e21035 Background: EMT renders neoplastic cancer cells the ability to migrate and to invade distant organs. The hallmark of EMT is the loss of E-cadherin, which is a prerequisite for epithelial tumor cell invasion. In pancreatic cancer, loss of tumor E-cadherin is an independent predictor of poor outcome. Aims: To analyze the effect of pancreatic fibroblasts (PF) on inducing EMT in pancreatic cancer cells and to identify the transcription factors (Snail, Slug, ZEB1, ZEB2) that mediate EMT process. Methods: Human PFs were isolated from human pancreatic specimens obtained from chronic pancreatitis and from unaffected margins of pancreatic adenocarcinoma and serous cistoadenoma. PF were cultured until complete cellular activation, as assessed by expression of α-smooth muscle actin, vimentin and fibronectin. Human pancreatic cancer cells Panc-1 were exposed to PF conditioned medium (PF-CM) and EMT analyzed by cell morphology, migration, and E-cadherin expression (quantitative RT-PCR and immunoblot). Gene expression of Snail, Slug, ZEB1, and ZEB2 was analyzed by quantitative RT-PCR, and their activity modulated by siRNA Results: Conditioned media from all types of activated PFs induced EMT changes in Panc-1 cells, as shown by 1) morphological transition from cobblestone shaped to fibroblast-like cells, 2) stimulation of cell migration, and 3) E-cadherin down–regulation; mRNA expression of Snail transiently increased at 30 min after exposure to PF returning to basal levels afterwards; mRNA levels of ZEB1 were not up-regulated upon exposure to PF-CM. However, ZEB1 protein greatly accumulated after 48h incubation with PF-CM, suggesting that PF prevent ZEB1 degradation in Panc-1 cells. Combined RNA downregulation of ZEB1 and ZEB2, but not of Snail and/or Slug, suppressed E-cadherin repression induced by PF. Conclusions: Activated PFs promote the invasive phenotype of pancreatic cancer cells through ZEB1 and ZEB2 activation.
Murine bone marrow macrophages were able to recognize gold nanoparticle peptide conjugates, while peptides or nanoparticles alone were not recognized. Consequently, in the presence of conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, as well as nitric oxide synthase (NOS2) were induced. Furthermore, macrophage activation by gold nanoparticles conjugated to different peptides appeared to be rather independent of peptide length and polarity, but dependent on peptide pattern at the nanoparticle surface. Correspondingly, the biochemical type of response also depended on the type of conjugated peptide and could be correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical nanoparticle conjugate design to either activate the immune system or hide from it in order to reach their targets before being removed by phagocytes.
Abstract Macrophages perform essential functions in the infection and resolution of inflammation. IFN-γ is the main endogenous macrophage Th1 type activator. The classical IFN-γ signaling pathway involves activation of Stat-1. However, IFN-γ has also the capability to activate members of the MAPK family. In primary bone marrow-derived macrophages, we have observed strong activation of p38 at early time points of IFN-γ stimulation, whereas weak activation of ERK-1/2 and JNK-1 was detected at a more delayed stage. In parallel, IFN-γ exerted repressive effects on the expression of a number of MAPK phosphatases. By using selective inhibitors and knockout models, we have explored the contributions of MAPK activation to the macrophage response to IFN-γ. Our findings indicate that these kinases regulate IFN-γ-mediated gene expression in a rather selective way: p38 participates mainly in the regulation of the expression of genes required for the innate immune response, including chemokines such as CCL5, CXCL9, and CXCL10; cytokines such as TNF-α; and inducible NO synthase, whereas JNK-1 acts on genes involved in Ag presentation, including CIITA and genes encoding MHC class II molecules. Modest effects were observed for ERK-1/2 in these studies. Interestingly, some of the MAPK-dependent changes in gene expression observed in these studies are based on posttranscriptional regulation of mRNA stability.
Macrophages proliferate in the presence of their growth factor, macrophage colony-stimulating factor (M-CSF), in a process that is dependent on early and short ERK activation. Lipopolysaccharide (LPS) induces macrophage activation, stops proliferation, and delays ERK phosphorylation, thereby triggering an inflammatory response. Proliferating or activating responses are balanced by the kinetics of ERK phosphorylation, the inactivation of which correlates with Mkp1 induction. Here we show that the transcriptional induction of this phosphatase by M-CSF or LPS depends on JNK but not on the other MAPKs, ERK and p38. The lack of Mkp1 induction caused by JNK inhibition prolonged ERK-1/2 and p38 phosphorylation. The two JNK genes, jnk1 and jnk2, are constitutively expressed in macrophages. However, only the JNK1 isoform was phosphorylated and, as determined in single knock-out mice, was necessary for Mkp1 induction by M-CSF or LPS. JNK1 was also required for pro-inflammatory cytokine biosynthesis (tumor necrosis factor-α, interleukin-1β, and interleukin-6) and LPS-induced NO production. This requirement is independent of Mkp1 expression, as shown in Mkp1 knock-out mice. Our results demonstrate a critical role for JNK1 in the regulation of Mkp1 induction and in LPS-dependent macrophage activation. Macrophages proliferate in the presence of their growth factor, macrophage colony-stimulating factor (M-CSF), in a process that is dependent on early and short ERK activation. Lipopolysaccharide (LPS) induces macrophage activation, stops proliferation, and delays ERK phosphorylation, thereby triggering an inflammatory response. Proliferating or activating responses are balanced by the kinetics of ERK phosphorylation, the inactivation of which correlates with Mkp1 induction. Here we show that the transcriptional induction of this phosphatase by M-CSF or LPS depends on JNK but not on the other MAPKs, ERK and p38. The lack of Mkp1 induction caused by JNK inhibition prolonged ERK-1/2 and p38 phosphorylation. The two JNK genes, jnk1 and jnk2, are constitutively expressed in macrophages. However, only the JNK1 isoform was phosphorylated and, as determined in single knock-out mice, was necessary for Mkp1 induction by M-CSF or LPS. JNK1 was also required for pro-inflammatory cytokine biosynthesis (tumor necrosis factor-α, interleukin-1β, and interleukin-6) and LPS-induced NO production. This requirement is independent of Mkp1 expression, as shown in Mkp1 knock-out mice. Our results demonstrate a critical role for JNK1 in the regulation of Mkp1 induction and in LPS-dependent macrophage activation. The serine/threonine mitogen-activated protein kinase (MAPK) 2The abbreviations used are: MAPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal protein kinase; M-CSF, macrophage colony-stimulating factor; LPS, lipopolysaccharide; IFN, interferon; IL, interleukin; NOS2, nitric-oxide synthase 2; TNF, tumor necrosis factor; Ab, antibody; KO, knock-out; FACS, fluorescence-activated cell sorter; GPI, glycosylphosphatidylinositol; TLR, toll-like receptor. family includes extracellular signal-regulated protein kinase (ERK-1/2), stress-activated protein kinases (SAPK), p38, and c-Jun NH2-terminal protein kinases (JNK). These MAPKs are responsible for transmitting extracellular signals from the membrane to the nucleus, which leads to the phosphorylation of several transcription factors and the regulation of genes involved in the control of a number of fundamental cellular processes, including proliferation, survival, differentiation, apoptosis, motility, and metabolism (1Yang S.H. Sharrocks A.D. Whitmarsh A.J. Gene. 2003; 320: 3-21Crossref PubMed Scopus (424) Google Scholar, 2Kolch W. Nat. Rev. Mol. Cell Biol. 2005; 6: 827-837Crossref PubMed Scopus (882) Google Scholar). Although MAPKs are conserved evolutionary pathways present in eukaryotic cells, the kinetics of activation and their subcellular compartmentalization is cell type-specific, and they orchestrate differential cellular responses. For example, in neuronal cells, sustained MAPK activation of even days is required for cellular activation or differentiation, whereas in fibroblasts activation of this kinase family is very short. In contrast, to achieve proliferation, extended activation of MAPKs is required in these cells (2Kolch W. Nat. Rev. Mol. Cell Biol. 2005; 6: 827-837Crossref PubMed Scopus (882) Google Scholar, 3Pouyssegur J. Lenormand P. Eur. J. Biochem. 2003; 270: 3291-3299Crossref PubMed Scopus (155) Google Scholar). Macrophages differentiate and proliferate in the presence of macrophage colony-stimulating factor (M-CSF). However, the proliferation of these cells is blocked when they are activated by Gram-negative lipopolysaccharide (LPS) or by IFN-γ (4Xaus J. Cardo M. Valledor A.F. Soler C. Lloberas J. Celada A. Immunity. 1999; 11: 103-113Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Activation causes many biochemical and morphological modifications, such as in the expression of inducible nitric-oxide synthase (NOS2) and the biosynthesis and release of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1 (IL-1β), and IL-6. Macrophage response to M-CSF and LPS involves the phosphorylation of the three members of the MAPK family (5Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar). The correct spatiotemporal regulation of MAPK signaling activation is crucial in determining cellular responses to growth or activating factors (6Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar, 7Valledor A.F. Comalada M. Xaus J. Celada A. J. Biol. Chem. 2000; 275: 7403-7409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In our cellular model, an early peak of ERK activity (5 min) correlated with cellular proliferation, whereas a later peak (15 min) was associated with the activation program (7Valledor A.F. Comalada M. Xaus J. Celada A. J. Biol. Chem. 2000; 275: 7403-7409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Also, the length of ERK activation is critical. For proliferation, ERK must be dephosphorylated ∼15 min after activation (7Valledor A.F. Comalada M. Xaus J. Celada A. J. Biol. Chem. 2000; 275: 7403-7409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). A number of conditions that elongate ERK activity, for example extracellular matrix proteins such as decorin or fibrinogen or treatment with cyclosporin A or FK506, reduce proliferation (8Xaus J. Comalada M. Cardo M. Valledor A.F. Celada A. Blood. 2001; 98: 2124-2133Crossref PubMed Scopus (112) Google Scholar, 9Comalada M. Valledor A.F. Sanchez-Tillo E. Umbert I. Xaus J. Celada A. Eur. J. Immunol. 2003; 33: 3091-3100Crossref PubMed Scopus (23) Google Scholar). MAPK phosphatases (MKPs or DUSP) are responsible for dephosphorylating tyrosine and threonine residues of MAPKs. Eleven MKP family members have been identified thus far, these differing in tissue-specific expression, subcellular localization, post-translational regulation, and substrate specificity within the MAPK family (10Farooq A. Zhou M.M. Cell. Signal. 2004; 16: 769-779Crossref PubMed Scopus (383) Google Scholar). Of these, nuclear MKP1, also termed DUSP1, is encoded by an immediate-early response gene induced in macrophages upon stimulation with M-CSF (11Valledor A.F. Xaus J. Marques L. Celada A. J. Immunol. 1999; 163: 2452-2462PubMed Google Scholar) or LPS (12Valledor A.F. Xaus J. Comalada M. Soler C. Celada A. J. Immunol. 2000; 164: 29-37Crossref PubMed Scopus (100) Google Scholar). Although MKP1 was initially identified as an in vitro ERK-specific phosphatase, depending on the cell type it also dephosphorylates other members of the MAPK family such as JNK and p38, thus suppressing signaling downstream of these kinases (10Farooq A. Zhou M.M. Cell. Signal. 2004; 16: 769-779Crossref PubMed Scopus (383) Google Scholar). Genetic ablation of Mkp1 has shown that this phosphatase is a pivotal feedback control regulator of the innate immune response (13Chi H. Barry S.P. Roth R.J. Wu J.J. Jones E.A. Bennett A.M. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2274-2279Crossref PubMed Scopus (472) Google Scholar, 14Salojin K.V. Owusu I.B. Millerchip K.A. Potter M. Platt K.A. Oravecz T. J. Immunol. 2006; 176: 1899-1907Crossref PubMed Scopus (293) Google Scholar, 15Hammer M. Mages J. Dietrich H. Servatius A. Howells N. Cato A.C. Lang R. J. Exp. Med. 2006; 203: 15-20Crossref PubMed Scopus (278) Google Scholar, 16Zhao Q. Wang X. Nelin L.D. Yao Y. Matta R. Manson M.E. Baliga R.S. Meng X. Smith C.V. Bauer J.A. Chang C.H. Liu Y. J. Exp. Med. 2006; 203: 131-140Crossref PubMed Scopus (333) Google Scholar). Consequently, the mechanisms responsible for regulating Mkp1 expression are determinant for controlling the length of MAPK responses in macrophages. In some cell types, such as fibroblasts, Mkp1 induction is dependent on ERK-1/2 activation (17Brondello J.M. Brunet A. Pouyssegur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 18Cook S.J. Beltman J. Cadwallader K.A. McMahon M. McCormick F. J. Biol. Chem. 1997; 272: 13309-13319Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) or SAPK (19Bokemeyer D. Sorokin A. Yan M. Ahn N.G. Templeton D.J. Dunn M.J. J. Biol. Chem. 1996; 271: 639-642Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Results from our group show that the activation of the MEK/ERK-1/2 cascade is not required for the induction of Mkp1 in macrophages (11Valledor A.F. Xaus J. Marques L. Celada A. J. Immunol. 1999; 163: 2452-2462PubMed Google Scholar, 12Valledor A.F. Xaus J. Comalada M. Soler C. Celada A. J. Immunol. 2000; 164: 29-37Crossref PubMed Scopus (100) Google Scholar). In those studies, we also found that of all the protein kinase C isoforms expressed in macrophages, only PKCϵ was involved in Mkp1 transcriptional induction by M-CSF or LPS and, consequently, in the negative control of ERK activity (11Valledor A.F. Xaus J. Marques L. Celada A. J. Immunol. 1999; 163: 2452-2462PubMed Google Scholar, 12Valledor A.F. Xaus J. Comalada M. Soler C. Celada A. J. Immunol. 2000; 164: 29-37Crossref PubMed Scopus (100) Google Scholar, 20Stawowy P. Goetze S. Margeta C. Fleck E. Graf K. Biochem. Biophys. Res. Commun. 2003; 303: 74-80Crossref PubMed Scopus (27) Google Scholar). Recently, we reported that Raf-1 activation is required for Mkp1 expression in macrophages and follows the different kinetics of induction by M-CSF and LPS in these cells (21Sanchez-Tillo E. Comalada M. Farrera C. Valledor A.F. Lloberas J. Celada A. J. Immunol. 2006; 176: 6594-6602Crossref PubMed Scopus (26) Google Scholar). Here we studied the role of all MAPK family members in the regulation of MKP1 in primary cultures of macrophages. Only JNK, and specifically the JNK1 isoform, was involved in Mkp1 induction by both M-CSF and LPS, thus regulating the activation profile of the other MAPKs. In addition, we demonstrate that JNK1 mediates the expression of NOS2 and pro-inflammatory cytokines during LPS and TNF-α activation. Our results show that the JNK1 isoform plays a crucial role in macrophage biology. Reagents—LPS, actinomycin D, propidium iodide, wortmannin, LY294002, anti-ERKP Thr-183/Tyr-185, and anti-β-actin antibodies (Abs) were from Sigma. In several experiments, the results obtained with commercial LPS were compared with highly purified LPS from Salmonella abortus equi, kindly donated by Dr. C. Galanos (Max Planck Institute, Freiburg, Germany (22Huber M. Kalis C. Keck S. Jiang Z. Georgel P. Du X. Shamel L. Sovath S. Mudd S. Beutler B. Galanos C. Freudenberg M.A. Eur. J. Immunol. 2006; 36: 701-711Crossref PubMed Scopus (142) Google Scholar)) and no differences were found. Recombinant M-CSF and IFN-γ were from R&D Systems Inc. (Minneapolis, MN). Murine TNF-α was obtained from Peprotech (London, UK). The p38P (Thr-180/Tyr-182) MAPK Ab was obtained from Cell Signaling Technology (Beverly, MA). The anti-JNK1, JNK, and MKP1 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059, GF109203X, SB2030580, SP600125, JNK inhibitor II, and NOS2 Abs were from Calbiochem. Secondary horseradish peroxidase anti-mouse (MP Biomedicals, Irvine, CA) and anti-rabbit (Sigma) Abs were also used. Cell Culture—Bone marrow macrophages were obtained from 6-week-old BALB/c mice (Harlan Ibérica, Barcelona, Spain) and cultured as described previously (23Celada A. Gray P.W. Rinderknecht E. Schreiber R.D. J. Exp. Med. 1984; 160: 55-74Crossref PubMed Scopus (357) Google Scholar). Macrophages were cultured for 6 days in Dulbecco's modified Eagle's medium (BioWhittaker-Cambrex, Emerainville, France), supplemented with 20% fetal calf serum (Sigma-Aldrich) and 30% L-cell conditioned media. Macrophages (80–90% confluent) were synchronized by culture with 10% fetal calf serum for 18 h (23Celada A. Gray P.W. Rinderknecht E. Schreiber R.D. J. Exp. Med. 1984; 160: 55-74Crossref PubMed Scopus (357) Google Scholar). Macrophages from Jnk1, Jnk2, and Mkp1 knock-out (KO) mice (24Dorfman K. Carrasco D. Gruda M. Ryan C. Lira S.A. Bravo R. Oncogene. 1996; 13: 925-931PubMed Google Scholar, 25Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar) were obtained in the same way. For the experiments with these mice, we used the corresponding background mouse controls. Animal use was approved by the Animal Research Committee of the University of Barcelona (Approval Number 2523). Cell Surface Staining—Analysis of cell surface receptors was performed as described (26Herrero C. Marques L. Lloberas J. Celada A. J. Clin. Investig. 2001; 107: 485-493Crossref PubMed Scopus (111) Google Scholar). 1 × 106 cells/ml were incubated with primary antibodies against Ly-71 (F4/80) and CD11b (Mac-1) fluorescein isothiocyanate Abs (eBioscience, San Diego) after blocking Fcγ receptors with anti-CD16/CD32 (FcγIII/II receptor) Ab (Pharmingen). Detection was done directly or after incubation with fluorescein isothiocyanate anti-rabbit IgG (Sigma). Cells were fixed with paraformaldehyde solution before flow cytometry analysis (Epics XL, Coulter Corp., Hialeah, FL). Each figure is representative of three independent experiments with triplicates expressed as the mean ± S.D. Dead cells, detected through low forward and side light scatter, were excluded. Blocking and direct incubation with secondary Ab was used as negative control. Apoptosis Assay—Cell viability was assessed by particle counting using FACS (Coulter Multisizer II, Midland, Canada) and confirmed by trypan blue exclusion. Cell death was also assessed by FACS analysis using the rAnnex V-FITC kit (Bender MedSystems, Burlingame, CA) following the manufacturer's instructions. Actinomycin D was used as a positive control of apoptosis. Each point was performed in triplicate and the results were expressed as the mean value ± S.D. Proliferation Assay—After 24 h of treatment, cells were pulsed with [3H]dThd (1 μCi/ml) (Amersham Biosciences) for 6 h as described previously (27Celada A. Maki R.A. J. Immunol. 1992; 148: 1102-1105PubMed Google Scholar). Each point was performed in triplicate, and the results are expressed as the mean ± S.D. Cell Cycle Analysis—Cell cycle was analyzed as described (4Xaus J. Cardo M. Valledor A.F. Soler C. Lloberas J. Celada A. Immunity. 1999; 11: 103-113Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Cells were fixed with EtOH 95%, incubated with propidium iodide plus RNase A, and then analyzed by FACS. Cell cycle distributions were analyzed with the Multicycle program (Phoenix Flow Systems, Inc., San Diego). Western Blot Analysis—Total cytoplasmic extracts were made by lysing cells as described previously (4Xaus J. Cardo M. Valledor A.F. Soler C. Lloberas J. Celada A. Immunity. 1999; 11: 103-113Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). SDS-PAGE was performed and proteins transferred to nitrocellulose membranes (Hybond-C, Amersham Biosciences). After blocking, incubation with primary Abs and secondary Ab was performed. The detection of the bands was done using the EZ-ECL kit (Biological Industries, Kibbutz beit Haemek, Israel) and by exposure to x-ray films (Agfa, Mortsel, Belgium). β-Actin was used as a loading control. Analysis of maximal expression was determined with a molecular analyst system (Bio-Rad). In-gel Kinase Assay—ERK activity was analyzed as described previously using 50–100 μg of total protein obtained as described above and separated by 12.5% SPD-PAGE containing 0.1 mg/ml myelin basic protein (Sigma) as substrate co-polymerized in the gel (7Valledor A.F. Comalada M. Xaus J. Celada A. J. Biol. Chem. 2000; 275: 7403-7409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). After several washes, denaturing, and renaturing, a phosphorylation assay was performed with 50 μm ATP and 100 μCi of [γ-32P]ATP (Amersham Biosciences). JNK Activity Assay—JNK activity was measured as described previously (28Caelles C. Gonzalez-Sancho J.M. Munoz A. Genes Dev. 1997; 11: 3351-3364Crossref PubMed Scopus (292) Google Scholar). Briefly, cells were lysed and immunoprecipitated with protein A-Sepharose and anti-JNK1 or anti-JNK2 Ab. After several washes, the reaction was performed with 1 μg of GST·c-Jun-(1–169) (MBL, Woburn, MA) as JNK substrate, 20 μm ATP and 1μCi of [γ-32P]ATP. SDS-PAGE was performed and the gel exposed to Agfa x-ray films. RNA Extraction and Northern Blot Analysis—Total RNA was extracted with the RNA kit EZ-RNA (Biological Industries). 10–15 μg of the RNA extract was separated in agarose gel containing formaldehyde and then transferred to nitrocellulose membrane (Amersham Biosciences). Probes for Mkp1, IL-6, IL-1β, Tnf-α, and 18S were labeled with [α-32P]dCTP (Amersham Biosciences) using the random prime labeling system (Amersham Biosciences). 18S was used as the loading control. Real-time PCR—cDNA was obtained from 1 μg of total RNA using Moloney murine leukemia virus reverse transcriptase (M-MLV, Promega, Madison, WI) as described (29Marques L. Brucet M. Lloberas J. Celada A. J. Immunol. 2004; 173: 1103-1110Crossref PubMed Scopus (40) Google Scholar). The primer sequences used for Mkp1, Tnf-α, IL-1β, IL-6, and NOS2 were designed with the Primer Express software (Applied Biosystems). The primers were the following: for β-actin, 5′-ACTATTGGCAACGACCGGTTT-3′ and 5′-AAGGAAGGCTGGAAAAGAGGG-3′; for NOS2, 5′-GCCACCAACAATGGCAACA-3′ and 5′-CGTACCGGATGAGCTGTGAATT-3′; for c-jun, 5′-TGAAAGCGCAAAACTCCGAG-3′ and 5′-GCACCCACTGTTAACGTGGTTTC-3′; for Tnf-α, 5′-CCTTGTTGCCTCCTCTTTTGC-3′ and 5′-TCAGTGATGTAGCGACAGCCTG-3′; for IL-1β, 5′-CCTGTGTTTTCCTCCTTGCCT-3′ and 5′-GCCTAATGTCCCCTTGAATCAA-3′; for IL-6, 5′-CAGAAGGAGTGGCTAAGGACCA-3′ and 5′-ACGCACTAGGTTTGCCGAGTAG-3′; for Jnk1, 5′-GATTTTGGACTGGCGAGGACT-3′ and 5′-TAGCCCATGCCGAGAATGA-3′; for Jnk2, 5′-TGATTGATCCAGACAAGCGG-3′ and 5′-AAATTTGAGGTGGTGGCGC-3′; for Jnk-3, 5′-AAACTACGTGGAGAATGCGCC-3′ and 5′-TGGCTTGGCTGGCTTTAAGT-3′ and for Mkp1, 5′-GGACAACCACAAGGCAGACATC-3′ and 5′-GGCCTGGCAATGACAAACA-3′. Real-time PCR was carried out with 2× SYBR Green PCR Master Mix using the ABI Prism 7900 detection system (Applied Biosystems, Foster city, CA). Thermal cycling conditions were 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for 35 cycles. Data were expressed as relative mRNA values normalized to β-actin expression levels in each sample. Statistical Analysis—To calculate the statistical differences between control and treated samples, we used the Student's paired t test. Values of p < 0.05 or lower were considered significant. JNK Is Required for the Induction of Mkp1 Expression by M-CSF or LPS—Although ERK-1/2 activation is required for both macrophage proliferation and activation, the differential kinetics of their phosphorylation/dephosphorylation correlates with specific macrophage responses (7Valledor A.F. Comalada M. Xaus J. Celada A. J. Biol. Chem. 2000; 275: 7403-7409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In macrophages stimulated with M-CSF or LPS, the induction of MKP1 correlates with the dephosphorylation of ERK-1/2 (11Valledor A.F. Xaus J. Marques L. Celada A. J. Immunol. 1999; 163: 2452-2462PubMed Google Scholar, 12Valledor A.F. Xaus J. Comalada M. Soler C. Celada A. J. Immunol. 2000; 164: 29-37Crossref PubMed Scopus (100) Google Scholar). Because of inefficient transfection of primary cultures of macrophages (30Celada A. Borras F.E. Soler C. Lloberas J. Klemsz M. van Beveren C. McKercher S. Maki R.A. J. Exp. Med. 1996; 184: 61-69Crossref PubMed Scopus (122) Google Scholar), we studied the effect of MAPK activation on MKP1 induction by means of specific inhibitors. PD098059, SB203580, and SP600125 have been used extensively as selective inhibitors of the activation of MEK/ERK, p38, and all JNK isoforms, respectively (31Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar, 32Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1981) Google Scholar, 33Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Crossref PubMed Scopus (2244) Google Scholar). These compounds also inhibited MAPKs in our macrophage model (data not shown). To test the effect of MAPKs on Mkp1 induction by M-CSF or LPS, we inhibited each MAPK separately. No effect was found when p38 or ERK-1/2 was inhibited (Fig. 1, A and B). Interestingly, JNK activation was required for MKP1 protein expression (Fig. 1C and 1D). Northern blot analysis showed that the inhibition of JNK blocked the induction of Mkp1 mRNA (Fig. 1, E and F). Similar results were obtained by real-time PCR (data not shown). Because the half-life of Mkp1 mRNA is short (34Lin Y.W. Yang J.L. J. Biol. Chem. 2006; 281: 915-926Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), we examined whether the decrease in the level of mRNA was due to inhibition of mRNA production or to an increase in its degradation. For this purpose, we measured the rate of mRNA degradation in the presence or absence of JNK inhibitors. Cells were treated for 30 min with M-CSF or LPS, and actinomycin D was then added at a concentration (5 μg/ml) sufficient to block all further RNA synthesis as determined by [3H]UTP incorporation (35Celada A. Maki R.A. Eur. J. Immunol. 1989; 19: 205-208Crossref PubMed Scopus (14) Google Scholar). RNA was isolated at 15, 30, and 60 min after the addition of actinomycin D, which allowed us to estimate the half-life of Mkp1. These mRNAs are highly unstable, and JNK inhibition did not alter the half-life (Fig. 1, G and H), which indicates that the reduction in mRNA levels was due to a decrease at the transcriptional level. To determine the nonselective effects of SP600125, we used the inactive analogue JNK inhibitor II (33Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Crossref PubMed Scopus (2244) Google Scholar). At the same concentration (25 μm) at which SP600125 inhibited the expression of Mkp1, no effect was observed when we used the JNK inhibitor II (data not shown). Inhibition of JNK Activation Causes an Elongation of Phosphorylation of other MAPKs—In macrophages treated with either M-CSF or LPS, SP600125 did not modify the induction of ERK-1/2 activation, as determined by in-gel kinase assay (Fig. 2A) and by Western blot using specific antibodies against the phosphorylated form (Fig. 2B). Although the initial activation of these kinases was not modified, the length of activation was prolonged, thereby showing an effect of JNK inhibition on the other MAPKs. Similar effects were observed on p38 phosphorylation induced by M-CSF (Fig. 2C). For LPS, the SP600125 treatment modified the kinetics of activation by slowly delaying and prolonging activation of p38 (Fig. 2D). Cell incubation with SP600125 alone did not induce activation of ERK or p38 (data not shown). However, when we tested the involvement of ERK or p38 in JNK activation, no modifications were detected (Fig. 2, E and F). Taken together, these results indicate that JNK activity is not required for the initial activation of the other MAPKs, but it does control the duration of their activity through induction of Mkp1. JNK Is Required for LPS-dependent Activation—We also studied the role of JNK during LPS activation. Total RNA of macrophages pretreated with SP600125 and then stimulated with LPS was analyzed for pro-inflammatory cytokine expression (Fig. 3A). The expression of Tnf-α, IL-1β, and IL-6 was severely compromised when JNK activity was inhibited. These results were confirmed using real-time PCR (data not show). Furthermore, SP600125 treatment caused a substantial reduction of LPS-induced NOS2 protein expression (Fig. 3B). These data indicate that JNK activation mediates several events in the macrophage inflammatory response to LPS. To establish whether the role of JNK in cytokine induction was a direct effect or was mediated through MKP1 regulation, we used macrophages from mice with disrupted Mkp1 (24Dorfman K. Carrasco D. Gruda M. Ryan C. Lira S.A. Bravo R. Oncogene. 1996; 13: 925-931PubMed Google Scholar). In these cells, JNK activation in response to LPS was not impaired (Fig. 3C). The LPS-induced expression of pro-inflammatory cytokines was analyzed using Northern blots (Fig. 3D). An increased expression of Tnf-α was detected in Mkp1 KO macrophages, thereby confirming the results of other authors (13Chi H. Barry S.P. Roth R.J. Wu J.J. Jones E.A. Bennett A.M. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2274-2279Crossref PubMed Scopus (472) Google Scholar, 14Salojin K.V. Owusu I.B. Millerchip K.A. Potter M. Platt K.A. Oravecz T. J. Immunol. 2006; 176: 1899-1907Crossref PubMed Scopus (293) Google Scholar, 15Hammer M. Mages J. Dietrich H. Servatius A. Howells N. Cato A.C. Lang R. J. Exp. Med. 2006; 203: 15-20Crossref PubMed Scopus (278) Google Scholar, 16Zhao Q. Wang X. Nelin L.D. Yao Y. Matta R. Manson M.E. Baliga R.S. Meng X. Smith C.V. Bauer J.A. Chang C.H. Liu Y. J. Exp. Med. 2006; 203: 131-140Crossref PubMed Scopus (333) Google Scholar). This observation demonstrates that the effects of JNK during LPS activation are not due to impaired Mkp1 regulation. JNK1 Is Required for Mkp1 Induction by LPS or M-CSF—To date, 10 isoforms resulting from three encoding genes of Jnk, namely Jnk1, Jnk2, and Jnk3, have been characterized (36Barr R.K. Bogoyevitch M.A. Int. J. Biochem. Cell Biol. 2001; 33: 1047-1063Crossref PubMed Scopus (221) Google Scholar, 37Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1183) Google Scholar, 38Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar). Jnk1 and Jnk2 are expressed ubiquitously, whereas Jnk3 is restricted to brain, testis, and heart (37Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1183) Google Scholar, 39Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). We studied the JNK isoforms predominantly expressed when macrophages proliferate or become activated by LPS. Using PCR with specific primer pairs, the mRNA levels of each isoform were determined. Jnk1 and Jnk2 were constitutively expressed (Fig. 4A). However, Jnk3 was not detected in macrophages even after treatment with M-CSF (Fig. 4A) or LPS (data not shown). Therefore, we focused our studies on the activation of these isoforms. The specificity of the antibodies was tested using the KOs for Jnk1, Jnk2, and Jnk3. For JNK1, two distinct protein products were detected. Although p46 was constitutively present, p54 was detected preferentially after stimulation with M-CSF (Fig. 4B). For JNK2, only p54 was detected, and this protein was not modified by treatments with M-CSF, LPS, or TNF-α (Fig. 4C). To evaluate the JNK isoform that is active during proliferation or activation, we immunoprecipitated the anti-JNK isoform and then performed phosphorylation assays using c-Jun as substrate. Both stimuli, LPS and M-CSF, induced strong activation of the JNK1 isoform with a similar time course (Fig. 4D). Under these experimental conditions no JNK2 activity was detected. The results thus far have suggested that the JNK1 isoform is involved in the transcription of Mkp1 induced by M-CSF and LPS. To corroborate these results, we performed studies with Jnk1 and Jnk2 single KO mice. First, the maturation of bone marrow-derived macrophages was characterized measuring F4/80 and Mac-1 surface expression as markers of macrophage terminal differentiation (40McKnight A.J. Macfarlane A.J. Dri P. Turley L. Willis A.C. Gordon S. J. Biol. Chem. 1996; 271: 486-489Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 41Pahl H.L. Scheibe R.J. Zhang D.E. Chen H.M. Galson D.L. Maki R.A. Tenen D.G. J. Biol. Chem. 1993; 268: 5014-5020Abstract Full Text PDF PubMed Google Scholar). Cytometric analysis did not reveal significant differences in the differentiation state of macrophages from single isoform-specific Jnk KO m
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Abstract MAPK phosphatase‐1 (MKP‐1) is a protein phosphatase that plays a crucial role in innate immunity. This phosphatase inactivates ERK1/2, which are involved in two opposite functional activities of the macrophage, namely proliferation and activation. Here we found that although macrophage proliferation and activation induce MKP‐1 with different kinetics, gene expression is mediated by the proximal promoter sequences localized between −380 and −180 bp. Mutagenesis experiments of the proximal element determined that CRE/AP‐1 is required for LPS‐ or M‐CSF‐induced activation of the MKP‐1 gene. Moreover, the results from gel shift analysis and chromatin immunoprecipitation indicated that c‐Jun and CREB bind to the CRE/AP‐1 box. The distinct kinetics shown by M‐CSF and LPS correlates with the induction of JNK and c‐jun, as well as the requirement for Raf‐1. The signal transduction pathways that activate the induction of MKP‐1 correlate kinetically with induction by M‐CSF and LPS.
Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion. Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.
Mouse bone marrow-derived macrophages proliferate in the presence of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor, or IL-3, but undergo apoptosis in their absence. Inhibition of extracellular signal-regulated kinases (ERK)-1/2 blocks growth factor-dependent proliferation but not survival, indicating that the two processes require independent signaling pathways. Although M-CSF induces the activation of other kinase pathways, such as c-Jun N-terminal kinase, p38, and phosphatidylinositol 3-kinase (PI-3K), these pathways are not required for proliferation. However, PI-3K is the only one necessary for the induction of survival, as demonstrated using the inhibitors LY294002 and Wortmannin. Growth factors also activate Akt kinase and a transient expression of the cdk inhibitor p21(Waf1), which inhibits apoptosis but is not required for proliferation. PI-3K inhibitors also block growth factor-dependent expression of p21(Waf1) and the activation of Akt. Moreover, the survival induced by cyclosporin A or decorin is also dependent on the PI-3K/Akt kinases and p21(Waf1). These findings demonstrate that the induction of p21(Waf1) through the PI-3K/Akt pathway is a general survival response of macrophages. Our results show that growth factors in macrophages use two pathways: one for proliferation, mediated by ERK, and the other for survival, which requires the PI-3K/Akt kinases and p21(Waf1).
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