Identified risk polymorphisms affecting the Jak-STAT3 pathway in patients with Crohn’s disease could affect TGF-β1 and collagen I expression and in the pathway’s negative regulator, SOCS3. Genetic factors, however, account for only ~25% of disease. Epigenetic events also shape gene expression. Recent experiments showed that autocrine IL-6 production in mesenchymal cells, subepithelial myofibroblasts (SEMF) and muscle cells, of patients with fibrostenotic Crohn’s disease causes sustained Jak-STAT3 activity, excess TGF-β1 and Collagen I production and fibrosis. SOCS3 paradoxically decreased in these cells. We now identify epigenetic mechanisms that silence SOCS3 expression in SEMF of patients with fibrostenotic Crohn’s disease. In a previous experiment, using isolated SEMF of normal ileum and affected ileum from patients with each Crohn’s phenotype, inflammatory (Montreal B1), fibrostenotic (B2) and penetrating (B3), we confirmed decreased SOCS3 protein levels were unique to B2 patients. Expression of miR-19b increased in SEMF of affected ileum. SOCS3 transcriptional activity decreased after transfection of miR-19b mimic and increased when antagomiR-19b was expressed. Epigenetic silencing of SOCS3 in ileal SEMF of patients with fibrostenotic Crohn’s disease occurs by increased miR-19b mediated inhibition of SOCS3

Kuemmerle, John F

LI, Chao

Marshall, Emily T.

English

LI, CHAO

Kuemmerle, John K.

VCU Scholars Compass

Epigenetic silencing of SOCS3 expression contributes to fibrosis in Crohn’s disease

Virginia Commonwealth UniversityVCU Scholars CompassMD Student Summer Research Fellowship ProgramPosters School of Medicine2018"Epigenetic silencing of SOCS3 expressioncontributes to fibrosis in Crohn’s disease"Emily T. MarshallVirginia Commonwealth UniversityChao LIVirginia Commonwealth UniversityJohn F. KuemmerleVirginia Commonwealth UniversityFollow this and additional works at: https://scholarscompass.vcu.edu/mds_postersPart of the Medicine and Health Sciences CommonsThis Poster is brought to you for free and open access by the School of Medicine at VCU Scholars Compass. It has been accepted for inclusion in MDStudent Summer Research Fellowship Program Posters by an authorized administrator of VCU Scholars Compass. For more information, pleasecontact libcompass@vcu.edu.Downloaded fromhttps://scholarscompass.vcu.edu/mds_posters/14Epigenetic silencing of  SOCS3 expression contributes to fibrosis in Crohn’s disease Emily Marshall, Chao Li1, John F. Kuemmerle1,2Departments of Medicine1, Physiology and Biophysics2, and VCU Program in Enteric Neuromuscular SciencesMedical College of Virginia Campus, Virginia Commonwealth University, Richmond, VAResults: miR-19b regulates SOCS3 ConclusionAbstract/BackgroundMethodsIntroductionIdentified risk polymorphisms affecting the Jak-STAT3 pathway in patientswith Crohn’s disease could affect TGF-β1 and collagen I expression and in thepathway’s negative regulator, SOCS3. Genetic factors, however, account foronly ~25% of disease. Epigenetic events also shape gene expression. Recentexperiments showed that autocrine IL-6 production in mesenchymal cells,subepithelial myofibroblasts (SEMF) and muscle cells, of patients withfibrostenotic Crohn’s disease causes sustained Jak-STAT3 activity, excess TGF-β1 and Collagen I production and fibrosis. SOCS3 paradoxically decreased inthese cells. We now identify epigenetic mechanisms that silence SOCS3expression in SEMF of patients with fibrostenotic Crohn’s disease. In aprevious experiment, using isolated SEMF of normal ileum and affected ileumfrom patients with each Crohn’s phenotype, inflammatory (Montreal B1),fibrostenotic (B2) and penetrating (B3), we confirmed decreased SOCS3protein levels were unique to B2 patients. Expression of miR-19b increased inSEMF of affected ileum. SOCS3 transcriptional activity decreased aftertransfection of miR-19b mimic and increased when antagomiR-19b wasexpressed. Epigenetic silencing of SOCS3 in ileal SEMF of patients withfibrostenotic Crohn’s disease occurs by increased miR-19b mediatedinhibition of SOCS3.• Cytokines, including IL-6, are pivotal modulators of responses ininflammatory diseases and are secreted by numerous cells includingactivated intestinal subepithelial myofibroblasts.• Binding of IL-6 activates the constitutively associated JAK-1 and JAK-2 resulting in cross-phosphorylation and docking sites for STAT3.Specific STAT3 residues are acetylated and phosphorylated andSTAT3 translocates to the nucleus where it regulates transcriptionalactivity, including of TGF-β1 and COL1a1.• A consensus STAT3 binding element is present in the 5’-UTRpromoter region of the Suppressor of Cytokine Signaling-3 (SOCS3)gene (1).• Normally activation by IL-6, via Jak-STAT3, induces expression ofSOCS3, a crucial negative regulator of cytokine-induced Jak-STAT3signaling.• We recently demonstrated that in patients with Crohn’s disease witha Montreal B2 fibrostenotic phenotype, that despite increasedautocrine production of numerous pro-inflammatory cytokines,particularly IL-6, in mesenchymal cells of affected ileum, there is lossof the expected upregulation of SOCS3 protein.• Mesenchymal cells were isolated from Crohn’s disease patients• Human studies were approved by the VCU Institutional Review Board• Quantitative real-time PCR used to measure RNA transcripts with miR-19a-5p and miR-19b-5p primers• Results for qRT-PCR were calculated using the 2-∆∆Ct method based on U6for miR amplification (remains stable across Crohn’s phenotypesexamined)• Luciferase Reporter Assay was used to measure gene transcription.• Cells were transfected with anti-miR-19b or pre-miR-19b.• Statistic Analysis: values represent means + SE of n experiments, where nrepresents the number of experiments on cells derived from separatesubjects• Statistical significance was determined by Student’s t-test for either pairedor unpaired data and was assumed for p<0.05• Comparison between multiple groups was made using ANOVA with aTurkey test for post hoc comparisons• Transcription of miR-19b-5p from the miR-17~92 targets SOCS3 mRNA fordegradation• The epigenetic events explored in this experiment, miRs, are linked notonly to fibrostenotic Crohn’s disease but also to development of fibrosis inother organs including the skin, heart, liver, lung, and kidney (4, 5, 6)• Expression of miRs are altered in the fibroblasts of fibrotic organsincluding several members of the paralogous miR17~92 and miR106a~363clusters, miR-21 and miR-29 (7-11). These resulting epigenomic alterationsimpact expression of pro-fibrotic genes such as collagen, TGF-β1, matrixmetalloproteinases and α-smooth muscle actin resulting in their increasedexpression and development of fibrosis.• In summary, identified genetic factors and susceptibility loci account foronly 13.6% of disease variability and no more than 25% of the genetic riskin patients with Crohn’s disease (12,13). Epigenomic changes can furthercontribute to the ‘heritable’ risk of developing fibrostenotic Crohn’sdisease. This data demonstrates one of the mechanisms of epigeneticsilencing of SOCS3 that allow the sustained Jak-STAT3 activity that drivesexcess TGF-β1 and collagen expression and characterizes fibrostenoticMontreal B2 Crohn’s disease.1. Cheng X, et al. miR-19b downregulates intestinal SOCS3 to reduce intestinal inflammation in Crohn's disease. Sci Rep   5: 10397, 2015.2. Li C, et al. Noncanonical STAT3 Activation Regulates Excess TGF-β1 and Collagen I Expression in Muscle of StricturingCrohn’s Disease. The Journal of Immunology 194: 3422-3431, 2015.3. Brock M, Trenkmann M, Gay RE, Michel BA, Gay S, Fischler M, Ulrich S, Speich R, and Huber LC. Interleukin-6 Modulates the Expression of the Bone Morphogenic Protein Receptor Type II Through a Novel STAT3–microRNA Cluster 17/92 Pathway. Circulation Research 104: 1184-1191, 2009.25. 4. Li C, and Kuemmerle JF. Genetic and epigenetic regulation of intestinal fibrosis. United European Gastroenterology Journal 4: 496-505, 2016.5. Ghosh AK, and Varga J. The transcriptional coactivator and acetyltransferase p300 in fibroblast biology and fibrosis. Journal of Cellular Physiology 213: 663-671, 2007.6. Bhattacharyya S, et al. Fibroblast expression of the coactivator p300 governs the intensity of profibrotic response to transforming growth factor beta. Arthritis Rheum 52: 1248-1258, 2005.12. 7. Hayashita Y, et al. Polycistronic MicroRNA Cluster, miR-17-92, Is Overexpressed in Human Lung Cancers and Enhances Cell Proliferation. Cancer Research 65: 9628-9632, 2005.8. Mogilyansky E, and Rigoutsos I. The miR-17/92 cluster: a comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and disease. Cell Death Differ 20: 1603-1614, 2013.9. Lewis A, Mehta S, Hanna LN, Rogalski LA, Jeffery R, Nijhuis A, Kumagai T, Biancheri P, Bundy JG, Bishop CL, Feakins R, Di Sabatino A, Lee JC, Lindsay JO, and Silver A. Low Serum Levels of MicroRNA-19 Are Associated with a StricturingCrohn's Disease Phenotype. Inflamm Bowel Dis 21: 1926-1934, 2015.10. Wu F, Zhang S, Dassopoulos T, Harris ML, Bayless TM, Meltzer SJ, Brant SR, and Kwon JH. Identification of microRNAs associated with ileal and colonic Crohn's disease. Inflamm Bowel Dis 16: 1729-1738, 2010. 30. 11. Ji M, Rao E, Ramachandrareddy H, Shen Y, Jiang C, Chen J, Hu Y, Rizzino A, Chan WC, Fu K, and McKeithan TW. The miR-17-92 MicroRNA Cluster Is Regulated by Multiple Mechanisms in B-Cell Malignancies. The American Journal of Pathology 179: 1645-1656, 2011.12. Jostins L, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 491: 119-124, 2012.13. Liu JZ, et al. Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations. Nat Genet 47: 979-986, 2015.Special thanks to John F. Kuemmerle and Chao Li for their continued guidance and direction in both this experiment and current endeavors. MethodsParticipantsAcknowledgementsFigure 1. SOCS3 expression decreased in affected ileum of patients with stricturing (Montreal B2) and penetrating (B3) Crohn’s disease compared to normal ileum in the same patient as well as non-Crohn’s subjects. The opposite occurred in patient with inflammatory (B1) Crohn’s disease. Values represent the mean ± SEM of 5-6 separate experiments. *denotes p<0.05 vsnormal ileum in the same patient.Demographics Patient No. (% of total)Age (years) under 20 2 (8)20-29 9 (38)30-39 7 (29)40-49 4 (17)50-59 1 (4)over 60 1 (4)SexMale 9 (37)Female 15 (63)RaceWhite 14 (58)Black or African 9 (38)Other/unknown 1 (4)Non-Crohn’s Subjects 6 (100) CD Subjects Montreal Phenotype B1-non-stricturing, non penetrating 6 (33)B2-stricturing 6 (33)L1-ileal 4 (67%) 4 (67)L2-ileo-colic 2 (33%) 2 (33)B3-Penetrating 6 (33)Figure A. Hybridization analysis of SOCS3 and miR-19b identified aconserved 8-mer seed sequences complementary for both has-miR-19a and hsa-miR-19b at position 1561-1568 of SOCS3 3' UTR.Figure B. miR-19a and miR-19b expression was unchanged or decreasedin SEMF of affected ileum in patients with Montreal B1 and B3phenotype Crohn’s disease, respectively, compared to non Crohn’ssubjects. In contrast, miR-19b levels were significantly elevated in SEMFof affected ileum compared to normal ileum in patients with B2fibrostenotic disease. * denotes p<0.05 vs normal ileum.Figure C. Transcriptional activity of socs3 was decreased in SEMF ofnormal ileum of patients with fibrostenosis by transfection of miR-19b-5p mimic. Transcriptional activity of SOCS3 in SEMF of affected ileumwas increased by transfection of antagomiR-19b-5p. Transcriptionalactivity was measured by dual luciferase-SEAP reporter assay. Valuesrepresent the mean ± SEM of 3-6 separate experiments. * denotesp<0.05 vs normal ileum (B), or control scrambled miR (C).References• In mesenchymal cells, subepithelial myofibroblast (SEMF) and smoothmuscle cells, we observed sustained JAK-STAT3 signaling that accountedfor the excess TGF-β1 and collagen I production from these cells that is acharacteristic pathogenetic mechanism in fibrostenotic Crohn’s disease(2).• Epigenetic mechanisms are increasingly recognized as regulators of geneexpression and disease. Genetic factors account for at most 25% ofdisease in Crohn’s patients. One important epigenetic process regulatingexpression is small non-coding microRNAs (miRs) that target mRNA fordegradation.• A highly conserved STAT3 binding element is present in the promoterregion of the miR17~92 cluster gene, C13orf25 (3). Position 1561-1568 ofthe SOCS3 3' UTR is an 8-mer sequence for miR-19a-5p and miR-19b-5pbinding derived from this cluster (1).• This work identifies one of three epigenetic mechanisms leading to theunexpected low levels of SOCS3 expression in patients with fibrostenoticCrohn’s disease compared to other Crohn’s disease phenotypes.• SOCS3 expression is silenced in SEMF of patients with fibrostenosis viaincreased expression of miR-19a-5p and miR19b-5p that targets Socs3mRNA for degradation.

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Epigenetic silencing of SOCS3 expression contributes to fibrosis in Crohn’s disease

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