Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells

Abstract

Peptidylarginine deiminase 4 (PAD4) is a Ca 2+ -dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3β (GSK3β), both in vitro and in vivo. Among other functions, GSK3β is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3β protein levels, increased TGF-β signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3β is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3β is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.

A molecular signature of preclinical rheumatoid arthritis triggered by dysregulated PTPN22

“…Indeed, citrullination critically regulates embryogenesis (21,22), epithelial-to-mesenchymal transformation (23), and pluripotency of embryonic stem cells (24). In addition, PAD4-mediated citrullination of histones is essential for the formation of neutrophil extracellular traps (NETs) (25)(26)(27), which can induce the expression of inflammatory cytokines, such as IL-6 and IL-18, as well as CCL20 and ICAM-1 from fibroblast-like synoviocytes (28).…”

A simple but efficient electrochemical method to assay protein arginine deiminase 4

“…Besides, aberrant increase of PAD 4 has also been involved in multiple sclerosis, glaucoma and even cancers [10][11][12]. Therefore, with the deciphering the relation between PAD4 and different disease models [13,14], PAD4 has emerged as a potential therapeutic target for human diseases.…”

Histone deacetylase HDA6 enhances brassinosteroid signaling by inhibiting the BIN2 kinase

“…Other sites on GSK3β, such as Ser-48, -389, and -390, can also be phosphorylated, but their functions remain unknown (27). Besides phosphorylation, other modifications also regulate GSK3 activities, such as cleavage by calpain and by matrix metalloproteinase 2 leading to activated GSK3 fragments, mono-ADP ribosylation leading to GSK3 inhibition, and citrullination leading to nuclear localization of GSK3 (27,28). Recent work reported that mammalian GSK3β can be acetylated, and depletion of the deacetylase led to reduced phosphorylation and enhanced kinase activity of GSK3β (29).…”