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Lysophosphatidylcholine-induced mitochondrial fission contributes to collagen production in human cardiac fibroblasts

2019; Elsevier BV; Volume: 60; Issue: 9 Linguagem: Inglês

10.1194/jlr.ra119000141

ISSN

1539-7262

Autores

Hui‐Ching Tseng, Chih‐Chung Lin, Li‐Der Hsiao, Chuen‐Mao Yang,

Tópico(s)

Cardiovascular Function and Risk Factors

Resumo

Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation. Here, we evaluated the mechanisms underlying LPC-mediated collagen induction via mitochondrial events in human cardiac fibroblasts (HCFs), coupling application of the pharmacologic cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and genetic mutations in FOXO1 on the fibrosis pathway. In HCFs, LPC caused prostaglandin E2 (PGE2)/PGE2 receptor 4 (EP4)-dependent collagen induction via activation of transcriptional activity of forkhead box protein O1 (FoxO1) on COX-2 gene expression. These responses were mediated through LPC-induced generation of mitochondrial reactive oxygen species (mitoROS), as confirmed by ex vivo studies, which indicated that LPC increased COX-2 expression and oxidative stress. LPC-induced mitoROS mediated the activation of protein kinase C (PKC)α, which interacted with and phosphorylated dynamin-related protein 1 (Drp1) at Ser616, thereby increasing Drp1-mediated mitochondrial fission and mitochondrial depolarization. Furthermore, inhibition of PKCα and Drp1 reduced FoxO1-mediated phosphorylation at Ser256 and nuclear accumulation, which suppressed COX-2/PGE2 expression and collagen production. Moreover, pretreatment with celecoxib or COX-2 siRNA suppressed WT FoxO1; mutated Ser256-to-Asp256 FoxO1-enhanced collagen induction, which was reversed by addition of PGE2. Our results demonstrate that LPC-induced generation of mitoROS regulates PKCα-mediated Drp1-dependent mitochondrial fission and COX-2 expression via a PKCα/Drp1/FoxO1 cascade, leading to PGE2/EP4-mediated collagen induction. These findings provide new insights about the role of LPC in the pathway of fibrotic injury in HCFs. Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation. Here, we evaluated the mechanisms underlying LPC-mediated collagen induction via mitochondrial events in human cardiac fibroblasts (HCFs), coupling application of the pharmacologic cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and genetic mutations in FOXO1 on the fibrosis pathway. In HCFs, LPC caused prostaglandin E2 (PGE2)/PGE2 receptor 4 (EP4)-dependent collagen induction via activation of transcriptional activity of forkhead box protein O1 (FoxO1) on COX-2 gene expression. These responses were mediated through LPC-induced generation of mitochondrial reactive oxygen species (mitoROS), as confirmed by ex vivo studies, which indicated that LPC increased COX-2 expression and oxidative stress. LPC-induced mitoROS mediated the activation of protein kinase C (PKC)α, which interacted with and phosphorylated dynamin-related protein 1 (Drp1) at Ser616, thereby increasing Drp1-mediated mitochondrial fission and mitochondrial depolarization. Furthermore, inhibition of PKCα and Drp1 reduced FoxO1-mediated phosphorylation at Ser256 and nuclear accumulation, which suppressed COX-2/PGE2 expression and collagen production. Moreover, pretreatment with celecoxib or COX-2 siRNA suppressed WT FoxO1; mutated Ser256-to-Asp256 FoxO1-enhanced collagen induction, which was reversed by addition of PGE2. Our results demonstrate that LPC-induced generation of mitoROS regulates PKCα-mediated Drp1-dependent mitochondrial fission and COX-2 expression via a PKCα/Drp1/FoxO1 cascade, leading to PGE2/EP4-mediated collagen induction. These findings provide new insights about the role of LPC in the pathway of fibrotic injury in HCFs. Cardiac fibrosis is characterized by activation of cardiac fibroblasts (CFs), persistence of differentiated myofibroblasts, and synthesis of excessive extracellular matrix (ECM) triggered by various factors (1González A. Schelbert E.B. Diez J. Butler J. Myocardial interstitial fibrosis in heart failure: biological and translational perspectives.J. Am. Coll. Cardiol. 2018; 71: 1696-1706Crossref PubMed Scopus (261) Google Scholar, 2Leask A. Getting to the heart of the matter: new insights into cardiac fibrosis.Circ. Res. 2015; 116: 1269-1276Crossref PubMed Scopus (245) Google Scholar). Lysophosphatidylcholine (LPC) is hydrolyzed by phospholipase A2 that is generated from cell membrane-derived phosphatidylcholine and accumulates in ischemic and injured myocardium, associating with cardiomyocyte apoptosis in fibrotic hearts (3Huang J.P. Cheng M.L. Wang C.H. Shiao M.S. Chen J.K. Hung L.M. High-fructose and high-fat feeding correspondingly lead to the development of lysoPC-associated apoptotic cardiomyopathy and adrenergic signaling-related cardiac hypertrophy.Int. J. Cardiol. 2016; 215: 65-76Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 4Nam M. Jung Y. Ryu D.H. Hwang G.S. A metabolomics-driven approach reveals metabolic responses and mechanisms in the rat heart following myocardial infarction.Int. J. Cardiol. 2017; 227: 239-246Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). LPC acts as a pro-inflammatory mediator and induces interleukin-6 (IL-6) expression (5Tseng H.C. Lin C.C. Wang C.Y. Yang C.C. Hsiao L.D. Yang C.M. Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.Cell. Mol. Life Sci. 2018; 75: 4599-4617Crossref PubMed Scopus (8) Google Scholar), whereas IL-6 is involved in fibroblast activation (6Wang J.H. Zhao L. Pan X. Chen N.N. Chen J. Gong Q.L. Su F. Yan J. Zhang Y. Zhang S.H. Hypoxia-stimulated cardiac fibroblast production of IL-6 promotes myocardial fibrosis via the TGF-beta1 signaling pathway.Lab. Invest. 2016; 96: 1035Crossref PubMed Scopus (4) Google Scholar). Therefore, we investigated to determine whether LPC-regulated fibrotic events resulted from collagen production in human CFs (HCFs). Mitochondrial fission increases mitochondrial fragmentation that reflects mitochondrial membrane depolarization (7Twig G. Shirihai O.S. The interplay between mitochondrial dynamics and mitophagy.Antioxid. Redox Signal. 2011; 14: 1939-1951Crossref PubMed Scopus (517) Google Scholar) and excessive mitochondrial reactive oxygen species (mitoROS) production (8Huang M. Wei R. Wang Y. Su T. Li P. Chen X. The uremic toxin hippurate promotes endothelial dysfunction via the activation of Drp1-mediated mitochondrial fission.Redox Biol. 2018; 16: 303-313Crossref PubMed Scopus (50) Google Scholar, 9Sharp W.W. Fang Y.H. Han M. Zhang H.J. Hong Z. Banathy A. Morrow E. Ryan J.J. Archer S.L. Dynamin-related protein 1 (Drp1)-mediated diastolic dysfunction in myocardial ischemia-reperfusion injury: therapeutic benefits of Drp1 inhibition to reduce mitochondrial fission.FASEB J. 2014; 28: 316-326Crossref PubMed Scopus (247) Google Scholar, 10Zhou H. Wang S. Zhu P. Hu S. Chen Y. Ren J. Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission.Redox Biol. 2018; 15: 335-346Crossref PubMed Scopus (292) Google Scholar). Dynamin-related protein 1 (Drp1) serves as an initiator of mitochondrial fission when it is translocated from the cytosol to the mitochondrial outer membrane (10Zhou H. Wang S. Zhu P. Hu S. Chen Y. Ren J. Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission.Redox Biol. 2018; 15: 335-346Crossref PubMed Scopus (292) Google Scholar, 11Qi X. Disatnik M.H. Shen N. Sobel R.A. Mochly-Rosen D. Aberrant mitochondrial fission in neurons induced by protein kinase C{delta} under oxidative stress conditions in vivo.Mol. Biol. Cell. 2011; 22: 256-265Crossref PubMed Scopus (0) Google Scholar). Drp1 is regulated by posttranslational modifications, including phosphorylation and dephosphorylation (12Flippo K.H. Strack S. Mitochondrial dynamics in neuronal injury, development and plasticity.J. Cell Sci. 2017; 130: 671-681Crossref PubMed Scopus (149) Google Scholar). The phosphorylation of Drp1 at different amino acid residues plays opposite roles in mitochondrial fission: phosphorylation at Ser616 and Ser637 is responsible for the initiation and inhibition of fission, respectively (10Zhou H. Wang S. Zhu P. Hu S. Chen Y. Ren J. Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission.Redox Biol. 2018; 15: 335-346Crossref PubMed Scopus (292) Google Scholar, 13Tsushima K. Bugger H. Wende A.R. Soto J. Jenson G.A. Tor A.R. McGlauflin R. Kenny H.C. Zhang Y. Souvenir R. et al.Mitochondrial reactive oxygen species in lipotoxic hearts induce post-translational modifications of AKAP121, DRP1, and OPA1 that promote mitochondrial fission.Circ. Res. 2018; 122: 58-73Crossref PubMed Scopus (147) Google Scholar). In contrast, overproduction of ROS also contributes to Drp1-mediated mitochondrial fission (13Tsushima K. Bugger H. Wende A.R. Soto J. Jenson G.A. Tor A.R. McGlauflin R. Kenny H.C. Zhang Y. Souvenir R. et al.Mitochondrial reactive oxygen species in lipotoxic hearts induce post-translational modifications of AKAP121, DRP1, and OPA1 that promote mitochondrial fission.Circ. Res. 2018; 122: 58-73Crossref PubMed Scopus (147) Google Scholar). LPC has been reported to facilitate mitoROS generation (14Jiang S. Park D.W. Stigler W.S. Creighton J. Ravi S. Darley-Usmar V. Zmijewski J.W. Mitochondria and AMP-activated protein kinase-dependent mechanism of efferocytosis.J. Biol. Chem. 2013; 288: 26013-26026Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Li X. Fang P. Li Y. Kuo Y.M. Andrews A.J. Nanayakkara G. Johnson C. Fu H. Shan H. Du F. et al.Mitochondrial reactive oxygen species mediate lysophosphatidylcholine-induced endothelial cell activation.Arterioscler. Thromb. Vasc. Biol. 2016; 36: 1090-1100Crossref PubMed Scopus (138) Google Scholar) and mitochondrial membrane depolarization (14Jiang S. Park D.W. Stigler W.S. Creighton J. Ravi S. Darley-Usmar V. Zmijewski J.W. Mitochondria and AMP-activated protein kinase-dependent mechanism of efferocytosis.J. Biol. Chem. 2013; 288: 26013-26026Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Nonetheless, whether LPC impairs mitochondrial function, in particular, via Drp1-mediated mitochondrial fission in HCFs, remains unknown. Protein kinase Cs (PKCs) consist of a catalytic domain (16Steinberg S.F. Structural basis of protein kinase C isoform function.Physiol. Rev. 2008; 88: 1341-1378Crossref PubMed Scopus (644) Google Scholar), which requires phospholipids, such as LPC, to promote PKCα activation (17Motley E.D. Kabir S.M. Gardner C.D. Eguchi K. Frank G.D. Kuroki T. Ohba M. Yamakawa T. Eguchi S. Lysophosphatidylcholine inhibits insulin-induced Akt activation through protein kinase C-alpha in vascular smooth muscle cells.Hypertension. 2002; 39: 508-512Crossref PubMed Scopus (54) Google Scholar). However, the role of ROS in interaction between PKCα and Drp1 is not defined in HCFs. Therefore, we investigated the interaction between PKCα and Drp1 leading to Drp1-mediated mitochondrial fragmentation. Cyclooxygenase-2 (COX-2) is responsible for the synthesis of prostaglandins (PGs), including prostaglandin E2 (PGE2) (18Greenhough A. Smartt H.J. Moore A.E. Roberts H.R. Williams A.C. Paraskeva C. Kaidi A. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment.Carcinogenesis. 2009; 30: 377-386Crossref PubMed Scopus (981) Google Scholar). The COX-2/PGE2 axis is involved in various pathophysiological processes, including inflammation, tumorigenesis, and proliferation (18Greenhough A. Smartt H.J. Moore A.E. Roberts H.R. Williams A.C. Paraskeva C. Kaidi A. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment.Carcinogenesis. 2009; 30: 377-386Crossref PubMed Scopus (981) Google Scholar, 19Chien P.T. Hsieh H.L. Chi P.L. Yang C.M. PAR1-dependent COX-2/PGE2 production contributes to cell proliferation via EP2 receptors in primary human cardiomyocytes.Br. J. Pharmacol. 2014; 171: 4504-4519Crossref PubMed Scopus (17) Google Scholar, 20Hsu C.K. Lee I.T. Lin C.C. Hsiao L.D. Yang C.M. Sphingosine-1-phosphate mediates COX-2 expression and PGE2 /IL-6 secretion via c-Src-dependent AP-1 activation.J. Cell. Physiol. 2015; 230: 702-715Crossref PubMed Scopus (49) Google Scholar). The upregulation of COX-2 in the myocardium is associated with heart failure (21Wong S.C. Fukuchi M. Melnyk P. Rodger I. Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure.Circulation. 1998; 98: 100-103Crossref PubMed Scopus (271) Google Scholar). The levels of PGE2 at tissue sites are accompanied by collagen deposition (22Avendaño M.S. Martinez-Revelles S. Aguado A. Simoes M.R. Gonzalez-Amor M. Palacios R. Guillem-Llobat P. Vassallo D.V. Vila L. Garcia-Puig J. et al.Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension.Br. J. Pharmacol. 2016; 173: 1541-1555Crossref PubMed Scopus (44) Google Scholar, 23Charo C. Holla V. Arumugam T. Hwang R. Yang P. Dubois R.N. Menter D.G. Logsdon C.D. Ramachandran V. Prostaglandin E2 regulates pancreatic stellate cell activity via the EP4 receptor.Pancreas. 2013; 42: 467-474Crossref PubMed Scopus (37) Google Scholar). Although the contribution of the LPC-induced COX-2/PG axis in collagen production is not well-established, induction of PGE2 can auto-regulate PGE2 receptors (EPs), including EP1–EP4. EP2 and EP3 have been shown to inhibit collagen synthesis (24Liu S. Ji Y. Yao J. Zhao X. Xu H. Guan Y. Breyer R.M. Sheng H. Zhu J. Knockout of the prostaglandin E2 receptor subtype 3 promotes eccentric cardiac hypertrophy and fibrosis in mice.J. Cardiovasc. Pharmacol. Ther. 2017; 22: 71-82Crossref PubMed Scopus (14) Google Scholar, 25Pomianowska E. Sandnes D. Grzyb K. Schjolberg A.R. Aasrum M. Tveteraas I.H. Tjomsland V. Christoffersen T. Gladhaug I.P. Inhibitory effects of prostaglandin E2 on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma.BMC Cancer. 2014; 14: 413Crossref PubMed Scopus (24) Google Scholar, 26Zhao J. Shu B. Chen L. Tang J. Zhang L. Xie J. Liu X. Xu Y. Qi S. Prostaglandin E2 inhibits collagen synthesis in dermal fibroblasts and prevents hypertrophic scar formation in vivo.Exp. Dermatol. 2016; 25: 604-610Crossref PubMed Scopus (30) Google Scholar). In contrast, activation of EP4 can increase collagen synthesis (23Charo C. Holla V. Arumugam T. Hwang R. Yang P. Dubois R.N. Menter D.G. Logsdon C.D. Ramachandran V. Prostaglandin E2 regulates pancreatic stellate cell activity via the EP4 receptor.Pancreas. 2013; 42: 467-474Crossref PubMed Scopus (37) Google Scholar). However, whether LPC-induced COX-2/PGE2-dependent IL-6 expression could promote collagen induction is not completely elucidated in HCFs. Here, we demonstrated that LPC-induced mitoROS generation contributed to PGE2/EP4-dependent collagen induction. Mechanistically, mitoROS, induced by LPC, were found to regulate activation of PKCα that interacted with Drp1, leading to mitochondrial fragmentation and depolarization. In addition, LPC-regulated COX-2 expression was mediated via the mitoROS/PKCα/Drp1 cascade in HCFs. Our study provided new insights into a relationship between mitochondrial events and COX-2-dependent collagen induction in HCFs exposed to LPC. Anti-phospho-forkhead box protein O1 (FoxO1) (Ser256) (rabbit polyclonal antibody, Cat# 9461), anti-phospho-JNK1/2 (rabbit monoclonal antibody, Cat# 4668), anti-phospho-Drp1 (Ser616) (rabbit polyclonal antibody, Cat# 3455), anti-phospho-Drp1 (Ser637) (rabbit monoclonal antibody, Cat# 6319), anti-Drp1 (rabbit monoclonal antibody, Cat# 5391), and anti-FoxO1 (rabbit monoclonal antibody, Cat# 2880) antibodies were obtained from Cell Signaling (Danvers, MA). Anti-COX-2 (rabbit monoclonal antibody, Cat# ab62331), anti-phospho-PKCα (rabbit monoclonal antibody, Cat# ab180848), and anti-TOM20 (mouse monoclonal antibody, Cat# ab56783) antibodies were obtained from Abcam (Cambridge, UK). Anti-JNK1/2 (mouse monoclonal antibody, Cat# sc-137020), anti-PKCα (rabbit polyclonal antibody, Cat# sc-208), and anti-lamin A (rabbit polyclonal antibody, Cat# sc-20680) antibodies were obtained from Santa Cruz (Santa Cruz, CA). Anti-GAPDH (mouse monoclonal antibody, Cat# MCA-1D4) antibody was obtained from EnCor Biotechnology (Gainesville, FL). LPC (L-0906) was obtained from Sigma-Aldrich (St. Louis, MO). LPC was dissolved in 50% ethanol and filtered through a 0.22 μm syringe filter. A final concentration of 0.5% ethanol was used for the experiments. NS-398, Gö 6976, SP600125, and celecoxib were obtained from Biomol (Plymouth Meeting, PA). MitoQ and Gö 6983 were obtained from Cayman Chemicals (Ann Arbor, MI). AS1842856 was obtained from EMD Millipore (Billerica, MA). MitoTEMPO, dynasore, and mdivi-1 were obtained from Santa Cruz. The pharmacological inhibitors were dissolved in DMSO at a working concentration of 0.5% DMSO used for the all experiments. SDS-PAGE reagents were obtained from MDBio Inc. (Taipei, Taiwan). All animal care and experimental procedures complied with the UK Animals (Scientific Procedures) Act (19860, Directive 2010/63/EU) of the European Parliament and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (National Institutes of Health Publication No. 85-23, revised 1996). Animal studies are reported in compliance with the ARRIVE. Male Institute of Cancer Research mice (25–30 g, 8 weeks old) were purchased from the National Laboratory Animal Centre (Taipei, Taiwan) and randomly assigned to standard cages, with five animals per cage and kept in standard housing conditions with food and water ad libitum, according to the guidelines of Animal Care Committee of Chang Gung University (Approval Document No. CGU 16-046) and National Institutes of Health Guide for the Care and Use of Laboratory Animals. Institute of Cancer Research mice were anesthetized with one injection of Zoletil (40 mg/kg ip) and xylazine (10 mg/kg ip). After anesthesia, mice were withdrawn with lined forceps on the paws, and then their chests were opened and the hearts were quickly removed for the experiments. The cardiac apexes of the mice were sliced into three segments and assigned randomly into three groups: vehicle [containing 0.5% (v/v) ethanol and 0.5% (v/v) DMSO], LPC treatment [containing 40 μM LPC with 0.5% (v/v) ethanol and 0.5% (v/v) DMSO], and MitoTEMPO plus LPC treatment [containing 10 μM MitoTEMPO plus 40 μM LPC with 0.5% (v/v) ethanol and 0.5% (v/v) DMSO]; five slices were chosen from each group. The slices of cardiac apexes were pretreated with the inhibitors for 1 h, and then incubated with LPC for 6 h in Krebs solution (pH 7.4 at 37°C). The homogenates of cardiac apexes were prepared and lysed in a lysis buffer and subjected to Western blot analysis and RT-quantitative (q)PCR, as previously described (5Tseng H.C. Lin C.C. Wang C.Y. Yang C.C. Hsiao L.D. Yang C.M. Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.Cell. Mol. Life Sci. 2018; 75: 4599-4617Crossref PubMed Scopus (8) Google Scholar). The ex vivo heart apexes, with or without respective inhibitor treatment for 1 h, were incubated with 40 μM LPC for 6 h. The homogenates were used to measure the ratio of GSH/GSSG as the marker of oxidative stress in the heart tissues, which was determined using a glutathione detection kit according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY). HCFs were purchased from ScienCell Research Laboratories (San Diego, CA) and maintained in DMEM/nutrient mixture F-12 (DMEM/F-12) medium supplemented with 10% FBS, as previously described (27Lin C.C. Yang C.C. Wang C.Y. Tseng H.C. Pan C.S. Hsiao L.D. Yang C.M. NADPH oxidase/ROS-dependent VCAM-1 induction on TNF-α-challenged human cardiac fibroblasts enhances monocyte adhesion.Front. Pharmacol. 2016; 6: 310Crossref PubMed Scopus (24) Google Scholar). Growth-arrested HCFs were incubated without or with different concentrations of LPC at 37°C for the indicated time intervals. When pharmacological inhibitors were used, they were added 1 h prior to the exposure to LPC. After incubation, the cells were rapidly washed with ice-cold PBS and lysed with a sample buffer containing 125 mM Tris-HCl, 1.25% SDS, 6.25% glycerol, 3.2% β-mercaptoethanol, and 7.5 nM bromophenol blue with pH 6.8. Samples were denatured, subjected to SDS-PAGE using a 10% (w/v) running gel, and transferred to nitrocellulose membrane (BioTrace™ NT membrane, Pall Life Sciences, Ann Arbor, MI). The membranes were immunoblotted with one of the primary antibodies (1:1,000 dilution) overnight at 4°C, followed by incubation with a peroxidase-conjugated secondary antibody at room temperature for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence reagent (Western Lighting Plus; Perkin Elmer, Waltham, MA). The images of the immunoblots were acquired using a UVP BioSpectrum 500 imaging system (Upland, CA), and densitometric analysis was conducted using UN-SCAN-IT gel software (Orem, UT). Total RNA was extracted with TRIzol (Sigma-Aldrich) according to the manufacturer's instructions. First-strand cDNA synthesis was performed with 5 μg of total RNA using Oligo(dT)15 as primer in a final volume of 20 μl [25 ng/μl Oligo(dT)15, 0.5 mM dNTPs, 10 mM DTT, 2 units/μl RNase inhibitor, and 10 units/μl of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA)]. The synthesized cDNAs were used as templates for PCR reaction using Q-Amp™ 2× Screening Fire Taq Master Mix (Bio-Genesis Technologies, Taipei, Taiwan) and primers for the target genes. qPCR was performed using Luna Universal probe qPCR Master Mix (M3004; New England BioLabs, Beverly, MA) on a StepOnePlus™ real-time PCR system (Applied Biosystems, Foster City, CA). The relative amount of the target gene was calculated using 2(Ct test gene-Ct GAPDH) (Ct = threshold cycle). The sequences of the primers used are shown in supplemental Table S1. HCFs were seeded in 6-well culture plates. After reaching 90% confluence, the cells were shifted into serum-free DMEM/F-12 overnight and treated with 40 μM of LPC for the indicated time intervals in either the presence or the absence of pharmacological inhibitors. The media were collected and the levels of soluble collagens (type I–V collagen) were analyzed using a Sircol collagen assay kit (Biocolor, Northern Ireland, UK). HCFs were seeded in 6-well culture plates with coverslips. After the cells reached 90% confluence, they were shifted to serum-free DMEM/F-12 and then incubated with LPC for the indicated time intervals in either the presence or the absence of pharmacological inhibitors 1 h prior to LPC exposure. Then, cells were incubated in DMEM/F12 medium containing 5 μM of MitoSOX Red at 37°C for 10 min. The fluorescence signals were recorded (excitation/emission: 510 nm/570 nm) using a fluorescence plate reader (Synergy HT1; BioTek, Winooski, VT). To visualize mitoROS generation, the cells were washed thrice with media, and their fluorescence intensity was determined by fluorescence microscopy with a rhodamine filter (Axiovert 200M; Carl Zeiss, Thornwood, NY) and quantified using ImageJ software (1.41v; US National Institutes of Health). HCFs were seeded in 6-well culture plates with coverslips. After the cells reached 90% confluence, they were transferred to serum-free DMEM/F-12 overnight. When pharmacological inhibitors were used, they were added 1 h prior to the exposure to LPC. After LPC treatment, the cells were washed with media, and then incubated in DMEM/F12 medium containing 500 nM MitoTracker Green (Invitrogen) for 10 min. To visualize mitochondrial morphology, the cells were washed thrice with media, and then observed using a fluorescence microscope with a FITC filter (Axiovert 200M; Carl Zeiss). HCFs were seeded in 6-well culture plates with coverslips or 24-well plates. After the cells reached 90% confluence, they were cultured in serum-free DMEM/F-12, and then stimulated with LPC for the indicated time intervals in either the presence or the absence of pharmacological inhibitors for 1 h prior to LPC exposure. To detect mitochondrial membrane potential (Δψm), cells were subjected to a mitochondria staining kit (CS0390; Sigma-Aldrich). Cells were cultured in 10 μg/ml 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) in DMEM/F-12 containing 10% FBS at 37°C for 10 min. In healthy cells, JC-1 monomers accumulated as aggregates in the mitochondria due to existing mitochondrial polarization. These aggregates were visible on the red channel (rhodamine) when viewed with a fluorescence microscope. The JC-1 exists as a monomer and was visible on the green channel (FITC) when mitochondria were depolarized. The fluorescent images were captured with a fluorescence microscope (Axiovert 200M; Carl Zeiss) and quantified using ImageJ software (1.41v; US National Institutes of Health). Moreover, JC-1 fluorescence was also measured in a fluorescence plate reader (Synergy™M H1 Hybrid Reader; BioTek). The fluorescence intensities of JC-1 monomer were measured by excitation at 490 nm and emission at 530 nm, and fluorescence intensities of JC-1 aggregates were measured by excitation at 520 nm and emission at 590 nm. HCFs were plated in 12-well plates, 6-well plates, or 10 cm dishes and after reaching about 90% confluence, were transferred to fresh serum-free DMEM/F-12 medium before transfection. The siRNAs of COX-2 (SASI_Hs01_00152843), EP2 (SASI_Hs01_00158176), EP3 (SASI_Hs02_00303570), EP4 (SASI_Hs02_00105505), FoxO1 (SASI_Hs01_0076732), and scrambled siRNA were obtained from Sigma-Aldrich. The sequences of siRNAs are shown in supplemental Table S2. Transient transfection of siRNA was conducted using GenMute™ siRNA transfection reagent according to the manufacturer's instructions (SignaGen Laboratories, Gaithersburg, MD). The siRNA (100 nM) was added to each well, and then the cells were incubated at 37°C for 6 h. The cells were transferred to DMEM/F-12 medium containing 10% FBS for an additional 6 h, washed twice with PBS, and then maintained in serum-free DMEM/F-12 medium for 24 h before treatment with LPC. Ser256-to-Ala256 (S256A) FoxO1 mutant and Ser256-to-Asp256 (S256D) FoxO1 mutant were cloned into the EcoRV-HindIII site of the pCMV-Tag2B vector, as previously described (5Tseng H.C. Lin C.C. Wang C.Y. Yang C.C. Hsiao L.D. Yang C.M. Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.Cell. Mol. Life Sci. 2018; 75: 4599-4617Crossref PubMed Scopus (8) Google Scholar). HCFs were seeded in 6-well plates or 10 cm dishes and after they reached 90% confluence, they were transferred to serum-free DMEM/F-12 medium and transiently transfected with plasmid DNA using an X-tremeGENE™ HP DNA transfection reagent (Roche Applied Science, Indianapolis, IN), as previously described (5Tseng H.C. Lin C.C. Wang C.Y. Yang C.C. Hsiao L.D. Yang C.M. Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.Cell. Mol. Life Sci. 2018; 75: 4599-4617Crossref PubMed Scopus (8) Google Scholar). For construction of the COX-2-luc plasmid, a human COX-2 promoter, a region spanning from −484 to +37 was cloned into pGL3-basic vector, as previously described (5Tseng H.C. Lin C.C. Wang C.Y. Yang C.C. Hsiao L.D. Yang C.M. Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.Cell. Mol. Life Sci. 2018; 75: 4599-4617Crossref PubMed Scopus (8) Google Scholar). HCFs were cotransfected with pGL3b-cox-2 and pCMV-β-gal plasmid (as an internal control). Promoter activities of COX-2 were determined using a luciferase assay HIT kit (BioThema, Handen, Sweden) and normalized with β-Gal reporter gene as determined by using a Galacto-Light Plus™ system (Applied Biosystems, Bedford, MA). HCFs were seeded on coverslips in 6-well culture plates, and after they reached 90% confluence, they were transferred to serum-free DMEM/F-12 medium overnight, and then stimulated with 40 μM of LPC for the indicated time intervals. After washing twice with ice-cold PBS, the cells were fixed with 4% (w/v) paraformaldehyde in PBS for 30 min, and then permeabilized with 0.1% Triton X-100 in PBS for 15 min. The staining was performed by incubating with 5% BSA for 2 h at 37°C, followed by incubation with a primary anti-phospho-Drp1S616 rabbit polyclonal antibody (1:100 dilution) and anti-TOM20 mouse monoclonal antibody (1:1,000) overnight in PBS containing 1% BSA. The cells were washed thrice with PBS and incubated for 2 h with a FITC-conjugated goat anti-rabbit antibody and rhodamine-conjugated goat anti-mouse antibody (1:100 dilution; Jackson ImmunoResearch, West Grove, PA) in PBS containing 1% BSA. Finally, cells were washed thrice with PBS, and then mounted with aqueous mounting medium containing DAPI (H1200; Vector Laboratories, Burlingame, CA). Images were captured with a fluorescence microscope (Axiovert 200 M; Carl Zeiss). To detect the association of transcription factors with human COX-2 promoter, chromatin immunoprecipitation analysis was performed. Protein-DNA complexes were fixed by 1% formaldehyde in DMEM/F-12 medium and the reaction was terminated with 125 mM glycine. The sample was lysed, immunoprecipitated, washed, and eluted, as previously described (5Tseng H.C. Lin C.C. Wang C.Y. Yang C.C. Hsiao L.D. Yang C.M. Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.Cell. Mol. Life Sci. 2018; 75: 4599-4617Crossref PubMed Scopus (8) Google Scholar). The enrichment of specific DNA and input DNA (as an internal control) were subjected to PCR amplification. The primer sequences were: FoxO1 forward primer 5′-AAGACATCTGGCGGAAACC-3′ and reverse primer 5′-ACAATTGGTCGCTAACCG

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