Stretch-induced Expression of CYR61 Increases the Secretion of IL-8 in A549 Cells via the NF-κβ/lκβ Pathway

Abstract

Mechanical ventilation (MV) with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses, resulting in ventilator-induced lung injury (VILI). The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear. VILI-related genes such as cysteine-rich angiogenic inducer 61 (Cyr61) have been demonstrated to play a detrimental role in the aggressive ventilation strategies. In the present study, we investigated the involvement of Cyr61 in the VILI and the underlying mechanism. A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting, respectively. Additionally, after exposure of A549 cells to cyclic stretch for 5 min to 1 h,the expression levels of nuclear factor kappaB (NF-κB) and IL-8 were detected by E L I S A and Western blotting. Thereafter, Cyr61 expression was depressed in A549 cells with the siRNA pGenesil1.1-Cyr61-3 before the cyclic stretch, and IL-8 secretion and the activation of NF-κB pathways were probed by ELISA and Western blotting, respectively. Moreover, A NF-κB inhibitor (PDTC) and an activator (TNF) were used before mechanical stretch. Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8, respectively. The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells. Additionally, cyclic stretch also mobilized NF-κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells. The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch. Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA. It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells. These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

Keywords

cysteine-rich angiogenic inducer 61 / cyclic stretch / lung injury

References

[1] Dos SantosCC, SlutskyAS. Invited review: mechanisms of ventilator-induced lung injury: a perspective J Appl Physiol, 2000, 89(4): 1645-1655

[2] CoplandIB, KavanaghBP, EngelbertsD, et al.. Early changes in lung gene expression due to high tidal volume. Am J Respir Crit Care Med, 2003, 68(9): 1051-1059

[3] O’BrienTP, YangGP, SandersL, et al.. Expression of Cyr61, a growth factor-inducible immediate-early gene. Mol Cell Biol, 1990, 10(7): 3569-3577

[4] ZemansRL, McClendonJ, AschnerY, et al.. Role of beta-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury. Am J Physiol Lung Cell Mol Physiol, 2013

[5] ChambellanA, CruickshankPJ, McKenzieP, et al.. Gene expression profile of human airway epithelium induced by hyperoxia in vivo. Am J Respir Cell Mol 677 Biol, 2006, 35(4): 424-435

[6] KonigshoffM, KramerM, BalsaraN, et al.. WNT1-inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J Clin Investig, 2009, 119(4): 772-787

[7] CaoY, ChenHL, ChengS, et al.. Inhibitory effect of dexamethasone on expression of cysteine-rich 61 protein in airway epithelial cells of allergic mouse models. J Huazhong Univ Sei Technol Med Sci, 2013, 33(5): 628-631

[8] DolinayT, KaminskiN, FelgendreherM, et al.. Gene expression profiling of target genes in ventilator-induced lung injury. Physiol Genomics, 2006, 26(1): 68-75

[9] WallaceMJ, ProbynME, ZahraVA, et al.. Early biomarkers and potential mediators of ventilationinduced lung injury in very preterm lambs. Respir Res, 2009, 10(1): 1465-9921

[10] MaSF, GrigoryevDN, TaylorAD, et al.. Bioinformatic identification of novel early stress response genes in rodent models of lung injury. Am J Physiol Lung Cell Mol Physiol, 2005, 289: L468-L477

[11] GrazioliS, GilS, AnD, et al.. Cyr61 (CCN1) overexpression induces lung injury in mice. Am J Physiol Lung Cell Mol Physiol, 2015

[12] SilvaPL, NegriniD, RoccoPR, et al.. Mechanisms of ventilator-induced lung injury in healthy lungs. Best Pract Res Clin Anaesthesiol, 2015, 29(3): 301-313

[13] ChaqourB, Goppelt-StruebeM, et al.. Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins. FEBS Js, 20063639-3649

[14] ZhouD, HerrickDJ, RosenbloomJ, et al.. Cyr61 mediates the expression of VEGF, alphav-integrin, and alpha-actin genes through cytoskeletally based mechanotransduction mechanisms in bladder smooth muscle cells. JAppl Physiol, 2005, 98(6): 2344-2354

[15] RanieriVM, SuterPM, TortorellaC, et al.. Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial. Jama, 1999, 282(1): 54-61

[16] LiLF, Ouyang, ChoukrounG, et al.. Stretch-induced IL-8 depends on c-Jun NH2-terminal and nuclear factor-kappaB-inducing kinases. Am J Physiol Lung Cell Mol Physiol, 2003, 285: L464-L475

[17] NingQ, WangX. Role of Rel A and IkappaB of nuclear factor kappaB in the release of interleukin-8 by cyclic mechanical strain in human alveolar type II epithelial cells A549. Respirology, 2007, 12(6): 792-798

[18] TschumperlinDJ, MarguliesSS. Alveolar epithelial surface area volume relationship in isolated rat lungs. JAppl Physio, 1999, 86(6): 2026-2033

[19] HadjiargyrouM, AhrensW, RubinCT, et al.. Temporal expression of the chondrogenic and angiogenic growth factor CYR61 during fracture repair. J Bone Miner Res, 2000, 15(6): 1014-1023

[20] MoonHG, KimSH, GaoJ, et al.. CCN1 secretion and cleavage regulate the lung epithelial cell functions after cigarette smoke. Am J Physiol Lung Cell Mol Physiol, 2014L326-L337

[21] LiuHM, LiuYT, ZhangJ, et al.. Bone marrow mesenchymal stem cells ameliorate lung injury through anti-inflammatory and antibacterial effect in COPD mice. J Huazhong Univ Sei Technolog Med Sci, 2017, 37(4): 496-504