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• W2091868955 abstract "Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Gα12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Gα12 regulates apoptosis in epithelial cells. Inducible expression of Gα12 or constitutively active (QL)α12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLα12, but not Gα12. Inducing QLα12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IκBα protein levels, a potent stimulator of apoptosis. Furthermore, the QLα12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Gα12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Gα12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Gα12 were nearly identical to the mechanisms identified in QLα12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IκBα. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Gα12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes. Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Gα12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Gα12 regulates apoptosis in epithelial cells. Inducible expression of Gα12 or constitutively active (QL)α12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLα12, but not Gα12. Inducing QLα12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IκBα protein levels, a potent stimulator of apoptosis. Furthermore, the QLα12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Gα12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Gα12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Gα12 were nearly identical to the mechanisms identified in QLα12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IκBα. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Gα12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes. Signaling through G proteins 2The abbreviations used are: G protein, guanine nucleotide-binding protein; PP2A, protein phosphatase 2A; MDCK, Madin-Darby canine kidney; Tet, tetracycline; dox, doxycycline; QLα12, Q229LGα12; JNK, cJun N-terminal kinase; Bcl, B-cell lymphoma; HEK293, human embryonic kidney 293; TPR, tetratricopeptide repeat domain of protein phosphatase 5; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κB; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; GST, glutathione S-transferase; siRNA, small interfering RNA. 2The abbreviations used are: G protein, guanine nucleotide-binding protein; PP2A, protein phosphatase 2A; MDCK, Madin-Darby canine kidney; Tet, tetracycline; dox, doxycycline; QLα12, Q229LGα12; JNK, cJun N-terminal kinase; Bcl, B-cell lymphoma; HEK293, human embryonic kidney 293; TPR, tetratricopeptide repeat domain of protein phosphatase 5; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κB; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; GST, glutathione S-transferase; siRNA, small interfering RNA. is an important mechanism regulating apoptosis, a highly conserved process of programmed cell death fundamental to normal development and somatic maintenance in all multicellular organisms. Changes in apoptosis signaling pathways contribute to major disease processes including cancer, degenerative neurological diseases, such as Parkinsonism and Alzheimer, and kidney failure from numerous etiologies including polycystic kidney disease. There are two major signaling pathways leading to apoptosis. The intrinsic pathway is mediated by the permeabilization of mitochondria and caspase-9 activation and is a general response to cell damage or stress. The extrinsic pathway is activated by stimulation from specific death inducing factors, such as Fas (apoptosis-stimulating fragment) and tumor necrosis factor and is mediated by caspase-8 (1Zimmermann K.C. Bonzon C. Green D.R. Pharmacol. Ther. 2001; 92: 57-70Crossref PubMed Scopus (700) Google Scholar). In the intrinsic pathway, the Bcl family (including pro- and anti-apoptotic proteins) is central to determining whether cells will undergo apoptosis (reviewed in Ref. 2Sorenson C.M. Biochim. Biophys. Acta. 2004; 1644: 169-177Crossref PubMed Scopus (103) Google Scholar). Bcl-2 is localized in the outer mitochondrial, endoplasmic reticulum, and perinuclear membranes, and under proliferative and anti-apoptotic (pro-survival) conditions, Bcl-2 heterodimerizes with pro-apoptotic protein BAX to form a stable, non-conductive complex (2Sorenson C.M. Biochim. Biophys. Acta. 2004; 1644: 169-177Crossref PubMed Scopus (103) Google Scholar). With an apoptotic stimulus, Bcl-2 is phosphorylated; this prevents dimerization with Bax and also leads to Bcl-2 degradation, thus permitting BAX to homodimerize and catalyze the formation of the mitochondrial apoptosis-induced channel. This channel allows the efflux of cytochrome c, triggering Apaf-1 and the caspase cascade leading to apoptosis (2Sorenson C.M. Biochim. Biophys. Acta. 2004; 1644: 169-177Crossref PubMed Scopus (103) Google Scholar, 3Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar).Heterotrimeric G proteins regulate numerous cellular processes including proliferation, differentiation, junctional assembly, and apoptosis. G proteins consist of Gα and Gβγ subunits, and form a stable heterotrimeric complex with GDP bound to the α subunit in the resting state. In canonical G protein signaling, ligand binding to a seven-transmembrane domain receptor results in conformational changes in Gα that lead to dissociation of GDP and separation from Gβγ. GTP subsequently binds to Gα, and signal transduction occurs through Gα and Gβγ subunits until the intrinsic Gα GTPase activity hydrolyzes GTP to GDP. It is now appreciated that G protein signaling is quite complex with the identification of G proteins in subcellular microdomains and in interactions with numerous regulatory and scaffolding proteins. Several studies have implicated signaling through each of the four heterotrimeric G protein families (Gαs, Gαi/o, Gαq/11, and Gα12/13) to regulate apoptosis, but the mechanism(s) are not well defined (4Al-Rasheed N.M. Willars G.B. Brunskill N.J. J. Am. Soc. Nephrol. 2006; 17: 986-995Crossref PubMed Scopus (53) Google Scholar, 5Howes A.L. Miyamoto S. Adams J.W. Woodcock E.A. Brown J.H. J. Mol. Cell Cardiol. 2006; 40: 597-604Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 6Maudsley S. Davidson L. Pawson A.J. Chan R. Lopez de Maturana R. Millar R.P. Cancer Res. 2004; 64: 7533-7544Crossref PubMed Scopus (123) Google Scholar, 7Tobin A.B. Budd D.C. Biochem. Soc. Trans. 2003; 31: 1182-1185Crossref PubMed Google Scholar, 8Ueda H. Morishita R. Narumiya S. Kato K. Asano T. Exp. Cell Res. 2004; 298: 207-217Crossref PubMed Scopus (29) Google Scholar, 9Zhao C. Lai J.S. Warsh J.J. Li P.P. J. Neurosci. Res. 2006; 84: 389-397Crossref PubMed Scopus (5) Google Scholar).Gα12 and Gα13 regulate fundamental cellular processes that include proliferation (10Radhika V. Hee Ha J. Jayaraman M. Tsim S.T. Dhanasekaran N. Oncogene. 2005; 24: 4597-4603Crossref PubMed Scopus (36) Google Scholar), transformation (11Jiang H. Wu D. Simon M.I. FEBS Lett. 1993; 330: 319-322Crossref PubMed Scopus (101) Google Scholar), tight junction assembly (12Meyer T.N. Hunt J. Schwesinger C. Denker B.M. Am. J. Physiol. 2003; 285 (-C1293): C1281Crossref PubMed Scopus (52) Google Scholar, 13Meyer T.N. Schwesinger C. Denker B.M. J. Biol. Chem. 2002; 277: 24855-24858Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), directed cell migration (14Goulimari P. Kitzing T.M. Knieling H. Brandt D.T. Offermanns S. Grosse R. J. Biol. Chem. 2005; 280: 42242-42251Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and regulation of the actin cytoskeleton (15Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Gα12/13 are potent oncogenes in some cell types (16Voyno-Yasenetskaya T.A. Pace A.M. Bourne H.R. Oncogene. 1994; 9: 2559-2565PubMed Google Scholar, 17Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar), and may also stimulate apoptosis. In transiently transfected COS cells, Gα12 and Gα13 induced apoptosis through activation of MEKK1 and Ask1 in a Bcl-2-dependent manner (18Berestetskaya Y.V. Faure M.P. Ichijo H. Voyno-Yasenetskaya T.A. J. Biol. Chem. 1998; 273: 27816-27823Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), and in a human adenocarcinoma cell line, a mutant Gα13 stimulated apoptosis through JNK (19Adarichev V.A. Vaiskunaite R. Niu J. Balyasnikova I.V. Voyno-Yasenetskaya T.A. Am. J. Physiol. 2003; 285 (-C934): C922Crossref PubMed Scopus (14) Google Scholar). However, there is very little known about the role of Gα12 in regulating apoptosis in non-transfected cells, and few studies have investigated G protein regulation of apoptosis in epithelial cells. Recently, we identified an interaction of Gα12 with the regulatory subunit of the serine/threonine phosphatase protein phosphatase 2A (PP2A) and demonstrated that Gα12 stimulated PP2A activity in vitro and in MDCK cells (20Zhu D. Kosik K.S. Meigs T.E. Yanamadala V. Denker B.M. J. Biol. Chem. 2004; 279: 54983-54986Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21Zhu D. Tate R.I. Ruediger R. Meigs T.E. Denker B.M. Mol. Pharmacol. 2007; 71: 1268-1276Crossref PubMed Scopus (22) Google Scholar). PP2A is implicated in many of the same cellular processes that have been described for Gα12 (reviewed in Ref. 22Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar). PP2A regulates apoptosis by maintaining dephosphorylated Bcl-2 (23Lin S.S. Bassik M.C. Suh H. Nishino M. Arroyo J.D. Hahn W.C. Korsmeyer S.J. Roberts T.M. J. Biol. Chem. 2006; 281: 23003-23012Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) as well as regulating several other members of the Bcl-2 family. Identification of the Gα12-PP2A interaction prompted us to investigate whether these signaling proteins were linked to apoptosis in epithelial cells. Using a well characterized MDCK cell culture model of inducible expression of Gα12, we report that Gα12 is a potent activator of apoptosis in epithelial cells and identify critical regulation of Bcl-2 protein levels by Gα12, PP2A, and JNK. Furthermore, we demonstrate for the first time that activation of endogenous Gα12-coupled signaling pathways stimulates apoptosis in epithelial cells and recapitulates the signaling pathways identified in the inducible Gα12-MDCK cell model.EXPERIMENTAL PROCEDURESReagents—Rabbit polyclonal anti-Gα12, anti-JNK1, anti-ERK, anti-NF-κB, anti-IκBα, and goat anti-PP2A catalytic subunit were from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit anti-pThr183/pTyr185-JNK, anti-pThr202/pTyr204-ERK, and anti-Bcl-xL were from Cell Signaling Technology (Danvers, MA), mouse anti-Bcl-2 was from BD Biosciences (San Jose, CA), and rabbit anti-phospho-cJun was from Biovision (Mountain View, CA). Rabbit polyclonal anti-β-actin was from Sigma. Fostriecin and okadaic acid were from Calbiochem, SP600125 was from A.G. Scientific (San Diego, CA), and lactacystin was from Sigma. Human α-thrombin was from Enzyme Research Laboratories (South Bend, IN). Plasticware and culture slides were from BD Falcon (Lincoln Park, NJ).Cell Culture—Establishment, characterization, and culture conditions for Tet-Off MDCK-II cell lines (Clontech) with inducible wild type Gα12 and constitutively activated QLα12 expression were previously described (13Meyer T.N. Schwesinger C. Denker B.M. J. Biol. Chem. 2002; 277: 24855-24858Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, cells were maintained in Dulbecco’s modified Eagle’s medium (Cellgro, Herndon, VA) containing 5% Tet system-approved fetal bovine serum (Clontech), 100 μg/ml G418 (Cellgro), 50 IU/ml penicillin, and 50 μg/ml streptomycin (Invitrogen), 100 μg/ml hygromycin (Roche Applied Science), and 40 ng/ml doxycycline (dox) (Sigma). Non-transfected Tet-Off MDCK-II cell lines were maintained in the same culture medium without hygromycin. Human embryonic kidney (HEK293) 293 cell lines (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum.Flow Cytometry—Gα12-or QLα12-expressing MDCK cells were grown to confluence and cultured ± dox for 72 h. Adherent cells were collected by trypsinization and pooled with floating cells (1-2 × 106) by low speed centrifugation, washed with phosphate-buffered saline (PBS), and fixed with 70% ethanol in PBS at 4 °C for 30 min. Subsequently, cells were incubated with 1 μg/ml RNase A in PBS for 30 min at room temperature, after which cells were stained in 50 μg/ml propidium iodide (Invitrogen) in PBS for 30 min at room temperature and analyzed by flow cytometry in the propidium iodide/PE Texas Red channel. 10,000 cells were analyzed per experiment.DNA Fragmentation Assay—Gα12- or QLα12-expressing MDCK cells were cultured ± dox for 72 h. Adherent cells were collected by trypsinization and pooled with floating cells by low speed centrifugation, washed with PBS, and lysed with genomic DNA extraction buffer (100 mm NaCl, 10 mm Tris-HCl, pH 8.0, 25 mm EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K). Extracts were incubated for 24 h at 50 °C and subsequently for 1 h at 37 °C with 1 μg/ml RNase A (MP Biomedical, Solon, OH). DNA was extracted with an equal volume of phenol/chloroform (1:1) and precipitated at -70 °C for 24 h with 3 volume equivalents of absolute ethanol. DNA pellets were resuspended in 20 μl of 10 mm Tris (pH 7.8), 1 mm EDTA buffer and analyzed by electrophoresis on a 0.5% agarose gel run at 50 V for 4 h. Images were obtained using a Kodak DC290 Zoom Digital Camera (Eastman Kodak).Caspase 9 Activity—Gα12-or QLα12-expressing MDCK cells were cultured ± dox for 72 h. Cells were lysed and assessed for caspase-9 activation using LEHD-pNA as a substrate, as per the manufacturer’s instructions (Caspase 9 Colorimetric Assay Kit, Chemicon/Millipore, Billerica, MA).Cell Proliferation Assay—Gα12-or QLα12-expressing MDCK cells were cultured ± dox for 72 h in a 96-well plate. Subsequently, cells were incubated with the tetrazolium salt 4-[3-(4-idophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (Biovision, Mountain View, CA) in Electrocoupling Solution (Biovision) substrate for 2 h. The plate was read at 450 nm to quantify formazan produced, which correlates directly to mitochondrial dehydrogenase activity.Kinexus Kinetworks™ Phospho-site Phosphorylation Screen—1 × 107 QLα12-expressing MDCK cells were grown to confluence, cultured ± dox for 72 h. To obtain whole cell lysates, monolayers were washed with ice-cold PBS, scraped in lysis buffer (100 mm NaCl, 2 mm EDTA, 10 mm HEPES, pH 7.5, 1 mm Na3VO4, 25mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.5% sodium deoxycholate, and proteases inhibitors (Roche)), sonicated gently, and subjected to low speed centrifugation. Supernatants were collected and normalized for protein concentration. 750 μl of 1 mg/ml was analyzed for phosphorylation of target proteins (Kinexus Bioinformatics Corp., Vancouver, B.C., Canada).c-Jun N-terminal Kinase Activity—Gα12-or QLα12-expressing MDCK cells were cultured ± dox for 72 h. Floating cells and adherent cells were collected by trypsinization for 5 min and low speed centrifugation, washed with PBS, and lysed with JNK Extraction Buffer (Biovision). Lysates were normalized for protein concentration and analyzed according to the manufacturer’s instructions (KinaseSTAR JNK Activity Screening Kit, Biovision). Briefly, lysates were incubated with GST-cJun on glutathione-Sepharose beads to pull down total JNK, which was resuspended with 200 μm ATP and incubated at 30 °C for 30 min. Samples were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and Western blot for phospho-cJun.RNA Isolation and Semiquantitiative RT-PCR Analysis—Gα12-or QLα12-expressing MDCK cells were cultured ± dox for 72 h and lysed with TRIzol (Invitrogen). Total RNA was purified according to the manufacturer’s protocol and quantified. 5 μg of total RNA was reverse transcribed using the Transcriptor Reverse Transcriptase Kit (Roche). Equal aliquots of cDNA (5 μl) were subsequently amplified for Bcl-2 and β-actin. The oligonucleotide primer sequences used for Bcl-2 and β-actin (Operon, Huntsville, AL) were as follows: Bcl-2: sense, 5′-ATGGCGCACGCTGGGC-3′; antisense, 5′-TCACTTATGGCCCAGATAGGCAC-3′; β-actin: sense, 5′-ATGGACGATGAAATTGCGGCG-3′; antisense, 5′-CTAGAAGCATTTGCGATGGACG-3′. After 35 cycles, the amplified fragments were resolved by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining using a Kodak DC290 Zoom Digital Camera (Kodak).Immunoblot Analysis—Gα12-or QLα12-expressing MDCK cells were cultured ± dox for 72 h. Lysates were prepared as described above and analyzed by SDS-PAGE and Western blot with the following primary antibodies: Gα12 (1:1000), Bcl-2 (1:500), JNK1 (1:500), pThr183/pTyr185-JNK (1:500), ERK1 (1:500), Bcl-xL (1:500), IκBα (1:500), NF-κB (1:500), or β-actin (1:10,000). After washing and incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies for 60 min at room temperature, signal was detected with SuperSignal West Pico horseradish peroxidase substrate system (Pierce) and autoradiography (Biomax MR, Kodak).GST-TPR Pull Down Assay—The GST-TPR construct was kindly provided by Dr. M. Negishi, Kyoto University, Kyoto Japan. GST-TPR and GST were expressed in Escherichia coli and purified from bacterial lysates as described previously (24Luo Y. Denker B.M. J. Biol. Chem. 1999; 274: 10685-10688Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Cells were lysed in Lysis Buffer (100 mm NaCl, 2 mm EDTA, 10 mm HEPES, pH 7.5, 1 mm Na3VO4, 25mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride and proteases inhibitors (Roche)) and normalized for protein concentration. 1 μg of GST or GST-TPR coupled to glutathione-agarose beads (Amersham Biosciences) was added to 800 μg of total protein and rocked overnight at 4 °C. Beads were pelleted with low speed centrifugation and washed three times with PBS, 0.1% Triton X-100, resuspended in Laemmli sample buffer, and analyzed by SDS-PAGE and Western blot.siRNA Knockdowns—PP2A catalytic subunit was selectively knocked down using SMARTpool pooled siRNAs (Upstate, Lake Placid, NY) that contains four different siRNAs (catalog number D-001206-13-01). A nonspecific control pool (catalog number D-001206-13-01) was used for the negative control and also contained four different siRNAs. HEK293 cells were transfected with siRNA (200 nm) using Lipofectamine (Invitrogen) following the manufacturer’s instructions. After overnight incubation, fresh media was added to the cells, and the cells were cultured an additional 24 h. Lysates were prepared and analyzed by Western blot with goat anti-PP2A catalytic subunit antibody at 1:2000. For thrombin stimulation, cells were serum starved overnight prior to treatment with 2 units/ml. JNK phosphorylation and Bcl-2 expression were examined by Western blot at 8 h, and apoptosis was quantified after 24 h.Quantification and Statistics—Western blots and agarose gels were scanned using an Epson 1640 desktop scanner and band intensity quantified using NIH Image (Wayne Rasband) after subtracting background and determining linear range. Statistics were done in GraphPad Prism (San Diego, CA). Significance was determined by using the two-tailed t test.RESULTSExpression of QLα12 in MDCK-II Cells Induces Apoptosis—Several factors including the disruption of cell-matrix interactions and dome formation associated with confluence have been shown to trigger apoptosis in MDCK cells (25Chang Y.H. Lin H.H. Wang Y.K. Chiu W.T. Su H.W. Tang M.J. J. Cell. Physiol. 2007; 211: 174-182Crossref PubMed Scopus (8) Google Scholar, 26Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2749) Google Scholar). The pathways associated with these phenotypes are not known. The study of Gα12 has been limited by low levels of expression in cells, and by its novel protein characteristics that make it difficult to express in vitro and in cell culture models. We have established and extensively characterized the inducible expression of wild type Gα12 and constitutively active QLα12 using the Tet-off MDCK cell culture model (12Meyer T.N. Hunt J. Schwesinger C. Denker B.M. Am. J. Physiol. 2003; 285 (-C1293): C1281Crossref PubMed Scopus (52) Google Scholar, 13Meyer T.N. Schwesinger C. Denker B.M. J. Biol. Chem. 2002; 277: 24855-24858Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The removal of dox for 72 h induces Gα12 expression (Fig. 1A), and the re-addition of dox suppresses Gα12 protein expression to background levels within 48 h (not shown, but in Refs. 12Meyer T.N. Hunt J. Schwesinger C. Denker B.M. Am. J. Physiol. 2003; 285 (-C1293): C1281Crossref PubMed Scopus (52) Google Scholar and 13Meyer T.N. Schwesinger C. Denker B.M. J. Biol. Chem. 2002; 277: 24855-24858Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We have previously demonstrated that expression of QLα12, but not wild type Gα12 leads to disruption of junctional complexes and inhibits dome formation (12Meyer T.N. Hunt J. Schwesinger C. Denker B.M. Am. J. Physiol. 2003; 285 (-C1293): C1281Crossref PubMed Scopus (52) Google Scholar). To determine whether Gα12 regulated apoptosis in MDCK cells, Gα12- and QLα12-MDCK cells were cultured ± dox for 72 h, followed by overnight serum starvation. Apoptosis was determined by propidium iodide flow cytometry (Fig. 1B), DNA laddering (Fig. 1, C and D), and caspase 9 activity (Fig. 1E). In all assays, QLα12-MDCK (-dox) cells showed a marked increase in apoptosis compared with the other conditions. In flow cytometry analysis (Fig. 1B), there was a 4-5-fold increase in the percentage of apoptotic cells with QLα12 expression when compared with +dox and with Gα12-MDCK cells ± dox. Moreover, there was a parallel reduction in the percentage of cells in the G0/G1 and mitosis phases of the cell cycle. Fig. 1C shows the DNA laddering in Gα12- and QLα12-MDCK cells ± dox. Total intact genomic DNA was reduced to less than 50% in the QLα12 expressing cells (-dox) (quantified in Fig. 1D), whereas there was little effect in Gα12-MDCK cells ± dox. However, compared with Gα12-MDCK cells, there was a small reduction in genomic DNA of QLα12-MDCK cells in +dox conditions (about 10-15%). This may reflect incomplete suppression of the tetracycline responsive element leading to a small amount of QLα12 expression (although QLα12 protein is not detectable by Western in +dox, Fig. 1A). Finally, to confirm the increase in apoptosis and to determine the role of the intrinsic pathway, we assessed the activity of caspase 9. Consistent with the flow cytometry results and DNA laddering, there was a marked increase in caspase 9 activity in the QLα12 expressing cells that was not seen in other conditions (Fig. 1E). This suggests that QLα12 activates the intrinsic apoptotic pathway, and this is distinct from apoptosis induced by dome formation and confluence, which is associated with distal activation of caspase 8 but not caspase 9 (25Chang Y.H. Lin H.H. Wang Y.K. Chiu W.T. Su H.W. Tang M.J. J. Cell. Physiol. 2007; 211: 174-182Crossref PubMed Scopus (8) Google Scholar).The Effect of Gα12 Expression on Proliferation—The results from flow cytometry (Fig. 1B) revealed little change in the fraction of cells in the synthesis stage suggesting that proliferation may not be increased under these conditions. To address the effects of Gα12 on proliferation in the inducible MDCK cell model, proliferation was quantified by measuring mitochondrial dehydrogenase activity (Fig. 1F), a well established correlate of cell proliferation (27Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45551) Google Scholar). Proliferation was measured in confluent and subconfluent monolayers. When Gα12- and QLα12-MDCK cells were plated at confluence (identical to conditions for apoptosis assays) and switched to ±dox for 72 h, there were no significant differences in proliferation (Fig. 1F). However, when Gα12-MDCK cells were plated at subconfluent density (∼50%) and switched into ±dox after plating, there was increased proliferation in Gα12- and QLα12-MDCK cells (-dox) at 72 h (Fig. 1G). Fig. 1G also shows that at 24 and 48 h proliferation rates were minimally stimulated in Gα12- and Qα12-MDCK cells (±dox) compared with Tet-off control cells. These results indicate that Gα12 stimulates proliferation in MDCK cells when cultured under specific conditions.QLα12 Expression Results in Reduced Bcl-2 Expression—We next examined the effects of Gα12 expression on the protein levels of the two major anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL. Fig. 2 (A and B) shows that inducing QLα12 (-dox) leads to nearly complete loss of Bcl-2 protein, in comparison with +dox and with Gα12-MDCK cells (±dox). In contrast, there was no appreciable difference in the levels of Bcl-xL protein for any of the conditions (Fig. 2A). The absence of any effect of Gα12 expression on Bcl-2 and Bcl-xL protein levels is consistent with the lack of an effect in Gα12-MDCK cells on apoptosis. To establish whether the QLα12-stimulated loss of Bcl-2 was mediated by changes in gene expression, semi-quantitative RT-PCR was performed for each condition. Fig. 2 (C and D) reveals no significant differences in Bcl-2 transcript in the QLα12-MDCK cells in -dox when compared with the other conditions.FIGURE 2Expression of Bcl-2 is significantly reduced in QLα12 cells.A, expression of anti-apoptotic proteins Bcl-2 and Bcl-xL in Gα12-MDCK cells. Western blots Bcl-2 and Bcl-xL were performed on lysates from Gα12- and QLα12-MDCK cells cultured in ±dox for 72 h. The blot was stripped and reprobed for β-actin. B, Western blots were quantified for Bcl-2 expression after normalization to the β-actin using NIH Image. Results are the mean ± S.E. of 10 independent experiments. C, reverse transcriptase-PCR for Bcl-2 was performed from Gα12- and QLα12-MDCK cells cultured in ±dox for 72 h. RT-PCR for β-actin was used as a loading control. D, reverse transcriptase-PCR analyses were quantified using NIH Image after normalization to β-actin. Results are the mean ± S.E. of three independent experiments. *, significance at p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression of QLα12 Activates JNK1—To screen for candidate signaling molecules activated in QLα12-MDCK cells in -dox, a phosphoprotein screen comparing lysates from QLα12-MDCK cells ± dox was performed (Kinexus Kinetworks™). QLα12 expression (-dox) specifically increased the phosphorylation of JNK1 and completely inhibited the phosphorylation of ERK1, without significantly affecting other MAPK family members, JNK2, ERK2, or p38 (Fig. 3A). To confirm the increased JNK1 activity detected in the phosphoprotein screen, JNK1 activity was determined by phospho-JNK1 Western blot (Fig. 3, B and C) and by direct measurement of JNK activity (Fig. 3, D and E). Fig. 3 shows that inducing QLα12 protein expression (-dox) led to a nearly 15-fold induction of JNK1 activity as determined by p-JNK1 immunoreactivity, a value similar to the effect seen in the phosphoprotein screen. QLα12 expression did not significantly affect total JNK1 levels. Additionally, Fig." @default.
• W2091868955 created "2016-06-24" @default.
• W2091868955 creator A5002505000 @default.
• W2091868955 creator A5014643963 @default.
• W2091868955 creator A5028068504 @default.
• W2091868955 creator A5058382756 @default.
• W2091868955 creator A5070802925 @default.
• W2091868955 date "2007-08-01" @default.
• W2091868955 modified "2023-10-12" @default.
• W2091868955 title "Gα12 Stimulates Apoptosis in Epithelial Cells through JNK1-mediated Bcl-2 Degradation and Up-regulation of IκBα" @default.
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