EF Domains Are Sufficient for Nongenomic Mineralocorticoid Receptor Actions
2008; Elsevier BV; Volume: 283; Issue: 11 Linguagem: Inglês
10.1074/jbc.m708751200
ISSN1083-351X
AutoresClaudia Großmann, Ruth Freudinger, Sigrid Mildenberger, Britta Husse, Michael Gekle,
Tópico(s)Ion Transport and Channel Regulation
ResumoThe mineralocorticoid receptor (MR) is important for salt homeostasis and reno-cardiovascular pathophysiology. Signaling mechanisms include, besides classical genomic pathways, nongenomic pathways with putative pathophysiological relevance involving the mitogen-activated protein kinases ERK1/2. We determined the MR domains required for nongenomic signaling and their potential to elicit pathophysiological effects in cultured cells under defined conditions. The expression of full-length human MR or truncated MR consisting of the domains CDEF (MRCDEF), DEF (MRDEF), or EF (MREF) renders cells responsive for the MR ligand aldosterone with respect to nongenomic ERK1/2 phosphorylation, whereas only full-length MR and MRCDEF conferred genomic responsiveness. ERK1/2 phosphorylation depends on the EGF receptor and cSRC kinase. MREF expression is sufficient to evoke the aldosterone-induced increase of collagen III levels, similar to full-length MR expression. Our data suggest that nongenomic MR signaling is mediated by the EF domains and present the first proof of principle showing that nongenomic signaling can be sufficient for some pathophysiological effects. The minimum amino acid motif required for nongenomic MR signaling and its importance in various effects have yet to be determined. The mineralocorticoid receptor (MR) is important for salt homeostasis and reno-cardiovascular pathophysiology. Signaling mechanisms include, besides classical genomic pathways, nongenomic pathways with putative pathophysiological relevance involving the mitogen-activated protein kinases ERK1/2. We determined the MR domains required for nongenomic signaling and their potential to elicit pathophysiological effects in cultured cells under defined conditions. The expression of full-length human MR or truncated MR consisting of the domains CDEF (MRCDEF), DEF (MRDEF), or EF (MREF) renders cells responsive for the MR ligand aldosterone with respect to nongenomic ERK1/2 phosphorylation, whereas only full-length MR and MRCDEF conferred genomic responsiveness. ERK1/2 phosphorylation depends on the EGF receptor and cSRC kinase. MREF expression is sufficient to evoke the aldosterone-induced increase of collagen III levels, similar to full-length MR expression. Our data suggest that nongenomic MR signaling is mediated by the EF domains and present the first proof of principle showing that nongenomic signaling can be sufficient for some pathophysiological effects. The minimum amino acid motif required for nongenomic MR signaling and its importance in various effects have yet to be determined. The mineralocorticoid receptor (MR) 2The abbreviations used are:MRmineralocorticoid receptorEGFepidermal growth factorERKextracellular signal-regulated kinaseELISAenzyme-linked immunosorbent assayEGFPenhanced green fluorescent proteinPBSphosphate-buffered salineSEAPsecretory alkaline phosphataseGREglucocorticoid receptor element.2The abbreviations used are:MRmineralocorticoid receptorEGFepidermal growth factorERKextracellular signal-regulated kinaseELISAenzyme-linked immunosorbent assayEGFPenhanced green fluorescent proteinPBSphosphate-buffered salineSEAPsecretory alkaline phosphataseGREglucocorticoid receptor element. is usually described as a ligand-inducible transcription factor that controls expression of target genes involved in the regulation of Na+ and K+ homeostasis as well as blood pressure regulation (1Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1632) Google Scholar). MR also promotes cardiovascular and renal fibrosis caused by tissue remodeling as well as endothelial dysfunction, independent of its effects on blood pressure or NaCl homeostasis (2Bauersachs J. Heck M. Fraccarollo D. Hildemann S.K. Ertl G. Wehling M. J. Am. Coll. Cardiol. 2002; 39: 351-358Crossref PubMed Scopus (138) Google Scholar, 3Blasi E.R. Rocha R. Rudolph A.E. Blomme E.A. Polly M.L. McMahon E.G. Kidney Int. 2003; 63: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 4Pitt B. Zannad F. Remme W.J. Cody R. Castaigne A. Perez A. Palensky J. Wittes J. N. Engl. J. Med. 1999; 341: 709-717Crossref PubMed Scopus (7487) Google Scholar). The activation of MR modulates the expression of various proteins like the epithelial sodium channel, Na+-K+-ATPase, the SGK kinase, and the EGF receptor (1Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1632) Google Scholar, 5Stockand J.D. Am. J. Physiol. Renal Physiol. 2002; 282: F559-F576Crossref PubMed Scopus (161) Google Scholar, 6Krug A.W. Grossmann C. Schuster C. Freudinger R. Mildenberger S. Govindan M.V. Gekle M. J. Biol. Chem. 2003; 278: 43060-43066Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Grossmann C. Freudinger R. Mildenberger S. Krug A.W. Gekle M. Am. J. Physiol. Renal Physiol. 2004; 286: F1226-F1231Crossref PubMed Scopus (52) Google Scholar). There is increasing evidence that not all biological effects of MR are mediated by direct DNA binding and control of target gene expression (8Funder J.W. Endocr. Rev. 2005; 26: 313-321Crossref PubMed Scopus (152) Google Scholar, 9Grossmann C. Benesic A. Krug A.W. Freudinger R. Mildenberger S. Gassner B. Gekle M. Mol. Endocrinol. 2005; 19: 1697-1710Crossref PubMed Scopus (142) Google Scholar). Some actions of MR appear to be the result of a cross-talk with other signaling cascades, such as nongenomic regulation of intracellular calcium (10Doolan C.M. Harvey B.J. J. Biol. Chem. 1996; 271: 8763-8767Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 11Gekle M. Golenhofen N. Oberleithner H. Silbernagl S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10500-10504Crossref PubMed Scopus (130) Google Scholar), protein kinase C, cSRC kinase, EGF receptor (EGFR) activity, or extracellular-regulated kinase (ERK1/2) (12Cato A.C.B. Nestl A. Mink S. Science's STKE. 2002; 138: 1-11Google Scholar, 13Levin E.R. Mol. Endocrinol. 2003; 17: 309-317Crossref PubMed Scopus (281) Google Scholar, 14Losel R. Wehling M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 46-56Crossref PubMed Scopus (689) Google Scholar, 15Mazak I. Fiebeler A. Muller D.N. Park J.K. Shagdarsuren E. Lindschau C. Dechend R. Viedt C. Pilz B. Haller H. Luft F.C. Circulation. 2004; 109: 2792-2800Crossref PubMed Scopus (205) Google Scholar). Furthermore, there seems to exist a functional cross-talk between classical and nongenomic actions (9Grossmann C. Benesic A. Krug A.W. Freudinger R. Mildenberger S. Gassner B. Gekle M. Mol. Endocrinol. 2005; 19: 1697-1710Crossref PubMed Scopus (142) Google Scholar, 16Feng W. Webb P. Nguyen P. Liu X. Li J. Karin M. Kushner P.J. Mol. Endocrinol. 2001; 15: 32-45Crossref PubMed Scopus (108) Google Scholar, 17Vasudevan N. Kow L.M. Pfaff D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12267-12271Crossref PubMed Scopus (146) Google Scholar, 18Pfau A. Grossmann C. Freudinger R. Mildenberger S. Benesic A. Gekle M. Mol. Cell. Endocrinol. 2007; 264: 35-43Crossref PubMed Scopus (21) Google Scholar).The classical receptors for estrogen (ER), progesterone (PR), androgens (AR), and glucocorticoids (GR) also contribute to the nongenomic effects (12Cato A.C.B. Nestl A. Mink S. Science's STKE. 2002; 138: 1-11Google Scholar, 14Losel R. Wehling M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 46-56Crossref PubMed Scopus (689) Google Scholar, 19Boonyaratanakornkit V. Scott M.P. Ribon V. Sherman L. Anderson S.M. Maller J.L. Miller W.T. Edwards D.P. Mol. Cell. 2001; 8: 269-280Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 20Wyckoff M.H. Chambliss K.L. Mineo C. Yuhanna I.S. Mendelsohn M.E. Mumby S.M. Shaul P.W. J. Biol. Chem. 2001; 276: 27071-27076Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 21Song R.X. McPherson R.A. Adam L. Bao Y. Shupnik M. Kumar R. Santen R.J. Mol. Endocrinol. 2002; 16: 116-127Crossref PubMed Scopus (382) Google Scholar, 22Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 23Russell K.S. Haynes M.P. Sinha D. Clerisme E. Bender J.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5930-5935Crossref PubMed Scopus (336) Google Scholar, 24Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar, 25Hafezi-Moghadam A. Simoncini T. Yang E. Limbourg F.P. Plumier J.C. Rebsamen M.C. Hsieh C.M. Chui D.S. Thomas K.L. Prorock A.J. Laubach V.E. Moskowitz M.A. French B.A. Ley K. Liao J.K. Nat. Med. 2002; 8: 473-479Crossref PubMed Scopus (469) Google Scholar, 26Barletta F. Wong C.W. McNally C. Komm B.S. Katzenellenbogen B. Cheskis B.J. Mol. Endocrinol. 2004; 18: 1096-1108Crossref PubMed Scopus (148) Google Scholar) of these hormones, in many cases via ERK1/2 kinases. Some nongenomic effects arise from classical receptors in or at the plasma membrane (e.g. ER, Ref. 13Levin E.R. Mol. Endocrinol. 2003; 17: 309-317Crossref PubMed Scopus (281) Google Scholar) but specialized membrane receptors, like mPR and GPR30 have also been described (27Filardo E.J. Quinn J.A. Frackelton A.R. Bland K.I. Mol. Endocrinol. 2002; 16: 70-84Crossref PubMed Scopus (563) Google Scholar, 28Zhu Y. Rice C.D. Pang Y. Pace M. Thomas P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2231-2236Crossref PubMed Scopus (671) Google Scholar). However, the results for the specialized membrane receptors are controversial (29Krietsch T. Fernandes M.S. Kero J. Losel R. Heyens M. Lam E.W.F. Huhtaniemi I. Brosens J.J. Gellersen B. Mol. Endocrinol. 2006; 20: 3146-3164Crossref PubMed Scopus (98) Google Scholar, 30Revankar C.M. Cimino D.F. Sklar L.A. Arterburn J.B. Prossnitz E.R. Science. 2005; 307: 1625-1630Crossref PubMed Scopus (1828) Google Scholar). The mechanism(s) of action for ER and PR as well as the receptor domains involved have been explored in more detail. Receptor domains D, E, and F seem to be of special importance (19Boonyaratanakornkit V. Scott M.P. Ribon V. Sherman L. Anderson S.M. Maller J.L. Miller W.T. Edwards D.P. Mol. Cell. 2001; 8: 269-280Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 26Barletta F. Wong C.W. McNally C. Komm B.S. Katzenellenbogen B. Cheskis B.J. Mol. Endocrinol. 2004; 18: 1096-1108Crossref PubMed Scopus (148) Google Scholar, 31Migliaccio A. Castoria G. Domenico Di M. Falco de A. Bilancio A. Lombardi M. Barone M.V. Ametrano D. Zannini M.S. Abbondanza C. Auricchio F. EMBO J. 2000; 19: 5406-5417Crossref PubMed Google Scholar, 32Razandi M. Alton G. Pedram A. Ghonshani S. Webb P. Levin E.R. Mol. Cell. Biol. 2003; 23: 1633-1646Crossref PubMed Scopus (286) Google Scholar, 33Chambliss K.L. Simon L. Yuhanna I.S. Mineo C. Shaul P.W. Mol. Endocrinol. 2005; 19: 277-289Crossref PubMed Scopus (59) Google Scholar). For example, ERα interacts with the SH2 domain of cSRC via a phosphorylated tyrosine residue at position 537 in the EF domain, and this interaction is facilitated by MNAR (26Barletta F. Wong C.W. McNally C. Komm B.S. Katzenellenbogen B. Cheskis B.J. Mol. Endocrinol. 2004; 18: 1096-1108Crossref PubMed Scopus (148) Google Scholar, 31Migliaccio A. Castoria G. Domenico Di M. Falco de A. Bilancio A. Lombardi M. Barone M.V. Ametrano D. Zannini M.S. Abbondanza C. Auricchio F. EMBO J. 2000; 19: 5406-5417Crossref PubMed Google Scholar). Razandi et al. (32Razandi M. Alton G. Pedram A. Ghonshani S. Webb P. Levin E.R. Mol. Cell. Biol. 2003; 23: 1633-1646Crossref PubMed Scopus (286) Google Scholar) described an important role for serine 522 within the EF domain of ERα during nongenomic signaling. However, there are also reports ascribing a role to domain D (33Chambliss K.L. Simon L. Yuhanna I.S. Mineo C. Shaul P.W. Mol. Endocrinol. 2005; 19: 277-289Crossref PubMed Scopus (59) Google Scholar). Recently, the direct interaction of ERα with Gαi through domain C and Gβγ through DEF domains has been reported (34Kumar P. Wu Q. Chambliss K.L. Yuhanna I.S. Mumby S.M. Mineo C. Tall G.G. Shaul P.W. Mol. Endocrinol. 2007; 21: 1370-1380Crossref PubMed Scopus (123) Google Scholar). PR has the ability to interact with the SH3 domain of cSRC via a proline-rich region of domain D (19Boonyaratanakornkit V. Scott M.P. Ribon V. Sherman L. Anderson S.M. Maller J.L. Miller W.T. Edwards D.P. Mol. Cell. 2001; 8: 269-280Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar).A possible role for MR with respect to nongenomic effects has not yet been investigated as extensively as for other steroid receptors. Pharmacological evidence for a physiologically relevant role of the MR in nongenomic actions was presented, for example, with respect to the modulation of the plasma membrane Na+-transporters (35Alzamora R. Michea L. Marusic E.T. Hypertension. 2000; 35: 1099-1104Crossref PubMed Scopus (158) Google Scholar) or vascular reactivity and NO synthesis (36Liu S.L. Schmuck S. Chorazcyzewski J.Z. Gros R. Feldman R.D. Circulation. 2003; 108: 2400-2406Crossref PubMed Scopus (141) Google Scholar, 37Uhrenholt T.R. Schjerning J. Hansen P.B. Norregaard R. Jensen B.L. Sorensen G.L. Skott O. Circ. Res. 2003; 93: 1258-1266Crossref PubMed Scopus (106) Google Scholar, 38Arima S. Kohagura K. Xu H.L. Sugawara A. Uruno A. Satoh F. Takeuchi K. Ito S. Hypertension. 2004; 43: 352-357Crossref PubMed Scopus (83) Google Scholar). Furthermore, nongenomic MR actions were made responsible for pathophysiological effects of aldosterone (39Callera G.E. Montezano A.C.I. Yogi A. Tostes R.C. He Y. Schiffrin E.L. Touyz R.M. Hypertension. 2005; 46: 1032-1038Crossref PubMed Scopus (87) Google Scholar, 40Min L.J. Mogi M. Li J.M. Iwanami J. Iwai M. Horiuchi M. Circ. Res. 2005; 97: 434-442Crossref PubMed Scopus (144) Google Scholar, 41Nagai Y. Miyata K. Sun G.P. Rahman M. Kimura S. Miyatake A. Kiyomoto H. Kohno M. Abe Y. Yoshizumi M. Nishiyama A. Hypertension. 2005; 46: 1039-1045Crossref PubMed Scopus (135) Google Scholar, 42Tsybouleva N. Zhang L. Chen S. Patel R. Lutucuta S. Nemoto S. DeFreitas G. Entman M. Carabello B.A. Roberts R. Marian A.J. Circulation. 2004; 109: 1284-1291Crossref PubMed Scopus (212) Google Scholar, 43Ishizawa K. Izawa Y. Ito H. Miki C. Miyata K. Fujita Y. Kanematsu Y. Tsuchiya K. Tamaki T. Nishiyama A. Yoshizumi M. Hypertension. 2005; 46: 1046-1052Crossref PubMed Scopus (77) Google Scholar, 44Hitomi H. Kiyomoto H. Nishiyama A. Hara T. Moriwaki K. Kaifu K. Ihara G. Fujita Y. Ugawa T. Kohno M. Hypertension. 2007; 50: 750-755Crossref PubMed Scopus (122) Google Scholar), although the final proof is missing. Recently, the involvement of MR in nongenomic signaling at the cellular level was investigated in detail using a heterologous expression system (9Grossmann C. Benesic A. Krug A.W. Freudinger R. Mildenberger S. Gassner B. Gekle M. Mol. Endocrinol. 2005; 19: 1697-1710Crossref PubMed Scopus (142) Google Scholar). The data from this study showed that MR contributes to rapid aldosterone-induced activation of the ERK1/2 pathway via cSRC and EGFR activation. For MR, no information regarding the domain(s) necessary for nongenomic actions are available. In the present study, we determined which MR domains are required for nongenomic signaling and whether this pathway has the potential to elicit a pathophysiological effect in cultured cells under defined conditions.EXPERIMENTAL PROCEDURESCell Culture—Cell culture was performed as described previously (6Krug A.W. Grossmann C. Schuster C. Freudinger R. Mildenberger S. Govindan M.V. Gekle M. J. Biol. Chem. 2003; 278: 43060-43066Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 55Krug A.W. Schuster C. Gassner B. Freudinger R. Mildenberger S. Troppmair J. Gekle M. J. Biol. Chem. 2002; 277: 45892-45897Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 56Gekle M. Freudinger R. Mildenberger S. Silbernagl S. Am. J. Physiol. Renal Physiol. 2002; 282: F669-F679Crossref PubMed Scopus (70) Google Scholar). We used CHO-K1, HEK-293, and OK cells from ATCC. 24-48 h prior to the experiment, serum was removed. For the experiments presented, the cells were cultivated either on Petri dishes (Becton Dickinson GmbH, Heidelberg, Germany) in 24-well plates (for reporter assay and collagen determination), in 96-well plates (for ELISA), or on glass coverslips (fluorescence).Constructs and Transfection—Transfection of the cells was performed under serum-free conditions as described before (6Krug A.W. Grossmann C. Schuster C. Freudinger R. Mildenberger S. Govindan M.V. Gekle M. J. Biol. Chem. 2003; 278: 43060-43066Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 55Krug A.W. Schuster C. Gassner B. Freudinger R. Mildenberger S. Troppmair J. Gekle M. J. Biol. Chem. 2002; 277: 45892-45897Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) with the Qiagen Polyfect reagent (Qiagen, Hilden, Germany), according to the manufacturer's instructions. We used the hMR expression vector pEGFP-C1-hMR (kindly provided by Dr. N. Farman (57Ouvrard-Pascaud A. Puttini S. Sainte-Marie Y. Athman R. Fontaine V. Cluzeaud F. Farman N. Rafestin-Oblin M.E. Blot-Chabaud M. Jaisser F. Am. J. Physiol. Renal Physiol. 2004; 286: F180-F187Crossref PubMed Scopus (17) Google Scholar)) and pEGFP-C1 (Clontech). Truncated versions of the pEGFP-hMR, which lack either the N-terminal AB domain (hMRCDEF), the AB domain and the DNA-binding domain (hMRDEF), or additionally the hinge region (hMREF) were constructed by cutting pEGFP-hMR with BglII and Hin-dIII and inserting appropriate PCR fragments (Table 1). hMRAB was generated by restriction with EcoR1. To determine whether the EGFP tag changed the characteristics of the receptor, we compared its GRE activation and nuclear translocation properties with that of the untagged hMR and could not find significant differences (9Grossmann C. Benesic A. Krug A.W. Freudinger R. Mildenberger S. Gassner B. Gekle M. Mol. Endocrinol. 2005; 19: 1697-1710Crossref PubMed Scopus (142) Google Scholar, 57Ouvrard-Pascaud A. Puttini S. Sainte-Marie Y. Athman R. Fontaine V. Cluzeaud F. Farman N. Rafestin-Oblin M.E. Blot-Chabaud M. Jaisser F. Am. J. Physiol. Renal Physiol. 2004; 286: F180-F187Crossref PubMed Scopus (17) Google Scholar).TABLE 1Primers and restriction enzymes for deletion constructsDeletion constructPrimer sense (restriction site)Primer antisense (restriction site)CDEF-(601-984)agatctatatgtttggtgtgtggg (BglII)aagctttcacttccggtggaagtaga (HindIII)DEF-(682-984)agatctgggattcacgaggagcag (BglII)aagctttcacttccggtggaagtaga (HindIII)EF-(735-984)agatctacaccttcccccgttatg (BglII)aagctttcacttccggtggaagtaga (HindIII) Open table in a new tab Immunoprecipitation—Cells were washed, harvested, and lysed in radioimmune precipitation assay buffer{3551}. Lysates were then centrifuged at 11,000 rpm at 4 °C for 10 min, and the supernatant was incubated overnight with EGFP antibody (sc-8334, Santa Cruz Biotechnology) with end-over-end rotation and then with A/G plus-agarose for another 24 h. After 10 min of centrifugation at 10,000 rpm at 4 °C, the pellet was mixed with 40 μl of Laemmli buffer and separated by an 8% SDS-PAGE gel.Western Blot Analysis—Cells were lysed in ice-cold Triton X-100 lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 5 mm EDTA, 200 μm sodium orthovanadate, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μm pepstatin A, 40 mg/liter bestatin, 2 mg/liter aprotinin, 1% Triton X-100), lysis buffer according to Le Moellic et al. (59Moellic Le C. Ouvrard-Pascaud A. Capurro C. Cluzeaud F. Fay M. Jaisser F. Farman N. Blot-Chabaud M. J. Amer. Soc. Nephrol. 2004; 15: 1145-1160PubMed Google Scholar) (50 mmol/liter Tris-HCl, 150 mmol/liter NaCl, 1% Nonidet P40, 2.4 mmol/liter EDTA, protease inhibitor mixture) or radioimmune precipitation assay buffer at 4 °C. Cell lysates were matched for protein content, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. Subsequently membranes were blotted with either rabbit anti-phospho-ERK1/2 antibody (1:1000, Cell Signaling Technologies), rabbit anti-ERK1/2 antibody (1:1000, Cell Signaling Technologies), mouse anti-EGFP (1:1000, Clontech), rabbit anti-HSP90 (1:1000, Santa Cruz Biotechnology), rabbit anti-collagen III (1:1000; Biotrend, Köln, Germany), or rabbit anti-pcSRC (Tyr-416, 1:1000, Cell Signaling Technologies). The bound primary antibody was visualized using horseradish peroxidase-conjugated secondary IgG and the ECL system (Amersham Biosciences). Densitometry analysis was performed with ImageJ.EGFP Fluorescence—Cells were cultivated on glass coverslips. Images were obtained either using an inverted microscope (Zeiss IM 135) equipped with ×40 and ×100 fluorescence objectives. Fluorescence images were taken with an ICCD camera (Hamamatsu, Herrsching, Germany). Alternatively, cells were analyzed by confocal microscopy (Radiance 2000, Bio-Rad), and the images were processed using the software MetaMorph Imaging System (Microsoft).Determination of EGFP-hMR Expression by ELISA—EGFP-hMR expression was determined basically by the same method as ERK1/2 phosphorylation (see above). HEK cells were transfected, made quiescent, and after 48 h, expression was determined with an anti-EGFP primary antibody (Clontech) and anti-mouse-horseradish peroxidase antibody. pcDNA3.1-transfected cells were used as negative controls.Quantification of ERK1/2 Phosphorylation by ELISA—For the quantification of ERK1/2 phosphorylation, we performed ELISA according to Versteeg et al. (58Versteeg H.H. Nijhuis E. den Brink Van G.R. Evertzen M. Pynaert G.N. Deventer van S.J. Coffer P.J. Peppelenbosch M.P. Biochem. J. 2000; 350: 717-722Crossref PubMed Scopus (147) Google Scholar) with minor modifications that were described previously (55Krug A.W. Schuster C. Gassner B. Freudinger R. Mildenberger S. Troppmair J. Gekle M. J. Biol. Chem. 2002; 277: 45892-45897Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). After stimulation as indicated, the cells were fixed with 4% formaldehyde in PBS and permeabilized with 0.1% Triton X-100. Cells were blocked with 10% fetal calf serum in PBS/Triton for 1 h and incubated overnight with the primary antibody. Subsequently, cells were incubated with secondary antibody (peroxidase-conjugated mouse anti-rabbit antibody) in PBS/Triton with 5% bovine serum albumin for 1 h at room temperature. Finally, the cells were incubated with 50 μl of a solution containing 0.4 mg/ml o-phenylenediamine, 11.8 mg/ml Na2HPO4, 7.3 mg/ml citric acid, and 0.015% H2O2 for 15 min at room temperature in the dark. The resulting signal was detected at 490 nm with a multiwell multilabel counter (Victor2, Wallac, Turku, Finland). The protein content was determined with Trypan Blue (55Krug A.W. Schuster C. Gassner B. Freudinger R. Mildenberger S. Troppmair J. Gekle M. J. Biol. Chem. 2002; 277: 45892-45897Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The determination of total ERK1/2 in parallel experiments was used to correlate phosphorylated ERK1/2 to total ERK1/2.Quantification of Phospho-ERK1/2 by Sandwich-ELISA—For the quantification of phospho-ERK expression, we used the sandwich-ELISA from R&D Systems, Inc., Minneapolis R&D (SUV1018) according to the manufacturer's instructions.Determination of Extracellular Collagen III Abundance by ELISA—Collagen III abundance in the medium of CHO cells expressing HER1 (55Krug A.W. Schuster C. Gassner B. Freudinger R. Mildenberger S. Troppmair J. Gekle M. J. Biol. Chem. 2002; 277: 45892-45897Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) was determined by ELISA as described previously (51Gekle M. Mildenberger S. Freudinger R. Grossmann C. Pflugers Arch. 2007; 454: 403-413Crossref PubMed Scopus (26) Google Scholar). We tested the cross-reactivity of the primary antibodies (1:1000; Biotrend, Köln, Germany) using collagen standards and did not observe any significant cross-reactivity.Determination of Gelatinase Activity—Gelatinase activity in cell culture media was determined by the ENZ® Gelatinase/Collagenase Assay kit from Molecular Probes (Leiden, NL) using fluorescein-conjugated gelatin. The increase in fluorescence is a direct measure for gelatinase/collagenase activity. As a positive control, bacterial collagenase activity (Sigma) was measured, and the gelatinase values are expressed as collagenase-equivalents.GRE Reporter Gene Assay—Transactivation was assessed by the Mercury™ Pathway Profiling reporter gene assay system from Clontech Inc. using secretory alkaline phosphatase (SEAP) as reporter, essentially as described earlier (9Grossmann C. Benesic A. Krug A.W. Freudinger R. Mildenberger S. Gassner B. Gekle M. Mol. Endocrinol. 2005; 19: 1697-1710Crossref PubMed Scopus (142) Google Scholar). In brief, the cells were cotransfected with pGRE-SEAP and receptor constructs or empty vectors. SEAP activity in the media was determined with the AttoPhos® System from Promega (Mannheim, Germany) and normalized to a transfection control (β-galactosidase or EGFP).Materials—U0126, tyrphostin AG 1478, PP2, and the protease inhibitors were obtained from Calbiochem (Bad Soden, Germany). Unless otherwise stated, all other materials were from Sigma. Control Ringer solution was composed of (mmol/liter): NaCl 130.0, KCl 5.4, CaCl2 1.0, MgCl2 1.0, NaH2PO4 1.0, HEPES 10, and glucose 5 (pH 7.4 at 37 °C), plus the respective vehicles (ethanol or Me2SO ≤ 1‰).Statistics—The data are presented as mean values ± S.E. Significance of difference was tested by paired or unpaired Student's t test or analysis of variance as applicable. Differences were considered significant with p < 0.05. Cells from at least two different passages were used for each experimental series. N represents the number of tissue culture dishes investigated.RESULTSTranscriptional Activity of hMR Deletion Constructs—We compared transcriptional activity of the MR deletion constructs using a GRE-SEAP reporter containing three canonical GRE elements. Only cells transfected with hMR or hMRCDEF responded to aldosterone with increased GRE-SEAP activity (Fig. 1). Dexamethasone, a ligand for the endogenous glucocorticoid receptor (GR, Ref. 18Pfau A. Grossmann C. Freudinger R. Mildenberger S. Benesic A. Gekle M. Mol. Cell. Endocrinol. 2007; 264: 35-43Crossref PubMed Scopus (21) Google Scholar) stimulated GRE-SEAP activity under all conditions, demonstrating the functionality of the experimental system. For cells transfected with hMR or hMRCDEF, the effect of dexamethasone was stronger, because of the additional activation of the mineralocorticoid receptor. The relative transactivation potency of hMRCDEF was greater when compared with hMR, because the inhibitory domain located in AB is missing (45Rupprecht R. Arriza J.L. Spengler D. Reul J.M. Evans R.M. Holsboer F. Damm K. Mol. Endocrinol. 1993; 7: 597-603Crossref PubMed Scopus (120) Google Scholar). Half-maximal activation was obtained with similar concentrations of aldosterone (Fig. 1).Expression of hMR Deletion Constructs—Western blot analysis confirmed the expression of proteins of the expected size (Fig. 2A). The expression level of hMR was slightly lower compared with deletion constructs when determined by ELISA (Fig. 2B). The simplest explanation for this difference is the size of the protein. However, differences in the delivery to degradation sites cannot be excluded.FIGURE 2Expression of the different MR constructs. A, Western blot. B, quantitative expression analysis by ELISA (n = 12). C, fluorescence microscopy of cells exposed to vehicle (control) or 10 nmol/liter aldosterone for 24 h. The bar represents 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using fluorescence microscopy, we investigated the cellular distribution. As expected, hMR is located mainly in the cytosol in the absence of hormone (Fig. 2C). In the presence of aldosterone (10 nmol/liter), the receptor translocates almost completely to the nucleus. hMRCDEF behaves in a similar way. hMRDEF and hMREF, which are located in the cytosol in the absence of hormone, translocate partially into the nucleus after the addition of aldosterone.ERK1/2 Phosphorylation in Cells Transfected with hMR Deletion Constructs—We used ERK1/2 phosphorylation as a read-out for rapid, nongenomic effects, because it has been described continuously in different experimental systems. First, we investigated whether expression of the different hMR deletion constructs affects ERK1/2 phosphorylation in the absence of aldosterone. For this purpose, ERK phosphorylation was determined by two independent techniques, i.e. sandwich-ELISA and Western blot. Fig. 3 shows that expression of hMREF led to a ligand-independent increase in ERK1/2 phosphorylation. The expression levels of total ERK1/2 or HSP90 were not altered significantly (Fig. 3). This difference was not observed in the presence of 10 μmol/liter U0126 (inhibitor of ERK1/2 phosphorylation; Fig. 3A). Similar results were obtained with a different cell line, OK cells, where hMREF also enhanced the basal pERK1/2 level (145 ± 9% of control, n = 18, p < 0.05).FIGURE 3A, upper panel, Western blot analysis of pERK1/2, ERK1/2, and HSP90 expression levels 48 h after transfection in the absence of aldosterone. Lower panel, sandwich-ELISA of pERK1/2 48 h after transfection in the absence of aldosterone (n = 12; *, p < 0.05 versus EGFP). B, ERK1/2 phosphorylation after short term exposure (10 min) to 10 nmol/liter aldosterone or 100 μg/liter EGF. Representative blots of three exp
Referência(s)