Session Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's - Pathogenesis, Animal Models and Genetics I
Session Type: Abstract Submissions (ACR)
Systemic Sclerosis (SSc) is characterized by autoimmune activation, vasculopathy and tissue fibrosis. Recently, activation of the Wnt/β-catenin signaling pathway in SSc fibroblasts has been linked to the pathogenesis of SSc. However, the relative role of crosstalk between TGF-β and Wnt Pathways in SSc is still to be determined. Here we have aimed to evaluate the Wnt pathway in SSc fibroblasts and the effects of TGF-β on canonical Wnt signalling.
Dermal fibroblasts from 3 early diffuse cutaneous (dc)-SSc patients and 3 healthy controls (HC) were stimulated with recombinant human (rh)TGF-β and/or rhWnt-3a. mRNA stability was investigated using actimomycin D. mRNA levels were quantified by qRT-PCR and protein expression was measured by western blotting or immunohistochemistry. Canonical Wnt signaling was evaluated by TOPFlash luciferase reporter activity. In- vivoexpression studies were performed on TGF-β-RII DeltaK transgenic mice, which have constitutive activation of the TGF-β pathway in fibroblasts, and wild-type littermates.
In basal conditions, SSc fibroblasts did not show any increase in TOPFlash reporter activity compared to HC. On the contrary, the expression of Axin2, a Wnt target gene and negative regulator of the Wnt pathway, was reduced at both mRNA (58%; P<0.01) and protein levels. Indeed, SSc fibroblasts had an increased response to rhWnt-3a compared to HC fibroblasts (11.6 fold increase in TOPFlash activity and 2.5 fold in Axin-2mRNA vs. 4.2 and 1.8 fold, respectively, in HC). TGF-β treatment of HC fibroblasts decreased Axin-2 expression to levels similar to SSc fibroblasts, both at mRNA (38.7%; [p<0.001]) and protein levels. This effect was associated with a 3.7 fold increase in mRNA decay. Concordantly, TGF-β-RII DeltaK transgenic mice displayed a reduced expression of Axin-2 in the dermis. Similar to SSc, pretreatment of HC fibroblasts with TGF-β increased their responsiveness to Wnt-3a (16.1 vs. 7.7 fold increase in TOPFlash activity; P<0.01). Depletion of Axin-2 by siRNA was sufficient to mimic the effect of TGF-β pretreatment (P<0.05). Accordingly, XAV939-mediated Axin-2 stabilization ablated the Wnt-3a-induced canonical hyperactivation in ‘TGF-β primed’ fibroblasts.
TGF-β stimulation primes dermal fibroblasts to increase their responsiveness to Wnt ligand-induced canonical signaling. TGF-β mediates the downregualtion of Axin-2, which is required for canonical Wnt signaling hyperactivation in dermal fibroblasts. Our data suggest that the increased Wnt signaling observed in SSc is a consequence of TGF-β signaling and therefore targeting of the TGF-β pathway may also help to resolve the aberrant Wnt signaling observed in SSc.
E. C. Derrett-Smith,
C. P. Denton,
F. Del Galdo,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/priming-of-wnt-signalling-during-fibrosis-is-mediated-by-tgf-%ce%b2-induced-axin-2-downregulation/