FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2131462893> ?p ?o ?g. }

• W2131462893 endingPage "51816" @default.
• W2131462893 startingPage "51804" @default.
• W2131462893 abstract "Extracellular zinc promotes cell proliferation and its deficiency leads to impairment of this process, which is particularly important in epithelial cells. We have recently characterized a zinc-sensing receptor (ZnR) linking extracellular zinc to intracellular release of calcium. In the present study, we addressed the role of extracellular zinc, acting via the ZnR, in regulating the MAP kinase pathway and Na+/H+ exchange in colonocytes. We demonstrate that Ca2+ release, mediated by the ZnR, induces phosphorylation of ERK1/2, which is highly metal-specific, mediated by physiological concentrations of extracellular Zn2+ but not by Cd2+, Fe2+, Ni2+, or Mn2+. Desensitization of the ZnR by Zn2+, is followed by ∼90% inhibition of the Zn2+-dependent ERK1/2 phosphorylation, indicating that the ZnR is a principal link between extracellular Zn2+ and ERK1/2 activation. Application of both the IP3 pathway and PI 3-kinase antagonists largely inhibited Zn2+-dependent ERK1/2 phosphorylation. The physiological significance of the Zn2+-dependent activation of ERK1/2 was addressed by monitoring Na+/H+ exchanger activity in HT29 cells and in native colon epithelium. Preincubation of the cells with zinc was followed by robust activation of Na+/H+ exchange, which was eliminated by cariporide (0.5 μm); indicating that zinc enhances the activity of NHE1. Activation of NHE1 by zinc was totally blocked by the ERK1/2 inhibitor, U0126. Prolonged acidification, in contrast, stimulates NHE1 by a distinct pathway that is not affected by extracellular Zn2+ or inhibitors of the MAP kinase pathway. Desensitization of ZnR activity eliminates the Zn2+-dependent, but not the prolonged acidification-dependent activation of NHE1, indicating that Zn2+-dependent activation of H+ extrusion is specifically mediated by the ZnR. Our results support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways that affect pH homeostasis in colonocytes. Furthermore activation of both, ERK and NHE1, by extracellular zinc may provide the mechanism linking zinc to enhanced cell proliferation. Extracellular zinc promotes cell proliferation and its deficiency leads to impairment of this process, which is particularly important in epithelial cells. We have recently characterized a zinc-sensing receptor (ZnR) linking extracellular zinc to intracellular release of calcium. In the present study, we addressed the role of extracellular zinc, acting via the ZnR, in regulating the MAP kinase pathway and Na+/H+ exchange in colonocytes. We demonstrate that Ca2+ release, mediated by the ZnR, induces phosphorylation of ERK1/2, which is highly metal-specific, mediated by physiological concentrations of extracellular Zn2+ but not by Cd2+, Fe2+, Ni2+, or Mn2+. Desensitization of the ZnR by Zn2+, is followed by ∼90% inhibition of the Zn2+-dependent ERK1/2 phosphorylation, indicating that the ZnR is a principal link between extracellular Zn2+ and ERK1/2 activation. Application of both the IP3 pathway and PI 3-kinase antagonists largely inhibited Zn2+-dependent ERK1/2 phosphorylation. The physiological significance of the Zn2+-dependent activation of ERK1/2 was addressed by monitoring Na+/H+ exchanger activity in HT29 cells and in native colon epithelium. Preincubation of the cells with zinc was followed by robust activation of Na+/H+ exchange, which was eliminated by cariporide (0.5 μm); indicating that zinc enhances the activity of NHE1. Activation of NHE1 by zinc was totally blocked by the ERK1/2 inhibitor, U0126. Prolonged acidification, in contrast, stimulates NHE1 by a distinct pathway that is not affected by extracellular Zn2+ or inhibitors of the MAP kinase pathway. Desensitization of ZnR activity eliminates the Zn2+-dependent, but not the prolonged acidification-dependent activation of NHE1, indicating that Zn2+-dependent activation of H+ extrusion is specifically mediated by the ZnR. Our results support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways that affect pH homeostasis in colonocytes. Furthermore activation of both, ERK and NHE1, by extracellular zinc may provide the mechanism linking zinc to enhanced cell proliferation. Arrested cell proliferation is a hallmark of zinc deficiency. This is particularly true in gastrointestinal cells (1MacDonald R.S. J. Nutr. 2000; 130: 1500S-1508SCrossref PubMed Google Scholar, 2Kaiser G.C. Yan F. Polk D.B. Exp. Cell Res. 1999; 249: 349-358Crossref PubMed Scopus (40) Google Scholar, 3Wang Q. Ding Q. Dong Z. Ehlers R.A. Evers B.M. Anticancer Res. 2000; 20: 75-83PubMed Google Scholar, 4Aliaga J.C. Deschenes C. Beaulieu J.F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar), where insufficient dietary zinc attenuates the renewal of the epithelium leading to severe diarrhea (1MacDonald R.S. J. Nutr. 2000; 130: 1500S-1508SCrossref PubMed Google Scholar). Extracellular zinc has been shown to regulate cell proliferation via the MAP 1The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; ZnR, zinc-sensing receptor; PI, phosphatidylinositol; PLC, phospholipase C; PKC, protein kinase C; IP, inositol phosphate; CaM, calcium-calmodulin; PMA, phorbol 12-myristate 13-acetate. kinase pathway in several cell types (5Park K.S. Lee N.G. Lee K.H. Seo J.T. Choi K.Y. Am. J. Physiol. Gastrointest Liver Physiol. 2003; 19: 19Google Scholar, 6Samet J.M. Graves L.M. Quay J. Dailey L.A. Devlin R.B. Ghio A.J. Wu W. Bromberg P.A. Reed W. Am. J. Physiol. 1998; 275: L551-L558Crossref PubMed Google Scholar, 7Kiss Z. Crilly K.S. Tomono M. FEBS Lett. 1997; 415: 71-74Crossref PubMed Scopus (24) Google Scholar). Although the mitogenic and anti-apoptotic effects of zinc are well recognized (8Maret W. BioMetals. 2001; 14: 187-190Crossref Scopus (125) Google Scholar, 9Beyersmann D. Haase H. BioMetals. 2001; 14: 331-341Crossref PubMed Scopus (514) Google Scholar), and the treatment of severe diarrhea by addition of dietary zinc is common, the direct link between this ion and the cellular mechanisms regulating proliferation is not well understood. It has been suggested that a decrease in intracellular zinc may lead directly to a reduction in activity of various metalloenzymes involved in transcription and cell metabolism (1MacDonald R.S. J. Nutr. 2000; 130: 1500S-1508SCrossref PubMed Google Scholar, 10Wu F.Y. Wu C.W. Annu. Rev. Nutr. 1987; 7: 251-272Crossref PubMed Scopus (124) Google Scholar). Several studies, however, indicate that extracellular zinc acts as a signaling molecule. In tracheal cells, for example, extracellular zinc, through activation of Src, leads to transactivation of EGFR and subsequently to activation of ERK1/2 (11Wu W. Graves L.M. Gill G.N. Parsons S.J. Samet J.M. J. Biol. Chem. 2002; 277: 24252-24257Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In fibroblasts, extracellular zinc has been shown to trigger the activation of the PI3K pathway, subsequently leading to the activation of AKT and the S6 kinase (12Kim S. Jung Y. Kim D. Koh H. Chung J. J. Biol. Chem. 2000; 275: 25979-25984Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The signaling pathways linking extracellular zinc to these proteins and to subsequent regulation of cellular ion, pH or volume homeostasis, however, remain poorly understood. We have recently identified and characterized an extracellular zinc-sensing receptor (ZnR) that triggers, upon exposure to extracellular zinc, the release of Ca2+ from intracellular stores by activation of the IP3 pathway (13Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11749-11754Crossref PubMed Scopus (208) Google Scholar). The pharmacological profile of the calcium response triggered by the ZnR, particularly its sensitivity to the PLC inhibitor, U73122, and the inhibitory effect of the IP3 receptor blocker, 2-APB, indicates that the ZnR is a Gq-coupled receptor (GPCR). Both the Gα and the Gβγ dimer of various GPCRs have been linked to activation of the MAP kinase via multiple intracellular pathways (14Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (366) Google Scholar, 15Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 16Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). In intestinal cells, furthermore, the muscarinic receptor has been shown to play a key role in promoting ion transport through activation of MAP and PI 3-kinase signal transduction (17Keely S.J. Barrett K.E. J. Biol. Chem. 1999; 274: 33449-33454Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 18Keely S.J. Calandrella S.O. Barrett K.E. J. Biol. Chem. 2000; 275: 12619-12625Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A role for the ZnR in activation of the MAP kinase pathway is demonstrated in this work. The ZnR was initially characterized in the colonocytic cell line, HT29, where a robust calcium signal was generated following activation of the receptor by changes in the concentration of extracellular Zn2+ (13Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11749-11754Crossref PubMed Scopus (208) Google Scholar). Importantly, the Ca2+ response induced by the ZnR in HT29 cells is triggered by ∼80 μm zinc, i.e. within the physiological range of zinc concentrations found in the digestive tract (19Sandstrom B. Cederblad A. Kivisto B. Stenquist B. Andersson H. Am. J. Clin. Nutr. 1986; 44: 501-504Crossref PubMed Scopus (35) Google Scholar, 20Sandstrom B. Analyst. 1995; 120: 913-915Crossref PubMed Google Scholar). Interestingly, activation of the ZnR also resulted in activation of Na+/H+ exchange in colonocytes. Distinct NHE isoforms are expressed in the colonic mucosa, each playing a unique physiological role in colon physiology (21Dudeja 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: G483-G493PubMed Google Scholar, 22Bachmann O. Riederer B. Rossmann H. Groos S. Schultheis P.J. Shull G.E. Gregor M. Manns M.P. Seidler U. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G125-G133Crossref PubMed Scopus (75) Google Scholar). It was not known, however, which of the NHE isoforms is regulated by the ZnR. The first NHE isoform, NHE1, is mostly expressed on the basolateral membrane of colonocytes and is involved in regulating the pHi, volume homeostasis (22Bachmann O. Riederer B. Rossmann H. Groos S. Schultheis P.J. Shull G.E. Gregor M. Manns M.P. Seidler U. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G125-G133Crossref PubMed Scopus (75) Google Scholar, 23Hasselblatt P. Warth R. Schulz-Baldes A. Greger R. Bleich M. Pflugers Arch. 2000; 441: 118-124Crossref PubMed Scopus (28) Google Scholar, 24Chu J. Chu S. Montrose M.H. Am. J. Physiol. Cell Physiol. 2002; 283: C358-C372Crossref PubMed Scopus (53) Google Scholar, 25Gonda T. Maouyo D. Rees S.E. Montrose M.H. Am. J. Physiol. 1999; 276: G259-G270Crossref PubMed Google Scholar). Such activity is of prime importance considering the major changes in osmolarity and the acid load imposed by the permeation of short chain fatty acids generated by bacterial fermentation (24Chu J. Chu S. Montrose M.H. Am. J. Physiol. Cell Physiol. 2002; 283: C358-C372Crossref PubMed Scopus (53) Google Scholar, 26Chu S. Montrose M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3303-3307Crossref PubMed Scopus (64) Google Scholar). Activation of NHE1, moreover, enhances cell survival by inhibiting caspase activity and by enhancing the PI3K pathway (27Wu K.L. Khan S. Lakhe-Reddy S. Jarad G. Mukherjee A. Obejero-Paz C.A. Konieczkowski M. Sedor J.R. Schelling J.R. J. Biol. Chem. 2004; 279: 26280-26286Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The other NHE isoforms expressed in the colon are NHE3 which is involved in vectorial solute transport, but is not expressed in HT29 cells (25Gonda T. Maouyo D. Rees S.E. Montrose M.H. Am. J. Physiol. 1999; 276: G259-G270Crossref PubMed Google Scholar), and NHE2 which has been suggested to mediate butyrate-stimulated sodium absorption (22Bachmann O. Riederer B. Rossmann H. Groos S. Schultheis P.J. Shull G.E. Gregor M. Manns M.P. Seidler U. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G125-G133Crossref PubMed Scopus (75) Google Scholar, 28Krishnan S. Rajendran V.M. Binder H.J. Am. J. Physiol. Cell Physiol. 2003; 285: C1246-C1254Crossref PubMed Scopus (46) Google Scholar). In the current study, we sought to identify the principal pathways activated by extracellular Zn2+ in colonocytes and to determine what role, if any, ERK1/2 plays in mediating the above described zinc-dependent regulation of the Na+/H+ exchanger. Our results indicate that extracellular Zn2+, acting through the ZnR, triggers activation of ERK and PI 3-kinase pathways. Subsequently, the zinc-dependent activation of ERK1/2 leads to a robust and specific activation of NHE1 in HT29 colonocytes and similarly in murine native colon epithelium. Thus, our results provide a cellular mechanism linking the well known effects of zinc to cellular proliferation. Cell Culture—HT29-Cl cells were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich) as previously described (29Sekler I. Kopito R. Casey J.R. J. Biol. Chem. 1995; 270: 21028-21034Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 30Maouyo D. Chu S. Montrose M.H. Am. J. Physiol. Cell Physiol. 2000; 278: C973-C981Crossref PubMed Google Scholar). Cells grown on 60-mm plates were serum-starved for 24 h, and then washed in Ringer's solution for 30 min. The cells were subsequently stimulated in Ringer's solution supplemented with ZnSO4 (80 μm, unless otherwise indicated), for 10 min. The zinc chelator, Ca-EDTA (100 μm), was added to the Ringer's solution to reduce residual zinc in control cultures. Agonists and inhibitors used for the analysis of the various signal transduction pathways were added prior to the addition of zinc, during the wash in Ringer's solution, for the indicated times. Cells were then harvested into lysis buffer as previously described (31Hazan-Halevy I. Seger R. Levy R. J. Biol. Chem. 2000; 275: 12416-12423Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), in the presence of protease inhibitor mixture (Complete, Roche Applied Science) and centrifuged for 30 min (14,000 rpm). Supernatants (cytosolic fraction) were collected, SDS sample buffer was added, the samples were boiled for 5 min and then frozen at –80 °C until used. Tissue Preparation—Wistar rats (70–150 g, n = 4) were killed, and the colon removed and washed using Parsons solution as previously described (32Schultheiss G. Lan Kocks S. Diener M. Biol. Proc. Online. 2002; 3: 70-78Crossref PubMed Scopus (20) Google Scholar). Briefly, a longitudinal incision was made along the colon wall and about 5 tissue samples were cut from the distal part of the colon. The tissue was spread, keeping the mucosal-luminal side upwards, on coverslips using cyanoacrylate glue (24Chu J. Chu S. Montrose M.H. Am. J. Physiol. Cell Physiol. 2002; 283: C358-C372Crossref PubMed Scopus (53) Google Scholar). The tissue samples were kept at 37 °C in high K+ solution for no longer than 3 h (32Schultheiss G. Lan Kocks S. Diener M. Biol. Proc. Online. 2002; 3: 70-78Crossref PubMed Scopus (20) Google Scholar). In each experimental group, the results are the mean of at least 15 cells from three independent experiments from each animal. Monitoring Kinase Activation—Cell samples containing the cytosolic fraction (20 μg) were separated on 7.5% SDS-PAGE followed by immunoblotting (29Sekler I. Kopito R. Casey J.R. J. Biol. Chem. 1995; 270: 21028-21034Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 30Maouyo D. Chu S. Montrose M.H. Am. J. Physiol. Cell Physiol. 2000; 278: C973-C981Crossref PubMed Google Scholar). Antibodies against doubly phosphorylated ERK1/2 (33Wolf I. Rubinfeld H. Yoon S. Marmor G. Hanoch T. Seger R. J. Biol. Chem. 2001; 276: 24490-24497Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and total ERK1/2 or phosphorylated AKT and total AKT (Sigma and Cell Signaling) were detected digitally using ChemImager 5 (Alpha-Innotech, Labtrade), and the blots quantified using the ChemImager software. Phospho-ERK1/2 or AKT levels were normalized against the total ERK1/2 or AKT protein, respectively. Phosphorylation of ERK1/2 is presented as a percentage of the effect triggered by application of 80 μm zinc. Each graph represents an average of at least three independent experiments. Statistical analysis was performed using unpaired Student's t test assuming equal variance, comparing each treatment to zinc: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Fluorescent Imaging—Fluorescent imaging measurements were acquired as previously described (13Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11749-11754Crossref PubMed Scopus (208) Google Scholar). Briefly, for [Ca2+]i and [Zn2+]i measurements, cells were incubated with 5 μm Fura-2 AM (TEF) for 30 min in 0.1% bovine serum albumin in Ringer's solution. For pH measurements, cells were loaded with 1.25 μm BCECF-AM for 12 min. Following dye loading, the coverslips were mounted in a flow chamber. For pHi calibration, nigericin was added to KCl Ringer's (120 mm KCl replacing NaCl) solution at pH 6.8, 7, and 7.2, the relative fluorescence was monitored, and a linear calibration curve produced (34Boyarsky G. Ganz M.B. Sterzel R.B. Boron W.F. Am. J. Physiol. 1988; 255: C857-C869Crossref PubMed Google Scholar, 35Boyarsky G. Ganz M.B. Sterzel R.B. Boron W.F. Am. J. Physiol. 1988; 255: C844-C856Crossref PubMed Google Scholar). The NH4Cl prepulse paradigm was applied to estimate Na+/H+ exchanger activity. Briefly, cells were washed with Ringer's containing NH4Cl (30 mm), and an intracellular equilibrium of NH+4 and NH3 was reached. Replacing the extracellular buffer with Na+-free Ringer's (iso-osmotically replaced by NMG) caused rapid acidification of the cells. Na+/H+ exchanger activity was estimated by calculating the rate of recovery (dpHi/dt) following addition of Na+ to the Ringer's solution. The results shown are the means of 4–6 independent experiments, with averaged responses of 30 cells in each experiment. Statistical analysis was performed as described above, comparing the activity of NHE in treated cells versus control cells. For imaging experiments of the native colonic tissue, the coverslips were incubated with BCECF (5 μm) in the presence of 0.05% pluronic acid for 30 min and then washed with 0.1% bovine serum albumin serum in Ringer's solution for 10 min before mounting on the stage of an Olympus BX-50 microscope. The pHi measurements were performed only using crypts such that the in epithelial cells clearly surrounded the lumen. The NH4Cl prepulse paradigm and analysis was performed as described above. Specificity and Temporal Dependence of ERK1/2 Phosphorylation by Zinc—To determine if zinc, at physiological concentrations shown previously to activate the ZnR, will activate the MAP kinase pathway, analysis of time dependence was performed. Phosphorylation of ERK1/2 was monitored by Western blots using antibodies recognizing the doubly phosphorylated, active form of ERK1/2 in serum-starved HT29 cells. To determine the time course of ERK phosphorylation, zinc (80 μm) was applied for the indicated times and phosphorylation of ERK1/2 monitored (Fig. 1A). Zinc-dependent phosphorylation of ERK1/2 was apparent already 2 min following application, peaking at 30 min. Phosphorylation was reduced after 60 min by about 30%, and approached resting levels after 2 h. Only very slight staining of the two bands was detected when extracellular residual zinc was chelated by Ca-EDTA. No significant change was detected in the total-ERK1/2 following addition of zinc. Application of the MEK1/2 inhibitors (U0126, PD98095) for 10 min in Ringer's solution, inhibited Zn2+-dependent ERK1/2 phosphorylation (Fig. 1B), indicating that the effect is mediated through activation of MEK1/2. The MAP kinase pathway can be activated by a number of different heavy metals (6Samet J.M. Graves L.M. Quay J. Dailey L.A. Devlin R.B. Ghio A.J. Wu W. Bromberg P.A. Reed W. Am. J. Physiol. 1998; 275: L551-L558Crossref PubMed Google Scholar). We sought, therefore, to determine if ERK1/2 activation in colonocytes is metal-specific. Cells were exposed to the indicated heavy metals (100 μm, 10 min), and the resulting phosphorylation of ERK1/2 was compared with the effect triggered by zinc (Fig. 1C). As shown, application of Mn2+, Ni2+, Cd2+, or Fe2+ failed to trigger activation of ERK1/2. In contrast, Zn2+ was highly potent in triggering ERK phosphorylation, indicating that in colonocytes, zinc plays a prominent and highly specific role in the activation of this important intracellular signaling pathway. ERK1/2 Is Activated by Extracellular Zinc via the ZnR— While the above results suggest that zinc specifically triggers ERK1/2 phosphorylation, they do not specify the role of extracellular zinc. To address this, we examined whether cellular Zn2+ influx is apparent at concentrations and time intervals required for ERK1/2 phosphorylation in HT29 cells loaded with Fura-2 AM. This dye, known primarily as a Ca2+-sensitive indicator, has a 100-fold higher affinity to Zn2+ (Kd[Zn2+] ∼2 nm) (36Atar D. Backx P.H. Appel M.M. Gao W.D. Marban E. J. Biol. Chem. 1995; 270: 2473-2477Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), that is, at least 10-fold higher than the zinc probe previously used to monitor zinc in HT29 cells, Mag-fura (5Park K.S. Lee N.G. Lee K.H. Seo J.T. Choi K.Y. Am. J. Physiol. Gastrointest Liver Physiol. 2003; 19: 19Google Scholar, 37Sensi S.L. Canzoniero L.M. Yu S.P. Ying H.S. Koh J.Y. Kerchner G.A. Choi D.W. J. Neurosci. 1997; 17: 9554-9564Crossref PubMed Google Scholar). Thus, Fura-2 AM is a highly sensitive dye for monitoring even minute changes in intracellular zinc concentrations. Potential interference by intracellular Ca2+ was eliminated prior to application of zinc by depleting the intracellular Ca2+ stores with thapsigargin (200 nm, not shown) and by the use of nominally Ca2+-free solutions. As shown in Fig. 2A, there was no apparent rise in Fura-2 fluorescence when HT29 cells were perfused with zinc-containing Ringer's solution for 10 min following Ca2+ store depletion. The sensitivity of this approach is demonstrated by monitoring Zn2+ influx, induced by opening of L-type calcium channels by depolarization, of Min6 insulinoma cells. Application of the intracellular zinc chelator, TPEN (50 μm), reduced the fluorescent signal to resting levels (not shown), indicating that it is indeed triggered by zinc. At time intervals sufficient to trigger a robust activation of ERK, therefore, no zinc influx was apparent in HT29 cells, supporting our hypothesis that ERK1/2 phosphorylation is mediated by extracellular Zn2+. To study the physiological significance of ERK1/2 activation by zinc, a dose response analysis was performed. Previous studies have employed zinc in the presence of serum (5Park K.S. Lee N.G. Lee K.H. Seo J.T. Choi K.Y. Am. J. Physiol. Gastrointest Liver Physiol. 2003; 19: 19Google Scholar, 38Thamilselvan V. Fomby M. Walsh M. Basson M.D. J. Surg. Res. 2003; 110: 255-265Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). A major drawback of this approach is that the fraction of the zinc ions bound to serum components is difficult to estimate, the result being that the free zinc concentration under such conditions is unknown. We therefore used zinc in Ringer's solution, such that the free zinc concentrations obtained (calculated using Geochem software, Ref. 13Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11749-11754Crossref PubMed Scopus (208) Google Scholar) are similar to those present in the digestive tract (19Sandstrom B. Cederblad A. Kivisto B. Stenquist B. Andersson H. Am. J. Clin. Nutr. 1986; 44: 501-504Crossref PubMed Scopus (35) Google Scholar, 20Sandstrom B. Analyst. 1995; 120: 913-915Crossref PubMed Google Scholar). Phosphorylation of ERK1/2 was monitored in cells exposed to the indicated concentrations of free zinc for 10 min (Fig. 2B) and was already apparent using 40 μm zinc. The effect of zinc on ERK phosphorylation was maximal at 80 μm and at 150 μm was still 10 ± 0.2-fold higher than controls. This was reduced by ∼30% at higher zinc concentrations (200 μm). The results of this analysis show that zinc at concentrations found in the digestive tract (19Sandstrom B. Cederblad A. Kivisto B. Stenquist B. Andersson H. Am. J. Clin. Nutr. 1986; 44: 501-504Crossref PubMed Scopus (35) Google Scholar, 20Sandstrom B. Analyst. 1995; 120: 913-915Crossref PubMed Google Scholar, 39Knudsen E. Jensen M. Solgaard P. Sorensen S.S. Sandstrom B. J. Nutr. 1995; 125: 1274-1282PubMed Google Scholar), which induce Ca2+ release through the ZnR (13Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11749-11754Crossref PubMed Scopus (208) Google Scholar), also trigger phosphorylation of ERK1/2. We next sought to determine the specific role played by the ZnR in mediating zinc-dependent ERK1/2 phosphorylation. To do this, we exploited the fact that the ZnR undergoes a profound functional desensitization that remains refractory for 3 h following exposure to 100 μm Zn2+ for 30 min. The ZnR-dependent Ca2+ release following reapplication of Zn2+ recovered to ∼10 and 20% after 4 and 6 h, respectively (Fig. 3A). As shown in Fig. 3B, zinc desensitization of the ZnR was also followed by a similar pattern of inhibition of the zinc-dependent phosphorylation of ERK1/2. The inhibition of ERK1/2 phosphorylation, monitored 3 h after the desensitization, was almost complete (90 ± 5%) and was still apparent even after 6 h, yielding an ∼50% inhibition. Phosphorylation of ERK1/2 induced by PMA (Fig. 3C) was used as a positive control to determine if the MAPK pathway, following zinc desensitization, remained responsive. No significant changes were detected in ERK1/2 phosphorylation induced by PMA in cells that were desensitized versus control cells, indicating that the ERK1/2 pathway was functional. The striking effect of ZnR desensitization, and the similarity between the patterns of the Ca2+ response and the zinc-dependent ERK1/2 activation suggest that a functional ZnR is essential for mediating zinc-dependent activation of the MAP kinase pathway. ERK1/2 Activation by Extracellular Zn2+Is Mediated by Ca2+-dependent and -independent Pathways—Activation of ERK has previously been demonstrated to be mediated by intracellular Ca2+ released via the IP3 pathway, such as that triggered by zinc via the ZnR in epithelial cells, ATP in astrocytes or by the cholinergic agonist, carbachol, in intestinal cells (18Keely S.J. Calandrella S.O. Barrett K.E. J. Biol. Chem. 2000; 275: 12619-12625Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 40Neary J.T. Kang Y. Willoughby K.A. Ellis E.F. J. Neurosci. 2003; 23: 2348-2356Crossref PubMed Google Scholar, 41Cullen P.J. Lockyer P.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 339-348Crossref PubMed Scopus (304) Google Scholar, 42Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar). However, cell signaling upstream or distinct from the IP3 pathway may also play a role in Zn2+-mediated ERK activation. We have, therefore, addressed the role of the Ca2+ rise in ERK activation by chelating intracellular Ca2+ using BAPTA-AM (25 μm, applied for 15 min prior to addition of zinc). As shown in Fig. 4A, BAPTA partially reduced zinc-dependent ERK1/2 phosphorylation, resulting in 60 ± 3% inhibition of Zn2+-dependent activation. To further elucidate the role of upstream components of the IP3 pathway, cells were pretreated with the PLC inhibitor, U73122 (4 μm, applied for 15 min). Application of the PLC inhibitor resulted in 40 ± 4% inhibition of ERK phosphorylation (Fig. 4A), similar to the effect of Ca2+ chelation. These results indicate that a calcium rise, triggered by IP3 pathway activation, plays a role in mediating zinc-dependent ERK activation in HT29 cells. Calcium-calmodulin (CaM) kinase II is activated by intracellular calcium rise and has been suggested to trigger MAP kinase activation, possibly by inhibiting Ras-GTPase-activating protein (RAS-GAP), thereby leading to Ras activation (43Egea J. Espinet C. Soler R.M. Peiro S. Rocamora N. Comella J.X. Mol. Cell. Biol. 2000; 20: 1931-1946Crossref PubMed Scopus (47) Google Scholar, 44Chen H.J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 45Ginnan R. Singer H.A. Am. J. Physiol. Cell Physiol. 2002; 282: C754-C761Crossref PubMed Scopus (96) Google Scholar). We studied the role of CaM kinase II in Zn2+-dependent phosphorylation of ERK1/2 using the CaM kinase II inhibitors, KN93 and KN62 (applied for 30 min in Ringer's solution). As shown in Fig. 4A, the inhibitory effect of CaM kinase II inhibitors (40 ± 2%) was similar to that of the upstream inhibitors, U73122 and BAPTA, suggesting that calcium-dependent ERK1/2 phosphorylation by the ZnR is mediated by CaM kinase II. A calcium rise like that mediated by the ZnR may trigger PKC activation. However, the PKC inhibitor bisindolylmaleimide I (BI) did not attenuate the zinc-dependent activation of ERK (Fig. 4B), suggesting that Zn2+-dependent activation of ERK1/2 is not mediate" @default.
• W2131462893 created "2016-06-24" @default.
• W2131462893 creator A5045420328 @default.
• W2131462893 creator A5081699453 @default.
• W2131462893 creator A5088169519 @default.
• W2131462893 creator A5088840006 @default.
• W2131462893 date "2004-12-01" @default.
• W2131462893 modified "2023-10-14" @default.
• W2131462893 title "Extracellular Zinc Triggers ERK-dependent Activation of Na+/H+ Exchange in Colonocytes Mediated by the Zinc-sensing Receptor" @default.
• W2131462893 cites W1499097678 @default.
• W2131462893 cites W173488229 @default.
• W2131462893 cites W188399478 @default.
• W2131462893 cites W1915245168 @default.
• W2131462893 cites W1959422603 @default.
• W2131462893 cites W1978686430 @default.
• W2131462893 cites W1981336142 @default.
• W2131462893 cites W1983297072 @default.
• W2131462893 cites W1984670510 @default.
• W2131462893 cites W1984889921 @default.
• W2131462893 cites W1985475545 @default.
• W2131462893 cites W1985760529 @default.
• W2131462893 cites W1989678156 @default.
• W2131462893 cites W1994816216 @default.
• W2131462893 cites W1998932212 @default.
• W2131462893 cites W1999234400 @default.
• W2131462893 cites W1999956246 @default.
• W2131462893 cites W2001087525 @default.
• W2131462893 cites W2007275795 @default.
• W2131462893 cites W2015358345 @default.
• W2131462893 cites W2025067667 @default.
• W2131462893 cites W2031989685 @default.
• W2131462893 cites W2033785444 @default.
• W2131462893 cites W2035063007 @default.
• W2131462893 cites W2035831063 @default.
• W2131462893 cites W2036839645 @default.
• W2131462893 cites W2039293969 @default.
• W2131462893 cites W2042631205 @default.
• W2131462893 cites W2046164813 @default.
• W2131462893 cites W2046659481 @default.
• W2131462893 cites W2046861885 @default.
• W2131462893 cites W2047386002 @default.
• W2131462893 cites W2050141745 @default.
• W2131462893 cites W2052345143 @default.
• W2131462893 cites W2052938761 @default.
• W2131462893 cites W2053080879 @default.
• W2131462893 cites W2056925277 @default.
• W2131462893 cites W2057211548 @default.
• W2131462893 cites W2059237401 @default.
• W2131462893 cites W2068461173 @default.
• W2131462893 cites W2074548785 @default.
• W2131462893 cites W2077435537 @default.
• W2131462893 cites W2079051863 @default.
• W2131462893 cites W2080158664 @default.
• W2131462893 cites W2087444241 @default.
• W2131462893 cites W2087784543 @default.
• W2131462893 cites W2089449927 @default.
• W2131462893 cites W2091749240 @default.
• W2131462893 cites W2096164786 @default.
• W2131462893 cites W2104034706 @default.
• W2131462893 cites W2110652761 @default.
• W2131462893 cites W2111334366 @default.
• W2131462893 cites W2113755074 @default.
• W2131462893 cites W2114170740 @default.
• W2131462893 cites W2116683511 @default.
• W2131462893 cites W2138149556 @default.
• W2131462893 cites W2139198003 @default.
• W2131462893 cites W2140333191 @default.
• W2131462893 cites W2149948289 @default.
• W2131462893 cites W2154473726 @default.
• W2131462893 cites W2160500827 @default.
• W2131462893 cites W2160702055 @default.
• W2131462893 cites W2169650081 @default.
• W2131462893 cites W2174270021 @default.
• W2131462893 cites W2182568691 @default.
• W2131462893 cites W2183807052 @default.
• W2131462893 cites W2187511581 @default.
• W2131462893 cites W2397284138 @default.
• W2131462893 cites W2407398125 @default.
• W2131462893 cites W2569329648 @default.
• W2131462893 doi "https://doi.org/10.1074/jbc.m406581200" @default.
• W2131462893 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15355987" @default.
• W2131462893 hasPublicationYear "2004" @default.
• W2131462893 type Work @default.
• W2131462893 sameAs 2131462893 @default.
• W2131462893 citedByCount "97" @default.
• W2131462893 countsByYear W21314628932012 @default.
• W2131462893 countsByYear W21314628932013 @default.
• W2131462893 countsByYear W21314628932014 @default.
• W2131462893 countsByYear W21314628932015 @default.
• W2131462893 countsByYear W21314628932016 @default.
• W2131462893 countsByYear W21314628932017 @default.
• W2131462893 countsByYear W21314628932018 @default.
• W2131462893 countsByYear W21314628932019 @default.
• W2131462893 countsByYear W21314628932020 @default.
• W2131462893 countsByYear W21314628932021 @default.
• W2131462893 countsByYear W21314628932022 @default.
• W2131462893 countsByYear W21314628932023 @default.
• W2131462893 crossrefType "journal-article" @default.

• next