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• W2016302829 abstract "In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-κB, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IκB kinase (IKK)-β and stimulated its abilities to phosphorylate IκB and to activate NF-κB. MKK6(Glu) induced NF-κB-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-α, which activates both NF-κB and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-α was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-α-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-β activated protein kinase (Tak1), which is known to activate p38 and NF-κB in other cell types. Thus, by stimulating both p38 and NF-κB, Tak1-activating cytokines, like TNF-α, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38. In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-κB, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IκB kinase (IKK)-β and stimulated its abilities to phosphorylate IκB and to activate NF-κB. MKK6(Glu) induced NF-κB-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-α, which activates both NF-κB and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-α was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-α-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-β activated protein kinase (Tak1), which is known to activate p38 and NF-κB in other cell types. Thus, by stimulating both p38 and NF-κB, Tak1-activating cytokines, like TNF-α, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38. tumor necrosis factor interleukin nuclear factor mitogen-activated protein kinase MAPK kinase MAPKK kinase extracellular signal-regulated kinase c-Jun N-terminal kinase transforming growth factor inhibitor of κB IκB kinase Dulbecco's modified Eagle's medium nucleotide(s) cytomegalovirus green fluorescent protein dithiothreitol enzyme-linked immunosorbent assay phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis Tris-buffered saline signal transducers and activators of transcription mitogen-activated protein kinase/extracellular signal-regulated kinase kinase terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling Cytokines derived from either infiltrating cells, such as macrophages, or sometimes from cells comprising the tissue itself, play important roles in the wound healing process (1Hawkins H.K. Entman M.L. Zhu J.Y. Youker K.A. Berens K. Dore M. Smith C.W. Am. J. Pathol. 1996; 148: 1957-1969PubMed Google Scholar). Since some injuries, such as myocardial infarction, are frequently life-threatening, a better understanding of the roles of cytokines in tissues such as the heart is critical. Following a myocardial infarction there is an accumulation of tumor necrosis factor (TNFα),1 interleukin (IL)-1β, and IL-6 at or near the affected region (2Guillen I. Blanes M. Gomez-Lechon M.J. Castell J.V. Am. J. Pathol. 1995; 269: R229-R235Google Scholar, 3Neumann F.J. Ott I. Gawaz M. Tichardt G. Holzapfel H. Jochum M. Schoemig A. Circulation. 1995; 92: 748-755Crossref PubMed Scopus (466) Google Scholar, 4Mann D.L. Cytokine Growth Factor Rev. 1996; 7: 341-354Crossref PubMed Scopus (90) Google Scholar, 5Meldrum D.R. Am. J. Physiol. 1998; 274: R577-R595Crossref PubMed Google Scholar). Previously, production of these cytokines in the heart was attributed to infiltrating macrophages or leukocytes and endothelial cells (e.g. see Ref. 6Kukielka G.L. Smith W. Manning A.M. Youker K.A. Michael L.H. Entman M.L. Circulation. 1995; 92: 1866-1875Crossref PubMed Scopus (279) Google Scholar). However, recent findings have demonstrated the expression of all three of these cytokines in cardiac myocytes following ischemic stress (7Kapadia S. Lee J.R. Torre-Amione G. Birdsall H.H. Ma T.S. Mann D.L. J. Clin. Invest. 1995; 96: 1042-1052Crossref PubMed Scopus (405) Google Scholar, 8Yamauchi-Takihara K. Ihara Y. Ogata A. Yoshizaki K. Azuma J. Kishimoto T. Circulation. 1995; 91: 1520-1524Crossref PubMed Scopus (269) Google Scholar, 9Gwechenberger M. Mendoza L.H. Youker K.A. Fangogiannis N.G. Smith W. Michael L.H. Entman M.L. Circulation. 1999; 99: 546-551Crossref PubMed Scopus (281) Google Scholar). Moreover, certain cytokines are thought to promote cardiac tissue recovery after a brief ischemic insult (4Mann D.L. Cytokine Growth Factor Rev. 1996; 7: 341-354Crossref PubMed Scopus (90) Google Scholar, 9Gwechenberger M. Mendoza L.H. Youker K.A. Fangogiannis N.G. Smith W. Michael L.H. Entman M.L. Circulation. 1999; 99: 546-551Crossref PubMed Scopus (281) Google Scholar, 10Hirota H. Yoshida K. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4862-4866Crossref PubMed Scopus (451) Google Scholar, 11Sheng Z. Knowlton K. Chen J. Hoshijima M. Brown J.H. Chien K.R. J. Biol. Chem. 1997; 272: 5783-5791Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Accordingly, there is renewed interest in studying the possible beneficial effects of cytokines in the heart as well as elucidating the signal transduction pathways and mechanisms responsible for their induction. The induction of most cytokine genes requires activation of the transcription factor, nuclear factor (NF)-κB (12Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2940) Google Scholar, 13Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2515) Google Scholar, 14Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2452) Google Scholar, 15Vanden Berghe W. Plaisance S. Boone E. De Bosscher K. Schmitz M.L. Fiers W. Haegeman G. J. Biol. Chem. 1998; 273: 3285-3290Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). NF-κB is activated and cytokine expression is increased in rat hearts submitted to ischemic stress; interestingly, these events appear to require the mitogen-activated protein kinase (MAPK), p38 (16Maulik N. Sato M. Price B.D. Das D.K. FEBS Lett. 1998; 429: 365-369Crossref PubMed Scopus (222) Google Scholar). Additionally, in cultured cardiac myocytes, NF-κB-dependent reporter gene expression is activated following the selective stimulation of p38 by its upstream activator, MKK6 (17Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1998; 273: 8232-8239Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Moreover, this p38-dependent NF-κB activation contributes to protecting cultured myocardial cells from undergoing apoptosis (17Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1998; 273: 8232-8239Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). However, while p38 and NF-κB are both important for the maintenance of heart function following stress, neither the mechanism by which p38 activates NF-κB nor the role of p38-activated NF-κB in myocardial cell survival are well understood. Accordingly, the present study was undertaken to begin addressing these questions. Many signaling pathways can interact with each other through biochemical cross-talk; however, such interactions between p38 and NF-κB are not well understood. Like the other MAPK family members, p38 is part of a cascade of kinases. One of the best studied activators of p38 is the MAPKK, MKK6, which lies directly upstream of p38; among ERK, JNK, and p38, MKK6 activates only p38 (18Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 20Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Ioshi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). Although MKK6 itself can be activated by several upstream kinases, a particularly interesting finding is that in some cells, MKK6 can be activated by the MAPKKK, TGF-β-activated protein kinase (Tak1) (18Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 20Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Ioshi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). Tak1 was originally named for its ability to be activated by TGF-β. However, Tak1 can also be activated by cytokines, such as interleukin-1 or TNF-α (20Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Ioshi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar, 21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar, 22Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) (see Fig. 7 for reference). In comparison with the p38 pathway, the NF-κB pathway is activated through a series of events that are also mediated by a cascade of kinases, many of which are believed to be unique to that pathway. NF-κB, which is comprised of two subunits (p65 and p50), is retained in the cytoplasm by virtue of its interaction with the inhibitor of κB (IκB). IκB is phosphorylated in response to NF-κB-activating signals; this phosphorylation leads to the ubiquitination and subsequent degradation of IκB. This then allows NF-κB to translocate to the nucleus, where it binds to critical elements in cytokine genes and increases their transcription (23Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar, 24DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). The kinases responsible for the phosphorylation of IκB belong to the IκB kinase family, or the IKKs (25Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 26Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 27Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Wu Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1855) Google Scholar, 28Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 29Karin M. J. Biol. Chem. 1998; 274: 27339-27342Abstract Full Text Full Text PDF Scopus (621) Google Scholar). NF-κB-inducing kinase phosphorylates and activates IKK (26Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 30Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1166) Google Scholar, 31Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9806Crossref PubMed Scopus (506) Google Scholar). Although it preferentially activates IKK, NF-κB-inducing kinase, which bears strong sequence homology to, and is now considered a member of, the MAPKKK family, can also activate the JNK MAPK pathway (32Su Y.C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (219) Google Scholar). Interestingly, in addition to being activated by NF-κB-inducing kinase, IKK can also be activated by MEKK1 (27Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Wu Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1855) Google Scholar), a MAPKKK that is also known to activate JNK in some cell types (33Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and by Tak1 (refer to Fig. 5). Thus, at least at the MAPKKK level, there exists the potential for cross-talk between the NF-κB and MAPK pathways. In the present study, we examined the nature of the cross-talk between the NF-κB and p38 pathways. Because of the clear importance of these pathways in promoting the recovery, or maintenance of, heart tissue function following stress or injury, we used cardiac myocytes as the model system. To this end, we explored whether NF-κB and p38 collaboratively activate the IL-6 gene in cardiac myocytes and, further, whether IL-6 itself has roles that are consistent with functional recovery of the tissue following stress. We have found the following: 1) agonists, such as TNF-α, can activate IL-6 transcription in cardiac myocytes in a manner that requires both NF-κB and p38; 2) the coordinate activation of the NF-κB and p38 pathways by TNF-α takes place largely through the MAPKKK, Tak1; 3) the p38 pathway can influence NF-κB activation, at least partly, through the physical association of MKK6 and IKKβ; and 4) IL-6 can protect cardiac myocytes from undergoing apoptosis induced by sphingosine, a signaling lipid known to increase in the heart following ischemic stress. Primary ventricular myocytes were prepared from 1–4 day old Harlan Sprague-Dawley rats as described (34Sprenkle A.B. Murray S.F. Glembotski C.C. Circ. Res. 1995; 77: 1060-1069Crossref PubMed Scopus (98) Google Scholar, 35Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar). Briefly, hearts were dissected in DMEM/air; the apical two-thirds of the ventricles were dissected away from the atria. After mincing and washing the ventricles twice with air-compatible DMEM, cells were isolated by multiple rounds of 10-min-long tissue dissociation with 0.001% trypsin. After each incubation with trypsin, the supernatant was added to an equal volume of DMEM/F-12 (1:1) containing 20% fetal bovine serum, and all of the supernatants were combined. Plastic wells were treated for at least 1 h with 5 ng of fibronectin/ml of DMEM/F-12 (1:1). Myocytes were plated in DMEM/F-12 (1:1) containing 10% fetal bovine serum for approximately 16 h. After washing with DMEM/F-12 (1:1), the cultures were incubated in DMEM/F-12 (1:1) with or without any test agents for the indicated times. Immediately following the final dissociation step, myocardial cells were resuspended in serum-free DMEM/F-12 (1:1). Between 5 and 12 × 106 cells were combined with the combinations of plasmids indicated in the figure legends in a total volume of 300 μl of DMEM/F-12 (1:1). The total amount of plasmids used for each electroporation was equalized using pCMV6. Dose-response experiments were carried out to determine optimal quantities of plasmids for transfection and to verify that the results obtained were consistent over a range of plasmid levels. Generally, the dose-response experiments led to using the following quantities of each plasmid type in each electroporation: 1 μg each of pCMV6 (control) or p38β2; 15 μg of MKK6(Glu), Tak1, or Tab1; 20 μg each of luciferase and β-galactosidase reporters; 45 μg of IKKβ-M, MKK6-M1, MKK6-M2, and Tak1-M. Cells were electroporated at 500 V, 25 microfarads, and 100 ohms in a 0.2-cm gap electroporation cuvette (Bio-Rad) using a Gene Pulser II (Bio-Rad). Under these conditions, only cardiac myocytes are transfected (34Sprenkle A.B. Murray S.F. Glembotski C.C. Circ. Res. 1995; 77: 1060-1069Crossref PubMed Scopus (98) Google Scholar, 36LaPointe M.C. Wu G. Garami M. Yang X.P. Gardner D.G. Hypertension. 1996; 27: 715-722Crossref PubMed Google Scholar). For reporter assays, 1.5 × 106 cells were plated per 24-mm well, whereas 4.5 × 106 myocytes per 35-mm well were plated to perform kinase assays. pcDNA3 FLAG-MKK6 (Glu) and pcDNA3 FLAG-MKK6(K82A), the latter of which we call MKK6-M1, code for activated and kinase-inactive human MKK6, respectively (19Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar) and were obtained from R. J. Davis (University of Massachusetts, Worcester, MA). In some cases, instead of MKK6-M1 we used MKK6-M2 (FLAG-MKK6(K82A; S207A; T211A)), which is a kinase-inactive form of MKK6 that also has the sites normally phosphorylated by an upstream MAPKKK, Ser207 and Thr211, mutated to Ala. MKK6-M2 produced similar inhibitory effects on the p38 pathway as MKK6-M1. Srα3 HA-p38–2, which codes for wild type human p38–2, was obtained from B. Stein (37Stein B. Yang M.X. Young D.B. Janknecht R. Hunter T. Murray B.W. Barbosa M.S. J. Biol. Chem. 1997; 272: 19509-19517Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) (Signal Pharmaceuticals, Inc., San Diego, CA). p38-2 is distinct from p38α, -γ, and -δ; however, it is identical to human p38β2 (38Enslen H. Raingeaud J. Davis R.J. J. Biol. Chem. 1998; 273: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). In the present work, we have adopted the p38β2 nomenclature. pRK5 C-FLAG-IKKβ and pRK5 C-FLAG-IKKβ (K44A), which code for wild type human IKKβ and kinase-dead human IKKβ, were obtained from M. Rothe (26Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar) (Tularik, San Francisco, CA). p2X NF-κB, which codes for a luciferase reporter driven by a minimal prolactin promoter with two nearby, upstream NF-κB consensus sites, was obtained from M. Karin (39DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar) (University of California at San Diego, La Jolla, CA). p1168hu.IL6-luc and pmut1168hu.IL6-luc, which code for 1168 nt of the wild type or NF-κB-mutated human IL-6 promoter driving luciferase in pGL3, respectively (15Vanden Berghe W. Plaisance S. Boone E. De Bosscher K. Schmitz M.L. Fiers W. Haegeman G. J. Biol. Chem. 1998; 273: 3285-3290Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar), were obtained from G. Haegeman (University of Gent, Belgium). NF-κB/IL-6-luc was prepared by ligating three IL-6 NF-κB elements upstream of the noninducible IL-6 minimal promoter, which is composed of 50 nt of IL-6 5′-flanking sequence. This construct was also obtained from G. Haegeman. pFLAG-Tak1 and pFLAG-Tak1-M encode human, full-length wild type Tak1 and human full-length Tak1 K63W, respectively. pHA-Tab1 encodes human, full-length Tab1 (22Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). All Tak1 and Tab1 constructs used in this study were obtained from T. Sugita (Tanabe Seiyaku Co., Ltd., Osaka, Japan). The AdEasy system was used for preparing recombinant adenoviral strains using previously described methods (40He T.C. Zhou S. da Costa L.T., Yu, J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). Briefly, human MKK6(Glu) and human p38β2were polymerase chain reaction-amplified from the parent templates (see above) such as to create restriction sites that would facilitate cloning into pAdTrack-CMV, an adenoviral shuttle vector that harbors CMV-driven green fluorescent protein (GFP), and a CMV-flanked multiple cloning site for the insertion of the gene of interest. Polymerase chain reaction-amplified p38β2 and MKK6(Glu) were cloned into the EcoRI and NotI sites of pGEX-6P-1 (Amersham Pharmacia Biotech), which served as a shuttle cloning vector. pGEX-6P-1/p38β2 and pGEX-6P-1/MKK6(Glu) were then digested with BamHI and NotI, and the resulting products of interest were cloned into the BglII and NotI sites of pAdTrack-CMV to create pAdTrack-CMV-p38β2 and pAdTrack-CMV-MKK6(Glu). pAdTrack-CMV-p38β2 or pAdTrack-CMV-MKK6(Glu) was linearized and then co-transformed with the adenoviral vector, pAdEasy-1, into Escherichia coli strain BM5183. This strain of E. coli allows for homologous recombination of pAdEasy-1 and the pAdTrack-CMV shuttle vector containing the gene of interest. Recombinants were selected on kanamycin and screened by restriction digestion with PacI. Recombinant plasmids were then retransformed into E. coli DH5α for propagation purposes. Recombinant adenoviral plasmids were linearized with PacI and then transfected into 293 human embryonic kidney cells, using LipofectAMINE® (Life Technologies, Inc.). Transfection efficiency was determined by observing GFP fluorescence, as described previously (40He T.C. Zhou S. da Costa L.T., Yu, J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). The recombinant viruses were then harvested 7–10 days postinfection. Viral titers were determined by observing GFP fluorescence of primary neonatal cardiac myocytes; the minimum quantity of viral stock that afforded 100% transfection efficiency was selected for the experiments in this study. Following the appropriate time in culture, each well of myocytes was washed twice with phosphate-buffered saline and then lysed in 500 μl of ice-cold lysis buffer (25 mm Gly-Gly, pH 7.8, 15 mm MgSO4, 4 mm EDTA, 0.25% Triton X-100) containing 1 mmdithiothreitol. The cell debris was removed by centrifugation. To measure β-galactosidase activity, 200 μl of the cell extract was added to 400 μl of β-galactosidase buffer (60 mmNa2HPO4, 40 mmNaH2PO4, 10 mm KCl, 1 mm MgSO4) containing 1 mg/ml chlorophenol red-β-d-galactopyranoside and 50 mmβ-mercaptoethanol. The reaction was incubated for 2 h at 37 °C. After stopping each reaction by adding 100 μl of 1m Na2CO3, the absorbance was measured at 570 nm. To measure luciferase activity, 100 μl of buffer (25 mm Gly-Gly, pH 7.8, 15 mmMgSO4, 4 mm EGTA, 45 mmKPO4, pH 7.8, 1 mm DTT, 0.3 mmd-luciferin, 3 mm ATP) was added to 100 μl of cell lysate. Light emission of each sample was measured by a BioOrbit 1251 luminometer for 30 s. The relative luciferase activities were determined by dividing the relative luciferase activity by the relative β-galactosidase activity. To measure IL-6 secretion, IL-6 ELISAs were carried out using a kit according to the manufacturer's protocol (BIOSOURCE International). After the myocardial cells were preplated as described (17Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1998; 273: 8232-8239Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) to eliminate noncardiac myocytes, the myocytes were plated at a density of 1300 cells/mm2 in 10% fetal calf serum for 24 h. Cells were washed and cultured in media with or without recombinant TNF-α (Genzyme), with or without 5 μm SB203580, or the cells were infected with AdV-MKK6(Glu), AdV-p38β2, or AdV-Control. Following 48 h of incubation, media were collected and assayed using the IL-6 ELISA kit. Myocardial cells were cultured with or without recombinant IL-6 (1 ng/ml) (BIOSOURCEInternational) supplemented DMEM/F-12 with or without 5 μm SB203580 for 48 h prior to the addition of 10 μm sphingosine (Calbiochem) for 6 h. TUNEL analyses of fragmented DNA were performed on cultured cardiac myocytes as described previously (17Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1998; 273: 8232-8239Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) and according to the manufacturer's protocol (Roche Molecular Biochemicals). Cells were scored for TUNEL-positive nuclei in a researcher-blinded manner. Assessment of apoptosis was performed by DNA laddering essentially as described previously (17Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1998; 273: 8232-8239Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Approximately 106 cardiac myocytes were lysed in a digestion buffer containing Proteinase K, scraped, pipetted several times, and incubated at 55 °C for 5–14 h. Samples were then extracted with phenol/chloroform/isoamyl alcohol, and then DNA was isopropyl alcohol-precipitated and washed several times with ethanol and allowed to air-dry. After dissolving the DNA and incubating with RNase A, DNA fragments were fractionated on an agarose gel. To study the cellular localization of the p65 subunit of NF-κB, myocardial cells were co-transfected with test expression constructs (1 μg each of pCMV6, p38β2 or 15 μg of MKK6(Glu)) and 5 μg of a plasmid encoding GFP. Following 48 h of culture in minimal media, cells were fixed as described (35Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar), and immunocytofluorescence was carried out using a polyclonal antibody raised against p65/NF-κB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by Texas Red-conjugated anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR). Transfected cardiac myocytes were identified as GFP-positive cells, and p65 localization was visualized in only the transfected cells. Myocardial cells were treated with or without TNF-α (1 ng/ml) for 10 min and then extracted in 400 μl of buffer A (50 mm Tris (pH 7.5), 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 5 mm sodium pyrophosphate, 10 mm sodium glycerophosphate, 50 mm NaF, 0.5 mm sodium orthovanadate, 0.1% 2-mercaptoethanol, 0.1 mm PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin). Following removal of debris by centrifugation, MAPKAP-K2 was immunoprecipitated using 1.5 μg of anti-MAPKAP-K2 (Upstate Biotechnology, Inc., Lake Placid, NY; catalog no. 06-534) and submitted to a kinase assay using [γ-32P]ATP and hsp27 as the substrates, as described by the manufacturer's protocol. Labeled hsp27 was then resolved by 12% SDS-PAGE, and the gel was submitted to PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis. IKKβ kinase activity was assessed in myocardial cells by co-transfecting C-FLAG-IKKβ with or without test expression constructs with or without SB203580 (5 μm). After the appropriate times, cultures were extracted in a buffer containing 20 mm Tris, pH 7.6, 20 mmβ-glycerophosphate, 250 mm NaCl, 3 mm EGTA, 3 mm EDTA, 0.5% Nonidet P-40, 0.1 mm sodium orthovanadate, 10 μg/ml aprotinin, 2 mm DTT, 1 mm PMSF, 1 mm p-nitrophenyl phosphate, and 10 μg/ml of leupeptin. After brief centrifugation, extracts were incubated for 2 h at 4 °C with anti-IKKβ antibody (Santa Cruz Biotechnology), followed by protein G-Sepharose (Amersham Pharmacia Biotech) precipitation. Immunocomplex kinase assays were carried out using 1 μg of recombinant IκB-α-(1–317) (Santa Cruz Biotechnology) per sample and 10 μm[γ-32P]ATP (5000 Ci/mmol) in a final volume of 30 μl of kinase buffer (30 mm HEPES, pH 7.4, 10 mmMgCl2, 1 mm DTT) at 25 °C for 15 min. The reactions were terminated by the addition of Laemmli sample buffer, and the phosphorylation level of IκB-α was evaluated by SDS-PAGE followed by autoradiography and PhosphorImager analyses. The quantity of IKKβ in each sample was determined by Western analysis; the observed levels were used to normalize relative IκB-α phosphorylation levels found in PhosphorImager analyses. Cultures (approximately 2 × 106 myocytes) were lysed in 100 μl of supplemented Laemmli sample buffer supplemented with 0.1 mm sodium orthovanadate, 10 μg/ml aprotinin, 2 mm DTT, 1 mm PMSF, 1 mm p-nitrophenyl phosphate, and 10 μg/ml leupeptin; boiled for 5 min; and then submitted to 10% SDS-PAGE and transferred to a nitrocellulose membrane in methanol transfer buffer at 60 V for 5 h or 30 V overnight. Membranes were blocked for 30 min in 5% nonfat milk dissolved in TBS-Tween (0.01%) at room temperature. Western analyses were then performed using 1:1000 dilutions of antisera specific for phospho-p38 (New England Biolabs, Beverly, MA; catalog no. 9211S), phospho-JNK (Santa Cruz Biotechnology; catalog no. SC6254), or phospho-ERK (New England Biolabs; catalog no. 9101S). Blots were subsequently stripped with 6.25 mm Tris, 2% SDS, and 100 mm2-mercaptoethanol for 30 min at 50 °C, washed for 1 h in TBS-Tween, and reprobed with 1:1000 dilutions of antisera specific for either p38 (Stressgen Biotechnologies Corp., Victoria, Canada; catalog no. KAP-MAOO9E), JNK (Santa Cruz Biotechnology; catalog no. SC-474), or ERK (Santa Cruz Biotechnology; catalog no. SC-093) for normalization purposes. Myocytes were cultured as described above and then treated with or without various test agents for 15 min. The cells from 3–35-mm culture wells (4.5 × 106myocytes) were collected by scraping into 450 μl of lysis buffer, which consisted of 20 mm Tris-HCl (pH 7.6), 20 μm sodium glycerophosphate, 250 mm NaCl, 3 mm EGTA, 3 mm EDTA, 0.5% No" @default.
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• W2016302829 title "p38 MAPK and NF-κB Collaborate to Induce Interleukin-6 Gene Expression and Release" @default.
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• W2016302829 doi "https://doi.org/10.1074/jbc.m909695199" @default.
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