WT1 Transcripts Are Alternatively Spliced And M1 Cytokine Inducible In Palmar Fascia Fibroblasts
John Luo, Emmy Sun, Trisiah Tugade, Ana Pena Diaz, Bing Siang Gan, Ruby Grewal, Nina Suh, David B. O'Gorman.
Lawson Health Research Institute and University of Western Ontario, London, ON, Canada.
Background: Excessive fibroproliferation and matrix deposition into palmar fascia, known as Dupuytren’s disease (DD), can induce debilitating palmar-digital contractures. We have previously reported upregulated expression of WT1, an oncogene with roles in cancer progression and epithelial-mesenchymal transition, in DD. In tumors, alternative splicing of WT1 gene transcripts results in WT1 isoforms that act as transcription factors or RNA splicing factors. The roles of WT1 proteins in palmar fascia repair are currently unclear. Methods: Total WT1 transcript levels in primary fibroblasts derived from fibrotic palmar fascia (DD fibroblasts), syngeneic “pre-fibrotic” (PF) fibroblasts and allogeneic normal control (CT) palmar fascia fibroblasts were assessed by qPCR, whereas alternatively spliced WT1 transcripts were identified by Reverse Transcription-PCR. “Cytomix” treatments with pro-inflammatory (M1) cytokines (TNF, IL1ß and IFN gamma) were used to mimic the early (M1) phase of tissue repair. Alamar Blue assays were used to assess the proliferation of CT fibroblasts transduced with adenoviral constructs expressing WT1 splice variants or GFP (vector control). RNA sequencing analyses of adenoviral gain-of-function and CRISPR-mediated loss-of-function constructs are underway. Results: Unlike PF and CT fibroblasts, DD fibroblasts constitutively express alternatively spliced WT1 gene transcripts. Although WT1 RNA expression is normally low in PF and CT fibroblasts, pro-inflammatory (M1) cytokines induce the expression of (at least) 4 different alternatively spliced WT1 gene transcripts. Adenoviral expression of WT1 isoform B, predicted to be a transcription factor, promotes the proliferation of CT fibroblasts relative to vector-transduced controls. Conclusions: These findings reveal abnormally sustained expression of alternatively spliced WT1 transcripts in DD, and implicate roles for transient expression of WT1 isoforms during the early inflammation phase of normal palmar fascia repair. We anticipate that our ongoing gain-of-function and CRISPR-mediated loss-of-function analyses will identify novel WT1 targets with roles in the normal and abnormal repair of palmar fascia, and potentially, in other WT1-positive tissues or organs fibroses.
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