Laboratoire de PhysioMédecine Moléculaire

Here, we report the isolation of a human multipotent adipose-derived stem (hMADS) cell population from adipose tissue of young donors. hMADS cells display normal karyotype; have active telomerase; proliferate >200 population doublings; and differentiate into adipocytes, osteoblasts, and myoblasts. Flow cytometry analysis indicates that hMADS cells are CD44+, CD49b+, CD105+, CD90+, CD13+, Stro-1(-), CD34-, CD15-, CD117-, Flk-1(-), gly-A(-), CD133-, HLA-DR(-), and HLA-I(low). Transplantation of hMADS cells into the mdx mouse, an animal model of Duchenne muscular dystrophy, results in substantial expression of human dystrophin in the injected tibialis anterior and the adjacent gastrocnemius muscle. Long-term engraftment of hMADS cells takes place in nonimmunocompromised animals. Based on the small amounts of an easily available tissue source, their strong capacity for expansion ex vivo, their multipotent differentiation, and their immune-privileged behavior, our results suggest that hMADS cells will be an important tool for muscle cell-mediated therapy.

Na+/H+ exchanger reverse transcription-polymerase chain reaction mitogen-activated protein kinase NHE regulatory factor Maintaining intracellular pH values close to neutrality is a crucial task for a wide variety of cells. Hence, various mechanisms for pH regulation have been selected early in evolution and are ubiquitously distributed. Among the actors in this scene, the members of the Na+/H+ exchanger gene family (NHE1 isoforms) are widely expressed and constitute extremely efficient systems for protecting cells against internal acidification. To date, at least six genes have been identified in mammalian cells, and to various extents, the corresponding proteins have been molecularly and functionally characterized. In this short review, we will update our current knowledge on these NHE family members and highlight the most important aspects of the basic function of these transporters. Then, in a broader physiological context, we will present what we think are the most prominent specific features of the different NHE isoforms.Structural and Functional Domains of NHEsThe first cDNA encoding the NHE-1 isoform was cloned using an expression strategy based on the ability of Na+/H+ exchangers to protect antiporter-deficient cells (1Pouysségur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (438) Google Scholar) against otherwise lethal intracellular acidification (2Sardet C. Franchi A. Pouysségur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (668) Google Scholar). Variations in the hormonal regulation and pharmacological features of Na+/H+ exchange were the first indications that a large family of Na+/H+ exchange molecules existed (3Clark J.D. Limbird L.E. Am. J. Physiol. 1991; 261: C945-C953Crossref PubMed Google Scholar). Therefore, using the NHE-1 cDNA as a probe led to the molecular identification of the NHE-2, -3, and -4 (4Tse C.M. Brant S.R. Walker M.S. Pouysségur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 5Tse C.M. Levine S.A. Yun C.H. Montrose M.H. Little P.J. Pouysségur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, 6Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 7Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar) isoforms. In addition, non-epithelial isoforms such as NHE-5 (8Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. et al.Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar, 9Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 10Sun A.M. Liu Y. Centracchio J. Dworkin L.D. J. Membr. Biol. 1998; 164: 293-300Crossref PubMed Scopus (24) Google Scholar) and NHE-6 have been cloned recently. By contrast to the other Na+/H+ exchangers, NHE-6 is not expressed at the plasma membrane but in the mitochondria (11Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar).Topological Features and Sequence ConservationThe highly hydrophobic N-terminal region of the protein is predicted to span the membrane 10–12 times depending on the algorithm used to calculate the hydropathy plot of the protein. In particular, the region situated in the central part of the transmembrane domain (residues 226–281 in the human NHE-1) is quite hydrophobic but contains several negatively charged residues. By contrast the C-terminal region of the Na+/H+ exchangers is hydrophilic and has been shown to be located in the cell cytoplasm, at least for the NHE-1 isoform (12Shrode L.D. Gan B.S. D'Souza S.J. Orlowski J. Grinstein S. Am. J. Physiol. 1998; 275: C431-C439Crossref PubMed Google Scholar, 13Sardet C. Counillon L. Franchi A. Pouysségur J. Science. 1990; 247: 723-726Crossref PubMed Scopus (375) Google Scholar). Interestingly, recent experiments on the NHE-3 isoform seem to indicate that epitopes within the C-terminal region of this protein are extracellularly exposed (14Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 1998; 273: 12391-12396Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). For NHE-1 and -2, the loop between the putative transmembrane segments 1 and 2 is glycosylated and therefore extracellular (15Counillon L. Pouysségur J. Reithmeier R.A. Biochemistry. 1994; 33: 10463-10469Crossref PubMed Scopus (123) Google Scholar, 16Tse C.M. Levine S.A. Yun C.H. Khurana S. Donowitz M. Biochemistry. 1994; 33: 12954-12961Crossref PubMed Scopus (75) Google Scholar). Methods such as scanning N-glycosylation mutagenesis (17Popov M. Tam L.Y. Li J. Reithmeier R.A. J. Biol. Chem. 1997; 272: 18325-18332Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) will be necessary to gain further topological information.Sequence Comparison between NHE IsoformsThe transmembrane domain exhibits from 45 to 65% amino acid identity, although this score drops to about 25–35% for the cytoplasmic domain. A more detailed analysis of the sequence homology clusters reveals the presence of two subfamilies of isoforms that have probably diverged later in evolution: NHE-2 and -4 as well as NHE-3 and -5. The central part of the domain (putative transmembrane segments 5a and 5b between residues 226 and 281 in human NHE-1) is nearly identical between all the NHE isoforms. This part of the polypeptide possesses negatively charged residues (aspartates 226, 238, and 267 and glutamates 247, 248, 253, and 262 in the human NHE-1) included in a highly hydrophobic stretch of sequence, and the substitution of Glu-262 in the NHE-1 isoform results in the inactivation of the transporter (18Fafournoux P. Noel J. Pouysségur J. J. Biol. Chem. 1994; 269: 2589-2596Abstract Full Text PDF PubMed Google Scholar). Although it is not possible to rule out an indirect effect of this mutation, this result in association with the extreme sequence conservation of this region among the NHE members strongly suggests that these two transmembrane segments of the exchangers constitute the catalytic core of the Na+/H+ exchangers.By contrast, the first putative transmembrane segment of the Na+/H+ exchangers and the first extracellular loop are not well conserved in the NHE family. These N-terminal sequences are divergent even in the same isoform cloned from various mammalian species (19Counillon L. Pouysségur J. Biochim. Biophys. Acta. 1993; 1172: 343-345Crossref PubMed Scopus (23) Google Scholar), indicating that sequence conservation in the first extracellular loop is not crucial for the function of the protein. A closer analysis of the first stretch of hydrophobic residues using the von Heijne rules (20von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3686) Google Scholar, 21von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (854) Google Scholar) reveals that this first putative transmembrane segment has the features of a signal peptide, including a positively charged N-terminal end and a relatively short hydrophobic stretch.Although the cytosolic domain sequence seems to be more poorly conserved, alignment methods based on the presence of hydrophobic secondary structures (hydrophobic score analysis) (22Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar, 23Callebaut I. Labesse G. Durand P. Poupon A. Canard L. Chomilier J. Henrissat B. Mornon J.P. Cell. Mol. Life Sci. 1997; 53: 621-645Crossref PubMed Scopus (430) Google Scholar) show that these C-terminal domains clearly exhibit structural similarities. 2L. Counillon, unpublished results. Recently, circular dichroism measurements performed on the Escherichia coli-expressed NHE-1 C-terminal region confirmed that this part of the protein possesses a high degree of structural organization (24Gebreselassie D. Rajarathnam K. Fliegel L. Biochem. Cell Biol. 1998; 76: 837-842Crossref PubMed Scopus (20) Google Scholar).Physiological Roles and Regulation of NHE IsoformsThe NHE-1 isoform is expressed in virtually all cells and tissues, although the expression pattern of the other NHE isoforms exhibits striking variation among different tissues. The least ambiguous expression pattern is that of NHE-3, which is highly expressed in the kidney (proximal tubule, thin and thick limbs of the loop of Henle) (59Soleimani M. Singh G. Bizal G.L. Gullans S.R. McAteer J.A. J. Biol. Chem. 1994; 269: 27973-27978Abstract Full Text PDF PubMed Google Scholar) and intestine (jejunum, ileum, ascending and descending colon, and rectum) (60Dujeda P.K. Rao D.D. Syed I. Joshi V. Dahdal R.Y. Gardner C. Risk M.C. Schmidt L. Bavishi D. Kim K.E. Harig J.M. Goldstein J.L. Layden T.J. Ramaswamy K. Am. J. Physiol. 1996; 271: G438-G493PubMed Google Scholar). Whereas NHE-1 is found mostly on the basolateral membrane of epithelial cells (61Coupaye-Gerard B. Bookstein C. Duncan P. Chen X.Y. Smith P.R. Musch M. Ernst S.A. Chang E.B. Kleyman T.R. Am. J. Physiol. 1996; 271: C1639-C1645Crossref PubMed Google Scholar) or both in the basolateral and apical membranes in epithelial cell lines such as opossum kidney or Madin-Darby canine kidney cells (62Noel J. Roux D. Pouysségur J. J. Cell Sci. 1996; 109: 929-939PubMed Google Scholar), NHE-3 is specifically targeted to the apical membrane (62Noel J. Roux D. Pouysségur J. J. Cell Sci. 1996; 109: 929-939PubMed Google Scholar, 63Hoogerwerf W.A. Tsao S.C. Devuyst O. Levine S.A. Yun C.H. Yip J.W. Cohen M.E. Wilson P.D. Lazenby A.J. Tse C.M. Donowitz M. Am. J. Physiol. 1996; 270: G29-G41PubMed Google Scholar).NHE-2, like NHE-3, has been detected both in intestine and kidney and is also targeted to the apical membrane of epithelial cells. However, whereas the presence of NHE-2 in the intestine has been confirmed by independent investigations (see for example Ref. 60Dujeda P.K. Rao D.D. Syed I. Joshi V. Dahdal R.Y. Gardner C. Risk M.C. Schmidt L. Bavishi D. Kim K.E. Harig J.M. Goldstein J.L. Layden T.J. Ramaswamy K. Am. J. Physiol. 1996; 271: G438-G493PubMed Google Scholar), the expression of this protein in the kidney is somewhat controversial (59Soleimani M. Singh G. Bizal G.L. Gullans S.R. McAteer J.A. J. Biol. Chem. 1994; 269: 27973-27978Abstract Full Text PDF PubMed Google Scholar, 64Sun A.M. Liu Y. Dworkin L.D. Tse C.M. Donowitz M. Yip K.P. J. Membr. Biol. 1997; 160: 85-90Crossref PubMed Scopus (60) Google Scholar, 65Bookstein C. Xie Y. Rabenau K. Musch M.W. McSwine R.L. Rao M.C. Chang E.B. Am. J. Physiol. 1997; 273: C1496-C1505Crossref PubMed Google Scholar).NHE-4 mRNA can be found in the stomach, intestine, kidney, and in the cavi ammoni fields of the hippocampus. In the kidney, NHE-4 is mostly present in the inner medulla collecting duct and has also been found heterogeneously distributed on the basolateral membrane of cortical tubule cells (65Bookstein C. Xie Y. Rabenau K. Musch M.W. McSwine R.L. Rao M.C. Chang E.B. Am. J. Physiol. 1997; 273: C1496-C1505Crossref PubMed Google Scholar).The recently cloned NHE-5 isoform has been detected predominantly in brain but also in testis, spleen, and skeletal muscle by Northern blot (8Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. et al.Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar), whereas NHE-6, which is expressed in mitochondria, has a wide tissue distribution.The common mechanism by which intracellular signaling pathways modulate the Na+/H+ exchangers involves the C-terminal region of these proteins, as shown in a series of key experiments. For example, the expression of a chimera consisting of the transmembrane region of the cAMP-insensitive human NHE-1 and the cytosolic region of the cAMP-activable β-NHE-1 of trout red cells results in a protein which is activated by cAMP, identical to the way the complete β-NHE-1 isoform behaves (66Borgese F. Sardet C. Cappadoro M. Pouysségur J. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6765-6769Crossref PubMed Scopus (124) Google Scholar, 67Borgese F. Malapert M. Fievet B. Pouysségur J. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5431-5435Crossref PubMed Scopus (37) Google Scholar). Conversely, NHE-3 is inhibited by cAMP in epithelial cells, and a chimeric construct between the transmembrane region of NHE-1 and the cytosolic region of NHE-3 becomes inhibited by cAMP (68Cabado A.G. Yu F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These key findings indicate that the C-terminal domain dictates the type of hormonal regulation in a given cell.Molecular Dissection of NHE-1 ActivationNHE-1 activation by an extreme variety of extracellular stimuli, including hormones, integrins, and virtually all growth factors results from an increase in affinity of the transporter for intracellular protons. The simplest model that has been proposed is that the cytoplasmic tail cooperates with the central pH i sensor to decrease the pH i threshold value of NHE-1. In this regard, the cytoplasmic tail is seen as a signal integrator capable of transmitting hormonal signals to the transmembrane built-in pH i sensor (69Wakabayashi S. Fafournoux P. Sardet C. Pouysségur J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2424-2428Crossref PubMed Scopus (236) Google Scholar). Therefore, as for a promoter region of a regulated gene, it is not surprising to see that the NHE-1 cytoplasmic tail has “collected” regulatory boxes that convey specific extracellular signals. For example, all growth factors have been shown to induce a very rapid and transient rise in cytoplasmic calcium as well as a more or less sustained activation of the p42/p44 MAPK cascade. Interestingly and as presented below, the NHE-1 cytoplasmic domain intercepts these distinct signals for transmission into a cytoplasmic alkalinization. Bertrand et al. (70Bertrand B. Wakabayashi S. Ikeda T. Pouysségur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13703-13709Abstract Full Text PDF PubMed Google Scholar) demonstrated that calmodulin physically interacts with a particular subdomain of the NHE-1 cytosolic region (71Wakabayashi S. Bertrand B. Ikeda T. Pouysségur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13710-13715Abstract Full Text PDF PubMed Google Scholar) releasing a negative constraint, thus resulting in the activation of NHE-1 by increases in intracellular Ca2+. Therefore, this calmodulin-binding regulatory box is sufficient to account for the rapid and transient activation of NHE-1 in response to growth factors and other Ca2+-mobilizing agonists. By contrast, a similar sequence is not found in NHE-3, which is also regulated by calmodulin, both in a calmodulin kinase-dependent and -independent manner (72Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Crossref PubMed Scopus (103) Google Scholar).Direct phosphorylation of NHE1 and/or phosphorylation of ancillary proteins could account for more robust and sustained activation of NHE-1. Both mechanisms have been well documented. First, it was demonstrated that NHE-1 is a phosphoprotein and that its level of phosphorylation is increased in mitogen-stimulated cells when compared with unstimulated controls (13Sardet C. Counillon L. Franchi A. Pouysségur J. Science. 1990; 247: 723-726Crossref PubMed Scopus (375) Google Scholar, 73Livne A.A. Sardet C. Pouysségur J. FEBS Lett. 1991; 284: 219-222Crossref PubMed Scopus (43) Google Scholar, 74Wang H. Silva N.L. Lucchesi P.A. Haworth R. Wang K. Michalak M. Pelech S. Fliegel L. Biochemistry. 1997; 36: 9151-9158Crossref PubMed Scopus (85) Google Scholar, 75Sardet C. Fafournoux P. Pouysségur J. J. Biol. Chem. 1991; 266: 19166-19171Abstract Full Text PDF PubMed Google Scholar). Phosphopeptide mapping carried out on wild-type and deletion mutants of the cytoplasmic region (76Wakabayashi S. Bertrand B. Shigekawa M. Fafournoux P. Pouysségur J. J. Biol. Chem. 1994; 269: 5583-5588Abstract Full Text PDF PubMed Google Scholar) revealed that the phosphorylation sites are located in the C-terminal cytoplasmic region of the protein. Ser-703 was recently demonstrated to be phosphorylated in vivo by the p42/p44 MAPK-activated target, p90RSK (77Takahashi E. Abe J. Gallis B. Aebersold R. Spring D.J. Krebs E.G. Berk B.C. J. Biol. Chem. 1999; 274: 20206-20214Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and to represent a major site for serum activation. This result is in agreement with our demonstration, using a Raf-activable construct, that p42/p44 MAPK plays a key role in NHE1 activation (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Besides the MAPK pathway, NHE-1 has been shown to be phosphorylated by p160 ROCK (79Sahai E. Alberts A.S. Treisman R. EMBO J. 1998; 17: 1350-1361Crossref PubMed Scopus (229) Google Scholar), a Rho effector associated with the assembly of stress fibers and focal adhesions. However, p42/p44 MAPK-mediated NHE-1 activation cannot be entirely explained by the direct phosphorylation of NHE-1. First, deletion of the distal cytoplasmic tail containing Ser-703 and other major phosphorylation sites attenuates but does not abolish growth factor activation. The residual activation (about 50%) remains sensitive to the MEK inhibitor PD98059 (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The simplest working model taking into account this set of results is that additional regulatory proteins, which may themselves be phosphorylated, interact with various domains of the cytosolic region of the exchanger. Candidate proteins have been identified, such as p24 NHE-1 (80Goss G. Orlowski J. Grinstein S. Am. J. Physiol. 1996; 270: C1493-C1502Crossref PubMed Google Scholar), HSP70 (81Silva N.L. Haworth R.S. Singh D. Fliegel L. Biochemistry. 1995; 34: 10412-10420Crossref PubMed Scopus (84) Google Scholar), CHP (82Lin X. Barber D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12631-12636Crossref PubMed Scopus (151) Google Scholar), and other proteins obtained by double hybrid screening, such as myosin light chain phosphatase. 4P. Fafournoux and J. Pouysségur, unpublished results. Additionally, NHE-1 can be activated by different mechanical stimuli such as osmotic stress or cell spreading. Grinstein et al. (83Grinstein S. Woodside M. Sardet C. Pouysségur J. Rotin D. J. Biol. Chem. 1992; 267: 23823-23828Abstract Full Text PDF PubMed Google Scholar) have demonstrated that the mechanism of this activation is phosphorylation-independent. In view of their finding that NHE-1 is associated with the actin cytoskeleton in focal adhesion plaques (84Grinstein S. Woodside M. Waddell T.K. Downey G.P. Orlowski J. Pouysségur J. EMBO J. 1993; PubMed Scopus Google Scholar), these results that this activation be by direct with This is by the that the presence of relatively high of is for NHE-1 activation N. Orlowski J. Grinstein S. J. Physiol. 1997; 109: PubMed Scopus Google Scholar) and that results in a more plasma membrane of NHE-1. the NHE-1 phosphorylation level is not NHE-1 interact in an manner with an ancillary protein NHE-1 with Woodside M. Wakabayashi S. Pouysségur J. Waddell T. Downey G.P. Grinstein S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). al. (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) demonstrated that the MAPK is a major for NHE-1 activation by growth stimuli, whereas the and stress kinase pathways and are not in the activation of NHE-1 by the C-terminal domain of NHE-1 can be as a series of regulatory phosphorylation or of regulatory proteins, the affinity of the transporter for intracellular is NHE-3 regulation mechanism is different from the NHE-1 activation the in of the of the transporter of in its affinity for intracellular protons. and it is possible to NHE-3 in S. A. Yu F. K. Lukacs G. K. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). and S. 1997; Google Scholar) have the and of a acid NHE regulatory factor review, see Ref. S. 1997; Google Scholar). This which can be cytoplasmic or negatively NHE-3 by direct in cells, is to cAMP to This protein is present in various of both the intestine and the kidney it has been detected in the NHE-3 is not indicating that it could cAMP regulation of proteins other the NHE-3 proteins, such as have been shown to other transmembrane including the or can also cAMP regulation NHE-3, indicating that the NHE regulatory proteins be C.H. S. M. D. Tsao S. Tse C.M. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). et al. R.A. J.A. A. S. Grinstein S. 1998; PubMed Scopus Google Scholar) demonstrated that the can with its a direct regulation of NHE-3 Yun et al. C.H. G. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) have demonstrated that to an intracellular region within the C-terminal domain of NHE-3 and more the protein in this these results that or have a the of NHE-3 and kinase to the NHE-3 cytosolic region therefore to of various regulatory which seem to and signals from various signaling pathways (72Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Crossref PubMed Scopus (103) Google Scholar). In an independent and (68Cabado A.G. Yu F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) that the region situated between residues and cAMP In this which contains and are crucial for cAMP K. Yu F.H. A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, although of is is an to into the physiological role of proteins to gene from this are for NHE isoforms. Schultheis et al. P.J. Meneton P. M. T. Wang T. G. Aronson P.S. Shull G.E. 1998; PubMed Scopus Google Scholar) have for NHE-3, an isoform which was to as well as kidney to these exhibit a decrease in are and present both in kidney and This important result the predicted physiological role of NHE-3 and that this when compared with -2, and the of the in kidney and contrast, et al. C.M. D. A. Aronson P.S. J.L. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar) have the molecular of the present in and that these a mutation, which a in the sequence of resulting in the of a The inactivation of NHE-1 gene function C.M. Schultheis P.J. R.L. Shull G.E. Am. J. Physiol. 1999; PubMed Google Scholar) confirmed the of this mutation, a finding which was in light of the expression of systems such as Interestingly, these have in their or in their kidney or intestine show in brain this which is highly sensitive to was not as the of the NHE-1 gene is therefore to as for the of the other isoforms may have surprising physiological Hence, the recent of the NHE-2 isoform in not result in in function but in a resulting in acid in the P.J. Meneton P. M. G.P. G. T. Shull G.E. J. 1998; PubMed Scopus Google Scholar). Therefore, as has been for gene the of the resulting be of the possible of the NHE isoforms of the and the presence of mechanisms in In this the recent of gene and of to be very for the physiological of Maintaining intracellular pH values close to neutrality is a crucial task for a wide variety of cells. Hence, various mechanisms for pH regulation have been selected early in evolution and are ubiquitously distributed. Among the actors in this scene, the members of the Na+/H+ exchanger gene family (NHE1 isoforms) are widely expressed and constitute extremely efficient systems for protecting cells against internal acidification. To date, at least six genes have been identified in mammalian cells, and to various extents, the corresponding proteins have been molecularly and functionally characterized. In this short review, we will update our current knowledge on these NHE family members and highlight the most important aspects of the basic function of these transporters. Then, in a broader physiological context, we will present what we think are the most prominent specific features of the different NHE isoforms. and Functional Domains of NHEsThe first cDNA encoding the NHE-1 isoform was cloned using an expression strategy based on the ability of Na+/H+ exchangers to protect antiporter-deficient cells (1Pouysségur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (438) Google Scholar) against otherwise lethal intracellular acidification (2Sardet C. Franchi A. Pouysségur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (668) Google Scholar). Variations in the hormonal regulation and pharmacological features of Na+/H+ exchange were the first indications that a large family of Na+/H+ exchange molecules existed (3Clark J.D. Limbird L.E. Am. J. Physiol. 1991; 261: C945-C953Crossref PubMed Google Scholar). Therefore, using the NHE-1 cDNA as a probe led to the molecular identification of the NHE-2, -3, and -4 (4Tse C.M. Brant S.R. Walker M.S. Pouysségur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 5Tse C.M.

Osteoclasts (OCLs) are key players in controlling bone remodeling. Modifications in their differentiation or bone resorbing activity are associated with a number of pathologies ranging from osteopetrosis to osteoporosis, chronic inflammation and cancer, that are all characterized by immunological alterations. Therefore, the 2000s were marked by the emergence of osteoimmunology and by a growing number of studies focused on the control of OCL differentiation and function by the immune system. At the same time, it was discovered that OCLs are much more than bone resorbing cells. As monocytic lineage-derived cells, they belong to a family of cells that displays a wide heterogeneity and plasticity and that is involved in phagocytosis and innate immune responses. However, while OCLs have been extensively studied for their bone resorption capacity, their implication as immune cells was neglected for a long time. In recent years, new evidence pointed out that OCLs play important roles in the modulation of immune responses toward immune suppression or inflammation. They unlocked their capacity to modulate T cell activation, to efficiently process and present antigens as well as their ability to activate T cell responses in an antigen-dependent manner. Moreover, similar to other monocytic lineage cells such as macrophages, monocytes and dendritic cells, OCLs display a phenotypic and functional plasticity participating to their anti-inflammatory or pro-inflammatory effect depending on their cell origin and environment. This review will address this novel vision of the OCL, not only as a phagocyte specialized in bone resorption, but also as innate immune cell participating in the control of immune responses.

Na + /H + exchangers play pivotal roles in the control of cell and tissue pH by mediating the electroneutral exchange of Na + and H + across cellular membranes. They belong to an ancient family of highly evolutionarily conserved proteins, and they play essential physiological roles in all phyla. In this review, we focus on the mammalian Na + /H + exchangers (NHEs), the solute carrier (SLC) 9 family. This family of electroneutral transporters constitutes three branches: SLC9A, -B, and -C. Within these, each isoform exhibits distinct tissue expression profiles, regulation, and physiological roles. Some of these transporters are highly studied, with hundreds of original articles, and some are still only rudimentarily understood. In this review, we present and discuss the pioneering original work as well as the current state-of-the-art research on mammalian NHEs. We aim to provide the reader with a comprehensive view of core knowledge and recent insights into each family member, from gene organization over protein structure and regulation to physiological and pathophysiological roles. Particular attention is given to the integrated physiology of NHEs in the main organ systems. We provide several novel analyses and useful overviews, and we pinpoint main remaining enigmas, which we hope will inspire novel research on these highly versatile proteins.

Atherosclerosis is driven by the expansion of cholesterol-loaded 'foamy' macrophages in the arterial intima. Factors regulating foamy macrophage differentiation and survival in plaque remain poorly understood. Here we show, using trajectory analysis of integrated single-cell RNA sequencing data and a genome-wide CRISPR screen, that triggering receptor expressed on myeloid cells 2 (Trem2) is associated with foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up oxidized low-density lipoprotein (oxLDL). Myeloid-specific deletion of Trem2 showed an attenuation of plaque progression, even when targeted in established atherosclerotic lesions, and was independent of changes in circulating cytokines, monocyte recruitment or cholesterol levels. Mechanistically, we link Trem2-deficient macrophages with a failure to upregulate cholesterol efflux molecules, resulting in impaired proliferation and survival. Overall, we identify Trem2 as a regulator of foamy macrophage differentiation and atherosclerotic plaque growth and as a putative therapeutic target for atherosclerosis.

OBJECTIVE: Under both physiological and pathological conditions, bone volume is determined by the rate of bone formation by osteoblasts and bone resorption by osteoclasts. Excessive bone loss is a common complication of human IBD whose mechanisms are not yet completely understood. Despite the role of activated CD4(+) T cells in inflammatory bone loss, the nature of the T cell subsets involved in this process in vivo remains unknown. The aim of the present study was to identify the CD4(+) T cell subsets involved in the process of osteoclastogenesis in vivo, as well as their mechanism of action. DESIGN: CD4(+) T cells were studied in IL10-/- mice and Rag1-/- mice adoptively transferred with naive CD4(+)CD45RB(high) T cells, representing two well-characterised animal models of IBD and in patients with Crohn's disease. They were phenotypically and functionally characterised by flow cytometric and gene expression analysis, as well as in in vitro cocultures with osteoclast precursors. RESULTS: In mice, we identified bone marrow (BM) CD4(+) T cells producing interleukin (IL)-17 and tumour necrosis factor (TNF)-α as an osteoclastogenic T cell subset referred to as Th17 TNF-α(+) cells. During chronic inflammation, these cells migrate to the BM where they survive in an IL-7-dependent manner and where they promote the recruitment of inflammatory monocytes, the main osteoclast progenitors. A population equivalent to the Th17 TNF-α(+) cells was also detected in patients with Crohn's disease. CONCLUSIONS: Our results highlight the osteoclastogenic function of the Th17 TNF-α(+) cells that contribute to bone loss in vivo in IBD.

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H(+) via an unidentified pH-sensitive background K(+) channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K(+) channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or β-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n = 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2(-/-) mice (n = 49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2(-/-) mice selected based on β-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K(+) currents were reduced in amplitude in RTN neurons from TASK-2(-/-) mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart-brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2(-/-) mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold.

Abstract In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo. These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche. Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg. Cancer Res; 78(18); 5229–42. ©2018 AACR.

ABSTRACT Bone destruction is a hallmark of chronic rheumatic diseases. Although the role of osteoclasts in bone loss is clearly established, their implication in the inflammatory response has not been investigated despite their monocytic origin. Moreover, specific markers are lacking to characterize osteoclasts generated in inflammatory conditions. Here, we have explored the phenotype of inflammatory osteoclasts and their effect on CD4+ T cell responses in the context of bone destruction associated with inflammatory bowel disease. We used the well-characterized model of colitis induced by transfer of naive CD4+ T cells into Rag1–/– mice, which is associated with severe bone destruction. We set up a novel procedure to sort pure osteoclasts generated in vitro to analyze their phenotype and specific immune responses by FACS and qPCR. We demonstrated that osteoclasts generated from colitic mice induced the emergence of TNFα-producing CD4+ T cells, whereas those generated from healthy mice induced CD4+FoxP3+ regulatory T cells, in an antigen-dependent manner. This difference is related to the osteoclast origin from monocytes or dendritic cells, to their cytokine expression pattern, and their environment. We identified CX3CR1 as a marker of inflammatory osteoclasts and we demonstrated that the differentiation of CX3CR1+ osteoclasts is controlled by IL-17 in vitro. This work is the first demonstration that, in addition to participating to bone destruction, osteoclasts also induce immunogenic CD4+ T cell responses upon inflammation. They highlight CX3CR1 as a novel dual target for antiresorptive and anti-inflammatory treatment in inflammatory chronic diseases. © 2016 American Society for Bone and Mineral Research.

TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.

CONTEXT: Primary aldosteronism is a heterogeneous group of disorders comprising both sporadic and familial forms. Mutations in the KCNJ5 gene, which encodes the inward rectifier K(+) channel 4 (G protein-activated inward rectifier K(+) channel 4, Kir3.4), cause familial hyperaldosteronism type III (FH-III) and are involved in the pathogenesis of sporadic aldosterone-producing adenomas. OBJECTIVE: The objective of the study was to characterize the effects of a newly described KCNJ5 mutation in vitro. PATIENTS AND METHODS: The index case is a 62-year-old woman affected by primary aldosteronism, who underwent left adrenalectomy after workup for adrenal adenoma. Exon 1 of KCNJ5 was PCR amplified from adrenal tissue and peripheral blood and sequenced. Electrophysiological and gene expression studies were performed to establish the functional effects of the new mutation on the membrane potential and adrenal cell CYP11B2 expression. RESULTS: KCNJ5 sequencing in the index case revealed a new p.Y152C germline mutation; interestingly, the phenotype of the patient was milder than most of the previously described FH-III families. The tyrosine-to-cysteine substitution resulted in pathological Na(+) permeability, cell membrane depolarization, and disturbed intracellular Ca(2+) homeostasis, effects similar, albeit smaller, to the ones demonstrated for other KCNJ5 mutations. Gene expression studies revealed an increased expression of CYP11B2 and its transcriptional regulator NR4A2 in HAC15 adrenal cells overexpressing KCNJ5(Y152C) compared to the wild-type channel. The effect was clearly Ca(2+)-dependent, because it was abolished by the calcium channel blocker nifedipine. CONCLUSIONS: Herein we describe a new germline mutation in KCNJ5 responsible for FH-III.

In an attempt to verify the nature of amplification events at band q13 on chromosome 11 we surveyed the amplification status of ten molecular markers specific for this region (GSTP, SEA, D11S97, D11S146, BCLI, PRADI/CCNDI, HST/FGF4, INT2/FGF3, EMSI, and DIIS833E) in a panel of 389 primary breast carcinoma DNA samples. Eighty-eight tumors (23%) showed at least one of these markers amplified, but in a majority of the cases amplification encompassed more than one of the tested loci. Our data confirm that amplicons at 11q13 can cover large portions of DNA and are consistent with the existence of several cores of amplification. One important core seems to be, as previously described, centered around PRADI/CCNDI; 57 tumors (14.7%) showed amplification at PRADI/CCNDI either alone (one tumor) or along with amplification of BCLI or INT2/FGF3. The level of amplification of PRADI/CCNDI sometimes exceeded that of surrounding markers. Three additional amplification events occurring independently of amplification of PRADI/CCNDI were also detected. Centromeric to BCLI, probes to DIIS97, and DIIS146 detected amplification in 60 tumors (15.4%) and were often the only amplified markers. Telomeric to INT2/FGF3, DIIS833E was found amplified alone in ten tumors, and it was the most amplified marker in another six cases. At a shorter distance of INT2/FGF3, EMSI was the only amplified marker in two tumors, with a level of amplification that could exceed that of PRADI/CCNDI and DIIS833E. Our data thus suggest the existence of four independent amplified regions within band 11q13 in breast cancer.

Abstract Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4 + T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.

The sigma-1 receptor is an intracellular protein characterized as a tumor biomarker whose function remains mysterious. We demonstrate herein for the first time that highly selective sigma ligands inhibit volume-regulated chloride channels (VRCC) in small cell lung cancer and T-leukemia cells. Sigma ligands and VRCC blockers provoked a cell cycle arrest underlined by p27 accumulation. In stably sigma-1 receptor-transfected HEK cells, the proliferation rate was significantly lowered by sigma ligands when compared with control cells. Sigma ligands produced a strong inhibition of VRCC in HEK-transfected cells but not in control HEK. Surprisingly, the activation rate of VRCC was dramatically delayed in HEK-transfected cells in the absence of ligands, indicating that sigma-1 receptors per se modulate cell regulating volume processes in physiological conditions. Volume measurements in hypotonic conditions revealed indeed that the regulatory volume decrease was delayed in HEK-transfected cells and virtually abolished in the presence of igmesine in both HEK-transfected and T-leukemic cells. Moreover, HEK-transfected cells showed a significant resistance to staurosporine-induced apoptosis volume decrease, indicating that sigma-1 receptors protect cancer cells from apoptosis. Altogether, our results show for the first time that sigma-1 receptors modulate "cell destiny" through VRCC and cell volume regulation.

OBJECTIVE: Brite adipocytes are inducible energy-dissipating cells expressing UCP1 which appear within white adipose tissue of healthy adult individuals. Recruitment of these cells represents a potential strategy to fight obesity and associated diseases. METHODS/RESULTS: Using human Multipotent Adipose-Derived Stem cells, able to convert into brite adipocytes, we show that arachidonic acid strongly inhibits brite adipocyte formation via a cyclooxygenase pathway leading to secretion of PGE2 and PGF2α. Both prostaglandins induce an oscillatory Ca(++) signaling coupled to ERK pathway and trigger a decrease in UCP1 expression and in oxygen consumption without altering mitochondriogenesis. In mice fed a standard diet supplemented with ω6 arachidonic acid, PGF2α and PGE2 amounts are increased in subcutaneous white adipose tissue and associated with a decrease in the recruitment of brite adipocytes. CONCLUSION: Our results suggest that dietary excess of ω6 polyunsaturated fatty acids present in Western diets, may also favor obesity by preventing the "browning" process to take place.

Rheumatoid arthritis (RA) and periodontitis are chronic inflammatory diseases leading to increased bone resorption. Preventing this inflammatory bone resorption is a major health challenge. Both diseases share immunopathogenic similarities and a common inflammatory environment. The autoimmune response or periodontal infection stimulates certain immune actors, leading in both cases to chronic inflammation that perpetuates bone resorption. Moreover, RA and periodontitis have a strong epidemiological association that could be explained by periodontal microbial dysbiosis. This dysbiosis is believed to be involved in the initiation of RA via three mechanisms. (i) The dissemination of periodontal pathogens triggers systemic inflammation. (ii) Periodontal pathogens can induce the generation of citrullinated neoepitopes, leading to the generation of anti-citrullinated peptide autoantibodies. (iii) Intracellular danger-associated molecular patterns accelerate local and systemic inflammation. Therefore, periodontal dysbiosis could promote or sustain bone resorption in distant inflamed joints. Interestingly, in inflammatory conditions, the existence of osteoclasts distinct from "classical osteoclasts" has recently been reported. They have proinflammatory origins and functions. Several populations of osteoclast precursors have been described in RA, such as classical monocytes, a dendritic cell subtype, and arthritis-associated osteoclastogenic macrophages. The aim of this review is to synthesize knowledge on osteoclasts and their precursors in inflammatory conditions, especially in RA and periodontitis. Special attention will be given to recent data related to RA that could be of potential value in periodontitis due to the immunopathogenic similarities between the two diseases. Improving our understanding of these pathogenic mechanisms should lead to the identification of new therapeutic targets involved in the pathological inflammatory bone resorption associated with these diseases.

Tandem of P domains in a weak inwardly rectifying K(+) channel 1 (TWIK1) is a K(+) channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT(1)R or adrenoreceptor alpha2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G(i) protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.

Task1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K(+) channel-related acid-sensitive K(+) channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3(-/-) mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3(-/-) mice were strongly depolarized compared with wild-type (-52 vs. -79 mV), and in fresh adrenal slices Ca(2+) signaling of Task3(-/-) glomerulosa cells was abnormal. In living Task3(-/-) mice, the regulation of aldosterone secretion showed specific deficits: Under low Na(+) and high K(+) diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na(+) and low K(+) diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3(-/-) mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na(+) diets. Isolated adrenal glands of Task3(-/-) produced 2-fold more aldosterone. As a consequence, Task3(-/-) mice showed salt-sensitive arterial hypertension (plus 10 mm Hg). In conclusion, Task3 plays an important role in the adaptation of aldosterone secretion to dietary salt intake.

The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of Drosophila during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by N 1-guanyl-1,7-diaminoheptane (GC7) or RNA interference–mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion–induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

Somatic mutations of the potassium channel KCNJ5 are found in 40% of aldosterone producing adenomas (APAs). APA-related mutations of KCNJ5 lead to a pathological Na(+) permeability and a rise in cytosolic Ca(2+), the latter presumably by depolarizing the membrane and activating voltage-gated Ca(2+) channels. The aim of this study was to further investigate the effects of mutated KCNJ5 channels on intracellular Na(+) and Ca(2+) homeostasis in human adrenocortical NCI-H295R cells. Expression of mutant KCNJ5 led to a 2-fold increase in intracellular Na(+) and, in parallel, to a substantial rise in intracellular Ca(2+). The increase in Ca(2+) appeared to be caused by activation of voltage-gated Ca(2+) channels and by an impairment of Ca(2+) extrusion by Na(+)/Ca(2+) exchangers. The mutated KCNJ5 exhibited a pharmacological profile that differed from the one of wild-type channels. Mutated KCNJ5 was less Ba(2+) and tertiapin-Q sensitive but was inhibited by blockers of Na(+) and Ca(2+)-transporting proteins, such as verapamil and amiloride. The clinical use of these drugs might influence aldosterone levels in APA patients with KCNJ5 mutations. This might implicate diagnostic testing of APAs and could offer new therapeutic strategies.