Background: Intestinal fibrosis is a common complication of the inflammatory bowel diseases(IBD), contributing to tissue stiffening and luminal narrowing. NR4A1 was previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. NR4A1 gene variants are associated with IBD risk, and it has been shown to regulate intestinal inflammation. Here, we tested the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating myofibroblast function. Methods: Using the SAMP1/YitFc mouse, we tested whether two pharmacological agents known to enhance NR4A1 signaling: cytosporone B(Csn-B) or 6-mercaptopurine(6-MP); could reduce fibrosis. We also employed the dextran sulphate sodium(DSS) model of colitis and assessed the magnitude of colonic fibrosis in Nr4a1-/- and their wild-type littermates(Nr4a1+/+). Lastly, intestinal myofibroblasts isolated from Nr4a1-/- and Nr4a1+/+ mice or primary human intestinal myofibroblasts were stimulated with transforming growth factor-beta-1(TGF-β1), in the presence or absence of Csn-B or 6-MP, and proliferation and ECM gene expression assessed. Results: Csn-B or 6-MP treatment significantly reduced ileal thickness, collagen and overall ECM content in SAMP1/YitFc mice. This was associated with a reduction in proliferative markers within the mesenchymal compartment. Nr4a1-/- mice exposed to DSS exhibited increased colonic thickening and ECM content. Nr4a1-/- myofibroblasts displayed enhanced TGF-β1-induced proliferation. Furthermore, Csn-B or 6-MP treatment was anti-proliferative in Nr4a1+/+, but not Nr4a1-/- cells. Lastly, activating NR4A1 in human myofibroblasts reduced TGF-β1-induced collagen deposition and fibrosis-related gene expression. Conclusions: Our data suggest that NR4A1 can attenuate fibrotic processes in intestinal myofibroblasts and could provide a valuable clinical target to treat inflammation-associated intestinal fibrosis.
ABSTRACT. Gain of function mutation of Janus kinase 2 (JAK2V617F) has been identified in Philadelphia-negative myeloproliferative diseases; about half of essential thrombocythemia (ET) patients harbor this mutation. The activated JAK-STAT pathway promotes cell proliferation, differentiation and anti-apoptosis. We studied the role of negative regulators of the JAK-STAT pathway, PIAS, and SOCS in ET patients. Twenty ET patients and 20 healthy individuals were enrolled in the study. Thirteen of the ET patients harbored the JAK2V617F mutation based on mutation analysis. Quantitative-PCR was applied to assay the expression of SOCS1, SOCS3, PIAS1, PIAS3. The expression levels of PIAS1 and PIAS3 were significantly lower in ET groups than that in normal individuals. There was no significant difference between JAK2V617F (+) and JAK2V617F (-) patients. SOCS1 and SOCS3 expression did not differ between ET patients and normal individuals, or between JAK2V617F (+) and JAK2V617F (-) patients. We suggest that failed negative regulators of the JAK-STAT pathway take part in the pathomechanism of ET.
Background Intestinal fibrosis and stricture formation are common complications of Crohn’s disease (CD). Recently, the stricturing phenotype has been recognized to be primarily attributable to hypertrophy/hyperplasia of smooth muscle, 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 has been reported as 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, the mechanisms regulating NR4A1 expression and its role in intestinal SMC function has not been investigated. Aims To characterize and understand the role of NR4A1 activation in the regulation of mitogen-induced proliferation of intestinal SMCs. Methods Primary intestinal SMCs were isolated from Nr4a1+/+ and Nr4a1-/- mice. Furthermore, a commercially sourced human primary intestinal SMC line was used. To assess the response of SMCs to culture and identify growth differences, proliferation was measured via trypan blue exclusion and EdU incorporation. In mouse and human SMCs, proliferation was induced by platelet-derived growth factor-BB (PDGF-BB) and NR4A1 activation was assessed by pretreating with selective agonists, cytosporone B (Csn-B) and 6-mercaptopurine (6-MP), at various concentrations. Mass spectrometry was used to characterize proteomic differences between Nr4a1+/+ and Nr4a1-/- SMCs. Expression levels of NR4A1 were assessed by qPCR and western blot after mitogen exposure and Csn-B treatment. Results Nr4a1-/- cells exhibited a significantly higher rate of proliferation compared to Nr4a1+/+ cells, under both basal and mitogen-exposed conditions. Proteomic analysis showed that Nr4a1-/- SMCs exhibited increased expression of proteins related to the cell cycle and metabolism, compared to Nr4a1+/+ SMCs. Pretreating human intestinal SMCs with Csn-B and 6-MP significantly 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. Furthermore, NR4A1 expression was rapidly induced by Csn-B and PDGF-BB, the latter response suggesting the existence of a potential negative feedback mechanism to control mitogen-induced SMC proliferation. Conclusions Our results suggest that NR4A1 is a critical regulator of intestinal SMC proliferation and 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 CAG, CCC, CIHR
Intestinal fibrosis and stricture formation are common complications of Crohn’s disease (CD). Recently, the stricturing phenotype has been recognized to be primarily attributable to hypertrophy/hyperplasia of smooth muscle, 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 has been reported as 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 inflammatory bowel disease, however, the mechanisms regulating NR4A1 expression and its role in intestinal SMC function have not been investigated. We sought to characterize and understand the role of NR4A1 activation in the regulation of mitogen‐induced proliferation of intestinal SMCs. We hypothesized that the activation of NR4A1 negatively regulates proliferative signaling and cell survival in intestinal SMCs. To test the hypothesis, primary intestinal SMCs were isolated from Nr4a1+/+ and Nr4a1−/− mice. Furthermore, a commercially sourced human primary intestinal SMC line was used. To assess the response of SMCs to culture and identify growth differences, proliferation was measured via trypan blue exclusion and EdU incorporation. In mouse and human SMCs, proliferation was induced by platelet‐derived growth factor‐BB (PDGF‐BB) and NR4A1 activation was assessed by pretreating with selective agonists, cytosporone‐B (Csn‐B) and 6‐mercaptopurine (6‐MP), at various concentrations. Mass spectrometry was used to characterize proteomic differences between Nr4a1+/+ and Nr4a1−/− SMCs. Expression levels of NR4A1 were assessed by qPCR and western blot after mitogen exposure and Csn‐B treatment. Nr4a1−/− cells exhibited a significantly higher rate of proliferation compared to Nr4a1+/+ cells, under both basal and mitogen‐exposed conditions. Proteomic analysis showed that Nr4a1−/− SMCs exhibited increased expression of proteins related to the cell cycle and metabolism, compared to Nr4a1+/+ SMCs. Pretreating human intestinal SMCs with Csn‐B and 6‐MP significantly 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. Furthermore, NR4A1 expression was rapidly induced by Csn‐B and PDGF‐BB, the latter response suggesting the existence of a potential negative feedback mechanism to control mitogen‐induced SMC proliferation. Our results suggest that NR4A1 is a critical regulator of intestinal SMC proliferation and 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. Support or Funding Information Canadian Institutes of Health Research
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