Background/Purpose: Systemic Sclerosis (SSc) is a heterogeneous disease characterized by autoimmune activation, fibroproliferative vasculopathy, and tissue fibrosis of skin and multiple internal organs. Several studies have indicated that both caevolin-1 (CAV-1) and WNT/β-catenin signaling play important roles in the pathogenesis of tissue fibrosis.  Indeed, CAV-1 is downregulated by 40% in SSc skin compared to healthy controls and, intriguingly, tissue expression studies with SSc skin biopsies show both upregulation of canonical WNT ligands^1,2 and consistent upregulation of Frizzled-Related Protein 4 (SFRP4), a putative WNT antagonist, at both mRNA at protein level^3,4. To evaluate the role CAV-1 and TGFβ on the expression of SFRP4, and the effects of SFRP4 upon WNT signaling in fibroblasts.

Methods: Immortalized primary healthy (HC) and SSc fibroblasts were cultured in 10% DMEM and starved in 0.5% DMEM for 24hrs prior to stimulation with recombinant TGFβ (10ng/ml), WNT-3a/5a (100ng/ml) and/or SFRP4 (100-500ng/ml). Gene expression was quantified by SYBRgreen RTPCR and by western blot. CAV-1 siRNA was transfected at 5μM. Canonical WNT signaling was assessed by TOPFlash luciferase reporter activity. ELISA was used to measure both the level of Phospho-c-JUN from whole cell lysates, and the level of SFRP4 from SSc patient sera.

Results: In SSc fibroblasts, the basal expression of SFRP4 was increased at protein level and also by 264% at mRNA level compared to HC [p<0.001]. TGFβ stimulation upregulated SFRP4 mRNA by 170% [P<0.01] at 48hrs and by 348% [P<0.01] at 72hrs. TGFβ also induced a time-dependent increase of both SFRP4 and α-SMA protein expression, while reducing CAV-1. The siRNA-mediated silencing of CAV-1 was sufficient to induce a time-dependent increase in SFRP4 protein expression. WNT-3a induced a 600% increase in TOPFlash activity, while co-treatment with SFRP4 suppressed this activation by 59%. In contrast, SFRP4 induced a 260% increase [p<0.001] in c-JUN phosphorylation, a non-canonical WNT target, at 10 min in both HC and SSc fibroblasts. This was similar to the activity of the non-canonical WNT-5a ligand. Interestingly, basal Phospho-c-JUN was increased by 180% [P<0.005] in SSc compared to HC fibroblasts. Further, SFRP4 treatment increased COL1A1 expression by 26% in HCs at 500ng/ml, while in SSc fibroblasts SFRP4 induced both COL1A1 and α-SMA by 49-50% with 100-500ng/ml SFRP4, respectively. Additionally, SSc sera levels of SFRP4 inversely correlated with diffusion lung capacity of carbon monoxide % (r=-0.29, P=0.01) and positively with mRSS in patients with normal lung function (r=0.40, p=0.006).

Conclusion: The increased expression of SFRP4 observed in SSc skin biopsies may be a direct consequence of CAV-1 downregulation by TGFβ in tissue fibroblasts. Given the non-canonical WNT activity of SFRP4, a TGFβ primed microenvironment may be responsible for shaping the phenotype of both fibroblasts and neighboring cells, through aberrant WNT pathway activation. Additionally, circulating SFRP4 may have potential as a biomarker of fibrosis. Investigation of the mechanisms linking CAV-1 expression and SFRP4 function will improve our understanding of the pathogenic role that aberrant WNT activation plays in SSc.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/secreted-frizzled-related-protein-4-can-be-induced-by-transforming-growth-factor-%ce%b2-is-regulated-by-caveolin-1-and-can-induce-non-canonical-wnt-signaling-in-fibroblasts/