HuR modulation counteracts lipopolysaccharide response in murine macrophages

Abstract

Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA binding protein that binds and regulates the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS-induced response. Here we investigated how and to what extent small molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. We show TMs exist in solution in keto-enolic tautomerism and that, by molecular dynamic calculations, the orto quinone form is the bioactive species interacting with HuR and inhibiting its binding mode vs mRNA targets. A chemical blockage of the diphenolic, reduced form as a diacetate caused the loss of activity of TMs in vitro but resulted to prodrug-like activity in vivo. The murine macrophage cell line RAW264.7 was treated with LPS and TMs, and the modulation of cellular LPS-induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes, and that TMs reduced such interactions. Functional annotation addressed a specific set of genes involved in chemotaxis and immune response, such as Cxcl10, Il1b, Cd40, and Fas, with a decreased association with HuR, a reduction of their expression and protein secretion. The same effect was observed in primary murine bone marrow-derived macrophages, and in vivo in an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such as TM7nox with remarkable anti-inflammatory properties in vivo.

Multiple roles for AU-rich RNA binding proteins in the development of haematologic malignancies and their resistance to chemotherapy

“…The range of targets and their role in the regulation of cellular processes important for haematopoiesis implicates the significance of HuR in the view of leukaemia development and progression. Silencing of HuR or blocking its interaction with RNA by MS-444 caused reduced clonogenic potential of AML cells [ 29 ], and reduced activation of macrophages, decreasing chemotaxis and pro-inflammatory cytokine release [ 97 ]. Apart from that, HuR-mediated stabilization of target transcripts stimulated proliferation and inhibited apoptosis in model AML cell lines [ 30 ].…”

Old tricks and new trends to address tumors: a medicinal chemist’s perspective

“…The synthesis of 3-(2',6'-substituted)aryl ATs 11a-d required significant optimization of the Suzuki coupling, due to the steric hindrance of bis-methylphenyl boronic acid; once successfully obtained, ATs 11a-d were tested in vitro to confirm their preserved activity as disruptors of the mRNA-HuR interaction. Most potent analogues 11a and 11b were tested in vivo, and the significantly increased bioavailability of the latter enabled the determination of its anticancer and immunomodulating activity in mice models 38 .…”