MINIREVIEW Osmosensing and signaling in the regulation of mammalian cell function Freimut Schliess, Roland Reinehr and Dieter Ha ¨ ussinger Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, Du ¨ sseldorf, Germany Introduction Sudden exposure of cells to hypo- or hyperosmotic solutions induces a rapid osmotic swelling or shrink- age, respectively. Extensive swelling or shrinkage is counteracted by induction of a regulatory volume decrease (RVD) or increase, respectively [1–3]. Most hypoosmotically swollen cells perform RVD by a release of inorganic ions, including K + ,Na + ,Cl – , and HCO 3 – and organic osmolytes (e.g. taurine, betaine). Hyperosmotic regulatory volume increase (RVI) at a short-term time scale is performed by activation of electrolyte uptake (e.g. via Na + ⁄ K + ⁄ 2Cl – cotransport and Na + ⁄ H + exchange). Long-term adaption to hyperosmolarity includes an isoosmotic exchange of inorganic ions against compatible organic osmolytes, which preserve protein function even at high concen- trations [4]. Transport systems involved in RVD or RVI can be activated also by hormones, substrates, second messen- gers and oxidative stress under isoosmotic conditions. In these cases, moderate and well-tolerated cell volume changes are created. For example, insulin produces a phosphoinositide 3-kinase (PI 3-kinase)-dependent hepatocyte swelling by inducing a net accumulation of ions inside the cell, which results from a concerted activation of Na + ⁄ H + exchange, Na + ⁄ K + ⁄ 2Cl – sym- port and the Na + ⁄ K + -ATPase [5]. In the early 1990s, it was recognized, that cell vol- ume changes trigger signals involved in the regulation of metabolism, gene expression and the susceptibility to different kinds of stress [6]. For example, the inhibi- tion of autophagic proteolysis by insulin, glutamine and ethanol in the perfused liver critically depends on the degree of hepatocyte swelling induced by these stimuli and can be mimicked by hypoosmotic swelling Keywords apoptosis; bile acids; CD95; cell volume; epidermal growth factor; insulin; integrins; osmolytes; oxidative stress; proliferation Correspondence F. Schliess, Heinrich-Heine-Universita ¨ t, Universita ¨ tsklinikum, Klinik fu ¨ r Gastroenterologie und Infektiologie, Moorenstrasse 5, D-40225 Du ¨ sseldorf, Germany Fax: +49 211 81 17517 Tel: +49 211 81 18941 E-mail: schliess@med.uni-duesseldorf.de (Received 2 July 2007, accepted 29 August 2007) doi:10.1111/j.1742-4658.2007.06100.x Volume changes of mammalian cells as induced by either anisoosmolarity or under isoosmotic conditions by hormones, substrates and oxidative stress critically contribute to the regulation of metabolism, gene expression and the susceptibility to stress. Osmosensing (i.e. the registration of cell volume) triggers signal transduction pathways towards effector sites (osmo- signaling), which link alterations of cell volume to a functional outcome. This minireview summarizes recent progress in the understanding of how osmosensing and osmosignaling integrate into the overall context of growth factor signaling and the execution of apoptotic programs. Abbreviations EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; PI 3-kinase, phosphoinositide 3-kinase; RGD, arginine-glycine- aspartic acid; ROS, reactive oxygen species; RVD, regulatory volume decrease; RVI, regulatory volume increase. FEBS Journal 274 (2007) 5799–5803 ª 2007 The Authors Journal compilation ª 2007 FEBS 5799 [7]. On the other hand, hyperosmotic shrinkage pre- vents insulin-induced hepatocyte swelling and proteo- lysis inhibition, indicating that swelling-dependent signaling essentially contributes to the entire response to insulin [5]. In general, cell swelling stimulates ana- bolic metabolism and proliferation and provides cyto- protection, whereas cellular shrinkage leads to catabolism and insulin resistance and sensitizes cells to apoptotic stimuli. The influence of cell volume on cell function requires structures that register volume changes (‘osmosensing’) and trigger signaling pathways towards effector sites (‘osmosignaling’). Anisoosmotically exposed cells and tissues were frequently used as a model in order to study osmosensing and osmosignaling. By this approach, the activation of signaling pathways by cell volume changes could be linked to specific functional outcomes [8]. Here, we summarize some recent pro- gress concerning the understanding of how ‘osmosen- sing’ and ‘osmosignaling’ integrate into the overall context of signal transduction, which is activated by growth factors, substrates or (pro)-apoptotic stimuli in mammalian cells with some focus on the hepatocyte. A more general treatize on osmosensing and osmosignal- ing is provided elsewhere [9]. Osmosensing in mammalian cells The investigation of osmosensing processes and struc- tures in mammalian cells considers, among others, macromolecular crowding, stretch-activated ion chan- nels, cholesterol-enriched microdomains of the plasma membrane (caveolae), intracellular organelles, ligand- independent activation of growth factor- and cytokine receptors and autocrine stimulation of signal transduc- tion by release of mediators such as ATP [10,11]. Recent studies have identified the integrin system as one major sensor of hepatocyte swelling [12–14]. Integrin-inhibitory peptides exhibiting an arginine-gly- cine-aspartic acid (RGD) motif abolish hypoosmotic osmosignaling towards Src-type kinases, mitogen-acti- vated protein kinases (MAPKs) and downstream metabolic events, including the stimulation of bile for- mation, proteolysis inhibition and volume-regulatory K + -efflux [12,13]. It should be noted that RGD pep- tides do not inhibit hypoosmotic hepatocyte swelling [13], indicating that inhibition of osmosensing at the integrin level uncouples hepatocyte swelling from osmosignaling and its functional consequences. Like hypoosmotic hepatocyte swelling [12,13], insu- lin-induced hepatocyte swelling is registrated by the integrin system, leading to a Src-dependent activation of the p38-type MAPK and thereby inhibition of autophagic proteolysis [14]. Thus, integrin-dependent cell volume sensing and signaling integrates into the overall context of insulin signaling. Similarly, sensing of glutamine-induced hepatocyte swelling by integrins feeds into Src-dependent p38 activation, which is criti- cally required for autophagic proteolysis inhibition by glutamine [13]. As demonstrated recently [15], hyperosmotic hepato- cyte shrinkage may be sensed by the endosomal com- partment. Mild hyperosmolarity (405 mosmolÆL )1 )in rat hepatocytes induces a rapid endosomal acidification by activation of vacuolar-type H + -ATPase, probably driven by an increase in intracellular Cl – concentration due to osmotic water loss and Cl – accumulation in the course of a RVI, respectively [15]. The endosomal compartment acidified by hyperosmolarity colocalized with the acidic sphingomyelinase [15]. Hyperosmolarity in hepatocytes triggers a rapid production of reactive oxygen species (ROS), which critically depends on ser- ine phosphorylation of the NADPH oxidase regulatory subunit p47 phox , which again depends on a acid sphin- gomyelinase-catalyzed ceramide production and subse- quent activation of the PKCf [15]. Bafilomycin A1 (an inhibitor of vacuolar-type H + -ATPases) and the anion channel blocker 4,4¢-diisothiocyanostilbene-2,2¢-disulf- onic acid disodium salt not only prevent endosomal acidification by hyperosmolarity, but also block the hyperosmotic increase of ceramide, p47 phox phosphory- lation and ROS [15]. The findings localize endosomal acidification most upstream in the signaling cascade underlying hyperosmotic ROS production. Osmosignaling in proliferation and apoptosis Growth factors stimulate a rapid osmolyte uptake that is important for mitogenesis [16]. For example, a rapid and transient Na + and amino acid influx is essential for the mitogenic response of 3T3 fibroblasts to growth factors [17,18]. Activation of Na + ⁄ K + ⁄ 2Cl – cotrans- port via NKCC1 was shown to be essential for cell cycle progression in 3T3 fibroblasts [17,19]. The impor- tance of ion uptake for cell cycle progression was strengthened by the finding that NKCC1 overexpres- sion in 3T3 fibroblasts induces a transformed pheno- type [20]. Cell swelling due to isoosmotic osmolyte uptake could be one mechanism that contributes to cell cycle progression. It was shown that cell water increases during the cell cycle of 3T3 fibroblasts resulting from Na + ⁄ K + ⁄ 2Cl – cotransport and glutamine uptake [17]. Likewise, hepatocyte swelling due to the activation of system A-type amino acid transporters was observed Osmosensing and signaling in mammalian cell function F. Schliess et al. 5800 FEBS Journal 274 (2007) 5799–5803 ª 2007 The Authors Journal compilation ª 2007 FEBS in vivo following partial hepatectomy, and inhibition of cell swelling antagonized liver regeneration [21]. Hypo- osmolarity in many cell types activates the MAPKs Erk1 ⁄ Erk2 and the PI 3-kinase [8], which play a major role in mitogenic signaling. Hypoosmotic exposure of HepG2 cells potentiates proliferation by a PI 3-kinase- meditated activation of the transcription factor activa- tor protein AP-1 [22], corroborating a critical role of cell swelling for cell cycle progression. Consistently, the cell volume was increased in 3T3 cells expressing oncogenic Ha-ras [23] and cell hyperhydration has been linked to tumor growth [24]. A volume decrease resulting from osmolyte release through specific transport proteins at the beginning of apoptosis (apoptotic volume decrease) is an early pre- requisite for the execution of apoptotic programs [25]. Signaling mechanisms upstream of apoptotic volume decrease depend on the cell type and stimulus under investigation and have been discussed previously [26– 28]. The contribution of apoptotic volume decrease to apoptotic signal transduction is currently not well understood. Using hyperosmotically treated cells as a model, it was shown that efficient volume regulation can protect cells from apoptosis [25,29] and it was sug- gested that the impairment of mechanisms antagoniz- ing cell shrinkage may be a general feature of apoptosis. For example, renal tubular epithelial cell apoptosis was accompanied by a caspase-dependent cleavage of the Na + ⁄ H + exchanger NHE1 [30]. How- ever, cell shrinkage is not always sufficient to trigger apoptosis [25,29]. Mechanisms that could protect cells from shrinkage-induced apoptosis include activation of the protein kinase B survival pathway [31], p53 activa- tion [32], induction of the serum- and glucocorticoid- inducible kinase Sgk [33], expression of the heat shock protein Hsp70 [34] and cyclooxygenase-2 [35], and a high antioxidant capacity [36]. In hepatocytes, a close interrelation between osmotic shrinkage and ROS production has been established. On the one hand, hyperosmotic shrinkage induces ROS production (see above) and, on the other, ROS mediate cell shrinkage. Thus, a vicious circle results, which, when not interrupted, results in apoptosis. Such a vicious circle may be activated by osmotic shrinkage or pro-apoptotic bile acids [37]. As shown in Fig. 1, hepatocyte shrinkage and ROS production are con- nected in an autoamplificatory signaling loop. Mutual amplification of shrinkage and ROS triggers apoptosis, which could be prevented by NAPDH oxidase inhibi- tors and the availability of antioxidants and osmolytes. In rat hepatocytes, mild hyperosmolarity (405 mosmolÆL )1 ) activates the CD95 system, which local- izes downstream of the ROS production triggered by endosomal acidification mentioned above [15,38]. Hyp- erosmotic CD95 activation includes trafficking of the CD95 from inside the hepatocyte to the plasma mem- brane [39], which depends on a ROS-mediated tyrosine phosphorylation of the epidermal growth factor (EGF)-receptor, the association of CD95 with the EGF-receptor, and phosphorylation of CD95 on Tyr232 and Tyr291 by the EGF-receptor tyrosine kinase activity [15]. Although the appearance of CD95 at the plasma membrane was associated with death inducing signaling complex formation and activation of caspases 3 and 8, mild hyperosmolarity was not suf- ficient to induce hepatocyte apoptosis [39], suggesting that apoptotic signals under this condition are counter- balanced by yet unknown survival signals. However, more severe hyperosmolarity (‡ 505 mosmolÆL )1 ) shifts the balance towards hepatocyte apoptosis [15]. Like hyperosmolarity, CD95 ligand in hepatocytes via generation of ROS induced EGF-receptor tyrosine phosphorylation, CD95 ⁄ EGF-receptor association, CD95 tyrosine phosphorylation, trafficking of the CD95 to the plasma membrane surface, and death inducing signaling complex formation, leading to the execution of apoptosis in this case [38]. Although inef- fective to induce apoptosis by itself, hyperosmolarity (405 mosmolÆL )1 ) sensitized the hepatocytes towards CD95 ligand-induced apoptosis [39], indicating a Fig. 1. Hepatocyte shrinkage and the production of reactive oxygen species constitute an autoamplificatory signaling loop. Hyperosmot- ic shrinkage triggers a NADPH oxidase-catalyzed ROS formation. ROS, again by stimulating K + -efflux, antagonize processes under- lying the RVI, and thereby increase hepatocyte shrinkage by hyper- osmolarity. Mutual amplification of swelling and oxidative stress may be limited by the hepatocyte’s antioxidant and volume-regula- tory capacity. Adapted from [37]. F. Schliess et al. Osmosensing and signaling in mammalian cell function FEBS Journal 274 (2007) 5799–5803 ª 2007 The Authors Journal compilation ª 2007 FEBS 5801 synergistic interplay between signals triggered by hyperosmotic shrinkage and CD95 ligand, respectively. Likewise, hyperosmolarity sensitized H4IIE rat hepa- toma cells to pro-apoptotic signaling by the protea- some inhibitor MG-132 [40]. These and other studies on hyperosmotically shrun- ken cells support the view that the apoptotic volume decrease, if not even producing de novo death signals, at least can amplify apoptotic signals released by dif- ferent apoptotic stimuli. Thus, the apoptotic volume decrease may further disarrange the balance between survival and death signals, thereby promoting execu- tion of the apoptotic program. Concluding remarks It is well acknowledged that cell volume fluctuations release signals of (patho)physiological relevance. The understanding of how cell swelling integrates into the cell cycle machinery and how cell shrinkage sensitizes cells to apoptotic stimuli requires further scientific effort. Cell hydration may markedly affect the action of drugs. For example cell hydration changes may switch the outcome of proteasome inhibitors from a nontoxic or even protective one to injury and apopto- sis [40]. Although routine monitoring of cell hydration in patients would provide valuable information in clin- ical medicine, this is currently limited by methodologi- cal difficulties. Acknowledgements Our own studies were supported by Deutsche Fors- chungsgemeinschaft through Sonderforschungsbereich 575 ‘Experimentelle Hepatologie’ (Du ¨ sseldorf). References 1 Chamberlin ME & Strange K (1989) Anisosmotic cell volume regulation: a comparative view. Am J Physiol 257, C159–C173. 2 Parker JC (1993) In defense of cell volume? Am J Phys- iol 265, C1191–C1200. 3 Hoffmann EK & Pedersen SF (2006) Sensors and signal transduction pathways in vertebrate cell volume regula- tion. Contrib Nephrol 152, 54–104. 4 Burg MB (1995) Molecular basis of osmotic regulation. Am J Physiol 37, F983–F996. 5 Schliess F, vom Dahl S & Ha ¨ ussinger D (2001) Insulin resistance by loop diuretics and hyperosmolarity in perfused rat liver. Biol Chem 382, 1063–1069. 6Ha ¨ ussinger D & Lang F (1992) Cell volume and hor- mone action. Trends Pharmacol Sci 13, 371–373. 7 vom Dahl S & Ha ¨ ussinger D (1996) Nutritional state and the swelling-induced inhibition of proteolysis in perfused rat liver. J Nutr 126, 395–402. 8Ha ¨ ussinger D & Schliess F (1999) Osmotic induction of signaling cascades: role in regulation of cell function. Biochem Biophys Res Comm 255, 551–555. 9Ha ¨ ussinger D & Sies H, eds (2007) Osmosensing and osmosignaling. Methods Enzymol. Vol 428, Academic Press, in press. 10 Hamill OP & Martinac B (2001) Molecular basis of mechanotransduction in living cells. Physiol Rev 81, 685–740. 11 Ha ¨ ussinger D, Reinehr RM & Schliess F (2006) The hepatocyte integrin system and cell volume sensing. Acta Physiol 187, 249–255. 12 Ha ¨ ussinger D, Kurz AK, Wettstein M, Graf D, vom Dahl S & Schliess F (2003) Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis. Gastroenterology 124, 1476–1487. 13 vom Dahl S, Schliess F, Reissmann R, Go ¨ rg B, Weiergra ¨ ber O, Kocalkova M, Dombrowski F & Ha ¨ ussinger D (2003) Involvement of integrins in osmo- sensing and signaling toward autophagic proteolysis in rat liver. J Biol Chem 278, 27088–27095. 14 Schliess F, Reissmann R, Reinehr R, vom Dahl S & Ha ¨ ussinger D (2004) Involvement of integrins and Src in insulin signaling towards autophagic proteolysis in rat liver. J Biol Chem 279, 21294–21301. 15 Reinehr R, Becker S, Braun J, Eberle A, Grether-Beck S&Ha ¨ ussinger D (2006) Endosomal acidification and activation of NADPH oxidase isoforms are upstream events in hyperosmolarity-induced hepatocyte apoptosis. J Biol Chem 281, 23150–23166. 16 Rozengurt E (1986) Early signals in the mitogenic response. Science 234, 161–166. 17 Bussolati O, Uggeri J, Belletti S, Dallasta V & Gazzola GC (1996) The stimulation of Na ⁄ K ⁄ 2Cl cotransport and of system AS for neutral amino acid transport is a mechanism for cell volume increase during the cell cycle. FASEB J 10, 920–926. 18 Berman E, Sharon I & Atlan H (1995) An early tran- sient increase of intracellular Na + may be one of the first components of the mitogenic signal. Direct detec- tion by 23Na-NMR spectroscopy in quiescent 3T3 mouse fibroblasts stimulated by growth factors. Biochim Biophys Acta 1239, 177–185. 19 Koch KS & Leffert HL (1979) Increased sodium influx is necessary to initiate rat hepatocyte proliferation. Cell 18, 153–163. 20 Panet R & Atlan H (2000) Overexpression of the Na + ⁄ K + ⁄ 2Cl – cotransporter gene induces cell prolifera- tion and phenotypic transformation in mouse fibro- blasts. J Cell Physiol 182, 109–118. Osmosensing and signaling in mammalian cell function F. Schliess et al. 5802 FEBS Journal 274 (2007) 5799–5803 ª 2007 The Authors Journal compilation ª 2007 FEBS 21 Freeman TL, Ngo HQ & Mailliard ME (1999) Inhibi- tion of system A amino acid transport and hepatocyte proliferation following partial hepatectomy in the rat. Hepatology 30, 437–444. 22 Kim RD, Roth TP, Darling CE, Ricciardi R, Schaffer BK & Chari RS (2001) Hypoosmotic stress stimulates growth in HepG2 cells via protein kinase B-dependent activation of activator protein-1. J Gastrointest Surg 5, 546–555. 23 Fu ¨ rst J, Haller T, Chwatal S, Wo ¨ ll E, Dartsch PC, Gschwentner M, Dienstl A, Zwierzina H, Lang F, Paul- michl M et al. (2002) Simvastatin inhibits malignant transformation following expression of the Ha-ras onco- gene in NIH 3T3 fibroblasts. Cell Physiol Biochem 12, 19–30. 24 McIntyre GI (2006) Cell hydration as the primary factor in carcinogenesis: a unifying concept. Med Hypotheses 66, 518–526. 25 Maeno E, IshizakiY, Kanaseki T, Hazama A & OkadaY (2000) Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97, 9487–9492. 26 Schliess F & Ha ¨ ussinger D (2002) The cellular hydra- tion state: a critical determinant for cell death and sur- vival. Biol Chem 383, 577–583. 27 Lang F, Huber SM, Szabo I & Gulbins E (2007) Plasma membrane ion channels in suicidal cell death. Arch Biochem Biophys 462, 189–194. 28 Bortner CD & Cidlowski JA (2007) Cell shrinkage and monovalent cation fluxes: role in apoptosis. Arch Bio- chem Biophys 462, 176–188. 29 Maeno E, Takahashi N & OkadaY (2006) Dysfunction of regulatory volume increase is a key component of apoptosis. FEBS Lett 580, 6513–6517. 30 Wu KL, Khan S, Lakhe-Reddy S, Wang L, Jarad G, Miller RT, Konieczkowski M, Brown AM, Sedor JR & Schelling JR (2003) Renal tubular epithelial cell apopto- sis is associated with caspase cleavage of the NHE1 Na + ⁄ H + exchanger. Am J Physiol 284, F829–F839. 31 Wu KL, Khan S, Lakhe-Reddy S, Jarad G, Mukherjee A, Obejero-Paz CA, Konieczkowski M, Sedor JR & Schelling JR (2004) The NHE1 Na + ⁄ H + exchanger recruits ezrin ⁄ radixin ⁄ moesin proteins to regulate Akt- dependent cell survival. J Biol Chem 279, 26280–26286. 32 Dmitrieva NI & Burg MB (2005) Hypertonic stress response. Mutat Res 569, 65–74. 33 Leong ML, Maiyar AC, Kim B, O’Keeffe BA & Firestone GL (2003) Expression of the serum- and gluco- corticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells. J Biol Chem 278, 5871–5882. 34 Shim EH, Kim JI, Bang ES, Heo JS, Lee JS, Kim EY, Lee JE, Park WY, Kim SH, Kim HS et al. (2002) Tar- geted disruption of hsp70.1 sensitizes to osmotic stress. EMBO Rep 3, 857–861. 35 Neuhofer W, Holzapfel K, Fraek ML, Ouyang N, Lutz J & Beck FX (2004) Chronic COX-2 inhibition reduces medullary HSP70 expression and induces papillary apoptosis in dehydrated rats. Kidney Int 65, 431–441. 36 Schliess F & Ha ¨ ussinger D (2005) The cellular hydra- tion state: role in apoptosis and proliferation. Signal Transduct 6, 297–302. 37 Becker S, Reinehr R, Graf D, vom Dahl S & Ha ¨ ussinger D (2007) Hydrophobic bile acids induce hepatocyte shrinkage via NADPH oxidase activation. Cell Physiol Biochem 19, 89–98. 38 Reinehr R, Schliess F & Ha ¨ ussinger D (2003) Hyper- osmolarity and CD95L trigger CD95 ⁄ EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation. FASEB J 17, 731–733. 39 Reinehr RM, Graf D, Fischer R, Schliess F & Ha ¨ us- singer D (2002) Hyperosmolarity triggres CD95 mem- brane trafficking and sensitizes rat hepatocytes towards CD95L-induced apoptosis. Hepatology 36, 602–614. 40 Lornejad-Scha ¨ fer MR, Scha ¨ fer C, Richter L, Grune T, Ha ¨ ussinger D & Schliess F (2005) Osmotic regulation of MG-132-induced MAP-kinase phosphatase MKP-1 expression in H4IIE rat hepatoma cells. Cell Physiol Biochem 16, 193–206. F. Schliess et al. Osmosensing and signaling in mammalian cell function FEBS Journal 274 (2007) 5799–5803 ª 2007 The Authors Journal compilation ª 2007 FEBS 5803 . MINIREVIEW Osmosensing and signaling in the regulation of mammalian cell function Freimut Schliess, Roland Reinehr and Dieter Ha ¨ ussinger Clinic. and thereby inhibition of autophagic proteolysis [14]. Thus, integrin-dependent cell volume sensing and signaling integrates into the overall context of insulin