P0178 Lack of DNA methyltransferase DNMT3A in fibroblasts impairs cell growth and wound healing
Authors: Escudero-Hernández C , Hübner E , Núñez-Torvisco S , Rodríguez-Peña A , Montague J , Rosenstiel P 
Published: January 2025

Abstract

Background
Fibrosis in Crohn’s disease (CD) is a complication of chronic inflammatory processes that may result in impaired gastrointestinal function and bowel strictures. However, exact pathomechanisms leading to intestinal fibrosis in CD are not fully understood. Since the de novo DNA methyltransferase DNMT3A is involved in cellular differentiation processes and genetic variants have been associated with increased risk for CD, we sought to characterize how DNMT3A loss might affect fibroblasts, the key mesenchymal cells involved in fibrosis.

Methods
We stably deleted Dnmt3a in 3T3 fibroblast cell line using CRISPR-Cas9 technology and subsequently selected GPF-positive clones using FACS sorting. Cell cultures were performed in cell culture plates with or without 0.2% gelatin coat. Reverse transcription quantitative PCR, western blot, and immunofluorescence were used to confirm Dnmt3a gene deletion and characterize wild-type (WT) and knockout (KO) cells. Cell adhesion was evaluated by fluorescence levels of calcein acetoxymethyl ester (CAM)-labelled cells that attached to cell culture plates after 1h of culture and subsequent wash. Collagen lattice assays in presence of proinflammatory and profibrotic stimuli served to assess fibroblast contractibility.

Results
3T3 fibroblasts lacking Dnmt3a acquire an activated myofibroblast phenotype (increased a-SMA expression) that also overexpress tissue remodeling matrix metalloproteinase Mmp3, and the inflammation-induced necroptosis marker RIPK3 when compared to their WT counterparts. In addition, Dnmt3a-depleted 3T3 cells adhere more strongly to cell culture plates. In presence of transforming growth factor (TGF)-b, cells embedded in a 3D collagen matrix and lacking Dnmt3a build an interconnected cellular network and contract up to a 77.78% (compared to interconnected cells that reach up to 13.73% contraction by WT cells). This contraction was independent of the presence of interleukin (IL)-11. Profibrotic factors such as bleomycin prevent intercellular network formation in both WT and KO cells; hence, incapacitating cells to contract the collagen matrix.

Conclusion
We conclude that Dnmt3a is crucial for fibroblast homeostasis, including cell activation and functionality. Furthermore, TGF-b induces higher fibroblast contractibility when Dnmt3a is lacking. Thus, additional characterization of Dnmt3a depletion in fibroblasts will deepen the understanding of fibrosis and may point towards new therapeutic approaches for this untreatable complication.

References
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2.Fazio A, Bordoni D, Kuiper JWP, et al. DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon. Nat Commun. 2022;13(1):6266. doi:10.1038/s41467-022-33844-2