expression of integrin α5, αv, and β1 at mRNA and protein level, compared to non-adherent cells. Knockdown of integrin β1 reduced RVD of the adherent but not of the non-adherent cells. Efflux of taurine was unaffected. In contrast to non-adherent, adherent cells exhibited chemoresistance to chemotherapeutic drugs (cisplatin and gemcitabine). However, knockdown of integrin β1 promoted cisplatin-induced caspase activity in adherent cells.
Our data identifies integrin β1 as a part of the osmosensing machinery and regulator of cisplatin resistance in adherent Ehrlich cells. © 2015 S. Karger AG, Basel.
... PI3K is activated by cell swelling [114] possibly through activation of the epidermal growth factor receptor (EGFR) [115], G protein-coupled receptors and tyrosine kinase receptors, resulting in generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 , see [116]). Taurine release, activated by either cell swelling (Fig. 2D) or by cell swelling in combination with thrombininduced PAR-receptor activation, is significantly decreased by PI3K inhibition with wortmanin [18,94,115]. ...
... PI3K is activated by cell swelling [114] possibly through activation of the epidermal growth factor receptor (EGFR) [115], G protein-coupled receptors and tyrosine kinase receptors, resulting in generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 , see [116]). Taurine release, activated by either cell swelling (Fig. 2D) or by cell swelling in combination with thrombininduced PAR-receptor activation, is significantly decreased by PI3K inhibition with wortmanin [18,94,115]. Generation of PIP 3 , by PI3K results in activation of the so-called AGC-kinases including notably members such as PDK-1, PKC, Akt/PKB, PKA and serum and glycocorticoide kinase (SGK), several of which are involved in control of proliferation, programmed cell death (apoptosis) and ion transport (see [117]). ...
Maintaining cell volume is critical for cellular function yet shift in cell volume is a prerequisite for mitosis and apoptosis. The ubiquitously and evolutionary conserved serine/threonine kinase CK2 promotes cell survival and suppresses apoptosis. The present review describes how mammalian cells regulate the cellular content of the major cellular organic osmolyte, taurine with emphasis on CK2 mediated regulation of active taurine uptake and volume-sensitive taurine release. Furthermore, we discuss how CK2-mediated regulation of taurine homeostasis is potentially involved in cellular functions such as proliferation and survival.
... A significant body of evidence obtained from non-neural cells suggests that both receptor TKs (such as epidermal growth factor; Franco et al., 2004) and nonreceptor TKs may play an important role in GPCR regulation of osmolyte release, with members of the src family being the preferred candidate (Vazquez-Juarez et al., 2008b). These observations are consistent with the known "cross-talk" between GPCR and TK signaling pathways (Luttrell and Luttrell, 2004). ...
Cell swelling can have profound deleterious effects in the brain and is observed to occur during several pathological conditions. Upon swelling, cells regulate their volume through the extrusion of various osmolytes. Prior studies in both chronically hyponatremic rat brains and cultures of primary astrocytes suggest that individual osmolytes are differentially utilized during cell volume regulation. In these models organic osmolytes are depleted from cells, whereas inorganic osmolytes are comparatively retained. Although selective osmolyte depletion has been appreciated for a number of years, the mechanism whereby this occurs has remained unknown. Activation of certain G-protein-coupled receptors, including muscarinic cholinergic receptors (mAChRs), has been demonstrated to non-selectively stimulate the release of both organic and inorganic osmolytes. However, the ability of the same receptors to regulate osmolyte influx has not been examined. I discovered that hypotonicity and receptor activation stimulated both the efflux and influx of K+ (monitored with 86Rb+) in SH-SY5Y cells and cultures of primary rat astrocytes. Furthermore, in SH-SY5Y cells, these fluxes (mediated primarily by K+ channels for efflux, and the Na+/K+ATPase and NKCC transporters for influx) were found to be of a similar magnitude so as to permit the retention of intracellular K+ during physiologically-relevant reductions in osmolarity. In contrast, taurine uptake (mediated via the taurine transporter) was inhibited by hypotonicity and mAChR activation in SH-SY5Y cells and cultured astrocytes. This process, when combined with increased taurine efflux, would promote taurine depletion. I also demonstrated that activation of mAChRs on SH-SY5Y cells, under isotonic conditions, resulted in an increased glutamate uptake (monitored as 3H-D-aspartate) and redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. However, hypotonicity inhibited mAChR-mediated glutamate uptake and disrupted EAAT3 trafficking. Such a process may permit glutamate to be conserved within cells during small reductions in osmolarity, whereas depletion would occur under more hyposmotic conditions. Together, these findings suggest that GPCR-mediated regulation of osmolyte influx represents a potential mechanism whereby the selective depletion or retention of osmolytes is mediated.
... Several reports have pointed to a role for receptor tyrosine kinases in osmosensing, although, similar to integrins, these receptors have been implicated in both the swelling-and shrinkage-activated responses. The EGF rececptor in Swiss 3T3 fibroblasts (247) and keratocytes (449) and other cell types (771), and the ErbB4 EGF receptor in cerebellar granular neurons (548), are activated by hypotonic cell swelling, resulting in activation of the PI3K-PKB and MEK1/2-ERK1/2 pathways and volume regulatory taurine efflux (247). ...
... Several reports have pointed to a role for receptor tyrosine kinases in osmosensing, although, similar to integrins, these receptors have been implicated in both the swelling-and shrinkage-activated responses. The EGF rececptor in Swiss 3T3 fibroblasts (247) and keratocytes (449) and other cell types (771), and the ErbB4 EGF receptor in cerebellar granular neurons (548), are activated by hypotonic cell swelling, resulting in activation of the PI3K-PKB and MEK1/2-ERK1/2 pathways and volume regulatory taurine efflux (247). ...
... A role for tyrosine phosphorylation events in modulation of swelling-induced taurine release is suggested by the inhibitory effect of tyrosine kinase blockers (genistein, tyrphostins) and potentiating effect of tyrosine phosphatase inhibitors on the efflux (492,662,670,920). Pharmacological evidence indicates that the tyrosine kinases regulating the taurine efflux pathway include receptor tyrosine kinases (247,492), FAK, and members of the Src family (391,492). ...
The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review.
... There is general agreement that TK activity is required for the activation of VSOAC in neural tissues, based upon the ability of inhibitors such as genistein or tyrphostins to attenuate the efflux of organic osmolytes and Cl -, and of inhibitors of tyrosine phosphatases to potentiate VSOAC activation (Sinning et al. 1997;Crepel et al. 1998;Mongin et al. 1999a;Deleuze et al. 2000;Morales-Mulia et al. 2001;Heacock et al. 2004;Cohen 2005). A significant body of evidence obtained from non-neural cells suggests that both receptor TKs (such as epidermal growth factor; Franco et al. 2004) and non-receptor TKs and may also play an important role in GPCR regulation of osmolyte release, with a member of the src family being the preferred candidate (see Vazquez-Juarez et al. 2008b). These observations are consistent with the known 'cross-talk' between GPCR and TK signaling pathways (Luttrell and Luttrell 2004). ...
The CNS is particularly vulnerable to reductions in plasma osmolarity, such as occur during hyponatremia, the most commonly encountered electrolyte disorder in clinical practice. In response to a lowered plasma osmolarity, neural cells initially swell but then are able to restore their original volume through the release of osmolytes, both inorganic and organic, and the exit of osmotically obligated water. Given the importance of the maintenance of cell volume within the CNS, mechanisms underlying the release of osmolytes assume major significance. In this context, we review recent evidence obtained from our laboratory and others that indicates that the activation of specific G-protein-coupled receptors can markedly enhance the volume-dependent release of osmolytes from neural cells. Of particular significance is the observation that receptor activation significantly lowers the osmotic threshold at which osmolyte release occurs, thereby facilitating the ability of the cells to respond to small, more physiologically relevant, reductions in osmolarity. The mechanisms underlying G-protein-coupled receptor-mediated osmolyte release and the possibility that this efflux can result in both physiologically beneficial and potentially harmful pathophysiological consequences are discussed.
... So far, different candidates have been proposed including different members of the src family of tyrosine kinases (such as src, syk, lyn and lck) (Lepple-Wienhues et al., 1998;Musch et al., 1999;Hubert et al., 2000;Browe and Baumgarten, 2003;Walsh and Zhang, 2005;Vazquez-Juarez et al., 2008); and the focal adhesion kinase (FAK) (Tilly et al., 1996;Browe and Baumgarten, 2003;Walsh and Zhang, 2005;Lezama et al., 2005b). Other tyrosine phosphorylation-dependent signaling pathways have also been proposed to participate in these phenomenon including the phosphatidyl-inositide 3-kinase (PI3K) (Tilly et al., 1996;Feranchak et al., 1998;Morales-Mulia et al., 2001;Shi et al., 2002;de La Paz et al., 2002;Wang et al., 2004;Franco et al., 2004aFranco et al., , 2001Ren et al., 2008), and the mitogen-activated (ERK1/ERK2) and stress-activated protein kinases (JNK and p38) (Crepel et al., 1998;Shen et al., 2001;vom Dahl et al., 2003;Franco et al., 2004b;Pan et al., 2007). In any case, the role of these signaling pathways seems to vary according to the cell type studied. ...
... Growth factor receptor signaling has been recently reported to regulate cell volume regulation. Insulin and epidermal growth factor (EGF) have been reported to stimulate RVD, osmolyte and ionic fluxes upon hyposmotic shock (Tilly et al., 1993;Miyauchi et al., 2000;Shi et al., 2002;Abdullaev et al., 2003;Franco et al., 2004a;Lezama et al., 2005b). Moreover, agonistic stimulation of tyrosine kinase receptors activates osmolyte efflux in the absence of cell swelling (Tilly et al., 1993;Bali et al., 2001;Varela et al., 2004;Franco et al., 2004a;Browe and Baumgarten, 2006). ...
... Insulin and epidermal growth factor (EGF) have been reported to stimulate RVD, osmolyte and ionic fluxes upon hyposmotic shock (Tilly et al., 1993;Miyauchi et al., 2000;Shi et al., 2002;Abdullaev et al., 2003;Franco et al., 2004a;Lezama et al., 2005b). Moreover, agonistic stimulation of tyrosine kinase receptors activates osmolyte efflux in the absence of cell swelling (Tilly et al., 1993;Bali et al., 2001;Varela et al., 2004;Franco et al., 2004a;Browe and Baumgarten, 2006). Accordingly, inhibition of EGFR has been reported to inhibit swelling-induced Cl À and taurine release (Du et al., 2004;Franco et al., 2004a;Ren and Baumgarten, 2005;Lezama et al., 2005b;Browe and Baumgarten, 2006;Ren et al., 2008). ...
Cell volume regulation is a basic homeostatic mechanism transcendental for the normal physiology and function of cells. It is mediated principally by the activation of osmolyte transport pathways that result in net changes in solute concentration that counteract cell volume challenges in its constancy. This process has been described to be regulated by a complex assortment of intracellular signal transduction cascades. Recently, several studies have demonstrated that alterations in cell volume induce the release of a wide variety of transmitters including hormones, ATP and neurotransmitters, which have been proposed to act as extracellular signals that regulate the activation of cell volume regulatory mechanisms. In addition, changes in cell volume have also been reported to activate plasma membrane receptors (including tyrosine kinase receptors, G-protein coupled receptors and integrins) that have been demonstrated to participate in the regulatory process of cell volume. In this review, we summarize recent studies about the role of changes in cell volume in the regulation of transmitter release as well as in the activation of plasma membrane receptors and their further implications in the regulation of the signaling machinery that regulates the activation of osmolyte flux pathways. We propose that the autocrine regulation of Ca2+-dependent and tyrosine phosphorylation-dependent signaling pathways by the activation of plasma membrane receptors and swelling-induced transmitter release is necessary for the activation/regulation of osmolyte efflux pathways and cell volume recovery. Furthermore, we emphasize the importance of studying these extrinsic signals because of their significance in the understanding of the physiology of cell volume regulation and its role in cell biology in vivo, where the constraint of the extracellular space might enhance the autocrine or even paracrine signaling induced by these released transmitters.
... A number of reports have also implicated a role of RTKs in osmosensing and volume control as upstream regulators of, e.g., MAPKs and the PI3K/Akt pathway. A prominent example is that of the EGF receptor (EGFR) family, including the EGFR in Swiss 3T3 fibroblasts and ErbB4 in cerebellar granule neurons, which are activated by cell swelling induced by hypoosmotic stress following the activation of PI3K/Akt and/or MEK1/2-ERK1/2 pathways (17,41). Stimulation of the EGFR by cell swelling has also been reported in other cell types (41,54). ...
... It is presently unknown by which mechanisms hypertonic stress affects the activity of the receptor, but our data add further evidence to the role of RTKs in sensing osmotic stress and/or in the registration of changes in cell volume that impinge upon signaling in development and tissue homeostasis. Previously, members of the EGFR family have been shown to become activated upon cell swelling by hypotonic stress in fibroblasts (17,54), keratinocytes (32), kidney cells, and cerebellar granule neurons (41,54), in which an increase in cell volume activates EGFRs at a level comparable with that of its natural ligands. Since we were unable to detect any changes in PDGFR- activity in fibroblasts upon cell swelling induced by hypotonic stress in either the absence (Fig. 7, B and C) or presence of PDGF-BB (Fig. 7D), we suggest that diverse types of RTKs may respond differently to changes in cell volume and/or osmotic stress, although it is currently unknown whether ligand-dependent activation of the EGFR is affected by hypertonic stress in these cells. ...
Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-beta, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-beta activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand-dependent activation of PDGFR-beta and its downstream effectors Akt, MEK1/2, and ERK1/2 is strongly modulated (inhibited) by hyperosmotic cell shrinkage, whereas cell swelling does not seem to affect the activation of the receptor but rather to activate ERK1/2 via a different mechanism. It is thus likely that cell swelling via activation of ERK1/2 and cell shrinkage via activation of the p38 and JNK pathway and inhibition of the PDGFR signaling pathway may act as key players in the regulation of tissue homeostasis.
