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• W2022260308 abstract "A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by β2-adrenergic receptors (β2-ARs). However, the detailed mechanism of β2-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by β2-ARs in human embryonic kidney 293A cells. The β2-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of β-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of β-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by β-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47phox knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a β-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for β2-AR-mediated early activation of p38 MAPK. A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by β2-adrenergic receptors (β2-ARs). However, the detailed mechanism of β2-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by β2-ARs in human embryonic kidney 293A cells. The β2-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of β-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of β-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by β-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47phox knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a β-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for β2-AR-mediated early activation of p38 MAPK. Numerous data indicate that p38, a member of the mitogen-activated protein kinase (MAPK) 2The abbreviations used are: MAPK, mitogen-activated protein kinase; β2-AR, β2-adrenergic receptor; PKA, protein kinase A; GPCR, G protein-coupled receptor; ROS, reactive oxygen species; Epac, exchange protein directly activated by cAMP; ERK1/2, extracellular signal-regulated kinases; GRKs, G-protein-coupled receptor kinases; siRNA, small interfering RNA; GAPDH, glyceraldehyde phosphate dehydrogenase; 8-pCPT-2Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl cyclic AMP; DPI, diphenyleneiodonium chloride; CM-H2DCFDA, 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; HA, hemagglutinin; PBS, phosphate-buffered saline; HEK, human embryonic kidney; TRITC, tetramethylrhodamine isothiocyanate; MOPS, 4-morpholinepropanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate. family, is implicated in many biological responses mediated by β2-adrenergic receptors (β2-ARs), including modulation of immune, inflammatory, and cardiovascular pathologic processes (1Chi D. Fitzgerald S.M. Pitts S. Cantor K. King E. Lee S. Huang S.K. Krishnaswamy G. BMC Immunol. 2004; 5: 22Crossref PubMed Scopus (31) Google Scholar, 2Ashwell J.D. Nat. Rev. Immunol. 2006; 6: 532-540Crossref PubMed Scopus (308) Google Scholar, 3Wang Y. Circulation. 2007; 116: 1413-1423Crossref PubMed Scopus (243) Google Scholar, 4Magne S. Couchie D. Pecker F. Pavoine C. J. Biol. Chem. 2001; 276: 39539-39548Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). For example, in β2-ARs-overexpressing transgenic mice with cardiomyopathy, cardiac-specific expression of dominant-negative p38α MAPK improved cardiac function and reduces cardiac apoptosis and fibrosis (5Peter P.S. Brady J.E. Yan L. Chen W. Engelhardt S. Wang Y. Sadoshima J. Vatner S.F. Vatner D.E. J. Clin. Investig. 2007; 117: 1335-1343Crossref PubMed Scopus (49) Google Scholar). Generally, β2-ARs are believed to exert their effects through a classic Gs/cAMP/protein kinase A (PKA) pathway. Upon β2-ARs stimulation, cAMP/PKA-dependent p38 MAPK activation has been shown in various cells, including macrophages, PC12, MC3T3-E1 cells, Chinese hamster ovary cells, NIH 3T3 cells, adipocytes, and rat cardiac myocytes (6Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 7Delghandi M.P. Johannessen M. Moens U. Cell Signal. 2005; 17: 1343-1351Crossref PubMed Scopus (250) Google Scholar, 8Zheng M. Zhang S.J. Zhu W.Z. Ziman B. Kobilka B.K. Xiao R.P. J. Biol. Chem. 2000; 275: 40635-40640Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In addition, β2-ARs, also via coupling to Gi, induce activation of p38 MAPK, and subsequently attenuate β-AR-stimulated apoptosis in adult rat cardiac myocytes (9Zheng M. Zhu W. Han Q. Xiao R.P. Pharmacol. Ther. 2005; 108: 257-268Crossref PubMed Scopus (79) Google Scholar). However, emerging evidence suggests that in some settings, the classical cAMP/PKA-dependent pathway does not fully account for β2-AR-mediated p38 MAPK activation. The cAMP/PKA-independent activation of p38 MAPK has been shown to regulate the expressions of the cellular inhibitor of apoptosis protein-2 in colon cancer cells (10Nishihara H. Hwang M. Kizaka-Kondoh S. Eckmann L. Insel P.A. J. Biol. Chem. 2004; 279: 26176-26183Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and pituitary hormone prolactin in human T lymphocytes (11Gerlo S. Verdood P. Hooghe-Peters E. Kooijman R. Cell Mol. Life Sci. 2006; 63: 92-99Crossref PubMed Scopus (27) Google Scholar). In Raw264.7 cells, inductions of interleukin-1 and interleukin-6 by stimulation of β2-ARs occur mainly though the Exchange protein directly activated by cAMP (Epac), B-Raf/extracellular signal-regulated kinases (ERK1/2), and the p38 MAPK pathway (12Tan K.S. Nackley A.G. Satterfield K. Maixner W. Diatchenko L. Flood P.M. Cell Signal. 2007; 19: 251-260Crossref PubMed Scopus (167) Google Scholar). Furthermore, we also recently demonstrated that in mouse cardiac fibroblasts, both classic cAMP/PKA and Epac/Rap1 routes are not required for p38 MAPK-mediated secretion of interleukin-6 in response to β2-ARs stimulation (13Yin F. Wang Y.Y. Du J.H. Li C. Lu Z.Z. Han C. Zhang Y.Y. J. Mol. Cell Cardiol. 2006; 40: 384-393Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Although it is well established that β2-AR-activate p38 MAPK in numerous cells and tissues, a detailed understanding of the underlying mechanism remains unknown. This is necessary before the potential benefits of exploiting p38 MAPK as a target of β2-AR agonists can be extrapolated into the therapeutic arena. The mammalian arrestin family consists of four members: arrestin-1 and -4, and β-arrestin-1 and -2. Of these, β-arrestin-1 and -2 are ubiquitously expressed (14Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol. Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In addition to their well established role in terminating GPCR signal transduction, accumulating evidence indicates that they can modulate compartmentalization of intracellular cAMP signaling and initiate various cytoplasmic and nuclear signaling cascades (15Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 16Ma L. Pei G. J. Cell Sci. 2007; 120: 213-218Crossref PubMed Scopus (210) Google Scholar). For example, binding of β-arrestin-1/2 to components of the ERK1/2 and c-Jun N-terminal kinase 3 cascades allows them to function as scaffolding molecules, thus localizing these kinases activation (17Tohgo A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Importantly, β-arrestin-dependent ERK1/2 activation is implicated in the regulation of chemotaxis and cell survival by altering the profile of gene transcription, in a manner distinct from the classic G-protein-dependent pathway (14Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol. Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 18Wisler J.W. DeWire S.M. Whalen E.J. Violin J.D. Drake M.T. Ahn S. Shenoy S.K. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16657-16662Crossref PubMed Scopus (489) Google Scholar). In the present study we investigate whether β-arrestins are involved in β2-AR-mediated p38 MAPK activation. We show that β2-ARs stimulation induces a biphasic activation (early and delayed) of p38 MAPK, which is distinct from a previously described monophasic model in mast cells (1Chi D. Fitzgerald S.M. Pitts S. Cantor K. King E. Lee S. Huang S.K. Krishnaswamy G. BMC Immunol. 2004; 5: 22Crossref PubMed Scopus (31) Google Scholar), fat cells, embryonic chick ventricular cells (4Magne S. Couchie D. Pecker F. Pavoine C. J. Biol. Chem. 2001; 276: 39539-39548Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and adult rat cardiac myocytes (8Zheng M. Zhang S.J. Zhu W.Z. Ziman B. Kobilka B.K. Xiao R.P. J. Biol. Chem. 2000; 275: 40635-40640Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Interestingly, early phase activation of p38 MAPK can be prevented by reducing the expression of β-arrestin-1 by siRNA and inhibiting NADPH oxidase activity. By contrast, the classic cAMP/PKA pathway is involved in the delayed activation of p38 MAPK following exposure to isoproterenol. Antibodies, Reagents, and Plasmids—Antibodies used included phospho-p38 MAPK (Thr180/Tyr182), phospho-p42/44 MAPK (Thr202/Tyr204), phospho-HSP27 (Ser82), p47phox and p38β MAPK (Cell Signaling Technology, Beverly, MA), p38 MAPK (C-20), GAPDH (6C5), β-arrestin-1 (K-16), β-arrestin-2 (H-9), β2-AR (H-73), G-protein-coupled receptor kinase 2 (GRK2, C-15), GRK3 (H-43), GRK5 (H-64), and GRK6 (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-labeled secondary antibodies and chemiluminescence reagents were from Pierce. TRITC-conjugated anti-rabbit IgG were from Beijing Zhongshan Golden Bridge Biotechnology (Beijing, China). Isoproterenol, ICI 118551, propranolol, (Rp)-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl cyclic AMP (8-pCPT-2Me-cAMP), H89, KT5720, SQ22536, isobutylmethylxanthine, and SB202190 were from Sigma. Clostridium difficile Toxin B (Toxin B), NSC23766, Y27632, diphenyleneiodonium chloride (DPI), apocynin, and rotenone were from Calbiochem (La Jolla, CA). 5-(and 6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) and rhodamine-conjugated phalloidin were from Invitrogen. Plasmids encoding dominant-negative human β-arrestin-2 and dynamin-1 (K44A) were a gift from Dr. Ming Zhao (La Jolla Institute for Molecular Medicine, San Diego, CA) (19Zhao M. Wimmer A. Trieu K. DiScipio R.G. Schraufstatter I.U. J. Biol. Chem. 2004; 279: 49259-49267Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). A dominant-negative plasmid of human p38α MAPK was a gift from Dr. Haian Fu (Department of Pharmacology, Emory University School of Medicine). A plasmid encoding human β2-AR with a FLAG tag was a gift from Dr. Kenneth P. Minneman (Department of Pharmacology, Emory University School of Medicine). Several dominant negative expression plasmids cloned into pcDNA3.1, including Rac1 T17N 3×HA, RhoA T19N 3× HA, and Cdc42 T17N 3× HA were purchased from Missouri S&T cDNA Resource Center (Rolla, MO). Cell Culture and Transfection—HEK293A cells were grown in high-glucose Dulbecco's modified Eagle's medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany), 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a humidified atmosphere of 5% CO2. Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Intracellular cAMP Assay—Intercellular cAMP levels were determined as previously described (20Malki S. Nef S. Notarnicola C. Thevenet L. Gasca S. Méjean C. Berta P. Poulat F. Boizet-Bonhoure B. EMBO J. 2005; 24: 1798-1809Crossref PubMed Scopus (173) Google Scholar), using a commercial Parameter™ Cyclic AMP immunoassay kit (R & D Systems, Minneapolis, MN). In brief, prior to stimulation with isoproterenol, cells were preincubated with 0.5 mm isobutylmethylxanthine for 20 min, and then lysed in 100 μl of the lysis buffer kit and freeze/thawed three times. The suspension was then centrifuged at 600 × g for 10 min at 4 °C, and the protein concentration of the supernatant measured using a BCA assay (Pierce). cAMP levels in the supernatant were immediately determined according to the manufacturer's protocol. The intra-assay and inter-assay coefficient of variance were 12.4 and 9.7%, respectively. PKA Activities Assay—PKA activity was determined using a non-radioactive PKA kinase activity assay kit (Assay Designs, MI). The assay utilizes a specific synthetic peptide as a substrate for PKA, which is readily phosphorylated by PKA. Cells seeded onto 60-mm dishes were lysed in 200 μl of lysis buffer (20 mm MOPS, 50 mm β-glycerolphosphate, 50 mm sodium fluoride, 1 mm sodium vanadate, 5 mm EGTA, 2 mm EDTA, 1% Nonidet P-40, 1 mm dithiothreitol, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin and aprotinin), and then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was stored at –70 °C until required. The assay was performed according to the manufacturer's instructions. Briefly, the samples are added to the appropriate duplicated wells, followed by the addition of ATP to initiate the reaction. The kinase reaction is terminated and a specific phosphor-substrate antibody added to the wells. Following the addition of a peroxidase-conjugated secondary antibody, color development was initiated by the addition of a tetramethylbenzidine substrate. The color development was stopped with an acid stop solution and the intensity of the color measured in a microplate reader at 450 nm; color development being proportional to PKA phosphotransferase activity. The results are expressed as relative kinase activity (OD/mg·protein). The intra-assay and inter-assay coefficients of variance were 5.4 and 4.7%, respectively. RNA Interference—Chemically synthesized, double-stranded siRNAs, with 19-nucleotide duplex RNA and 2-nucleotide 3′-deoxyterminal deoxynucleotidyltransferase overhangs were from Shanghai GeneChem (Shanghai, China). The siRNA sequences targeting human β-arrestin-1 and -2 were 5′-AGCCUUCUGCGCGGAGAAUtt-3′ and 5′-GGACCGCAAAGUGUUUGUGtt-3′, respectively. The previously validated siRNA sequences targeting GRKs included: GRK2 (5′-AAGAAGUACGAGAAGCUGGAG-3′), GRK3 (5′-AAGCAAGCUGUAGAACACGUA-3′), GKR5 (5′-AAGCCGUGCAAAGAACUCUUU-3′), and GRK6 (5′-AACAGUAGGUUUGUAGUGAGC-3′) (21Violin J.D. Ren X.R. Lefkowitz R.J. J. Biol. Chem. 2006; 281: 20577-20588Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The previously confirmed siRNA sequences targeting p38α and p38β are 5′-AAGAAGCTCTCCAGACCATTT-3′ and 5′-AACTGGATGCATTACAACCAA-3′, respectively (22Shiromizu T. Goto H. Tomono Y. Bartek J. Totsukawa G. Inoko A. Nakanishi M. Matsumura F. Inagaki M. Genes Cells. 2006; 11: 477-485Crossref PubMed Scopus (43) Google Scholar). siRNA for human GAPDH was used as a positive control (5′-GUGGAUAUUGUUGCCAUCAtt-3′). A non-silencing scramble RNA duplex was used as a negative control (5′-UUCUCCGAACGUGUCACGUtt-3′). Additionally, to knock down the expression of human p47phox in a complementary experiment, p47phox siRNA duplex (sc-29422) was purchased from Santa Cruz Biotechnology. For siRNA experiments, early passage HEK293A cells at 40–50% confluence on 35-mm plates were transfected with siRNA by use of Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. After 48 to 72 h of transfection, cells were treated with isoproterenol for the appropriate times and harvested. The inhibitory efficiency of these siRNA probes was assessed by measuring knockdown of their respective proteins by Western blot analysis. Western Blot Analysis—Protein expression was analyzed by Western blot as previously described (23Yin F. Li P. Zheng M. Chen L. Xu Q. Chen K. Wang Y. Zhang Y. Han C. J. Biol. Chem. 2003; 278: 21070-21075Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Briefly, samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking, blots were probed with the appropriate primary antibodies overnight at 4 °C or for 2 h at room temperature, then washed and incubated with horseradish peroxidase-conjugated secondary antibody. Bands were visualized by use of a super-Western sensitivity chemiluminescence detection system (Pierce). Autoradiographs were quantitated by densitometry (Science Imaging System, Bio-Rad). Analysis of in Vitro p38 MAPK Kinase Activity—p38 MAPK kinase activity was determined using a non-radioactive p38 kinase assay kit (Cell Signaling Technology, Beverly, MA). Briefly, cells were lysed in the kit lysis buffer containing 1 mm phenylmethylsulfonyl fluoride. Cell lysates (600 μg of protein) were immunoprecipitated with an immobilized phosphor-p38 MAPK (Thr180/Tyr182) monoclonal antibody, and then centrifuged at 14,000 × g for 30 s. The immunocomplexes were washed (2 times with lysis buffer and 3 times with the kit kinase buffer), centrifuged, and suspended in 50 μl of the kinase buffer. Following addition of ATP (200 μm) and the phosphor-p38 MAPK substrate (ATF-2 fusion protein, 1 μg), samples were incubated at 30 °C for 30 min, and the reaction terminated by addition of 25 μl of 3× SDS sample buffer. Samples were then boiled, centrifuged, and separated by 10% SDS-PAGE. Phosphorylated ATF-2 was detected by immunoblotting with a phospho-ATF-2 (Thr76) antibody. Rap1 and Rac1 Activity Pulldown Assay—As previously reported (13Yin F. Wang Y.Y. Du J.H. Li C. Lu Z.Z. Han C. Zhang Y.Y. J. Mol. Cell Cardiol. 2006; 40: 384-393Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), Rap1 activation was determined using a commercially available kit (Millipore-Upstate). Rac1 activation was measured using a Stressgen StressXpress® Rac1 activation kit (Assay Designs, MI), according to the manufacturer's instructions. In brief, following cell lysis, 600 μg of total protein was incubated with 20 μg of glutathione S-transferase-Pak-PBD in the spin cup containing an immobilized glutathione disc for 1 h at 4 °C with gentle rocking. Bound proteins were washed, mixed with 2× SDS sample buffer, boiled, and centrifuged at 7200 × g for 2 min. Pulled down active or GTP-Rac1 was separated by 12% SDS-PAGE and immunoblotted with a Rac1 monoclonal antibody. Flow Cytometric Analysis of ROS—Serum-starved cells were loaded with 10 μm CM-H2DCFDA in Dulbecco's modified Eagle's medium without phenol red in the dark for 30 min at 37 °C. Cells were then washed gently with Dulbecco's modified Eagle's medium and incubated in the presence of isoproterenol. CM-H2DCFDA is a cell permeant indicator for ROS, which is non-fluorescent until oxidization by intracellular oxidation. Treated cells were washed twice with ice-cold phosphate-buffered saline (PBS), then detached by trypsinization and pelleted by centrifugation (800 × g for 5 min) at 4 °C. Cell pellets were resuspended in 0.6 ml of ice-cold PBS and cellular fluorescence intensity immediately measured using a flow cytometer (BD FACS Calibur, CA) equipped with a laser lamp (emission 480 nm; band pass filter 530 nm). Data were normalized to values obtained from mock-treated controls. Approximately 10,000 cells were analyzed for each sample. Fluorescence Microscopy—To assess F-actin rearrangement induced by isoproterenol, cells grown on glass coverslips were cultured without serum for 6 h. Following exposure to the appropriate inhibitors and isoproterenol, cells were washed, fixed, and permeabilized with 4% formaldehyde and 0.5% Triton X-100. Following washing with PBS, F-actin was stained with rhodamine-conjugated phalloidin (1.0 μg/ml) for 45 min at room temperature, washed, mounted on slides with 90% glycerol in PBS, and visualized by fluorescence microscopy (×200). To provide a valid comparison, identical acquisition parameters were used for all observations. Images were randomly acquired from 10 different fields per dish or coverslip. Confocal Imaging—To monitor intracellular distribution of phosphorylated p38 MAPK, cells were seeded on glass coverslips. The serum-starved treated cells were washed, fixed, and permeabilized as above. Following washing with PBS, cells were blocked with 10% normal goat serum, and then incubated with primary antibodies overnight, and a TRITC-conjugated secondary antibody (1:100) for 2 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (50 ng/ml) in PBS for 15 min. Coverslips were washed, mounted with 90% glycerol, and viewed on a confocal laser scanning microscope (TCS SP2; Leica, Wetzlar, Germany) with a ×40 oil immersion objective lens. Statistics—Values are expressed as mean ± S.E. One-way analysis of variance or Student's t test was used for statistical analysis as appropriate. A p < 0.05 was considered statistically significant. Biphasic Activation of p38 MAPK in Response to Isoproterenol Stimulation—To elucidate the mechanism of p38 MAPK activation by β2-ARs, HEK293A cells were incubated with 1 μm isoproterenol (β-adrenergic receptor agonist) for 2.5 min to 24 h. Phosphorylated p38 MAPK was determined by Western blot analysis with a specific antibody against p38 MAPK phosphorylated at residues Thr180/Tyr182. Isoproterenol caused a rapid increase in p38 MAPK phosphorylation, which was detected as early as 2.5 min and peaked at 10 min (i.e. early activation) before returning to basal level by 60 min. Surprisingly, isoproterenol also caused a secondary increase in phosphorylation of p38 MAPK after 90 min (i.e. delayed activation), which lasted for at least 6 h (Fig. 1A). Stripping and reprobing the blots with antibody against p38 MAPK demonstrated that equal amounts of p38 MAPK were detectable before and after isoproterenol treatment. In vitro kinase analysis indicated that the kinetics of p38 MAPK activity paralleled changes in its phosphorylation (Fig. 1B). In addition, intracellular immunofluorescence staining with specific phosphor-p38 MAPK antibodies and confocal imaging also illustrated the biphasic activation of p38 MAPK. Interestingly, during the early phase of the response, activated p38 MAPK was localized to the cytoplasm and plasma membrane, whereas during the delayed phase it was uniformly distributed within the cytoplasm and nucleus (Fig. 1C). The difference in distribution of activated p38 MAPK at distinct phases suggests the existence of different physiological end points. Furthermore, isoproterenol dose dependently increased phosphorylation of p38 MAPK during both phases of the response, with EC50 of 1.094 and 4.405 nm for early and delayed activation, respectively (supplemental data Fig. S1, A and B). Previous studies indicate that β2- but not β1-ARs are endogenously expressed in HEK293 cells. As expected, cells pretreated for 30 min with either propranolol, a non-selective β-AR antagonist, or ICI 118551, a selective β2-AR antagonist, reduced both early and delayed p38 MAPK activation (supplemental data Fig. S2). Therefore, the stimulation of endogenous β2-ARs is sufficient to evoke biphasic activation of p38 MAPK in a time- and dose-dependent fashion. It is noted that the similar pattern of p38 MAPK phosphorylation was also obtained in cells with transient overexpression of β2-ARs (data not shown). Compared with those mock-transfected cells, isoproterenol stimulation readily evoked the biphasic phosphorylation of p38 MAPK, as evidenced by the overall p38 MAPK phosphorylation that was increased by 2.5-fold at 10 min and 3.2-fold at 90 min (supplemental data Fig. S3, A and B). Both p38α and p38β Are Implicated in the Biphasic Activation of p38 MAPK by β2-ARs—The mammalian p38 MAPK subfamily includes four known isoforms: p38α, p38β, p38γ, and p38δ. p38α and p38β are ubiquitously expressed, whereas p38γ and p38δ appear to have more restricted tissue-specific expression (2Ashwell J.D. Nat. Rev. Immunol. 2006; 6: 532-540Crossref PubMed Scopus (308) Google Scholar, 3Wang Y. Circulation. 2007; 116: 1413-1423Crossref PubMed Scopus (243) Google Scholar). To elucidate which p38 isoform(s) is involved in the biphasic activation of p38 MAPK in response to isoproterenol, we focused on p38α and p38β MAPK. In cells transfected with a dominant negative mutant of p38α MAPK, both the early and delayed activation of p38 MAPK by isoproterenol was partially attenuated (supplemental data Fig. S4). In addition, in cells transfected with siRNA directed toward p38α, p38β MAPK, or scramble control there was selective knockdown of their endogenously expressed target protein compared with scramble control (Fig. 2A). Furthermore, silencing either p38α or p38β MAPK inhibited the early and delayed activation of MAPK (Fig. 2, B and C). Collectively, these results suggest that p38α and p38β MAPK are both implicated in the biphasic activation of p38 MAPK following stimulation with isoproterenol in HEK293 cells. Effects of the cAMP/PKA Signaling Pathway in the Biphasic Activation of p38 MAPK—β2-ARs classically couple to Gs proteins to initiate signaling via a cAMP/PKA pathway. To determine the involvement of the cAMP/PKA pathway in the biphasic activation of p38 MAPK by β2-ARs, cells were pretreated with a potent adenyl cyclase inhibitor. To our surprise, even though 100 μm SQ22536 inhibited the isoproterenol-induced increase in cAMP as determined by a competitive nonradioactive enzyme-linked assay (supplemental data Fig. S5), it did not affect the early activation of p38 MAPK. However, it did inhibit phosphorylation of p38 MAPK during the delayed phase (Fig. 3A). PKA is the primary downstream target for cAMP. As in previous studies (12Tan K.S. Nackley A.G. Satterfield K. Maixner W. Diatchenko L. Flood P.M. Cell Signal. 2007; 19: 251-260Crossref PubMed Scopus (167) Google Scholar, 24Shenoy S.K. Drake M.T. Nelson C.D. Houtz D.A. Xiao K. Madabushi S. Reiter E. Premont R.T. Lichtarge O. Lefkowitz R.J. J. Biol. Chem. 2006; 281: 1261-1273Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar), isoproterenol-induced PKA activation and ensuing ERK1/2 phosphorylation were inhibited by selective inhibitors, including (Rp)-cAMP (10 μm), H89 (10 μm), and KT5720 (0.3 μm) (supplemental data Fig. S6, A and B). Interestingly, in cells preincubated for 30 min with 10 μm (Rp)-cAMP, the early activation of p38 MAPK was unaffected. However, delayed activation of p38 MAPK was completely abolished (Fig. 3B). Similar results were also obtained from cells pretreated with H89 or KT5720 (Fig. 3, C and D). Together, these results indicate that the Gs/cAMP/PKA pathway is only responsible for the delayed activation of p38 MAPK by β2-ARs. cAMP/Epac Signaling Is Not Involved in Isoproterenol-induced p38 MAPK Activation—In addition to PKA, cAMP can also active the Epac/Rap1 pathway and has been implicated in β2-AR-induced activation of several effectors including protein kinase Cϵ in dorsal root ganglion neurons (25Hucho T.B. Dina O.A. Levine J.D. J. Neurosci. 2005; 25: 6119-6126Crossref PubMed Scopus (190) Google Scholar) and ERK1/2 in HEK293 cells (26Keiper M. Stope M.B. Szatkowski D. Bohm A. Tysack K. vom Dorp F. Saur O. Oude W.P. Evellin S. Jakobs K.H. Schmidt M. J. Biol. Chem. 2004; 279: 46497-46508Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Activation of Epac by specific cAMP analog 8-pCPT-2Me-cAMP (8-pCPT) can activate p38 MAPK in cultured cerebellar granule cells (27Ster J. De, Bock F. Guérineau N.C. Janossy A. Barrère-Lemaire S. Bos J.L. Bockaert J. Fagni L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2519-2524Crossref PubMed Scopus (94) Google Scholar). In this study, 8-pCPT, as a direct activator of Epac, and ISO activated Rap1 as determined using pulldown assays (Fig. 4A), 8-pCPT did not elicit p38 MAPK phosphorylation during the first 90 min of exposure (Fig. 4B). Therefore, these results suggest that β2-AR-mediated biphasic activation of p38 MAPK involves different signaling mechanisms. The classical cAMP/PKA pat" @default.
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• W2022260308 date "2008-10-01" @default.
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