FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2885341302> ?p ?o ?g. }

• W2885341302 endingPage "1189" @default.
• W2885341302 startingPage "1176" @default.
• W2885341302 abstract "•FXR1 interacts with HuR via mRNA tethering on the 3′ UTR of inflammatory transcripts•FXR1 is a negative regulator of inflammatory transcript mRNA stability•FXR1 binds canonical and non-canonical sequences in the 3′ UTR of TNFα•FXR1 is required for IL-19-dependent reduction of HuR mRNA stability and abundance This work identifies the fragile-X-related protein (FXR1) as a reciprocal regulator of HuR target transcripts in vascular smooth muscle cells (VSMCs). FXR1 was identified as an HuR-interacting protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HuR-FXR1 interaction is abrogated in RNase-treated extracts, indicating that their association is tethered by mRNAs. FXR1 expression is induced in diseased but not normal arteries. siRNA knockdown of FXR1 increases the abundance and stability of inflammatory mRNAs, while overexpression of FXR1 reduces their abundance and stability. Conditioned media from FXR1 siRNA-treated VSMCs enhance activation of naive VSMCs. RNA EMSA and RIP demonstrate that FXR1 interacts with an ARE and an element in the 3′ UTR of TNFα. FXR1 expression is increased in VSMCs challenged with the anti-inflammatory cytokine IL-19, and FXR1 is required for IL-19 reduction of HuR. This suggests that FXR1 is an anti-inflammation responsive, HuR counter-regulatory protein that reduces abundance of pro-inflammatory transcripts. This work identifies the fragile-X-related protein (FXR1) as a reciprocal regulator of HuR target transcripts in vascular smooth muscle cells (VSMCs). FXR1 was identified as an HuR-interacting protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HuR-FXR1 interaction is abrogated in RNase-treated extracts, indicating that their association is tethered by mRNAs. FXR1 expression is induced in diseased but not normal arteries. siRNA knockdown of FXR1 increases the abundance and stability of inflammatory mRNAs, while overexpression of FXR1 reduces their abundance and stability. Conditioned media from FXR1 siRNA-treated VSMCs enhance activation of naive VSMCs. RNA EMSA and RIP demonstrate that FXR1 interacts with an ARE and an element in the 3′ UTR of TNFα. FXR1 expression is increased in VSMCs challenged with the anti-inflammatory cytokine IL-19, and FXR1 is required for IL-19 reduction of HuR. This suggests that FXR1 is an anti-inflammation responsive, HuR counter-regulatory protein that reduces abundance of pro-inflammatory transcripts. Despite nutritional modification and lipid-reducing medications, atherosclerotic and other vascular syndromes account for 50% of all mortality and is increasing in the developing world. The injurious effects of pro-inflammatory cytokines resulting in vascular smooth muscle cell (VSMC) activation and development of multiple vascular diseases are well described (Allahverdian et al., 2014Allahverdian S. Chehroudi A.C. McManus B.M. Abraham T. Francis G.A. Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis.Circulation. 2014; 129: 1551-1559Crossref PubMed Scopus (379) Google Scholar, Ross, 1999Ross R. Atherosclerosis--an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19195) Google Scholar). Results from the recent CANTOS trial support the preeminent role of inflammation in vascular disease (Weber and von Hundelshausen, 2017Weber C. von Hundelshausen P. CANTOS Trial Validates the Inflammatory Pathogenesis of Atherosclerosis: Setting the Stage for a New Chapter in Therapeutic Targeting.Circ. Res. 2017; 121: 1119-1121Crossref PubMed Google Scholar). VSMCs respond to and synthesize pro-inflammatory immune modulators (Doran et al., 2008Doran A.C. Meller N. McNamara C.A. Role of smooth muscle cells in the initiation and early progression of atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 812-819Crossref PubMed Scopus (625) Google Scholar, Hansson and Libby, 2006Hansson G.K. Libby P. The immune response in atherosclerosis: a double-edged sword.Nat. Rev. Immunol. 2006; 6: 508-519Crossref PubMed Scopus (1781) Google Scholar, Singer et al., 2004Singer C.A. Salinthone S. Baker K.J. Gerthoffer W.T. Synthesis of immune modulators by smooth muscles.BioEssays. 2004; 26: 646-655Crossref PubMed Scopus (52) Google Scholar) and promulgate the recruitment of leukocytes to the lesion (Hansson and Libby, 2006Hansson G.K. Libby P. The immune response in atherosclerosis: a double-edged sword.Nat. Rev. Immunol. 2006; 6: 508-519Crossref PubMed Scopus (1781) Google Scholar, Libby et al., 1997Libby P. Geng Y.J. Sukhova G.K. Simon D.I. Lee R.T. Molecular determinants of atherosclerotic plaque vulnerability.Ann. N Y Acad. Sci. 1997; 811: 134-142Crossref PubMed Scopus (82) Google Scholar), leading to a localized vascular inflammatory lesion. In many vascular diseases, VSMCs migrate into the intima, where they proliferate and synthesize cytokines and matrix proteins leading to loss of lumen and subsequent tissue ischemia. Resolution of inflammation is a dynamic and tightly regulated process, and much attention has been aimed at identification of countervailing mechanisms that modulate inflammatory processes (Fredman and Tabas, 2017Fredman G. Tabas I. Boosting inflammation resolution in atherosclerosis: the next frontier for therapy.Am. J. Pathol. 2017; 187: 1211-1221Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Libby et al., 2014Libby P. Tabas I. Fredman G. Fisher E.A. Inflammation and its resolution as determinants of acute coronary syndromes.Circ. Res. 2014; 114: 1867-1879Crossref PubMed Scopus (350) Google Scholar). A better understanding of countervailing mechanisms that modulate inflammatory processes, and identification of proteins and pathways that modulate the VSMC response to injury is key to the development of therapeutics to combat multiple vascular diseases. The regulation of mRNA stability and translation are two levels of post-transcriptional regulation that permit VSMCs to rapidly respond to inflammatory stimuli (Barreau et al., 2006Barreau C. Paillard L. Osborne H.B. AU-rich elements and associated factors: are there unifying principles?.Nucleic Acids Res. 2006; 33: 7138-7150Crossref PubMed Scopus (781) Google Scholar). AU-rich elements (AREs) in the 3′ UTR of mammalian mRNA appear to be the target sequence for degradation or stabilization of transcripts. Most of the transcripts targeted for rapid degradation encode key regulatory proteins involved in cell growth, inflammation, and other responses to external stimuli (Bakheet et al., 2001Bakheet T. Frevel M. Williams B.R. Greer W. Khabar K.S. ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins.Nucleic Acids Res. 2001; 29: 246-254Crossref PubMed Scopus (342) Google Scholar). Importantly, most inflammatory cytokines contain conserved or semi-conserved AU-rich elements in their 3′ UTR, imparting target specificity for a potential anti-inflammatory modality (Peng et al., 1996Peng S.S. Chen C.Y. Shyu A.B. Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements.Mol. Cell. Biol. 1996; 16: 1490-1499Crossref PubMed Scopus (125) Google Scholar). Controlling mRNA decay allows the cell to fine-tune mRNA abundance and translation for rapid adaptation to environmental conditions, especially inflammation (Schoenberg and Maquat, 2012Schoenberg D.R. Maquat L.E. Regulation of cytoplasmic mRNA decay.Nat. Rev. Genet. 2012; 13: 246-259Crossref PubMed Scopus (2) Google Scholar). An essential regulatory protein involved in this process is human antigen R (HuR), a member of the Elav protein family and one of the best characterized, ARE-recognizing, RNA-binding proteins (RBPs) involved in mRNA stability and regulation of pro-inflammatory gene expression (Doller et al., 2008Doller A. Pfeilschifter J. Eberhardt W. Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR.Cell. Signal. 2008; 20: 2165-2173Crossref PubMed Scopus (180) Google Scholar, Palanisamy et al., 2012Palanisamy V. Jakymiw A. Van Tubergen E.A. D’Silva N.J. Kirkwood K.L. Control of cytokine mRNA expression by RNA-binding proteins and microRNAs.J. Dent. Res. 2012; 91: 651-658Crossref PubMed Scopus (85) Google Scholar). While HuR is ubiquitously expressed, it is activated in response to inflammatory signals to stabilize inflammatory mediators (Doller et al., 2008Doller A. Pfeilschifter J. Eberhardt W. Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR.Cell. Signal. 2008; 20: 2165-2173Crossref PubMed Scopus (180) Google Scholar, Palanisamy et al., 2012Palanisamy V. Jakymiw A. Van Tubergen E.A. D’Silva N.J. Kirkwood K.L. Control of cytokine mRNA expression by RNA-binding proteins and microRNAs.J. Dent. Res. 2012; 91: 651-658Crossref PubMed Scopus (85) Google Scholar). Since most pro-inflammatory transcripts contain AREs in their 3′ UTR, this is a crucial and specific mechanism for the initiation and maintenance of the pro-inflammatory phenotype observed in vascular diseases. The exact mechanisms of HuR regulation have yet to be characterized; however, they could represent key targets in regulating inflammation (Gallouzi and Steitz, 2001Gallouzi I.E. Steitz J.A. Delineation of mRNA export pathways by the use of cell-permeable peptides.Science. 2001; 294: 1895-1901Crossref PubMed Scopus (234) Google Scholar). Even though modulation of mRNA stability has been posited as a possible therapeutic strategy (Eberhardt et al., 2007Eberhardt W. Doller A. Akool S. Pfeilschifter J. Modulation of mRNA stability as a novel therapeutic approach.Pharmacol. Ther. 2007; 114: 56-73Crossref PubMed Scopus (139) Google Scholar), surprisingly, there is negligible literature exploring the concept that it could be directly regulated or possibly reduced by anti-inflammatory stimuli. We posit that the regulation of HuR and other RBPs is a critical, and understudied, step in the regulation of vascular inflammation. We previously reported that interleukin (IL)-19, an anti-inflammatory cytokine, reduced inflammatory transcript mRNA stability in VSMCs (Cuneo et al., 2010Cuneo A.A. Herrick D. Autieri M.V. Il-19 reduces VSMC activation by regulation of mRNA regulatory factor HuR and reduction of mRNA stability.J. Mol. Cell. Cardiol. 2010; 49: 647-654Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and reduced HuR abundance in several cell types (Ellison et al., 2013Ellison S. Gabunia K. Kelemen S.E. England R.N. Scalia R. Richards J.M. Orr A.W. Traylor Jr., J.G. Rogers T. Cornwell W. et al.Attenuation of experimental atherosclerosis by interleukin-19.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 2316-2324Crossref PubMed Scopus (48) Google Scholar). In this report, we identify and characterize one protein, termed fragile X-related protein (FXR1), a muscle-enhanced, autosomal homolog of the FMR (fragile X mental retardation) neural protein, which interacts with HuR in inflammatory, but not basal, conditions. We report here that FXR1 expression is induced by IL-19 in VSMCs and that modulation of FXR1 regulates ARE-containing transcripts in VSMCs. RNA EMSA (electrophoretic mobility shift assay) and RNA immunoprecipitation demonstrate that FXR1 interacts with the canonical AREs and a previously uncharacterized element in the 3′ UTR of tumor necrosis factor alpha (TNFα. This work implicates FXR1 as a previously unrecognized negative regulator of inflammation and suggests that IL-19 induction of FXR1 expression is a negative compensatory, counter-regulatory mechanism used by VSMCs to respond to and resolve inflammation. It is presumed that HuR activity is regulated by interacting proteins (Doller et al., 2008Doller A. Pfeilschifter J. Eberhardt W. Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR.Cell. Signal. 2008; 20: 2165-2173Crossref PubMed Scopus (180) Google Scholar, Gallouzi and Steitz, 2001Gallouzi I.E. Steitz J.A. Delineation of mRNA export pathways by the use of cell-permeable peptides.Science. 2001; 294: 1895-1901Crossref PubMed Scopus (234) Google Scholar, Pullmann et al., 2005Pullmann Jr., R. Juhaszova M. López de Silanes I. Kawai T. Mazan-Mamczarz K. Halushka M.K. Gorospe M. Enhanced proliferation of cultured human vascular smooth muscle cells linked to increased function of RNA-binding protein HuR.J. Biol. Chem. 2005; 280: 22819-22826Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Human vascular smooth muscle cells (hVSMCs) were transfected with a FLAG-tagged HuR or FLAG-tagged empty control vector and then starved for 48 hr in 0.1% fetal bovine serum (FBS) before stimulation with TNFα. HuR pull-down was followed by un-biased liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the proteins that immunoprecipitated with HuR. HuR-interacting candidates were identified by eliminating any protein with a raw peptide count below ten (Table S1). Interacting proteins were also scrutinized in the Contaminant Repository for Affinity Purification (CRAPome) database (Mellacheruvu et al., 2013Mellacheruvu D. Wright Z. Couzens A.L. Lambert J.-P. St-Denis N.A. Li T. Miteva Y.V. Hauri S. Sardiu M.E. Low T.Y. et al.The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.Nat. Methods. 2013; 10: 730-736Crossref PubMed Scopus (912) Google Scholar) to determine the occurrence of proteins in control experiments to eliminate “sticky proteins” that may non-specifically interact with HuR. The final list of proteins that met these criteria were examined for Gene Ontology (GO) annotation. A number of interacting proteins were identified; most were involved in various aspects of mRNA processing (Table S2). The last row of Table S2 includes Elav1 (HuR), as it was the bait protein used to perform LC-MS/MS, although nothing is known about HuR and the FLAG-tag epitope according to the CRAPome database. FXR1 was chosen for further study because of the novelty of its interaction; because FXR1 expression is muscle enhanced (Garnon et al., 2005Garnon J. Lachance C. Di Marco S. Hel Z. Marion D. Ruiz M.C. Newkirk M.M. Khandjian E.W. Radzioch D. Fragile X-related protein FXR1P regulates proinflammatory cytokine tumor necrosis factor expression at the post-transcriptional level.J. Biol. Chem. 2005; 280: 5750-5763Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, Mientjes et al., 2004Mientjes E.J. Willemsen R. Kirkpatrick L.L. Nieuwenhuizen I.M. Hoogeveen-Westerveld M. Verweij M. Reis S. Bardoni B. Hoogeveen A.T. Oostra B.A. Nelson D.L. Fxr1 knockout mice show a striated muscle phenotype: implications for Fxr1p function in vivo.Hum. Mol. Genet. 2004; 13: 1291-1302Crossref PubMed Scopus (105) Google Scholar); and because no literature exists on FXR1 inducibility by inflammatory stimuli or VSMC, making FXR1 a particularly attractive target to study in the context of vascular disease. Finally, similar to HuR, FXR1 is presumed to be an RBP (Adinolfi et al., 1999Adinolfi S. Bagni C. Musco G. Gibson T. Mazzarella L. Pastore A. Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.RNA. 1999; 5: 1248-1258Crossref PubMed Scopus (74) Google Scholar). FXR1 exists in several isoforms in mouse and is predicted to have three isoforms in human (Dubé et al., 2000Dubé M. Huot M.E. Khandjian E.W. Muscle specific fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast.BMC Genet. 2000; 1: 4Crossref PubMed Scopus (40) Google Scholar). We were only able to detect isoform 1 in human VSMCs by western blot and transcript-specific qRT-PCR (data not shown). Subsequent experiments utilized isoform 1 to ensure that all domains were represented. A series of immunoprecipitations were performed in order to confirm the interaction between HuR and FXR1. First, we performed a co-immunoprecipitation of endogenous FXR1 for HuR in hVSMCs that were either serum starved or serum starved and stimulated with TNFα for 8 hr (Figure 1A). We also overexpressed FXR1 using a FLAG-tagged adenovirus (adeno-FXR1 [AdFXR1]) and concurrently overexpressed HuR also using an adenovirus (AdHuR) in hVSMCs and performed immunoprecipitation using anti-FLAG-conjugated beads (Figure 1B). Next, hVSMCs were treated as described, but after serum starvation, they were stimulated with TNFα for 8 hr. Figure 1C shows that the HuR/FXR1 interaction was enhanced in TNFα-stimulated cells. Figure 1D shows that the FXR1-HuR interaction is abrogated by the addition of RNase A, suggesting that the interaction we identified via proteomics may be mediated by RNA tethering. The increased FXR1-HuR interaction observed in TNFα-stimulated VSMCs may be due to an increase in transcripts that harbor both FXR1 and HuR binding elements. We next used confocal microscopy to determine HuR and FXR1 localization under basal and inflammatory conditions in hVSMCs. HuR nucleocytoplasmic shuttling has been reported (Wu et al., 2016Wu T. Shi J.-X. Geng S. Zhou W. Shi Y. Su X. The MK2/HuR signaling pathway regulates TNF-α-induced ICAM-1 expression by promoting the stabilization of ICAM-1 mRNA.BMC Pulm. Med. 2016; 16: 84Crossref PubMed Scopus (17) Google Scholar), and consistent with the literature, in TNFα-stimulated VSMCs, HuR translocated from the nucleus to the cytoplasm. FXR1 remained predominantly cytoplasmic in both unstimulated and stimulated conditions. Interestingly, HuR and FXR1 co-localized in the cytoplasm following 8-hr TNFα stimulation, which is consistent with literature showing HuR nucleocytoplasmic translocation upon inflammatory stimuli (Figures 1E and 1F). RNA processing often occurs in phase-dense structures that form in the cytoplasm of eukaryotic cells in response to environmental stresses. The composition of stress granules suggests that regulation of labile ARE-containing inflammatory transcripts could be occurring there. To further associate a relationship between FXR1 and HuR in RNA processing, we used immunostaining and confocal microscopy to co-localize HuR and FXR1 within punctate stress granules in hVSMCs. Clotrimazole was used to induce stress granule formation, and poly(A)-binding protein (PABP) was used as a marker for stress granules (Kedersha et al., 2008Kedersha N. Tisdale S. Hickman T. Anderson P. Real-time and quantitative imaging of mammalian stress granules and processing bodies.Methods Enzymol. 2008; 448: 521-552Crossref PubMed Scopus (81) Google Scholar). Figure 1G shows that hVSMCs stimulated with 20 μM clotrimazole demonstrated well-defined, punctate co-localization of FXR1 and HuR in stress granules, which further suggests a role for FXR1 along with HuR in RNA processing in VSMCs. There is no literature describing FXR1 induction in VSMCs or models of vascular injury. We examined FXR1 expression in mouse and human atherosclerotic and restenotic tissue and detected inducible FXR1 expression in VSMCs in multiple models of vascular injury. Figure 2A indicates that FXR1 expression is increased in neointimal, compared with medial, VSMCs in the carotid artery from ligated mice. Similarly, FXR1 expression is increased in VSMCs in atherosclerotic plaque and cap, but much lower in non-diseased medial VSMCs in the aortic arch from LDLR−/− mice fed an atherogenic diet (Figure 2B). FXR1 expression is negligible in normal, non-diseased arteries from these mice (Figures S1A and S1B). Importantly, FXR1 expression is barely detectible in a coronary artery from a non-failing human heart, but expression is enhanced in myofibrous atherosclerotic plaque from a human coronary artery (Figures 2C and D). Figure 2E shows dual-color immunohistochemistry indicating that FXR1 expression co-localizes in plaque SMCs in the human coronary artery. Together, these data suggest that FXR1 induction is a VSMC response to inflammatory stimuli in vivo. Literature on FXR1 function is inconsistent and appears to be cell type specific. To more definitively link FXR1 function with vascular inflammation, we transfected hVSMCs with FXR1-specific small interfering RNA (siRNA) and then stimulated the cells with TNFα. Knockdown of FXR1 resulted in a dramatic and significant increase in the abundance of inflammatory transcripts in hVSMCs (IL-1 β, ICAM1, and MCP-1 are shown as examples). Interestingly, these transcripts have been previously shown to be stabilized by HuR in other cell types (Aguado et al., 2015Aguado A. Rodríguez C. Martínez-Revelles S. Avendaño M.S. Zhenyukh O. Orriols M. Martínez-González J. Alonso M.J. Briones A.M. Dixon D.A. Salaices M. HuR mediates the synergistic effects of angiotensin II and IL-1β on vascular COX-2 expression and cell migration.Br. J. Pharmacol. 2015; 172: 3028-3042Crossref PubMed Scopus (26) Google Scholar, Chen et al., 2006Chen Y.-L. Huang Y.-L. Lin N.-Y. Chen H.-C. Chiu W.-C. Chang C.-J. Differential regulation of ARE-mediated TNFalpha and IL-1beta mRNA stability by lipopolysaccharide in RAW264.7 cells.Biochem. Biophys. Res. Commun. 2006; 346: 160-168Crossref PubMed Scopus (78) Google Scholar, Krishnamurthy et al., 2010Krishnamurthy P. Lambers E. Verma S. Thorne T. Qin G. Losordo D.W. Kishore R. Myocardial knockdown of mRNA-stabilizing protein HuR attenuates post-MI inflammatory response and left ventricular dysfunction in IL-10-null mice.FASEB J. 2010; 24: 2484-2494Crossref PubMed Scopus (60) Google Scholar, Wu et al., 2016Wu T. Shi J.-X. Geng S. Zhou W. Shi Y. Su X. The MK2/HuR signaling pathway regulates TNF-α-induced ICAM-1 expression by promoting the stabilization of ICAM-1 mRNA.BMC Pulm. Med. 2016; 16: 84Crossref PubMed Scopus (17) Google Scholar) (Figure 3A). Correspondingly, Figure 3B shows that siRNA reduction of FXR1 also increases the abundance of inflammatory proteins as well as HuR in TNFα-stimulated human VSMCs. As FXR1 knockdown increased inflammatory transcripts, we reasoned that FXR1 overexpression would reduce abundance of inflammatory mRNA and protein. Adenoviral overexpression of FXR1 decreases the abundance of inflammatory mRNA (Figure 3C) and protein (Figure 3D) in a dose-dependent fashion compared with AdGFP control. The siRNA knockdown and overexpression data are complementary and strongly suggest that FXR1 expression may regulate abundance of inflammatory proteins as well as HuR in VSMCs. FMR1 family members are putative RBPs (Adinolfi et al., 1999Adinolfi S. Bagni C. Musco G. Gibson T. Mazzarella L. Pastore A. Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.RNA. 1999; 5: 1248-1258Crossref PubMed Scopus (74) Google Scholar), and we next determined whether modulation of FXR1 would affect mRNA stability. Using the transcription inhibitor actinomycin D in TNFα-stimulated VSMCs, we determined that the mRNA stability of ARE-containing transcripts IL-1 β, ICAM1, and HuR is significantly increased when FXR1 is knocked down and, importantly, significantly decreased when FXR1 is overexpressed (Figures 4A and 4B ). The mRNA stability of peroxisome proliferator-activated receptor alpha (PPARα), the expression of which is not regulated by AREs in its 3′ UTR, was not affected by FXR1 knockdown or overexpression, demonstrating target transcript specificity for FXR1 activity. Of particular importance was the finding that FXR1 appears to have a reciprocal relationship with HuR abundance, suggesting important, possibly competitive roles for these proteins in regulation of mRNA stability. Together, these results suggest a previously unrecognized function for FXR1 in regulation of mRNA stability and subsequent abundance of pro-inflammatory proteins. Maladaptive VSMC proliferation is a hallmark of several vascular pathologies and is driven by inflammatory gene expression (Hansson and Libby, 2006Hansson G.K. Libby P. The immune response in atherosclerosis: a double-edged sword.Nat. Rev. Immunol. 2006; 6: 508-519Crossref PubMed Scopus (1781) Google Scholar, Libby, 2002Libby P. Inflammation in atherosclerosis.Nature. 2002; 420: 868-874Crossref PubMed Scopus (6937) Google Scholar, Libby et al., 2014Libby P. Tabas I. Fredman G. Fisher E.A. Inflammation and its resolution as determinants of acute coronary syndromes.Circ. Res. 2014; 114: 1867-1879Crossref PubMed Scopus (350) Google Scholar, Ross, 1999Ross R. Atherosclerosis--an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19195) Google Scholar). Knockdown of FXR1 in hVSMCs significantly increased cell proliferation compared to scrambled control cells (Figure 5A). Concordantly, VSMC proliferation was significantly decreased in a manner inversely proportional with FXR1 expression, confirming that FXR1 can regulate VSMC proliferation (Figure 5B). VSMC paracrine signaling is also a characteristic of many vascular diseases. Since FXR1 appeared to regulate the abundance of cytokines, it was important to determine whether this participated in paracrine signaling. First, serum-starved VSMCs were stimulated with conditioned media collected from VSMCs transfected with FXR1 siRNA or scrambled controls for 4 hr; then, inflammatory transcript mRNA was quantitated. Figure 5C shows that hVSMCs treated with FXR1 siRNA knockdown conditioned media had increased inflammatory gene expression compared to scrambled control. Next, using conditioned media from scrambled control or FXR1 siRNA knockdown VSMCs, we performed a proliferation assay to demonstrate the autocrine and paracrine effects on cell growth (Figure 5D). hVSMCs treated with FXR1 siRNA conditioned media had significantly increased proliferation compared to scrambled media control. These data suggest that the reduction of FXR1 results in increased cytokine production that has potential autocrine and paracrine effects on other hVSMCs. Various complementary methods were used to determine whether FXR1 binds mRNA. First, glutathione S-transferase (GST) and human GST-FXR1 fusion proteins were used in RNA EMSAs (cRNA EMSAs) with a biotinylated probe consisting of a 50-bp region of the human TNFα 3′ UTR (position 1,333–1,380). The addition of recombinant FXR1 to this probe suggested that it bound to RNA (Figure 6A). The AUUUA monomer (as negative control probe) did not form a complex with FXR1. Interaction specificity was demonstrated by super-shift of the FXR1-probe complex by the addition of anti-FXR1 antibody (Figure 6B). FXR1 binding affinity for this region was calculated, and, in our hands, FXR1 has similar affinity to the TNFα probe as HuR (Figures S2A and S2B). To determine the site or sites on the TNFα 3′ UTR recognized by FXR1, four probes representing different regions of the 50-bp TNFα 3′ UTR (probe A) were synthesized; a 25-mer of (AUUUA)5 (probe B), a 27-mer of (UUAUUUAUU)3 (probe C), a 36-mer of (CUUGUGAUU)4 (probe D), and a 40-mer of (CAGAGAUGAA)4 (probe E), were added to the cRNA EMSAs to compete with the biotinylated 50-bp TNFα 3′ UTR probe. The GST negative control protein did not interact with any probes (Figures S3A and S3B). FXR1 binding with various amounts of cold competitor probes was performed to determine probe input concentrations (Figures S4A and S4B). Figure 6C shows that probes B and C, which contained recognized AREs, were capable of competing with the full-length probe for FXR1 binding and nearly ablated the gel shift. Interestingly, probe D, which does not contain a canonical ARE, also successfully competed with the full-length TNFα 3′ UTR for FXR1 binding but did not compete with the full-length probe for HuR, suggesting an additional, previously unrecognized region on the TNFα 3′ UTR recognized by FXR1. These results were quantified using densitometry as a percentage of GST-protein bound to probe A (Figure 6C, lower panel). An additional binding element termed the G quadruplex has been implicated as a binding site for FXR1 (Bechara et al., 2007Bechara E. Davidovic L. Melko M. Bensaid M. Tremblay S. Grosgeorge J. Khandjian E.W. Lalli E. Bardoni B. Fragile X related protein 1 isoforms differentially modulate the affinity of fragile X mental retardation protein for G-quartet RNA structure.Nucleic Acids Res. 2007; 35: 299-306Crossref PubMed Scopus (45) Google Scholar). Using this element as a cold competitor to the biotinylated TNFα 3′ UTR probe, we found that the G quadruplex was able to bind FXR1 (Figures S4C and S4D). Using RNA immunoprecipitation (RIP), we determined whether FXR1 directly binds RNAs that were shown to be regulated by FXR1. VSMCs were transduced with FLAG-tagged adeno-FXR1, serum starved for 48 hr, and then stimulated with TNFα for 8 hr. Figure 6D shows that several transcripts were identified as interacting with FXR1 compared to immunoglobulin G (IgG) control antibody. Importantly, mRNA transcripts not regulated by AREs in 3′ UTR such as PPARα were not amplified. It was possible that FXR1 could compete with HuR for occupancy on 3′ UTR of transcripts that contained these regions. A constitutively driven luciferase reporter representing the TNFα 3′ UTR and containing ARE tandem repeats was transfected into HEK cells and also transduced with plasmid encoding FXR1 cDNA or a control empty-vector plasmid. Figure 6E shows that FXR1 reduced luciferase activity, suggesting that FXR1 may compete with HuR for ARE occupancy. We also used an adenovirus expressing the TNFα 3′ UTR luciferase construct to perform the experiment in hVSMCs. Adeno-FXR1 and AdHuR were co-transduced with the adeno-expression TNFα 3′ UTR luciferase, as well as an AdGFP for control. Figure 6F demonstrates that, in hVSMCs, FXR1 overexpression reduced TNFα 3′ UTR luciferase activity, while HuR was able to increase it, supporting the concept that FXR1 is a negative regulator of inflammatory transcripts. IL-19 is anti-proliferative for VSMCs and reduce" @default.
• W2885341302 created "2018-08-22" @default.
• W2885341302 creator A5003137481 @default.
• W2885341302 creator A5005917520 @default.
• W2885341302 creator A5007039952 @default.
• W2885341302 creator A5013940811 @default.
• W2885341302 creator A5031893896 @default.
• W2885341302 creator A5058433588 @default.
• W2885341302 creator A5078708669 @default.
• W2885341302 creator A5087702098 @default.
• W2885341302 date "2018-07-01" @default.
• W2885341302 modified "2023-10-11" @default.
• W2885341302 title "FXR1 Is an IL-19-Responsive RNA-Binding Protein that Destabilizes Pro-inflammatory Transcripts in Vascular Smooth Muscle Cells" @default.
• W2885341302 cites W1493026183 @default.
• W2885341302 cites W1605399551 @default.
• W2885341302 cites W1649119959 @default.
• W2885341302 cites W19715912 @default.
• W2885341302 cites W1985643407 @default.
• W2885341302 cites W1989804578 @default.
• W2885341302 cites W1996866382 @default.
• W2885341302 cites W2003230246 @default.
• W2885341302 cites W2003350590 @default.
• W2885341302 cites W2003972847 @default.
• W2885341302 cites W2015725486 @default.
• W2885341302 cites W2026273230 @default.
• W2885341302 cites W2030117457 @default.
• W2885341302 cites W2039422436 @default.
• W2885341302 cites W2045714098 @default.
• W2885341302 cites W2049885842 @default.
• W2885341302 cites W2058252917 @default.
• W2885341302 cites W2064513293 @default.
• W2885341302 cites W2066963386 @default.
• W2885341302 cites W2069338832 @default.
• W2885341302 cites W2073507583 @default.
• W2885341302 cites W2087250116 @default.
• W2885341302 cites W2095709591 @default.
• W2885341302 cites W2102606027 @default.
• W2885341302 cites W2116973505 @default.
• W2885341302 cites W2118956966 @default.
• W2885341302 cites W2125366349 @default.
• W2885341302 cites W2135709931 @default.
• W2885341302 cites W2137138056 @default.
• W2885341302 cites W2142803556 @default.
• W2885341302 cites W2146917818 @default.
• W2885341302 cites W2152003287 @default.
• W2885341302 cites W2154325818 @default.
• W2885341302 cites W2157280773 @default.
• W2885341302 cites W2162563495 @default.
• W2885341302 cites W2167844024 @default.
• W2885341302 cites W2168759699 @default.
• W2885341302 cites W2288467024 @default.
• W2885341302 cites W2324841714 @default.
• W2885341302 cites W2395030726 @default.
• W2885341302 cites W2594848225 @default.
• W2885341302 cites W2616120362 @default.
• W2885341302 cites W2767179147 @default.
• W2885341302 doi "https://doi.org/10.1016/j.celrep.2018.07.002" @default.
• W2885341302 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30067974" @default.
• W2885341302 hasPublicationYear "2018" @default.
• W2885341302 type Work @default.
• W2885341302 sameAs 2885341302 @default.
• W2885341302 citedByCount "28" @default.
• W2885341302 countsByYear W28853413022018 @default.
• W2885341302 countsByYear W28853413022019 @default.
• W2885341302 countsByYear W28853413022020 @default.
• W2885341302 countsByYear W28853413022021 @default.
• W2885341302 countsByYear W28853413022022 @default.
• W2885341302 countsByYear W28853413022023 @default.
• W2885341302 crossrefType "journal-article" @default.
• W2885341302 hasAuthorship W2885341302A5003137481 @default.
• W2885341302 hasAuthorship W2885341302A5005917520 @default.
• W2885341302 hasAuthorship W2885341302A5007039952 @default.
• W2885341302 hasAuthorship W2885341302A5013940811 @default.
• W2885341302 hasAuthorship W2885341302A5031893896 @default.
• W2885341302 hasAuthorship W2885341302A5058433588 @default.
• W2885341302 hasAuthorship W2885341302A5078708669 @default.
• W2885341302 hasAuthorship W2885341302A5087702098 @default.
• W2885341302 hasBestOaLocation W28853413021 @default.
• W2885341302 hasConcept C104317684 @default.
• W2885341302 hasConcept C134018914 @default.
• W2885341302 hasConcept C185592680 @default.
• W2885341302 hasConcept C203014093 @default.
• W2885341302 hasConcept C2776914184 @default.
• W2885341302 hasConcept C2779395532 @default.
• W2885341302 hasConcept C2992686903 @default.
• W2885341302 hasConcept C41282012 @default.
• W2885341302 hasConcept C55493867 @default.
• W2885341302 hasConcept C67705224 @default.
• W2885341302 hasConcept C86803240 @default.
• W2885341302 hasConcept C95444343 @default.
• W2885341302 hasConceptScore W2885341302C104317684 @default.
• W2885341302 hasConceptScore W2885341302C134018914 @default.
• W2885341302 hasConceptScore W2885341302C185592680 @default.
• W2885341302 hasConceptScore W2885341302C203014093 @default.
• W2885341302 hasConceptScore W2885341302C2776914184 @default.
• W2885341302 hasConceptScore W2885341302C2779395532 @default.
• W2885341302 hasConceptScore W2885341302C2992686903 @default.
• W2885341302 hasConceptScore W2885341302C41282012 @default.

• next