Background: Fibrosis is caused by an excessive accumulation of extracellular matrix proteins released from persistently activated fibroblasts and is a major hallmark of systemic sclerosis (SSc). Notch signaling plays a key role in development of many tissues and cell types, and aberrant Notch signaling has been shown to contribute to fibroblast activation and tissue fibrosis in the past. However, the precise role of Notch signaling in the molecular network of fibrotic pathways remains unknown.

Objectives: The aims of the present study were to evaluate whether constitutive activation of Notch signaling in fibroblasts is sufficient to induce fibrosis, and to identify the regulatory effects of Notch signaling on other profibrotic pathways.

Methods: Mice with alleles for the active Notch1 ^ICD inserted into the GT(ROSA)26Sor locus were crossbred with tamoxifen-inducible Col1a2-CreER mice to yield mice expressing both GT(ROSA)26Sor ^Notch1ICD and Col1a2-CreER (Notch1 ^ICD ;Col1a2-CreER). Fibrosis outcomes were analysed 4, 12, and 20 weeks after Cre induction with tamoxifen. Expression profiles and cellular processes were analysed by RNAseq and bioinformatical modeling. Identified differentially expressed genes were confirmed by qPCR and immunofluorescence co-staining.

Results: Fibroblast-specific activation of Notch signaling induced dermal fibrosis in a time-dependent manner with median increases in dermal thickness by 72 %, of the hydroxyproline content by 77 %, and the myofibroblast counts by 148 %. The expression of the classical Notch target genes Hes1 and Hey1 increased by 2.1- and 3.8-fold, respectively. In addition to dermal fibrosis, mice with conditional activation of Notch1 signaling also displayed fibrotic changes in the lungs and the colon. RNA sequencing analysis of the skin showed an upregulation of pro-fibrotic genes such as different types of collagens, MAP kinases, and components of JAK-STAT signaling amongst others. On the contrary, induction of Notch signaling in fibroblasts decreased the expression of anti-fibrotic genes such as the Wnt pathway inhibitors Wif1 and Sfrp1, the inhibitory Smad7, and vitamin D receptor Vdr.

Conclusion: We demonstrate that activation of Notch signaling in fibroblasts is sufficient to induce fibrosis in vivo . Cross-activation of different pro-fibrotic pathways with parallel repression of anti-fibrotic mediators might contribute to Notch1-induced fibrogenesis. Fibroblast-specific activation of Notch signaling might be an additional experimental model for testing anti-fibrotic molecules.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: None declared.