Der TGFβ1-IFNγ-Crosstalk wird bidirektional durch YB-1 mediiert — Schlüssel zur Pathogenese der Palmarfibromatose?

Abstract

Introduction: Fibrosis of the palmar aponeurosis (Dupuytren’s disease) results in severe disfigurement and impairment of hand function. Previously, we have demonstrated that inhibitory Smad7 acts via manipulation of the TGFβ1-IFNγ crosstalk on cellular and nuclear levels, thereby down regulating endogenous expression of TGFβ1. It has already been shown in other fibrotic diseases that Y-box protein 1 (YB-1) acts as a connecting link between both signaling pathways. However, only a unidirectional interaction via phosphorylation of YAK1-STAT1 to activate Smad7 expression has already been identified. The aim of our study was the identification of a postulated bi-directional interaction of YB-1 during fibrogenesis of the palmar fascia on morphological and molecular levels. Methods: Fibroblasts were isolated from excised tissues of Dupuytren’s disease (Tubiana Score I-IV) by explant culture, cultivated and incubated on chamberslides. One part of the cells was stimulated with TGFβ1 and IFNγ after 24 h; other cells were infected with adenoviral caTβRI, Smad7 or IFNγ. Starved cells and fibroblasts transfected with Jak1-, STAT1-, YB-1-siRNA served as controls. For morphological investigations, cells were incubated on sterile microscopic slides and subsequently stimulated with recombinant IFNγ or TGFβ1. Afterwards, every 10 minutes 1 slide was removed from culture, fixed and incubated with antibodies against YAK1, STAT1, YB-1 and Smad7 and subjected to fluorescence microscopy. Additionally, cell lysates were investigated by western blot and PCR. Results: Stimulation of Dupuytren’s fibroblasts with TGFβ1 and infection with ad-caTβRI resulted in significant upregulation of functional proteins and endogenous expression of INFγ. Infection with ad-Smad7 completely blocked TGFγ1 specific signalling and expression of profibrotic proteins. Surprisingly, specific signal proteins of the IFNγ pathway, STAT1 and YAK1, as well as YB-1 were upregulated. Consecutively, increased expression levels of IFNγ were shown. In vitro stimulation with recombinant IFNγ and infection with ad IFNγ resulted in massive upregulation of YB-1 and endogenous Smad7. Subsequently, TGF1-signalling was blocked effectively. Cellular knock-down of STAT1, Jak1 and YB-1 with specific siRNA suppressed expression of endogenous Smad7. Further, YB-1 siRNA blocked reverse activation of Jak-STAT-signalling. These results were confirmed by immunofluorescence, western blot and PCR analysis. Conclusion: Our results demonstrate that YB-1 is a central element of bi-directional TGFβ1-IFNγ signalling. This may explain specific phenomena during final stages of Dupuytren’s disease and provide a possible basis for future therapeutic strategies.