Inhibition of Glycogen Synthase Kinase-3β Is Sufficient for Airway Smooth Muscle Hypertrophy
2008; Elsevier BV; Volume: 283; Issue: 15 Linguagem: Inglês
10.1074/jbc.m800624200
ISSN1083-351X
AutoresHuan Deng, Gregoriy A. Dokshin, Jing Lei, Adam M. Goldsmith, Khalil N. Bitar, Diane C. Fingar, Marc B. Hershenson, J. Kelley Bentley,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoWe examined the role of glycogen synthase kinase-3β (GSK-3β) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3β, each of which inhibit GSK-3β activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins α-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-β, a proasthmatic cytokine. GSK-3β inhibition increased mRNA expression of α-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bϵ (eIF2Bϵ) attenuated LiCl- and SB216763-induced protein synthesis and expression of α-actin and SM22, indicating that eIF2B is required for GSK-3β-mediated airway smooth muscle hypertrophy. eIF2Bϵ siRNA also blocked CT-1- but not TGF-β-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3β-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3β, blocked protein synthesis and α-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-β. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased α-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3β phosphorylation. These data suggest that inhibition of the GSK-3β/eIF2Bϵ translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-β-induced hypertrophy does not depend on GSK-3β/eIF2B signaling. We examined the role of glycogen synthase kinase-3β (GSK-3β) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3β, each of which inhibit GSK-3β activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins α-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-β, a proasthmatic cytokine. GSK-3β inhibition increased mRNA expression of α-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bϵ (eIF2Bϵ) attenuated LiCl- and SB216763-induced protein synthesis and expression of α-actin and SM22, indicating that eIF2B is required for GSK-3β-mediated airway smooth muscle hypertrophy. eIF2Bϵ siRNA also blocked CT-1- but not TGF-β-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3β-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3β, blocked protein synthesis and α-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-β. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased α-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3β phosphorylation. These data suggest that inhibition of the GSK-3β/eIF2Bϵ translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-β-induced hypertrophy does not depend on GSK-3β/eIF2B signaling. Increased smooth muscle mass is the most prominent pathologic change observed in the airways of patients with asthma. Clinical studies examining the underlying cellular mechanism are limited but suggest that both hypertrophy (1Ebina M. Takahashi T. Chiba T. Motomiya M. Am. Rev. Respir. Dis. 1993; 148: 720-726Crossref PubMed Scopus (562) Google Scholar, 2Benayoun L. Druilhe A. Dombret M.C. Aubier M. Pretolani M. Am. J. Respir. Crit. Care Med. 2003; 167: 1360-1368Crossref PubMed Scopus (658) Google Scholar) and hyperplasia (1Ebina M. Takahashi T. Chiba T. Motomiya M. Am. Rev. Respir. Dis. 1993; 148: 720-726Crossref PubMed Scopus (562) Google Scholar, 3Woodruff P.G. Dolganov G.M. Ferrando R.E. Donnelly S. Hays S.R. Solberg O.D. Carter R. Wong H.H. Cadbury P.S. Fahy J.V. Am. J. Respir. Crit. Care Med. 2004; 169: 1001-1006Crossref PubMed Google Scholar) play a role. Despite evidence that smooth muscle hypertrophy contributes to airway remodeling in asthma, little is known about the biochemical mechanisms regulating this process. Glycogen synthase kinase (GSK)-3β 2The abbreviations used are:GSKglycogen synthase kinaseNFATnuclear factors of activated T cellseIFeukaryotic initiation factorCTcardiotrophinTGFtransforming growth factor4E-BPeIF-4E-binding proteinDMEMDulbecco's modified Eagle's mediumSRFserum response factorsmMHCsmooth muscle myosin heavy chainMLCKmyosin light chain kinaseERKextracellular signal-regulated kinaseGAPDHglyceraldehyde 3-phosphate dehydrogenasePBSphosphate-buffered salinesiRNAsmall interfering RNAFBSfetal bovine serumIBIowa BlackANOVAanalysis of varianceFAM6-carboxyfluoresceinTAMRAtetramethyl-6-carboxyrhodamine.2The abbreviations used are:GSKglycogen synthase kinaseNFATnuclear factors of activated T cellseIFeukaryotic initiation factorCTcardiotrophinTGFtransforming growth factor4E-BPeIF-4E-binding proteinDMEMDulbecco's modified Eagle's mediumSRFserum response factorsmMHCsmooth muscle myosin heavy chainMLCKmyosin light chain kinaseERKextracellular signal-regulated kinaseGAPDHglyceraldehyde 3-phosphate dehydrogenasePBSphosphate-buffered salinesiRNAsmall interfering RNAFBSfetal bovine serumIBIowa BlackANOVAanalysis of varianceFAM6-carboxyfluoresceinTAMRAtetramethyl-6-carboxyrhodamine. is a serine/threonine kinase that is constitutively active in unstimulated cells and becomes inactivated upon phosphorylation at Ser9 (4Cohen P. Frame S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776Crossref PubMed Scopus (1271) Google Scholar). The serine/threonine kinase Akt is the major GSK-3β kinase, but others exist, including mitogen-activated protein kinase kinase 1 (5Shaw M. Cohen P. FEBS Lett. 1999; 461: 120-124Crossref PubMed Scopus (81) Google Scholar) and protein kinase A (6Fang X. Yu S.X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (618) Google Scholar). GSK-3β activity is also inhibited by Wingless/Wnt signaling, independently of phosphorylation at serine 9 (7Dominguez I. Green J.B. Dev. Biol. 2001; 235: 303-313Crossref PubMed Scopus (59) Google Scholar). Accumulated evidence suggests that GSK-3β negatively regulates cardiac (8Morisco C. Zebrowski D. Condorelli G. Tsichlis P. Vatner S.F. Sadoshima J. J. Biol. Chem. 2000; 275: 14466-14475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 9Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (331) Google Scholar, 10Badorff C. Ruetten H. Mueller S. Stahmer M. Gehring D. Jung F. Ihling C. Zeiher A.M. Dimmeler S. J. Clin. Invest. 2002; 109: 373-381Crossref PubMed Scopus (145) Google Scholar, 11Antos C.L. McKinsey T.A. Frey N. Kutschke W. McAnally J. Shelton J.M. Richardson J.A. Hill J.A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 907-912Crossref PubMed Scopus (409) Google Scholar) and skeletal muscle (12Vyas D.R. Spangenburg E.E. Abraha T.W. Childs T.E. Booth F.W. Am. J. Physiol. 2002; 283: C545-C551Crossref PubMed Scopus (124) Google Scholar, 13Rochat A. Fernandez A. Vandromme M. Moles J-P. Bouschet T. Carnac G. Lamb N.J. C. Mol. Biol. Cell. 2004; 15: 4544-4555Crossref PubMed Scopus (112) Google Scholar) hypertrophy. The mechanisms underlying GSK-3β-mediated inhibition of hypertrophy are not completely understood. GSK-3β negatively regulates transcription factors involved in muscle-specific gene expression, including nuclear factors of activated T cells (NFAT), GATA4, and β-catenin (9Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (331) Google Scholar, 11Antos C.L. McKinsey T.A. Frey N. Kutschke W. McAnally J. Shelton J.M. Richardson J.A. Hill J.A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 907-912Crossref PubMed Scopus (409) Google Scholar, 12Vyas D.R. Spangenburg E.E. Abraha T.W. Childs T.E. Booth F.W. Am. J. Physiol. 2002; 283: C545-C551Crossref PubMed Scopus (124) Google Scholar, 14Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 15Haq S. Michael A. Andreucci M. Bhattacharya K. Dotto P. Walters B. Woodgett J. Kilter H. Force T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4610-4615Crossref PubMed Scopus (196) Google Scholar, 16Armstrong D.D. Esser K.A. Am. J. Physiol. 2005; 289: C853-C859Crossref PubMed Scopus (118) Google Scholar). Phosphorylation of the GTPase-activating protein tuberous sclerosis complex-2 by GSK-3β increases the ability of tuberous sclerosis complex-2 to inhibit mammalian target of rapamycin signaling (17Inoki K. Ouyang H. Zhu T. Lindvall C. Wang Y. Zhang X. Yang Q. Bennett C. Harada Y. Stankunas K. Wang C. He X. MacDougald O.A. You M. Williams B.O. Guan K-L. Cell. 2006; 126: 955-968Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar). Other downstream targets of GSK-3β regulate muscle hypertrophy via the translational process. One of the critical steps controlling the initiation of protein translation is formation of the 43 S preinitiation complex. Eukaryotic initiation factor-2 (eIF2), a multimer consisting of α, β, and γ subunits, functions to recruit methionyl-tRNA and conduct it as a tRNA-eIF2-GTP ternary complex to the 40 S ribosomal subunit. eIF2 GTP loading is determined by the activity of eIF2B, a guanine nucleotide exchange factor. eIF2Bϵ Ser539 phosphorylation by GSK-3β inhibits its GDP/GTP exchange activity, thereby limiting binding of methionyl-tRNA to the 40 S ribosomal subunit. However, phosphorylation of GSK-3β by Akt inactivates it, leading to eIF2B dephosphorylation and activation and a general enhancement of translation initiation (18Welsh G.I. Miller C.M. Loughlin A.J. Price N.T. Proud C.G. FEBS Lett. 1998; 421: 125-130Crossref PubMed Scopus (246) Google Scholar, 19Hardt S.E. Tomita H. Katus H.A. Sadoshima J. Circ. Res. 2004; 94: 926-935Crossref PubMed Scopus (68) Google Scholar). Accordingly, overexpression of a nonphosphorylatable eIF2Bϵ increases cardiac myocyte cell size and abolishes the antihypertrophic effect of GSK-3β (19Hardt S.E. Tomita H. Katus H.A. Sadoshima J. Circ. Res. 2004; 94: 926-935Crossref PubMed Scopus (68) Google Scholar). glycogen synthase kinase nuclear factors of activated T cells eukaryotic initiation factor cardiotrophin transforming growth factor eIF-4E-binding protein Dulbecco's modified Eagle's medium serum response factor smooth muscle myosin heavy chain myosin light chain kinase extracellular signal-regulated kinase glyceraldehyde 3-phosphate dehydrogenase phosphate-buffered saline small interfering RNA fetal bovine serum Iowa Black analysis of variance 6-carboxyfluorescein tetramethyl-6-carboxyrhodamine. glycogen synthase kinase nuclear factors of activated T cells eukaryotic initiation factor cardiotrophin transforming growth factor eIF-4E-binding protein Dulbecco's modified Eagle's medium serum response factor smooth muscle myosin heavy chain myosin light chain kinase extracellular signal-regulated kinase glyceraldehyde 3-phosphate dehydrogenase phosphate-buffered saline small interfering RNA fetal bovine serum Iowa Black analysis of variance 6-carboxyfluorescein tetramethyl-6-carboxyrhodamine. The role of GSK-3β in smooth muscle hypertrophy has not been studied. Unlike cardiac myocytes, which withdraw from the cell cycle early in development, smooth muscle cells may proliferate or hypertrophy, depending on the stimulus. A number of peptide growth factors and bronchoconstrictor agonists have been shown to induce airway smooth muscle proliferation in vitro (20Zhou L. Hershenson M.B. Respir. Physiol. Neurobiol. 2003; 137: 295-308Crossref PubMed Scopus (47) Google Scholar). More recently, cardiotrophin (CT)-1, a member of the IL-6 superfamily present in human lungs, has been shown to induce protein synthesis and cell enlargement, but not DNA synthesis, in cultured human bronchial smooth muscle cells (21Zhou D. Zheng X. Wang L. Stelmack G. Halayko A.J. Dorscheid D. Bai T.R. Br. J. Pharmacol. 2003; 140: 1237-1244Crossref PubMed Scopus (25) Google Scholar) and guinea pig airway explants (22Zheng X. Zhou D. Seow C.Y. Bai T.R. Am. J. Physiol. 2004; 287: L1165-L1171Crossref PubMed Scopus (15) Google Scholar). In addition, we showed that transforming growth factor (TGF)-β, a proasthmatic cytokine (23Ohno I. Nitta Y. Yamauchi K. Hoshi H. Honma M. Woolley K. O'Byrne P. Tamura G. Jordana M. Shirato K. Am. J. Respir. Cell Mol. Biol. 1996; 15: 404-409Crossref PubMed Scopus (239) Google Scholar, 24Minshall E.M. Leung D.Y.M. Matin R.J. Song Y.L. Cameron L. Ernst P. Hamid Q. Am. J. Respir. Cell Mol. Biol. 1997; 17: 326-333Crossref PubMed Scopus (594) Google Scholar, 25Tillie-Leblond I. Pugin J. Marquette C-H. Lamblin C. Saulnier F. Brichet A. Wallaert B. Tonnel A.-B. Gosset P. Am. J. Respir. Crit. Care Med. 1999; 159: 487-494Crossref PubMed Scopus (214) Google Scholar, 26Nomura A. Uchida Y. Sakamoto T. Ishii Y. Masuyama K. Morishima Y. Hirano K. Sekizawa K. Clin. Exp. Allergy. 2002; 32: 860-865Crossref PubMed Scopus (49) Google Scholar), increased human bronchial smooth muscle cell size, protein synthesis, expression of α-smooth muscle actin and smooth muscle myosin heavy chain (smMHC), formation of actomyosin filaments, and cell shortening to acetylcholine (27Goldsmith A.M. Bentley J.K. Zhou L. Jia Y. Bitar K.N. Fingar D.C. Hershenson M.B. Am. J. Respir. Cell Mol. Biol. 2006; 34: 247-254Crossref PubMed Scopus (84) Google Scholar). Further, TGF-β induced the phosphorylation of eIF-4E-binding protein (4E-BP), and inhibitors of 4E-BP phosphorylation blocked TGF-β-induced α-actin expression and cell enlargement, suggesting that eIF4E-, cap-dependent translation is necessary for TGF-β-induced hypertrophy. In this report, we investigate the contribution of the GSK-3β translational control pathway to airway smooth muscle hypertrophy. We found that inhibition of GSK-3β is sufficient for human airway smooth hypertrophy and that inhibition of GSK-3β/eIF2B signaling is required for CT-1- but not TGF-β-induced hypertrophy. Finally, we provide evidence for airway smooth muscle GSK-3β phosphorylation in a mouse model of asthma. Cell Culture—Primary human airway smooth muscle cells were isolated by enzymatic digestion from lung donor tissue unsuitable for transplantation (from Julian Solway, University of Chicago). This protocol was approved by the relevant institutional review boards. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and penicillin/streptomycin. Cells were seeded on uncoated plastic culture plates at ∼50% confluence. Prior to experiments, cells were serum-deprived for 24 h. Cells were treated with LiCl (10 mm), SB216763 (50 nm), CT-1 (10 ng/ml), or TGF-β (10 ng/ml) for 6 days. Fresh medium and chemicals were added 48 h after initial treatment. Experiments were performed in the absence of serum. Finally, for selected experiments, A7R5 rat vascular smooth muscle cells (American Type Culture Collection, Manassas, VA) were studied. Immunoblotting—Human bronchial smooth muscle cell lysates were matched for protein concentration, resolved by SDS-PAGE, and transferred to nitrocellulose or polyvinylidene difluoride membrane. Membranes were blocked in 5% milk for 1 h and probed with mouse anti-α-smooth muscle actin (Calbiochem), mouse anti-myosin light chain kinase (MLCK) (Sigma), mouse anti-SM-22 (Abcam, Cambridge, MA), mouse anti-smMHC (Sigma), rabbit anti-extracellular signal regulated kinase (ERK) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), rabbit anti-phospho-Ser9 GSK-3β, rabbit anti-GSK-3β, rabbit anti-eIF2Bϵ (each from Cell Signaling, Danvers, MA), or rabbit anti-phospho-Ser539 eIF2Bϵ (Biosource, Camarillo, CA). Antibody binding was detected with a peroxidase-conjugated anti-rabbit or anti-mouse IgG and chemiluminescence. In Vitro GSK-3β Kinase Assay—GSK-3β activity was measured by immunoprecipitating cell lysates with mouse anti-GSK-3β (clone GSK-4B; Sigma) and incubating immunoprecipitates with the GSK-3β substrate Tau (1 μg/μl; Sigma), ATP (1 mm), and [γ-32P]ATP (10 μCi) for 30 min at 30 °C, as described (28Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (740) Google Scholar). Because immunoprecipitation of human airway smooth muscle cell lysates brought down a contaminating phosphorylated protein of similar molecular weight to the substrate, A7R5 vascular smooth muscle cells were used for these experiments. Cells were treated with LiCl, SB216763, CT-1, or TGF-β for 6 days. Cells were then washed with ice-cold PBS and then lysed. Reaction mixtures were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to film. Total GSK-3β in the immunoprecipitates was examined using rabbit anti-GSK-3β (Cell Signaling). Transfection of GSK-3β and eIF2Bϵ siRNA—21-bp duplexes of either GSK-3β siRNA or eIF2Bϵ siRNA (both from Dharmacon, Lafayette, CO) were transfected into subconfluent primary human airway smooth muscle cells using Oligofectamine in OptiMEM (Invitrogen). For GSK-3β, a pool of double-stranded siRNAs containing equal parts of the following antisense sequences was used: 1, 5′-PUAUACCACACCAAAUGAUCUU-3′; 2, 5′-PUAUGUUACAGUGAUCUAGCUU-3′; 3, 5′-PAUAGGCUAAACUUCGGAACUU-3′; 4, 5′-PCAAAGAUCAACUCUGGUGCUU-3′. For eIF2Bϵ siRNA, a pool of double-stranded siRNAs containing equal parts of the following antisense sequences was used: 1, 5′-PUAAAUCAUAUCGAACCUCCUU; 2, 5′-PUUAGACUUAUGUUAUAGGCUU; 3, 5′-PCCAUAUUCCUUAGCUGUUAUU; 4, 5′-PUCUUGCGCAACUGCUGGCCUU. The corresponding nontargeting siRNA sequence was 5′-UAGCGACUAAACACAUCAA-3′. Six hours later, DMEM and FBS were added. The next morning, cells were incubated in fresh DMEM containing 10% FBS for 24 h. Finally, cells were treated with the relevant stimulus in serum-free medium for 2 days prior to harvest. Cell Size Analysis—Human bronchial smooth muscle cell size was measured by fluorescence-activated cell sorting. Cells were treated with LiCl, SB216763, GSK-3β siRNA, CT-1, or TGF-β. Cells were collected and fixed with 75% ethanol and stored at -20 °C before staining. Cells were centrifuged and stained with propidium iodide (50 μg/ml), RNase (100 μg/ml) solution for 1 h. Cells in G0/G1 phase were gated for forward scatter measurement using a FACSCalibur flow cytometer (BD Biosciences). Protein Synthesis—Cells were serum-starved for 24 h before experiments. Cells were plated at 5 × 105 cells/well (or 3 × 105 cells/well for experiments involving transfection) and incubated in [3H]leucine (0.5 μCi; PerkinElmer Life Sciences) for 48 h. Cells were lysed, and proteins were precipitated with 10% trichloroacetic acid. After washing with cold ethanol and solubilization with 1% Triton X-100 in 0.5 m NaOH, radioactivity was measured by a scintillation counter. Fluorescence Microscopy—Human bronchial smooth muscle cells were grown on collagen-coated glass slides (BD Biosciences) and fixed in 1% paraformaldehyde. To stain filamentous actin, slides were incubated with Alexa Fluor 488-conjugated phalloidin (Molecular Probes, Eugene, OR). For immunocytochemistry, slides were probed with Cy3-conjugated mouse anti-α-smooth muscle actin-Cy3 (Sigma) or anti-MLCK, followed by Alexa Fluor 594-labeled goat anti-mouse IgG (Molecular Probes) and with phospho-GSK-3β antibody followed by Alexa Fluor 488-labeled goat anti-rabbit IgG (Molecular Probes). Retroviral Transduction of Human Bronchial Cell Lines—cDNA encoding a nonphosphorylatable GSK-3β (GSK-3β-A9), with Ser9 replaced by alanine, was provided by Dr. Anne Vojtek (University of Michigan). GSK-3β-A9 cDNA was subcloned into the pMSCVpuro retroviral vector (BD Biosciences). The Phoenix-GP retrovirus packaging cell line, a 293-cell derivative line that expresses only the gag-pol viral components (provided by G. Nolan, Stanford University) was transiently transfected with pHCMV-G, which contains the vesicular stomatitis virus envelope glycoprotein, and either pMSCVpuro-AA-GSK-3β-A9 or pMSCV alone. Viral supernatant was collected, filtered, and supplemented with Polybrene (8 μg/ml). Human bronchial smooth muscle cells were infected with viral supernatant (four times for 4 h each). Infected cells were selected with puromycin (2 μg/ml). After selection, cells were grown to confluence, split into 6-well plates, and incubated in the absence or presence of LiCl, CT-1, or TGF-β. Cell Contraction—Individual cell length before and after contraction with KCl was measured by computerized image micrometry. Cells were seeded in 100-mm dishes and grown to confluence in serum-free medium or medium supplemented with LiCl, CT-1, or TGF-β. At confluence, cells were scraped off with a rubber policeman, triturated, and transferred to polypropylene tubes. At this stage, cells tend to maintain a contracted state due to mechanical stimulation. The cells were treated with 8-bromo-cAMP and then allowed to float freely and relax for 24 h with occasional swirling to prevent settling or sticking to the sides of the tube. During this period, cells regain a spindle shape and extend processes. Aliquots of cultured cell suspension (2.5 × 104 cells/0.5 ml) were stimulated with KCl (75 mm). The reaction was allowed to proceed for 4 min and stopped by the addition of 0.1 ml of glutaraldehyde at a final concentration of 1% (v/v). Fixed cells were allowed to settle and then transferred by wide mouth pipette to a microscope slide for analysis. The average length of cells before or after the addition of test agents was obtained from 20 cells encountered in successive microscopic fields. Real Time PCR—Quantitative two-step real time PCR for human α-smooth muscle actin, human 18 S rRNA, mouse α-actin, mouse β-actin, and mouse CT-1 was conducted using specific primers and probes. Primers were from IDT (Coralville, IA) and used 6-carboxyfluorescien (FAM) as a reporter fluorochrome and either tetramethyl-6-carboxyrhodamine (TAMRA) or Iowa Black (IB) as fluorescent quenchers. Human α-actin forward primer was 5′-GAC CCT GAA GTA CCC GAT AGA AC-3′, reverse primer was 5′-GGG CAA CAC GAA GCT CAT TG-3′, and probe was 5′-FAM-TGG CAT CAT CAC CAA CTG GGA CG-IB-3′. Human 18 S rRNA forward primer was 5′-CGC CGC TAG AGG TGA AAT TCT-3′, reverse primer was 5′-CAT TCT TGG CAA ATG CTT TCG-3′, and probe was 5′-FAM-ACC GGC GCA AGA CGG ACC AGA-TAMRA-3′. Mouse α-actin forward primer was 5′-CCA GGC ATT GCT GAC AGG AT-3′, reverse primer was 5′-CCA CCG ATC CAG ACA GAG TAC-3′, and probe was 5′-FAM-AAG GAG ATC ACA GCC CTC GCA CC-IB-3′. Mouse β-actin forward primer was 5′-TGA CAG GAT GCA GAA GGA GAT-3′, reverse primer was 5′-GCG CTC AGG AGG AGC AAT-3′, and probe was 5′FAM-ACT GCT CTG GCT CCT AGC ACC AT-IB-3′. Mouse CT-1 forward primer was 5′-GCC TCA GCC TTT GAG AGG AAA-3′, reverse primer was 5′-AGC CCG GTC TGT CCA GTG A-3′, and probe was 5′-FAM-TGC AGA GGC TAC ATA GTG ACC CGA-IB-3′. Reactions were performed on an Eppendorf Realplex2 thermocycler (Westbury, NY). Reporter Assays—A7R5 cells were used for these experiments because of their superior transfection efficiency. Cells were transiently transfected with the 200 ng of NFAT-luc (BD Biosciences) or serum response factor (SRF)-luc (29Camoretti-Mercado B. Fernandes D.J. Dewundara S. Churchill J. Ma L. Kogut P.C. McConville J.F. Parmacek M.S. Solway J. J. Biol. Chem. 2006; 281: 20383-20392Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) (from J. Solway, University of Chicago). Three nanograms of the SV40 Renilla luciferase vector was used as transfection control. Cells were transfected using a Lipofectamine 2000 (Invitrogen). The following day, cells were serum-deprived for 2 h and treated with LiCl, SB216763, CT-1, or TGF-β for 48 h. Cells were subsequently lysed, and luciferase activity was measured using the Promega luciferase assay system (Madison, WI). Ovalbumin Sensitization/Challenge Model and Isolation of Murine Airway Smooth Muscle Cells—BALB/c mice (Charles River Laboratories, Wilmington, MA) were sensitized and challenged to endotoxin-free ovalbumin (Pierce), as previously described (30Leigh R. Ellis R. Wattie J. Southam D.S. De Hoogh M. Gauldie J. O'Byrne P.M. Inman M.D. Am. J. Respir. Cell Mol. Biol. 2002; 27: 526-535Crossref PubMed Scopus (174) Google Scholar). On day 0, mice were anesthetized and injected intraperitoneally with 200 μl of a suspension of 25% (w/v) alum (Pierce) in either PBS or ovalbumin (5 mg/ml). On day 11, animals were given an identical intraperitoneal injection as well as a 50-μl intranasal instillation of PBS or ovalbumin (20 mg/ml). On days 18, 21, 22, and 23, intranasal instillations were repeated. Twenty-four hours after the final challenge, mice were euthanized, and the lungs were processed for RNA quantitation or airway smooth muscle cell isolation. Cells were isolated by dissection of major bronchi from the lung, mincing well, and incubating for 1 h at 21 °C on a rotary mixer in DMEM with 0.1% trypsin and 0.1% collagenase. Proteolysis was stopped by the addition of 9 volumes of DMEM with 10% FBS. Intact cells and tissue debris were sedimented at 1000 × g for 5 min, dispersed with further mincing, and plated in growth media with antibiotics. Medium was changed every day for the next week to remove floating cells, and all tissue fragments were removed at the end of 1 week. GSK-3β Inhibitors Decrease GSK-3β Activity in Intact Cells—LiCl attenuates GSK-3β activity by increasing the phosphorylation of the inhibitory Ser9 residue, probably by inhibiting a protein phosphatase, and by acting as a competitive inhibitor for Mg2+, which is needed for maximal kinase activity (31Jope R.S. Trends Pharmacol. Sci. 2003; 24: 441-443Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). LiCl has been shown to decrease cardiac myocyte GSK-3β activity and eIF2Bϵ phosphorylation (19Hardt S.E. Tomita H. Katus H.A. Sadoshima J. Circ. Res. 2004; 94: 926-935Crossref PubMed Scopus (68) Google Scholar) while increasing protein synthesis (9Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (331) Google Scholar). SB216763 is a permeable, structurally distinct maleimide that inhibits GSK-3 activity (32Coghlan M.P. Culbert A.A. Cross D.A. Corcoran S.L. Yates J.W. Pearce N.J. Rausch O.L. Murphy G.J. Carter P.S. Roxbee Cox L. Mills D. Brown M.J. Haigh D. Ward R.W. Smith D.G. Murray K.J. Reith A.D. Holder J.C. Chem. Biol. 2000; 7: 793-803Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar). We examined the effect of these inhibitors, as well as CT-1 and TGF-β, on GSK-3β phosphorylation and kinase activity. First, early passage human bronchial smooth muscle cells were treated with LiCl (10 mm), SB216763 (50 nm), CT-1 (10 ng/ml), or TGF-β (10 ng/ml), and GSK-3β phosphorylation was assessed by immunoblotting with a phosphospecific antibody. LiCl, CT-1, and TGF-β each enhanced GSK-3β phosphorylation without affecting the expression of total GSK-3β (Fig. 1A). As expected, SB216763 had no effect on GSK-3β phosphorylation. Next, we examined the effects of LiCl, SB216763, CT-1, and TGF-β on GSK-3β kinase activity by in vitro assay. A7R5 cell lysates were immunoprecipitated with anti-GSK3β antibody and incubated with [γ-32P]ATP and Tau protein, a GSK-3β substrate. Treatment with LiCl, SB216763, CT-1, and TGF-β each inhibited GSK-3β activity, as indicated by a decrease in Tau phosphorylation (Fig. 1B). GSK-3β Inhibitors Increase Human Airway Smooth Muscle Cell Size and Protein Synthesis—We determined the effect of GSK-3β inhibition on human airway smooth muscle cell size, protein synthesis, and contractile protein expression. Flow cytometry data showed that cells treated with LiCl or SB216763 displayed a rightward shift in forward scatter, indicating an increase in cell size (Fig. 2A). Changes in cell size were accompanied by an increase in protein synthesis, since the [3H]leucine incorporation was enhanced (Fig. 2B). We also examined the effect of GSK-3β knockdown on cell size and protein synthesis using specific siRNA. Cells treated with GSK-3β siRNA exhibited decreased GSK-3β expression (Fig. 2C). GSK-3β siRNA-treated cells also exhibited a rightward shift in the forward scatter compared with the cells treated with nontargeting siRNA (Fig. 2D). Like LiCl and SB216763, treatment with GSK-3β siRNA also enhanced protein synthesis (Fig. 2E). CT-1 and TGF-β also induced cell hypertrophy, as indicated by a rightward shift in forward scatter and increased protein synthesis (Fig. 2, F-H). Finally, there was no effect of GSK-3β inhibition or CT-1 on human airway smooth muscle DNA synthesis (Fig. 2I). GSK-3β Inhibition Increases Contractile Protein Expression—Cellular proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for α-smooth muscle actin, MLCK, smMHC, and SM22. LiCl
Referência(s)