A28 CHARACTERIZING THE ROLE OF NR4A1 IN THE REGULATION OF INTESTINAL SMOOTH MUSCLE CELL PHENOTYPE AND FUNCTION. (4th March 2021)

• Record Type: Journal Article
• Authors: Szczepanski, H E; Flannigan, K L; Mainoli, B; Alston, L; Lee, J; Baruta, G M; Venu, V P; Shearer, J; Dufour, A; Hirota, S A
• Is Part Of: Journal of the Canadian Association of Gastroenterology. Volume 4(2021)Supplement 1
• Page Start: 147
• Page End: 148
• Publication Date: 2021-03-04
• Subjects: Gastroenterology -- Periodicals; 616.33005
• DOI: 10.1093/jcag/gwab002.027
• Languages: English
• ISSNs: 2515-2084

Abstract

Abstract: Background: Intestinal fibrosis and stricture formation are common complications of Crohn's disease (CD). Recently, smooth muscle hypertrophy/hyperplasia have gained greater recognition as a driver of stricture formation, rather than an increase in fibrosis alone. Despite advances in treatment of CD, current therapies do little to prevent or reverse strictures. NR4A1 is an orphan nuclear receptor that is anti-fibrotic in non-intestinal systems and exhibits anti-proliferative effects in smooth muscle cells (SMCs). NR4A1 gene variants have been associated with increased risk of IBD, however, mechanisms regulating NR4A1 expression and its role in intestinal SMC function have not been investigated. Aims: To characterize the role of NR4A1 presence and activation in modulating intestinal SMC phenotype. Methods: Primary intestinal SMCs were isolated from Nr4a1 +/+ and Nr4a1 -/ - mice. A commercially sourced human primary intestinal SMC line was also used. Mass spectrometry identified proteomic differences between Nr4a1 +/+ and Nr4a1 -/ - SMCs. To assess the role of NR4A1 in regulating SMC growth, basal and platelet-derived growth factor-BB (PDGF-BB)-induced proliferation were quantified. NR4A1 activation was induced by selective agonists, cytosporone B (Csn-B) and 6-mercaptopurine (6-MP). Cellular respirometry was used to determine metabolism in Nr4a1 +/+ and Nr4a1 -/ - SMCs. Expression of NR4A1 and phenotypic switching mediators were assessed by qPCR. Immunofluorescence was used to assess contractile markers and cell morphology. Results: Proteomic analysis revealed increased expression of proteins related to the cell cycle, metabolism, and extracellular matrix synthesis in Nr4a1 -/ - SMCs. Nr4a1 +/+ SMCs upregulated proteins involved in smooth muscle contraction and was supported by higher mRNA levels of myocardin and lower Krüppel-like factor 4. Nr4a1 -/ - cells were more proliferative compared to Nr4a1 +/+ cells under basal conditions. Treating human intestinal SMCs with Csn-B attenuated proliferation induced by PDGF-BB. Similar effects were observed in Nr4a1 +/+ SMCs, however, the anti-proliferative effect of Csn-B was absent in Nr4a1 -/ - cells. Nr4a1 -/ - SMCs had higher maximal respiration, spare respiratory capacity, and glycolysis. NR4A1 expression was rapidly induced by Csn-B/6-MP and PDGF-BB, the latter suggesting a potential negative feedback mechanism to control mitogen-induced SMC proliferation. Conclusions: Our results suggest that NR4A1 is a regulator of intestinal SMC proliferation, bioenergetics, and phenotype. Its induction by mitogens may contribute to a negative feedback loop to control smooth muscle growth. These data support targeting NR4A1 to treat excessive smooth muscle hypertrophy/hyperplasia that contributes to tissue remodelling observed in fibrostenotic CD. Funding Agencies: CCC, CIHR