Title: Differential Activities of Glucocorticoid-induced Leucine Zipper Protein Isoforms
Abstract: Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of ∼17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation. Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of ∼17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation. Glucocorticoid-induced leucine zipper (GILZ) 3The abbreviations used are:GILZglucocorticoid-induced leucine zipper proteinCCDcortical collecting ductCDcollecting ductEGFepidermal growth factorENaCepithelial sodium channelERKextracellular signal-regulated kinase 1/2MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseORFopen reading frameGREglucocorticoid response elementFHREforkhead response elementRACErapid amplification of cDNA endsmGILZmouse GILZBrdUrdbromodeoxyuridineP1promoter 1P2promoter 2. 3The abbreviations used are:GILZglucocorticoid-induced leucine zipper proteinCCDcortical collecting ductCDcollecting ductEGFepidermal growth factorENaCepithelial sodium channelERKextracellular signal-regulated kinase 1/2MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseORFopen reading frameGREglucocorticoid response elementFHREforkhead response elementRACErapid amplification of cDNA endsmGILZmouse GILZBrdUrdbromodeoxyuridineP1promoter 1P2promoter 2. is a small leucine zipper protein of ∼17 kDa. As its name implies, GILZ was first discovered as a dexamethasone-induced transcript in murine thymocytes, which it protects from apoptosis induced by treatment with anti-CD3 antibody (1D'Adamio F. Zollo O. Moraca R. Ayroldi E. Bruscoli S. Bartoli A. Cannarile L. Migliorati G. Riccardi C. Immunity. 1997; 7: 803-812Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). It is a member of the TSC22D (transforming growth factor β1-stimulated clone 22 domain) family of proteins that are widely expressed and appear to impact multiple biological processes (2Ayroldi E. Zollo O. Macchiarulo A. Di Marco B. Marchetti C. Riccardi C. Mol. Cell. Biol. 2002; 22: 7929-7941Crossref PubMed Scopus (150) Google Scholar, 3Bhalla V. Soundararajan R. Pao A.C. Li H. Pearce D. Am. J. Physiol. 2006; 291: F714-F721Crossref PubMed Scopus (85) Google Scholar, 4Fiol D.F. Mak S.K. Kultz D. FEBS J. 2007; 274: 109-124Crossref PubMed Scopus (50) Google Scholar, 5Treisman J.E. Lai Z.C. Rubin G.M. 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Commun. 1996; 222: 821-826Crossref PubMed Scopus (43) Google Scholar) and, more recently, its detection at sites of epithelial-mesenchymal interactions during mouse embryogenesis (10Dohrmann C.E. Belaoussoff M. Raftery L.A. Mech. Dev. 1999; 84: 147-151Crossref PubMed Scopus (31) Google Scholar) suggest an important role for this protein during vertebrate development. A similar role has been identified for the TSC22 homologue, bunched, in developing Drosophila larvae (11Levine B. Jean-Francois M. Bernardi F. Gargiulo G. Dobens L. Dev. Biol. 2007; 305: 217-231Crossref PubMed Scopus (15) Google Scholar). TSC22D2 and TSC22D4 are expressed in renal cortex, medulla, and papilla and are involved in adaptation of these cells to hypertonicity (4Fiol D.F. Mak S.K. Kultz D. FEBS J. 2007; 274: 109-124Crossref PubMed Scopus (50) Google Scholar). These two transcripts are significantly up-regulated by hyperosmolality, and overexpression of a specific splice variant of TSC22D2 (TSC22D2-4) in mIMCD3 cells confers protection against osmotic stress-induced cell death (4Fiol D.F. Mak S.K. Kultz D. FEBS J. 2007; 274: 109-124Crossref PubMed Scopus (50) Google Scholar). glucocorticoid-induced leucine zipper protein cortical collecting duct collecting duct epidermal growth factor epithelial sodium channel extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase/extracellular signal-regulated kinase kinase open reading frame glucocorticoid response element forkhead response element rapid amplification of cDNA ends mouse GILZ bromodeoxyuridine promoter 1 promoter 2 glucocorticoid-induced leucine zipper protein cortical collecting duct collecting duct epidermal growth factor epithelial sodium channel extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase/extracellular signal-regulated kinase kinase open reading frame glucocorticoid response element forkhead response element rapid amplification of cDNA ends mouse GILZ bromodeoxyuridine promoter 1 promoter 2 TSC22D3 (also known as GILZ) is expressed in numerous tissues and is rapidly induced by glucocorticoids in T lymphocytes, in which it inhibits anti-CD3-induced interleukin-2 production, interleukin-2 receptor expression, Fas and Fas-ligand up-regulation, and cell death consequent to CD3-induced activation (1D'Adamio F. Zollo O. Moraca R. Ayroldi E. Bruscoli S. Bartoli A. Cannarile L. Migliorati G. Riccardi C. Immunity. 1997; 7: 803-812Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 12Riccardi C. Cifone M.G. Migliorati G. Cell Death Differ. 1999; 6: 1182-1189Crossref PubMed Scopus (82) Google Scholar, 13Ayroldi E. Migliorati G. Bruscoli S. Marchetti C. Zollo O. Cannarile L. D'Adamio F. Riccardi C. Blood. 2001; 98: 743-753Crossref PubMed Scopus (253) Google Scholar, 14Riccardi C. Zollo O. Nocentini G. Bruscoli S. Bartoli A. D'Adamio F. Cannarile L. Delfino D. Ayroldi E. Migliorati G. Therapie. 2000; 55: 165-169PubMed Google Scholar). Moreover, GILZ expression is down-regulated by anti-CD3 stimulation, further suggesting that GILZ contributes to the control of T-cell activation and development (12Riccardi C. Cifone M.G. Migliorati G. Cell Death Differ. 1999; 6: 1182-1189Crossref PubMed Scopus (82) Google Scholar, 13Ayroldi E. Migliorati G. Bruscoli S. Marchetti C. Zollo O. Cannarile L. D'Adamio F. Riccardi C. Blood. 2001; 98: 743-753Crossref PubMed Scopus (253) Google Scholar). Recent evidence also supports a role for GILZ in the antiproliferative effects of glucocorticoids in T lymphocytes (15Ayroldi E. Zollo O. Bastianelli A. Marchetti C. Agostini M. Di Virgilio R. Riccardi C. J. Clin. Invest. 2007; 117: 1605-1615Crossref PubMed Scopus (127) Google Scholar). Glucocorticoids also up-regulate GILZ expression in macrophages, and GILZ overexpression, in turn, inhibits production of inflammatory mediators and proinflammatory chemokines as well as Toll-like receptor expression (16Berrebi D. Bruscoli S. Cohen N. Foussat A. Migliorati G. Bouchet-Delbos L. Maillot M.C. Portier A. Couderc J. Galanaud P. Peuchmaur M. Riccardi C. Emilie D. Blood. 2003; 101: 729-738Crossref PubMed Scopus (225) Google Scholar). Although the role of GILZ in immune cell function has been well studied, its role outside of the immune system, until recently, was unknown. In kidney cortical collecting duct (CCD), as well as in mpkCCDc14 cells, a highly differentiated mouse CCD cell line, its expression was shown to be robustly induced by aldosterone (17Robert-Nicoud M. Flahaut M. Elalouf J.M. Nicod M. Salinas M. Bens M. Doucet A. Wincker P. Artiguenave F. Horisberger J.D. Vandewalle A. Rossier B.C. Firsov D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2712-2716Crossref PubMed Scopus (185) Google Scholar). In this cell line, GILZ markedly augments epithelial sodium channel (ENaC)-mediated sodium (Na+) transport (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), a process that is central to Na+ homeostasis. Transepithelial Na+ transport involves the regulated functioning of many different pathways and elements that are interestingly shared in several remarkably diverse cellular processes, such as cell proliferation, differentiation, and apoptosis. Importantly, GILZ appears to stimulate ENaC cell surface expression at least in part by inhibiting extracellular signal-regulated kinase 1/2 (ERK). In the course of experiments aimed at characterizing GILZ function and expression in a kidney collecting duct cell line (mpkCCDc14) using a newly developed antiserum, it became clear that several protein species were expressed, including one prominent species with a molecular mass of ∼28 kDa, substantially larger than the originally identified GILZ isoform, GILZ1. Mass spectrometric analysis indicated that this species probably represented a distinct GILZ isoform, and we therefore set out to identify and functionally characterize cDNAs corresponding to this and other expressed GILZ isoforms. Production of Anti-GILZ Antiserum—A rabbit polyclonal antiserum recognizing mouse GILZ was generated to the peptide sequence corresponding to amino acids 111–125 in GILZ1. Peptide synthesis, purification, verification of peptide sequence (by mass spectrometry), conjugation to carrier (keyhole limpet hemocyanin), injection of animals (New Zealand White rabbits), and subsequent boosters were all carried out by Quality Controlled Biochemicals (Hopkinton, MA). Following extensive laboratory characterization, selected bleeds were affinity-purified and used for all subsequent analyses. mpkCCDc14 Cell Culture and Electrophysiological Measurements—mpkCCDc14 cells were maintained in plastic tissue culture flasks in a modified Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) medium ("regular medium"), as described previously (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. RafestinOblin M.E. Rossier B.C. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar). For electrophysiological and biochemical experiments, the cells were seeded and grown in regular medium on collagen-coated filters (Transwell; pore size 0.4 μm; Corning Costar) until the cell monolayers reached transepithelial resistance greater than 1000 ohms·cm2. They were then maintained in steroid hormone-free, serum-free medium (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) for at least 24 h prior to treatment with aldosterone (10–6m, apical and basolateral sides) or an equal volume of vehicle as control for specified periods of time. Following electrophysiological measurements, cells were harvested and processed for protein analysis. U0126 treatment (10 μm) was performed as described previously (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). All experiments were performed in parallel with appropriate vehicle controls (aldosterone was dissolved in ethanol; U0126 was dissolved in Me2SO). Transepithelial resistance and potential difference across the cell monolayers were measured using a mini-volt ohmmeter (MilliCell ERS; Millipore Corp.) at the specified time periods following treatment. The equivalent short circuit current (Ieq) was calculated using Ohm's law (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. RafestinOblin M.E. Rossier B.C. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar). Amiloride (1 μm) added to the medium completely inhibited this current, thereby indicating its ENaC dependence (data not shown). 5′-Rapid Amplification of cDNA Ends (RACE) PCR and Cloning of GILZ Isoforms—Full-length cDNA corresponding to various mouse GILZ (mGILZ) isoforms were cloned by rapid amplification of cDNA ends using the SMART-RACE cDNA amplification kit (Clontech), according to the manufacturer's instructions. Total RNA isolated from mpkCCDc14 cells was used for reverse transcription, followed by 5′-RACE-PCR (using 5′-TTA CAC CGC AGA ACC ACC AGG GGC TTC CGG GG-3′ as the gene-specific primer) and subsequent cloning into the TOPOII TA cloning vector (Invitrogen). Following sequence analyses, the ORFs were subcloned into the in vitro expression vector, pMO (described previously (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar)). An N-terminal Myc tag (EQKLISEEDL) was added to facilitate analysis of expression. In Silico Analysis of the Tsc22d3 Gene—The obtained 5′-RACE products were searched via BLAST against the Mus musculus genome on the NCBI site on the World Wide Web, and a region 12 kb upstream and 2 kb downstream of the two most distal matched BLAST results was used for further in silico analysis. Exons were defined by the exact matched BLAST results to the 5′-RACE products and previously reported Tsc22d3 mRNA sequences (available on the World Wide Web), and introns were defined as the genomic sequence between individual exons. The GC content of the Tsc22d3 gene was analyzed using two World Wide Web-based programs, "CpG Island Searcher" (available on the World Wide Web) (20Takai D. Jones P.A. In Silico Biol. 2003; 3: 235-240PubMed Google Scholar) and "DNA Base Composition Analysis Tool" (available on the World Wide Web). Initial promoter description of the Tsc22d3 gene was performed previously by other workers (21Asselin-Labat M.L. David M. Biola-Vidamment A. Lecoeuche D. Zennaro M.C. Bertoglio J. Pallardy M. Blood. 2004; 104: 215-223Crossref PubMed Scopus (114) Google Scholar, 22Asselin-Labat M.L. Biola-Vidamment A. Kerbrat S. Lombes M. Bertoglio J. Pallardy M. Mol. Endocrinol. 2005; 19: 1752-1764Crossref PubMed Scopus (45) Google Scholar, 23Wang J.C. Derynck M.K. Nonaka D.F. Khodabakhsh D.B. Haqq C. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15603-15608Crossref PubMed Scopus (253) Google Scholar), identifying functional glucocorticoid response elements (GREs) and forkhead response elements (FHREs). For further analysis of the promoter for other putative transcription factor binding sites, TFsearch (available on the World Wide Web), an application on the Helmholtz Network for Bioinformatics (available on the World Wide Web), and MatInspector (available on the World Wide Web) (24Cartharius K. Frech K. Grote K. Klocke B. Haltmeier M. Klingenhoff A. Frisch M. Bayerlein M. Werner T. Bioinformatics. 2005; 21: 2933-2942Crossref PubMed Scopus (1632) Google Scholar) were used. mpkCCDc14 Transient Transfections—mpkCCDc14 cells (at 70–80% confluence) were transfected with various Myc-mGILZ constructs (5 μg of DNA/2 × 106 cells) or the empty vector alone (vector control) as specified, using a high efficiency electroporation protocol (nucleofection; Amaxa Biosystems Inc.) according to the manufacturer's instructions. Mass Spectrometric Analysis—GILZ was immunoprecipitated from aldosterone-treated (10–6m) mpkCCDc14 cell lysates using the new anti-GILZ antibody. Immune complexes were resolved by SDS-PAGE, and protein bands were visualized by Coomassie staining. The 28-kDa band of interest was carefully excised and prepared for in-gel digest with trypsin followed by liquid chromatography tandem mass spectrometric analysis, according to techniques standardized by the Mass Spectrometric Facility at the University of California (San Francisco, CA) (available on the World Wide Web). Liquid chromatography tandem mass spectrometric analysis per se was performed by Custom Biologics (Toronto, Canada), according to procedures standardized by the company. Purified eluted tryptic peptides were detected by an LCQ DECA XP mass spectrometer (Thermo), equipped with an electrospray ionization source, a low flow metal needle assembly operating in data-dependent mode. The method consisted of two scan events, a full scan and a second data-dependent tandem mass spectrometry scan. The dynamic mass range of the full scan was set at 300–3000 m/z. The resulting tandem mass spectrometry data-dependent scan rejected known "contaminant" masses of 371.0, 391.0, 445.0, 462.0, 1221.89, 1321.9, 1421.9, 1521.8, 1521.9, 1621.9, 1721.9, and 1821.9 m/z. Other method settings included a default charge state set to 4, dynamic exclusion with repeat count set to 2, repeat duration 1 min, an exclusion list size of 25, and exclusion duration of 3 min. All other method parameters were default values set by the Xcaliber software, version 1.2 (Thermo). Using this software, raw sequence data files were searched against the extensive mouse/rat species-specific data base developed by Custom Biologics. Any protein with a peptide match was manually inspected and only accepted if a contiguous y and/or b ion series of greater than 7 residues was obtained. Sequences so obtained were reanalyzed using the NCBI BLAST search in order to confirm identity of the protein. HEK 293 Cell Culture and Transient Transfections—Human Embryonic Kidney (HEK 293) cells were regularly maintained in plastic tissue culture flasks at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. For the transfection experiments, cells were seeded on 6-well dishes and allowed to grow overnight in antibiotic-free medium. They were then transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Following 24 h of transfection, cell lysates were harvested for protein analysis. For experiments involving immunofluorescent staining of cell surface and total cellular ENaC, cells were seeded on poly-l-lysine-coated coverslips in 6-well dishes (∼1.8 × 105 cells/well) and allowed to grow until optimal cell density was reached. Cells were then transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen) and optimized DNA concentrations (200 ng each of α-, β-, and γ-FLAG-xENaC and 250 ng of various GILZ constructs/well). After treatment, the coverslips were removed and processed to detect expression of cell surface ENaC using live cell staining or using permeabilized staining to detect total cellular ENaC, as described previously (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 25Kikonyogo A. Bouamr F. Vana M.L. Xiang Y. Aiyar A. Carter C. Leis J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11199-11204Crossref PubMed Scopus (192) Google Scholar). Cell surface ENaC was visualized using FLAG monoclonal antibody M2 (Sigma) and goat anti-mouse secondary antibody conjugated to Alexa-594 fluorophore (Molecular Probes, Inc., Eugene, OR) followed by nuclear co-staining with 4′,6-diamidino-2-phenylindole. Images were obtained at ×630 magnification using a Zeiss Axioscope epifluorescence microscope with Metamorph imaging software (Universal Imaging) and filter sets for 4′,6-diamidino-2-phenylindole (nuclei) and Alexa-594 (FLAG-ENaC). Each experiment was repeated at least three times with similar results. For quantitation, a blinded observer counted the number of cells with detectable surface ENaC, divided by the number of 4′,6-diamidino-2-phenylindole-positive cells, in at least three independent experiments of cells subjected to live cell staining. The typical number of ENaC-positive cells in controls ranged from 40 to 50 per 100 cells. Cell Proliferation Assays—HEK 293 cells were seeded on 96-well tissue culture plates at a concentration of 1 × 104 cells/well and allowed to grow overnight in the absence of antibiotics. They were then transfected with 200 ng of the specified GILZ constructs or the empty vector (control) using Lipofectamine 2000. 24 h following transfection, the cells were treated with 10 μm 5-bromo-2′-deoxyuridine (BrdUrd) for an additional 24 h. Cell proliferation was then assessed by (colorimetric) enzyme-linked immunosorbent assay using a commercially available BrdUrd incorporation kit according to the manufacturer's instructions (Roche Applied Science). All transfections were performed in triplicate, and all experiments were repeated at least four times with similar results. NF-κB Luciferase Reporter Assays—CV1-b mouse kidney epithelial cells (Cell Culture Facility, University of California, San Francisco) were regularly maintained in plastic tissue culture flasks at 37 °C in Dulbecco's modified Eagle's medium H-16 supplemented with 5% fetal bovine serum and 100 units/ml penicillin/streptomycin, as described previously (26Liu W. Wang J. Yu G. Pearce D. Mol. Endocrinol. 1996; 10: 1399-1406PubMed Google Scholar). For NF-κB luciferase reporter assays, cells were seeded on 12-well dishes, allowed to grow to optimal cell density, and co-transfected with 200 ng of the specified GILZ constructs, 250 ng of a reporter vector (NF-κB-LUC) containing tandem repeats of the murine NF-κB site in which the promoter drives the expression of the firefly luciferase (a kind gift from Dr. Warner Greene (Gladstone Institutes)), and 20 ng of the constitutively transcribed RSV-β-galactosidase plasmid, Δ6RL (which serves as an internal control for transfection efficiency (27Meijer O.C. Williamson A. Dallman M.F. Pearce D. J. Neuroendocrinol. 2000; 12: 245-254Crossref PubMed Scopus (68) Google Scholar)), using Lipofectamine, according to the manufacturer's instructions (Invitrogen). 48 h after transfection, cell lysates were harvested and processed to assess protein content, luciferase, and β-galactosidase activity, as described previously (26Liu W. Wang J. Yu G. Pearce D. Mol. Endocrinol. 1996; 10: 1399-1406PubMed Google Scholar, 27Meijer O.C. Williamson A. Dallman M.F. Pearce D. J. Neuroendocrinol. 2000; 12: 245-254Crossref PubMed Scopus (68) Google Scholar). Since activation of corticosteroid receptors may strongly influence the Rous sarcoma virus promoter, values are presented normalized to total protein content. Each transfection was performed in triplicate, and each experiment was repeated at least three times with similar results. Immunoprecipitation and Western Blot Analyses—Protein lysates from cells/tissue samples were prepared as previously described (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Immunoprecipitation of transfected FLAG-c-Raf was carried out using anti-FLAG-agarose conjugate (Sigma) according to the manufacturer's instructions. Immunoblotting was performed as previously described (18Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), using either our new anti-GILZ antibody or one of the following commercially available antibodies, as specified throughout. phospho-ERK antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA). Total ERK antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-SGK1 (serum and glucocorticoid-induced kinase-1) antibody was a kind gift from Prof. Gary Firestone (University of California, Berkeley, CA). Anti-Myc antibody (Santa Cruz Biotechnology) was used to assess heterologous expression of Myc-tagged GILZ isoforms. Anti-FLAG antibody was purchased from Sigma. Blots were stripped and reprobed for Actin to ensure equality of protein loading (anti-actin antibody; Chemicon International, Temecula, CA). Peptide Competition Assay—In order to check the specificity of signals obtained, the anti-GILZ antibody was preincubated overnight with a 50 μg/ml concentration of the immunizing peptide and then centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was used as primary antibody for overnight incubation with the protein blot (at the same dilution as the original antibody). Statistical Analysis—Data are represented as mean ± S.E. In all experiments involving Na+ current measurements, 3–6 samples were tested, and at least three independent experiments were performed with the same treatment protocol. Unless otherwise specified, all statistical comparisons were evaluated using Student's unpaired two-tailed t test, and significance was defined as p < 0.05. Identification of Novel GILZ Isoforms in Cultured Kidney Collecting Duct (CD) Cells and Animal Tissues—In order to begin to characterize endogenous GILZ protein in CD cells, we developed a rabbit polyclonal antiserum to mouse GILZ (see "Experimental Procedures"). As shown in Fig. 1A, the antiserum recognizes GILZ in whole cell lysates of HEK 293 kidney epithelial cells transiently transfected with Myc-tagged or untagged forms of mGILZ. Preimmune serum or antiserum precleared with the immunizing peptide (Peptide Competition Assay) gave little-to-no signal, thereby verifying the authenticity of the new antiserum. As a further test of GILZ specificity, we analyzed possible cross-reactivity with its closest relative in the TSC22D family, TSC22D1 (Fig. 1B). The antiserum was found to specifically recognize GILZ but not TSC22D1. Fig. 1, A and B, shows that this new antiserum specifically recognizes heterologously expressed GILZ. We next determined if the new antiserum would recognize endogenous GILZ in mouse kidney epithelial cells. For this purpose, mpkCCDc14 cells were grown on Transwell filters and treated with 10–6m aldosterone for specified periods of time, as described under "Experimental Procedures." Harvested protein lysates were subjected to SDS-PAGE. Western blot analysis indicated that the new antiserum did indeed recognize a 17 kDa band, which comigrated with transfected mGILZ, (Fig. 1C, panel 1); however, several additional bands were apparent, including a prominent ∼28 kDa band, which was also consistently aldosterone-induced (Fig. 1, C and D). A peptide competition assay suggested that these signa