Uncovering the Regulation of Fibrotic Signaling in Scleroderma by the Ubiquitin E3 Ligase KLHL42

Abstract

Systemic scleroderma (SSc) is an autoimmune disease of unknown cause that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive pro‐fibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three‐quarters of SSc patients develop pulmonary fibrosis within five years; excessive scarring in SSc lung fibrosis is the main driver of mortality. There are no approved medicines to treat lung SSc. Recent research suggests that TGFb signaling is directly tied to SSc disease, through its pro‐fibrotic transcription factor signaling. Previous studies have shown ubiquitin e3 ligases potently control TGFb signaling through the targeted degradation of key regulator proteins, however their role in SSc‐TGFb signaling remains unclear. We utilized primary SSc patient lung cells for High Throughput Screening of modulators of TGFb signaling through High Content Imaging of the fibrotic transcription factor SMAD2/3 nuclear translocation. We screened an RNAi library targeting Ubiquitin E3 ligases and observed that the knockdown of the E3 ligase KLHL42 impaired TGFb‐dependent fibrotic signaling. KLHL42 knockdown reduced the production of fibrotic material and decreased TGFb‐mediated SMAD activation. We utilized unbiased ubiquitin proteomics to identify the phosphatase PP2A regulator PPP2R5e to be a KLHL42 substrate. Mechanistic studies validated the ubiquitin‐mediated control of PPP2R5e stability through KLHL42. Knockdown of PPP2R5e exacerbated TGFb‐mediated fibrotic signaling, demonstrating a role for PPP2R5e in the regulation of fibrotic signaling in SSc. Finally, we observed KLHL42 affected overall PP2A phosphatase activity through its targeted degradation of the subunit PPP2R5e. Here we report the characterization of the KLHL42‐PPP2R5e axis regulating fibrotic signaling in SSc lung fibroblasts. Future studies will investigate the potential for chemical inhibition of KLHL42 to ameliorate fibrotic signaling in SSc.Support or Funding Information5F31HL143843, 1R01DK119627, 5P50AR060780‐08, 1K08HL144820, 5R01HL142777, 5R35HL139860, and 5R01HL133184.