Session Information
Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud’s – Pathogenesis, Animal Models and Genetics
Session Type: Abstract Submissions (ACR)
Background/Purpose: Systemic Sclerosis (SSc) is a chronic fibrotic disease involving autoimmune activation, fibroproliferative vasculopathy and tissue fibrosis of skin and multiple internal organs. Several studies have indicated that activation of WNT/β-catenin signaling pathway plays an important role in the pathogenesis of tissue fibrosis. Intriguingly, tissue expression studies on SSc skin show both upregulation of canonical WNT ligands1,2 and consistent upregulation of a putative WNT antagonist, Secreted Frizzled-Related Protein 4 (SFRP4) both at mRNA at protein level3,4.
Objectives: To determine the role of SFRP4 in modulating WNT signaling and the profibrotic phenotype of SSc fibroblasts.
Methods: Immortalized primary control and SSc fibroblasts were cultured in 10% DMEM and starved in 0.5% DMEM for 24hrs prior to stimulation with recombinant WNT-3a, WNT-5a (100ng/ml) and/or SFRP4 (70ng/ml). Gene expression was quantified by SYBR green QPCR. Canonical WNT signaling activation was assessed by TOPflash TCF/LEF luciferase reporter activity. Whole cell lysates were used to assess protein expression by Western Blot and c-Jun phosphorylation by Phospho-c-Jun ELISA (Cell Signaling Technology, UK). Mean ± SEM of 4 independent experiments were analyzed using Graph Prism 5.0.
Results: Basal SFRP4 gene expression in SSc fibroblasts was increased on average by 264% compared to normal controls [P<0.0001]. Stimulation with WNT-3a upregulated SFRP4 expression by 56% [P<0.001]. WNT-3a upregualted COL1A1 and α–SMA gene expression by 160% [P<0.001] and 230% [P<0.0001] in SSc fibroblasts, in contrast to 18% [P<0.05] and 72% [P<0.01] in normal fibroblasts, respectively. Consistent with canonical WNT signaling activation, WNT-3a caused an upregulation of axin-2 by 200% [P<0.0001] in SSc and 90% [P<0.001] in normal fibroblasts. TOPflash activity was also increased [P<0.0001] by 44% and 38%, respectively. Co-stimulation with SFRP4 showed no inhibitory activity of the WNT-3a effects. However, SFRP4 treatment alone was able to induce both COL1A1 by 43% [P<0.01] and α-SMA by 46% [P<0.001] only in SSc fibroblasts without any significant changes in either axin-2 expression or TOPflash activity. Similarly, stimulation with WNT-5a, a classic non-canonical WNT ligand, increased COL1A1 in SSc fibroblasts by 42% [P<0.0001] without any increase in axin-2 expression or TOPflash activity. Additionally, SSc fibroblasts treated with SFRP4 showed an increase in c-Jun phosphorylation reaching maximum levels at 10min by 167% [P<0.001] and again at 24hrs by 184% [P<0.0001].
Conclusion: Increased SFRP4 in SSc may contribute to the fibrotic process through non-canonical WNT activation instead of functioning as a WNT inhibitor. The molecular events underlying this paradox response of SSc fibroblasts may reveal insightful mechanisms in the pathogenesis and perpetuation of the fibrotic response in SSc.
1Wei, J. et al., 2011. Arthritis and Rheumatism, 63(6), pp.1707–1717.
2Beyer, C. et al., 2012. Annals of the Rheumatic Diseases, 71(5), pp.761–767.
3Milano, A. et al., 2008. PloS One, 3(7), p.e2696.
4Bayle, J. et al., 2007. J Invest Dermatol, 128(4), pp.871–881.
Disclosure:
J. Gillespie,
None;
P. Emery,
None;
F. Del Galdo,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/secreted-frizzled-related-protein-4-induces-a-profibrotic-phenotype-in-systemic-sclerosis-fibroblasts-by-activating-a-non-canonical-wnt-signaling-pathway/