FRI0400 The ap1 transcription factor cjun amplifies hedgehog-induced fibroblast activation and tissue fibrosis

Abstract

BackgroundThe pathologic activation of fibroblasts is a key feature of fibrotic disorders such as Systemic Sclerosis (SSc). Deregulation of TGFβ- and Hedgehog signalling has been shown to be critical for the persistent, uncontrolled activation of fibroblasts in SSc.1–3 However, the consequences of the concomitant upregulation of multiple profibrotic pathways are unknown and cross-talk between individual pathways in fibrotic diseases is currently poorly characterised. Mutual activation and amplification of profibrotic signals might be central for the persistent activation of fibroblasts.ObjectivesThe aim of this study is to characterise the crosstalk between AP1- and hedgehog signalling in fibrotic tissue disease in SSc.MethodsCo-localization of cJUN and GLI2 in fibrotic skin was analysed by confocal microscopy and interaction was shown by Co-IP. cJUN/AP1 signalling and GLI2 signalling were inhibited in vitro and in vivo using the pharmacological inhibitors T5224 and GANT61. Hedgehog signalling was activated in mice by fibroblast-specific overexpression of constitutively active Smoothend (SmoACT mice).ResultsExpression profiling of all AP1 family members revealed most pronounced differences for cJUN in SmoACT mice. The expression of cJUN colocalized with the hedgehog transcription factor GLI2. Overexpression and colocalization of cJUN and GLI2 were also observed in fibroblasts in the skin of SSc patients. The number of GLI2- and cJUN double-positive fibroblasts was strongly increased in involved SSc skin compared to healthy controls and was particularly high in samples of SSc patients with diffuse and progressive disease. Based on the upregulation of both cJun and GLI2 and their colocalization, we hypothesised that cJUN and GLI2 might interact with each other to amplify fibroblast activation and tissue fibrosis. Stimulation of resting fibroblasts with TGFβ induces both cJUN and GLI2 mRNA and protein in a time-dependent manner. cJUN and GLI2 are also induced upon stimulation with Sonic hedgehog (SHH), demonstrating that TGFβ and SHH are both capable to activate cJUN and GLI2-dependent transcription. This crosstalk occurs by direct interaction of cJUN and GLI2, which amplifies the transactivation potential of both transcription factors. Co-immunoprecipitation demonstrated that stimulation of fibroblasts with TGFβ or SHH induces direct interaction of cJUN and GLI2. Overexpression of cJun and Gli2 resulted in activation in both AP1 and Hedgheog target genes. The central role of the crosstalk between cJUN and GLI2 for tissue fibrosis was further highlighted by the finding that hedgehog-induced fibrosis was strongly reduced by AP1 inhibition. SmoACT mice developed extensive skin fibrosis. Treatment with a cJUN/AP1 inhibitor T5224, however, strongly ameliorated hedgehog-induced fibrosis in SmoACT mice.ConclusionsWe demonstrate in the present study that the concomitant activation of AP1- and hedgehog signalling amplifies signalling through both cascades to promote excessive fibroblast activation and tissue fibrosis. This f...