- Short report
- Open Access
Src mediates cytokine-stimulated gene expression in airway myocytes through ERK MAPK
© Singer et al; licensee BioMed Central Ltd. 2011
- Received: 5 January 2011
- Accepted: 20 May 2011
- Published: 20 May 2011
The p38 and extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) participate in cytokine-stimulated inflammatory gene expression in airway smooth muscle cells. The following study was undertaken to determine whether Src tyrosine kinases are signaling intermediaries upstream of cytokine-stimulated MAPK activation and gene expression. Treating human airway myocytes with interleukin (IL)-1β, tumor necrosis factor (TNF) α and interferon (IFN) γ caused a rapid 1.8-fold increase in Src family tyrosine kinase activity within 1 minute that remained 2.3 to 2.7 fold above basal conditions for 15 minutes. This activity was blocked by addition of 30 μM PP1, a pyrimidine inhibitor specific for Src family tyrosine kinases, in immune-complex assays to confirm that this stimulus activates Src tyrosine kinase. Addition of PP1 also blocked cytokine-stimulated expression of IL-1β, IL-6 and IL-8, while decreasing phosphorylation of ERK, but not p38 MAPK. Since this inflammatory stimulus may activate additional inflammatory signaling pathways downstream of Src, we tested the effects of PP1 on phosphorylation of signal transducers and activators of transcription (STAT). PP1 had no effect on cytokine-stimulated STAT 1 or STAT 3 phosphorylation. These results demonstrate that Src tyrosine kinases participate in the regulation of IL-1β, IL-6 and IL-8 expression and that these effects of Src are mediated through activation of ERK MAPK and not p38 MAPK or STAT1/STAT3 phosphorylation.
- MAPK Activation
- Airway Smooth Muscle Cell
- Inflammatory Gene Expression
- Human Airway Smooth Muscle Cell
- Upstream Mediator
Our laboratory has examined signaling pathways regulating secretion of inflammatory mediators by human airway smooth muscle cells. The synthesis and secretion of Th1/Th2 cytokines, along with CC and C-X-C chemokines, chemotactic proteins, peptide growth factors and their receptors can be induced in these myocytes by exposure to, among others, interleukin (IL)-1β, tumor necrosis factor (TNF) α, interferon (IFN) γ, or transforming growth factor β [1, 2] and contributes to inflammatory airway disease. In previous studies, we used a complementary DNA expression array to analyze expression of inflammatory mediators following treatment with a pro-inflammatory stimulus consisting of IL-1β, TNFα and IFNγ and established that this stimulus induces expression of multiple inflammatory mediators including IL-1β, IL-6, and IL-8 . Pharmacological inhibitors of mitogen-activated protein kinase (MAPK) activation were used to further demonstrate that both p38 and ERK MAPK are upstream mediators of IL-6 and IL-8 expression, while ERK MAPK alone was involved in mediating IL-1β expression.
Src tyrosine kinases are one of the signaling intermediaries linking multiple types of receptors to MAPK activation in smooth muscle. In colonic myocytes, the G-protein coupled M2 muscarinic receptor is coupled to ERK MAPK through Src activation  and expression of IL-1β, IL-6, IL-8, and cyclooxygenase (COX-2) mRNA is reduced by inhibition of Src with the pyrimidine inhibitor PP1 . In vascular smooth muscle cells, angiotensin II stimulates Src-dependent p38 MAPK activation  and CD40 ligation initiates Src activation of p38 and ERK MAPK, resulting in the induction of IL-8 and monocyte chemotactic protein-1 . In airway myocytes, the PDGF receptor signals to ERK MAPK through Src activation via a pertussis-toxin sensitive mechanism that suggests the involvement of Gi-protein subunits  and sphingosine-1-phosphate stimulation of a Gi-coupled receptor also stimulates ERK MAPK through Src . In addition to acting as an upstream mediator of MAPK, Src may also activate other signaling pathways that could affect inflammatory gene expression, such as the Janus kinase-signal transducers and activators of transcription (JAK-STAT). This is supported by evidence that inhibition of Src in PDGF-stimulated airway myocytes with PP2 blocks both ERK MAPK and JAK2 activation, resulting in decreased phosphorylation of STAT1 and STAT3  that requires an interaction with the small GTPase Rac1 .
Clearly, the functions of Src tyrosine kinases in smooth muscle cells are varied and likely involve interactions with multiple signaling pathways. In the airway, Src mediates serotonin-evoked peak Ca2+ responses by affecting phosphoinositide levels to alter cell contraction . Src also mediates PDGF and thrombin-induced proliferative responses, possibly through stimulation of cyclin D1 expression [10, 13]. While previous studies have established that both ERK and p38 MAPK are involved in regulating expression of many inflammatory genes in airway myocytes [3, 16, 17], this work demonstrates that Src tyrosine kinases also play a role in regulating inflammatory gene expression by signaling upstream of ERK but not p38 MAPK. This is supported by studies of inflammatory gene expression in other cell types. In pulmonary epithelial cells, both ERK and p38 MAPK contribute to silica-induced IL-8 release but only ERK MAPK activation is dependent on Src . CD40 stimulation of IL-8 and MCP-1 production in vascular myocytes is also dependent on ERK and p38 MAPK activation but the inhibitor PP2 was found to inhibit only ERK MAPK . In this same study, PP2 also decreased activation of IκB kinase, indicating that Src stimulates NF-κB signaling to affect chemokine production and suggesting that NF-κB activation could link cytokine stimulation to MAPK activation. We have previously shown that IL-1β and TNFα but not IFNγ activates NF-κB in airway myocytes and inhibition of NF-κB activity reduces expression of IL-1β, IL-6, IL-8 and COX-2 in a manner independent of p38 MAPK [5, 16].
The link between inflammatory signaling and Src-dependent MAPK activation in our studies remains to be determined. One possibility may be the TNF receptor-associated proteins (TRAFs). Binding of IL-1β to the IL-1 receptor activates IL-1 receptor associated kinases that recruit TRAFs. TNF receptors also recruit TRAFs through the TNF receptor DEATH domain (TRADD). In fibroblasts, Src interacting with TRAF2 links TNFα stimulation to ERK MAPK  and TRAF proteins also activate NF-κB through recruitment of IκB kinase (see review by ). The contribution of IFNγ signaling to Src-dependent ERK MAPK activation is less clear. Interactions between Src tyrosine kinases and receptor-associated JAKs are often required for complete STAT activation  but downstream activation of ERK MAPK has not been demonstrated. It has been proposed that IFNγ can directly activate ERK MAPK pathways through MEKK1  but the signaling intermediates, if any, are unknown. Another possibility could involve heterotrimeric Gi subunits implicated in Src-dependent activation of ERK MAPK in airway myocytes stimulated with PDGF . The contribution of these proteins have not been widely examined in the context of IL-1β TNFα or IFNγ signaling, which are more likely to utilize small GTPases such as Rac1  and RhoA . Thus, while these results demonstrate that Src participate in the regulation of inflammatory gene expression through activation of ERK MAPK, further studies will explore the signaling pathways linking Src-dependent cytokine stimulation to MAPK activation.
Acknowledgements and Funding
The authors would like to thank Ms. Shanti Rawat and Ms. Michelle Deetken for their excellent technical assistance. This work was supported by a Minority Investigator Research Supplement from the NIH/NHLBI to CAS and by the NIH/NHLBI HL48183-10 to WTG.
- Singer CA, Salinthone S, Baker KJ, Gerthoffer WT: Synthesis of immune modulators by smooth muscles. Bioessays. 2004, 26: 646-655. 10.1002/bies.20041.View ArticlePubMedGoogle Scholar
- Knox AJ, Pang L, Johnson S, Hamad A: Airway smooth muscle function in asthma. Clin Exp Allergy. 2000, 30: 606-614. 10.1046/j.1365-2222.2000.00762.x.View ArticlePubMedGoogle Scholar
- Hedges JC, Singer CA, Gerthoffer WT: Mitogen-activated protein kinases regulate cytokine gene expression in human airway myocytes. Am J Respir Cell Mol Biol. 2000, 23: 86-94.View ArticlePubMedGoogle Scholar
- Singer CA, Vang S, Gerthoffer WT: Coupling of M(2) muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells. Am J Physiol Gastrointest Liver Physiol. 2002, 282: G61-G68.View ArticlePubMedGoogle Scholar
- Salinthone S, Singer CA, Gerthoffer WT: Inflammatory gene expression by human colonic smooth muscle cells. Am J Physiol Gastrointest Liver Physiol. 2004Google Scholar
- Touyz RM, He G, El MM, Diep Q, Mardigyan V, Schiffrin EL: Differential activation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen activated-protein kinase by AT1 receptors in vascular smooth muscle cells from Wistar-Kyoto rats and spontaneously hypertensive rats. J Hypertens. 2001, 19: 553-559. 10.1097/00004872-200103001-00006.View ArticlePubMedGoogle Scholar
- Mukundan L, Milhorn DM, Matta B, Suttles J: CD40-mediated activation of vascular smooth muscle cell chemokine production through a Src-initiated, MAPK-dependent pathway. Cell Signal. 2004, 16: 375-384. 10.1016/j.cellsig.2003.08.008.View ArticlePubMedGoogle Scholar
- Conway AM, Rakhit S, Pyne S, Pyne NJ: Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase. Biochem J. 1999, 337: 171-177. 10.1042/0264-6021:3370171.PubMed CentralView ArticlePubMedGoogle Scholar
- Rakhit S, Conway AM, Tate R, Bower T, Pyne NJ, Pyne S: Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase. Biochem J. 1999, 338 (Pt 3): 643-649.PubMed CentralView ArticlePubMedGoogle Scholar
- Simon AR, Takahashi S, Severgnini M, Fanburg BL, Cochran BH: Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2002, 282: L1296-L1304.View ArticlePubMedGoogle Scholar
- Simeone-Penney MC, Severgnini M, Rozo L, Takahashi S, Cochran BH, Simon AR: PDGF-induced human airway smooth muscle cell proliferation requires STAT3 and the small GTPase Rac1. Am J Physiol Lung Cell Mol Physiol. 2008, 294: L698-704. 10.1152/ajplung.00529.2007.View ArticlePubMedGoogle Scholar
- Cheng HC, Nishio H, Hatase O, Ralph S, Wang JH: A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases. J Biol Chem. 1992, 267: 9248-9256.PubMedGoogle Scholar
- Tsang F, Choo HH, Dawe GS, Wong WS: Inhibitors of the tyrosine kinase signaling cascade attenuated thrombin-induced guinea pig airway smooth muscle cell proliferation. Biochem Biophys Res Commun. 2002, 293: 72-78. 10.1016/S0006-291X(02)00170-5.View ArticlePubMedGoogle Scholar
- Ovrevik J, Lag M, Schwarze P, Refsnes M: p38 and Src-ERK1/2 pathways regulate crystalline silica-induced chemokine release in pulmonary epithelial cells. Toxicol Sci. 2004, 81: 480-490. 10.1093/toxsci/kfh214.View ArticlePubMedGoogle Scholar
- Tolloczko B, Turkewitsch P, Choudry S, Bisotto S, Fixman ED, Martin JG: Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate. Am J Physiol Lung Cell Mol Physiol. 2002, 282: L1305-L1313.View ArticlePubMedGoogle Scholar
- Singer CA, Baker KJ, McCaffrey A, AuCoin DP, Dechert MA, Gerthoffer WT: p38 MAPK and NF-κB mediate COX-2 expression in human airway myocytes. Am J Physiol Lung Cell Mol Physiol. 2003, 285: L1087-L1098.View ArticlePubMedGoogle Scholar
- Laporte JD, Moore PE, Abraham JH, Maksym GN, Fabry B, Panettieri RA Jr, Shore SA: Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1β. Am J Physiol. 1999, 277: L943-L951.PubMedGoogle Scholar
- van Vliet C, Bukczynska PE, Puryer MA, Sadek CM, Shields BJ, Tremblay ML, Tiganis T: Selective regulation of tumor necrosis factor-induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase. Nat Immunol. 2005, 6: 253-260.View ArticlePubMedGoogle Scholar
- Lee NK, Lee SY: Modulation of life and death by the tumor necrosis factor receptor-associated factors (TRAFs). J Biochem Mol Biol. 2002, 35: 61-66. 10.5483/BMBRep.2002.35.1.061.View ArticlePubMedGoogle Scholar
- Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA, Cox CE, Falcone R, Fairclough R, Parsons S, Laudano A, Gazit A, Levitzki A, Kraker A, Jove R: Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene. 2001, 20: 2499-2513. 10.1038/sj.onc.1204349.View ArticlePubMedGoogle Scholar
- Roy SK, Hu J, Meng Q, Xia Y, Shapiro PS, Reddy SP, Platanias LC, Lindner DJ, Johnson PF, Pritchard C, Pagés G, Pouyssegur J, Kalvakolanu DV: MEKK1 plays a critical role in activating the transcription factor C/EBP-beta-dependent gene expression in response to IFN-γ. Proc Natl Acad Sci USA. 2002, 99: 7945-7950. 10.1073/pnas.122075799.PubMed CentralView ArticlePubMedGoogle Scholar
- Hunter I, Nixon GF: Spatial compartmentalization of tumor necrosis factor (TNF) receptor 1-dependent signaling pathways in human airway smooth muscle cells. Lipid rafts are essential for TNF-α-mediated activation of RhoA but dispensable for the activation of the NF-κB and MAPK pathways. J Biol Chem. 2006, 281: 34705-34715. 10.1074/jbc.M605738200.PubMed CentralView ArticlePubMedGoogle Scholar
- Rahman MS, Shan LY, Yang J, Unruh H, Yang X, Halayko AJ, Gounni AS: IL-17R activation of human airway smooth muscle cells induces CXCL-8 production via transcriptional dependent mechanism. Clin Immunol. 2005, 115: 268-276. 10.1016/j.clim.2005.01.014.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.