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• W2017609900 abstract "The interleukin-6 (IL-6) family of cytokines is a family of structurally and functionally related proteins, including IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These proteins are also known as gp130 cytokines because they all share gp130 as a common transducer protein within their functional receptor complexes. Several of these cytokines (LIF, OSM, CNTF, and CT-1) also utilize the LIF receptor (LIFR) as a component of their receptor complex. We have shown that all of these cytokines are capable of activating both the JAK/STAT and p42/44 mitogen-activated protein kinase signaling pathways in 3T3-L1 adipocytes. By performing a variety of preincubation studies and examining the ability of these cytokines to activate STATs, ERKs, and induce transcription of SOCS-3 mRNA, we have also examined the ability of gp130 cytokines to modulate the action of their family members. Our results indicate that a subset of gp130 cytokines, in particular CT-1, LIF, and OSM, has the ability to impair subsequent signaling activity initiated by gp130 cytokines. However, IL-6 and CNTF do not exhibit this cross-talk ability. Moreover, our results indicate that the cross-talk among gp130 cytokines is mediated by the ability of these cytokines to induce ligand-dependent degradation of the LIFR, in a proteasome-independent manner, which coincides with decreased levels of LIFR at the plasma membrane. In summary, our results demonstrate that an inhibitory cross-talk among specific gp130 cytokines in 3T3-L1 adipocytes occurs as a result of specific degradation of LIFR via a lysosome-mediated pathway. The interleukin-6 (IL-6) family of cytokines is a family of structurally and functionally related proteins, including IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These proteins are also known as gp130 cytokines because they all share gp130 as a common transducer protein within their functional receptor complexes. Several of these cytokines (LIF, OSM, CNTF, and CT-1) also utilize the LIF receptor (LIFR) as a component of their receptor complex. We have shown that all of these cytokines are capable of activating both the JAK/STAT and p42/44 mitogen-activated protein kinase signaling pathways in 3T3-L1 adipocytes. By performing a variety of preincubation studies and examining the ability of these cytokines to activate STATs, ERKs, and induce transcription of SOCS-3 mRNA, we have also examined the ability of gp130 cytokines to modulate the action of their family members. Our results indicate that a subset of gp130 cytokines, in particular CT-1, LIF, and OSM, has the ability to impair subsequent signaling activity initiated by gp130 cytokines. However, IL-6 and CNTF do not exhibit this cross-talk ability. Moreover, our results indicate that the cross-talk among gp130 cytokines is mediated by the ability of these cytokines to induce ligand-dependent degradation of the LIFR, in a proteasome-independent manner, which coincides with decreased levels of LIFR at the plasma membrane. In summary, our results demonstrate that an inhibitory cross-talk among specific gp130 cytokines in 3T3-L1 adipocytes occurs as a result of specific degradation of LIFR via a lysosome-mediated pathway. The interleukin 6 (IL-6) 2The abbreviations used are: IL-6, interleukin-6; CNTF, ciliary neurotrophic factor; CNTFRα, α-receptor for CNTF; CT-1, cardiotrophin-1; ERK, extracellular signal-regulated kinase; GH, growth hormone; LDM, low density microsomes; LIF, leukemia inhibitory factor; LIFR, LIF receptor; MAPK, mitogen-activated protein kinase; OSM, oncostatin M; SOCS-3, suppressor of cytokine signaling-3; STAT, signal transducers and activators of transcription. 2The abbreviations used are: IL-6, interleukin-6; CNTF, ciliary neurotrophic factor; CNTFRα, α-receptor for CNTF; CT-1, cardiotrophin-1; ERK, extracellular signal-regulated kinase; GH, growth hormone; LDM, low density microsomes; LIF, leukemia inhibitory factor; LIFR, LIF receptor; MAPK, mitogen-activated protein kinase; OSM, oncostatin M; SOCS-3, suppressor of cytokine signaling-3; STAT, signal transducers and activators of transcription. cytokine family is a group of functionally and structurally related proteins including IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1) (1Hirano T. 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In fact, members of the IL-6 cytokine family play pivotal roles in the immune, hematopoietic, nervous, cardiovascular, and endocrine systems, as well as in bone metabolism, inflammation, and acute phase responses (11Sehgal P.B. Helfgott D.C. Santhanam U. Tatter S.B. Clarick R.H. Ghrayeb J. May L.T. J. Exp. Med. 1988; 167: 1951-1956Crossref PubMed Scopus (93) Google Scholar, 12Hirano T. Int. Rev. Immunol. 1998; 16: 249-284Crossref PubMed Scopus (655) Google Scholar, 13Metcalf D. Int. J. Cell Cloning. 1991; 9: 95-108Crossref PubMed Scopus (125) Google Scholar, 14Patterson P.H. Nawa H. Cell. 1993; 72: 123-137Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 15Pennica D. Wood W.I. Chien K.R. Cytokine Growth Factor Rev. 1996; 7: 81-91Crossref PubMed Scopus (91) Google Scholar, 16Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar, 17Hirano T. Chem. Immunol. 1992; 51: 153-180Crossref PubMed Google Scholar, 18Hilton D.J. Trends Biochem. 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Saito T. Miki D. Hamaguchi M. Davis S. Shoyab M. Yancopoulos G.D. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10998-11001Crossref PubMed Scopus (191) Google Scholar). Hence, IL-6 family cytokines are often referred to as gp130 cytokines. Several gp130 cytokines (LIF, OSM, CNTF, and CT-1) also require the gp130-related protein LIFR (LIF receptor) as a part of their functional receptor complexes (25Gearing D.P. Thut C.J. VandeBos T. Gimpel S.D. Delaney P.B. King J. Price V. Cosman D. Beckmann M.P. EMBO J. 1991; 10: 2839-2848Crossref PubMed Scopus (513) Google Scholar, 26Gearing D.P. Comeau M.R. Friend D.J. Gimpel S.D. Thut C.J. McGourty J. Brasher K.K. King J.A. Gillis S. Mosley B. Science. 1992; 255: 1434-1437Crossref PubMed Scopus (790) Google Scholar, 27Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Li Y. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Cell. 1992; 69: 1121-1132Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 28Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Crossref PubMed Scopus (587) Google Scholar, 29Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). In fact, these cytokines have very low affinity to gp130 until it becomes a part of the LIFR/gp130 heterodimer. Apart from the shared functional receptor components, IL-6, IL-11, OSM, and CNTF also have cytokine-specific receptor components, often referred to as the α-receptors (30Murakami M. Hibi M. Nakagawa N. Nakagawa T. Yasukawa K. Yamanishi K. Taga T. Kishimoto T. Science. 1993; 260: 1808-1810Crossref PubMed Scopus (638) Google Scholar, 31Yin T. Taga T. Tsang M.L. Yasukawa K. Kishimoto T. Yang Y.C. J. Immunol. 1993; 151: 2555-2561PubMed Google Scholar, 32Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Harrison-Smith M. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO J. 1994; 13: 4765-4775Crossref PubMed Scopus (253) Google Scholar).Although LIF and CT-1 signal through a LIFR/gp130 heterodimer, other gp130 cytokines have more distinct receptor complex structures. IL-6 first binds to its α-receptor, IL-6R, and then two of these IL-6·IL-6R complexes associate with gp130 each, allowing the formation of a gp130 homodimer (33Ward L.D. Howlett G.J. Discolo G. Yasukawa K. Hammacher A. Moritz R.L. Simpson R.J. J. Biol. Chem. 1994; 269: 23286-23289Abstract Full Text PDF PubMed Google Scholar, 34Paonessa G. Graziani R. De Serio A. Savino R. Ciapponi L. Lahm A. Salvati A.L. Toniatti C. Ciliberto G. EMBO J. 1995; 14: 1942-1951Crossref PubMed Scopus (206) Google Scholar). A similar pattern has also been proposed for the formation of IL-11 functional receptor complexes (31Yin T. Taga T. Tsang M.L. Yasukawa K. Kishimoto T. Yang Y.C. J. Immunol. 1993; 151: 2555-2561PubMed Google Scholar). OSM can either signal through the LIFR/gp130 heterodimer (26Gearing D.P. Comeau M.R. Friend D.J. Gimpel S.D. Thut C.J. McGourty J. Brasher K.K. King J.A. Gillis S. Mosley B. Science. 1992; 255: 1434-1437Crossref PubMed Scopus (790) Google Scholar, 35Liu J. Modrell B. Aruffo A. Marken J.S. Taga T. Yasukawa K. Murakami M. Kishimoto T. Shoyab M. J. Biol. Chem. 1992; 267: 16763-16766Abstract Full Text PDF PubMed Google Scholar) or through its α-receptor OSMR associated with a gp130 subunit (36Thoma B. Bird T.A. Friend D.J. Gearing D.P. Dower S.K. J. Biol. Chem. 1994; 269: 6215-6222Abstract Full Text PDF PubMed Google Scholar, 37Murakami-Mori K. Taga T. Kishimoto T. Nakamura S. J. Clin. Invest. 1995; 96: 1319-1327Crossref PubMed Scopus (49) Google Scholar). Structurally, OSMR is closely related to both LIFR and gp130. However, neither OSMR nor LIFR can bind OSM in the absence of gp130 subunit. The α-receptor for CNTF, CNTFRα, is structurally closely related to extracellular region of IL-6R but is anchored to the cell membrane via a glycosylphosphatidylinositol linkage. CNTF binds to CNTFRα, followed by the recruitment of gp130 and LIFR by two of these dimers, resulting in a hexameric receptor complex (38De Serio A. Graziani R. Laufer R. Ciliberto G. Paonessa G. J. Mol. Biol. 1995; 254: 795-800Crossref PubMed Scopus (49) Google Scholar). The α-receptors for IL-6, IL-11, and CNTF can also be found in a soluble form, and these soluble receptors can bind their ligands in a manner identical to their membrane-associated forms (39Baumann H. Wang Y. Morella K.K. Lai C.F. Dams H. Hilton D.J. Hawley R.G. Mackiewicz A. J. Immunol. 1996; 157: 284-290PubMed Google Scholar, 40Taga T. Hibi M. Hirata Y. Yamasaki K. Yasukawa K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1186) Google Scholar). These soluble receptors have the potential to confer cytokine responsiveness to cells expressing gp130 and LIFR that do not express membrane-associated forms of the α-receptors (41Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1090) Google Scholar, 42Davis S. Aldrich T.H. Ip N.Y. Stahl N. Scherer S. Farruggella T. DiStefano P.S. Curtis R. Panayotatos N. Gascan H. Science. 1993; 259: 1736-1739Crossref PubMed Scopus (327) Google Scholar).The ubiquitous expression of gp130 in every cell type examined explains the pleiotropic nature of gp130 cytokine action, whereas shared usage of gp130 by all these cytokines in part explains the redundancy of their actions. Unlike gp130, LIFR and the specific α-receptors exhibit a more tissue-specific expression (43Saito M. Yoshida K. Hibi M. Taga T. Kishimoto T. J. Immunol. 1992; 148: 4066-4071PubMed Google Scholar) and therefore contribute to the specificity of gp130 cytokine actions. Spatial and temporal expression of the individual cytokines is another factor that contributes to the specificity of their actions (44Hirota H. Kiyama H. Kishimoto T. Taga T. J. Exp. Med. 1996; 183: 2627-2634Crossref PubMed Scopus (280) Google Scholar).Functionally, it is the formation of the gp130/gp130 homodimer or the LIFR/gp130 heterodimer that is essential for the downstream signal transduction of gp130 cytokines. The function of the α-receptor is to recruit the ligand and aid in the formation of signal-transducing dimers in response to the ligands that are themselves incapable of doing so. After stimulation by gp130 cytokines, target cells undergo a wide variety of fates: growth promotion, growth arrest, differentiation, or the expression of specific genes. The specificity of cytokine actions in a given cell type arises not only from the differences in the receptor and cytokine expression, but also from the differences in downstream signaling pathways activated by the cytokine.Upon stimulation by gp130 cytokines, the gp130 receptor itself undergoes homo- or heterodimerization governed by the phosphorylated tyrosine residues on the cytoplasmic region of the protein. This dimerization triggers the activation of cytoplasmic protein kinases associated with gp130 receptor. In the case of gp130, those kinases are JAK1, JAK2, and TYK2 (45Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar, 46Darnell J.E.J. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4938) Google Scholar, 47Lutticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Science. 1994; 263: 89-92Crossref PubMed Scopus (704) Google Scholar, 48Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Science. 1994; 263: 92-95Crossref PubMed Scopus (841) Google Scholar, 49Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell J.E.J. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (864) Google Scholar, 50Narazaki M. Witthuhn B.A. Yoshida K. Silvennoinen O. Yasukawa K. Ihle J.N. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2285-2289Crossref PubMed Scopus (250) Google Scholar). Activated JAKs phosphorylate tyrosine residues in the distal part of gp130 and LIFR and provide docking sites for Src homology 2 domain-containing proteins such as STATs. Even though the STAT family consists of 7 members, it is generally recognized that gp130 cytokines activate STAT 3 and, to a lesser extent, STAT 1 (51Akira S. Nishio Y. Inoue M. Wang X.J. Wei S. Matsusaka T. Yoshida K. Sudo T. Naruto M. Kishimoto T. Cell. 1994; 77: 63-71Abstract Full Text PDF PubMed Scopus (863) Google Scholar, 52Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1688) Google Scholar). Once recruited to the receptor, STATs are phosphorylated and form dimers that translocate to the nucleus, bind DNA, and modulate transcription. Stimulation by gp130 cytokines can also lead to the activation of the p42/44 MAPK pathway (53Satoh T. Nakafuku M. Kaziro Y. J. Biol. Chem. 1992; 267: 24149-24152Abstract Full Text PDF PubMed Google Scholar).Recent studies have shown that three different gp130 cytokines activate both the JAK/STAT and p42/44 MAPK signaling pathways in fat cells (54Zvonic S. Cornelius P. Stewart W.C. Mynatt R.L. Stephens J.M. J. Biol. Chem. 2003; 278: 2228-2235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 55Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5104) Google Scholar, 56Balhoff J.P. Stephens J.M. Biochem. Biophys. Res. Commun. 1998; 247: 894-900Crossref PubMed Scopus (47) Google Scholar, 57Stephens J.M. Lumpkin S.J. Fishman J.B. J. Biol. Chem. 1998; 273: 31408-31416Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Because these cytokines utilize many of the same signaling proteins, we examined the cross-talk among LIF, CT-1, OSM, CNTF, and IL-6 in adipocytes. We also examined the ability of growth hormone (GH), a potent STAT activator, to modulate gp130 cytokine signaling. Our results demonstrate that a 2-h pretreatment with the gp130 cytokines examined leads to the inhibition of their own subsequent signaling. Interestingly, LIF could also inhibit the signaling of CT-1, CNTF, and OSM, but not IL-6. Similarly, CT-1 could inhibit its own signaling or a cellular response to LIF and OSM. OSM also exhibited similar properties. Inhibition of signaling was measured by three parameters: activation of STATs, activation of ERKs, and the induction of SOCS-3 mRNA. Moreover, we observed that the cross-talk that resulted in an inhibition of signaling correlated with the degradation of the LIFR in a proteasome-independent manner and a loss of the LIFR at the plasma membrane. In summary, our findings strongly suggest that inhibitory cross-talk among specific gp130 cytokines in fat cells is mediated by the ligand-dependent lysosome-mediated degradation of the LIFR.EXPERIMENTAL PROCEDURESMaterials—Dulbecco's modified Eagle's medium was purchased from Invitrogen. Bovine and fetal bovine sera were purchased from Sigma and Invitrogen, respectively. Rat recombinant CNTF and human recombinant CT-1 were purchased from Calbiochem. Recombinant mouse IL-6 and recombinant human OSM were purchased from Bio Source International. Mouse recombinant LIF was purchased from Chemicon International. Insulin, human recombinant GH, leupeptin, chloroquine, and cycloheximide were all purchased from Sigma. Epoxomicin and MG132 were purchased from Boston Biochem. TRIzol was purchased from Invitrogen. All STAT antibodies were monoclonal IgGs purchased from Transduction Laboratories or polyclonal IgGs purchased from Santa Cruz. The highly phospho-specific polyclonal antibodies for STAT 1 (Tyr701), STAT 3 (Tyr705), and STAT 5 (Tyr694) were IgGs purchased from BD Transduction Laboratories and Upstate Biotechnology, Inc. LIFR and ERK1/ERK2 antibodies were rabbit polyclonal IgGs purchased from Santa Cruz. Active ERK antibody was a rabbit polyclonal IgG purchased from Cell Signaling Technology. Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immunoresearch. An Enhanced Chemiluminescence (ECL) kit was purchased from Pierce. Nitrocellulose and Zeta Probe-GT membranes were purchased from Bio-Rad.Preparation of Whole Cell Extracts—Monolayers of 3T3-L1 preadipocytes or adipocytes were rinsed with phosphate-buffered saline and then harvested in a nondenaturing buffer containing 150 mm NaCl, 10 mm Tris, pH 7.4, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 μm phenylmethylsulfonyl fluoride, 1 μm pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 μm leupeptin, and 2 mm sodium vanadate. Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4 °C for 15 min. Supernatants containing whole cell extracts were analyzed for protein content using a BCA kit (Pierce) according to the manufacturer's instructions.Gel Electrophoresis and Western Blot Analysis—Proteins were separated in 5, 7.5, 10, or 12% polyacrylamide (acrylamide from National Diagnostics) gels containing SDS according to Laemmli (58Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205903) Google Scholar) and transferred to nitrocellulose membrane in 25 mm Tris, 192 mm glycine, and 20% methanol. After transfer, the membrane was blocked in 4% fat-free milk for 1 h at room temperature. Results were visualized with horse-radish peroxidase-conjugated secondary antibodies and ECL.RNA Analysis—Total RNA was isolated from cell monolayers with TRIzol according to the manufacturer's instructions with minor modifications. For Northern blot analysis, 20 μg of total RNA was denatured in formamide and electrophoresed through a formaldehyde/agarose gel. The RNA was transferred to Zeta Probe-GT, cross-linked, hybridized, and washed as described previously (59Stephens J.M. Pekala P.H. J. Biol. Chem. 1991; 266: 21839-21845Abstract Full Text PDF PubMed Google Scholar). Probes were labeled by random priming using the Klenow fragment and [α-32P]dATP.Rodent Adipose Tissue Isolation—Animals were euthanized by cervical dislocation, and tissues were immediately removed and frozen in liquid nitrogen. Frozen tissues were homogenized in a buffer containing 150 mm NaCl, 10 mm Tris, pH 7.4, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 μm phenylmethylsulfonyl fluoride, 1 μm pepstatin, 50 trypsin inhibitory milliunits of aprotinin, and 10 μm leupeptin, and 2 mm sodium vanadate. Homogenates were centrifuged for 10 min at 5,000 rpm to remove any debris and insoluble material and then analyzed for protein content. All C57BL/6J mice were obtained from a colony at the Pennington Biomedical Research Center. All animal studies were carried out with protocols that were reviewed and approved by institutional IACUCs.3T3-L1 Cell Membrane Fractionation—Untreated and LIF-treated serum-deprived 3T3-L1 adipocytes were rinsed with buffer A (250 mm sucrose, 20 mm HEPES, 1 mm EDTA, pH 7.4) at 37 °C and then harvested at 4 °C in buffer A and homogenized with a Teflon pestle. Total membranes were pelleted at 250,000 × g for 90 min and resuspended in buffer B (20 mm HEPES, 1 mm EDTA, pH 7.4). For some experiments, membranes were fractionated into plasma membrane, intracellular membranes (low density microsomes, LDM), and a nuclear/mitochondrial fraction as we have described previously (59Stephens J.M. Pekala P.H. J. Biol. Chem. 1991; 266: 21839-21845Abstract Full Text PDF PubMed Google Scholar). Membrane and cytosolic fractions were divided and immediately stored at –70 °C. The protein content for all fractions was determined with a BCA kit according to the manufacturer's instructions.RESULTSOne of the unifying features of all gp130 cytokines is their ability to activate JAK/STAT and MAPK (ERK1/2) signaling pathways. We therefore wanted to determine whether gp130 cytokines, which affect these pathways in fat cells, can work synergistically to initiate these signals, or whether their actions antagonize each other. To address this question, we pretreated fully differentiated 3T3-L1 adipocytes with several different gp130 cytokines (IL-6, LIF, OSM, CNTF, and CT-1), as well as with GH for 2 h. Next, we treated the cells for 15 min with either CT-1, CNTF, LIF, or GH. As shown in Fig. 1, gp130 cytokines interacted primarily in an antagonistic fashion. The results in Fig. 1A represent an experiment in which 3T3-L1 adipocytes were pretreated with CT-1, which is known to activate STAT 1 and 3 in these cells potently (60Zvonic S. Hogan J.C. Arbour-Reily P. Mynatt R.L. Stephens J.M. J. Biol. Chem. 2004; 279: 47572-47579Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We observed that CT-1 signaling was impaired by a 2-h pretreatment with several gp130 cytokines, including CT-1. Our results demonstrate that a 2-h pretreatment with LIF, OSM, or CT-1 completely blocked the STAT 1 activation induced by a 15-min CT-1 treatment. These same cytokines also interfered with the activation of STAT 3 by CT-1. In this case, LIF acted as the most potent inhibitor, whereas CT-1 and OSM pretreatments consistently resulted in a partial, but significant, inhibition of STAT activation. No substantial STAT 5 activation occurred with CT-1 treatment, whereas GH pretreatment caused a robust STAT 5 activation that was detectable even after the 2-h pretreatment period. The ability of LIF, OSM, and CT-1 to block further actions of CT-1 was also evident through their effects on MAPK (ERK1/2) signaling because the activation of this pathway by acute CT-1 was attenuated through pretreatment with these cytokines.Even though CNTF is not a potent activator of JAK/STAT or MAPK signaling in adipocytes (54Zvonic S. Cornelius P. Stewart W.C. Mynatt R.L. Stephens J.M. J. Biol. Chem. 2003; 278: 2228-2235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 60Zvonic S. Hogan J.C. Arbour-Reily P. Mynatt R.L. Stephens J.M. J. Biol. Chem. 2004; 279: 47572-47579Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), its ability to activate STAT 3 (first lane, Fig. 1B) was affected by the pretreatment with all other gp130 cytokines examined, except for IL-6. As was the case with the acute CT-1 treatment, a 2-h pretreatment of adipocytes with LIF, OSM, or CT-1 decreased the magnitude of STAT 3 activation by CNTF. This pattern of regulation was not observed with active MAPK, which we attribute to the weak activation of this kinase by CNTF.The data in Fig. 1C verify that LIF acts as a potent activator of JAK/STAT and MAPK signaling in 3T3-L1 adipocytes. However, the ability of LIF to activate STAT 1 and 5 was completely abolished by a 2-h pretreatment with LIF, OSM, or CT-1. An identical pattern of inhibition was also observed with the activation of MAPK (ERK1/2) by a 15-min LIF treatment. Acute LIF administration resulted in a robust STAT 3 activation, and only the 2-h pretreatment with LIF resulted in an attenuation of STAT 3 activation. Hence, the gp130 cytokines LIF, OSM, and CT-1 pretreatment inhibited the LIF-induced activation of STAT 1 and 5, but not of STAT 3.Acute treatment with GH, shown in Fig. 1D, also leads to a robust induction of JAK/STAT and MAPK signaling, as evident by the activation of STAT 1, 3, and 5 and ERK1/2. However, unlike gp130 cytokines, the ability of GH to activate these pathways was not affected by a 2-h preincubation with any of the gp130 cytokines examined. In fact, LIF, OSM, and CT-1 mildly enhanced the GH-induced activation of STAT 3, but not of STAT 1 and 5. The only inhibitory effect observed for GH singling was the inhibition of STAT and MAPK activation after a 2-h pretreatment with GH itself. STAT5A levels are shown in all panels of Fig. 1 to demonstrate equivalent loading of whole cell extracts. The expression of STAT 3 and total level of ERK1/2 (MAPK) also remained unchanged by all of the treatments shown in Fig. 1 (data not shown).We utilized a similar experimental approach in an in vivo model to examine further the nature of interactions among gp130 cytokines. C57BL/6J mice were injected with either 0.05 nm CT-1 or the appropriate vehicle (saline) control, then injected again either four (Fig. 2A) or eight (Fig. 2B) h later, and then sacrificed 15 min after the second round of injections. As shown in Fig. 2, acute intraperitoneal administration of CT-1 lead to the activation of STAT 3 in mouse epididymal fat pads. Interestingly, MAPK (ERK1/2) was active even in animals injected with vehicle (saline) control. As expected, phosphorylated STAT 3 proteins were present in the adipose tissue extracts from mice after a 15-min injection with CT-1. We still observed active STAT 3 at 4 h after CT-1 administration (Fig. 2A) at levels comparable with that following a 15-min treatment. In an independent experiment, six animals were pretreated with CT-1 after an intraperitoneal injection with CT-1 or vehicle, and epididymal fat pads were harvested for analysis. We observed that the active forms of both STAT 3 and MAPK were absent from the fat pads 8 h after a CT-1 injection (Fig. 2B). The animals injected with CT-1 for 4 h, and then again for 15 min (Fig. 2A), showed no additional increase in STAT 3 or MAPK activation compared with animals injected with CT-1 for 15 min or 4 h only. Yet, in the fat pads of mice injected with CT-1 for 8 h, and then again for 15 min (Fig. 2B), we observed no increase in STAT 3 or MAPK activation compared with animals injected with CT" @default.
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• W2017609900 title "Cross-talk among gp130 Cytokines in Adipocytes" @default.
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