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A4344 - Secreted Frizzled Related Protein 1 (SFRP1) as a Novel Regulator of the RhoA/Rock1 Pathway Is Expressed in a Distinct Subset of Fibroblasts in the Fibrotic Lung
Author Block: A. Sengupta1, B. Oehrle1, M. Strunz1, H. B. Schiller1, O. Eickelberg2, G. Burgstaller1; 1Comprehensive Pneumology Center, Munich, Germany, 2Pulmonary Sciences and Critical Care Medicine, Univ of Colorado Denver, Denver, CO, United States.
Rationale
The activation, invasion and differentiation of fibroblasts into myofibroblasts are hallmarks of progressing lung fibrogenesis. Here, we present new evidence that SFRP1 is expressed in a very distinct subpopulation of fibroblasts, which are alpha-smooth muscle actin (αSMA) negative, during the onset and progression of fibrosis. Furthermore, our data support SFRP1's novel role as a regulator of the non-canonical Wnt pathway along the RhoA-Rock1 axis altering the actin cytoskeleton and cellular morphology.
Methods
Single cell suspensions from whole lung tissues of healthy and Bleomycin (Bleo) instilled mice from day 14 were applied to a drop-seq single cell RNA-sequencing (scRNAseq) platform. SFRP1's protein expression was quantified from whole lung lysates on day 3, 7 14, 28, and 56, healthy and fibrotic mice. Immunofluorescent stainings were performed on Formalin fixed, paraffin embedded (FFPE) tissue sections. Mouse precision-cut lung slices (PCLS) were stimulated with Transforming growth factor beta 1 (TGFß1) for 72 hours to mimic early fibrotic conditions ex vivo. Depletion of SFRP1 was achieved in vitro by small interfering RNA (siRNA) mediated gene silencing.
Results
We found that expression of SFRP1 in fibrotic mouse lungs started on day 3, peaking on day 14. scRNAseq data from fibrotic mouse lungs revealed that SFRP1 expressing cells represented a specific subpopulation of pulmonary fibroblasts and that these are distinct from αSMA expressing (myo)fibroblasts, a finding which was further corroborated by immunofluorescent stainings. We tried to mimic early fibrotic conditions by treating ex vivo PCLS with TGFß1, and observed, similar to the in vivo fibrotic mouse model, an upregulation of SFRP1 on protein level after 72 hours. In vitro cultured fibroblasts strongly expressed SFRP1, which was enriched in the cytoplasm as well as in exosomes. Strikingly, TGFß1 treated fibroblasts decreased SFRP1's transcript and protein levels upon their transdifferentiation into myofibroblasts. Depletion of SFRP1 by siRNAs did not alter the canonical Wnt pathway, but caused a significant reduction in protein levels of RhoA and Rock1, both known to be members of the non-canonical Wnt pathway. Consequently, SFRP1 depleted fibroblasts revealed striking morphological and actin related cytoskeletal changes, with small-elongated cell bodies and a reduction of stress fibers.
Conclusion
SFRP1, expressed by a distinct sub-population of fibroblasts in fibrotic lungs, might stand out as a novel biomarker for early events in lung fibrosis. Furthermore, SFRP1 might act as a new regulator of the non-canonical Wnt-pathway along the RhoA-Rock1 axis interfering with actin cytoskeletal and cellular morphological changes.