Trimethylangelicin reduces IL-8 transcription and potentiates CFTR function

Abstract

Chronic inflammatory response in the airway tract of patients affected by cystic fibrosis is characterized by an excessive recruitment of neutrophils to the bronchial lumina, driven by the chemokine interleukin (IL)-8. We previously found that 5-methoxypsoralen reduces Pseudomonas aeruginosa-dependent IL-8 transcription in bronchial epithelial cell lines, with an IC(50) of 10 μM (Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, Cabrini G. Int Immunopharmacol 9: 1411-1422, 2009). Here, we extended the investigation to analogs of 5-methoxypsoralen, and we found that the most potent effect is obtained with 4,6,4'-trimethylangelicin (TMA), which inhibits P. aeruginosa-dependent IL-8 transcription at nanomolar concentration in IB3-1, CuFi-1, CFBE41o-, and Calu-3 bronchial epithelial cell lines. Analysis of phosphoproteins involved in proinflammatory transmembrane signaling evidenced that TMA reduces the phosphorylation of ribosomal S6 kinase-1 and AKT2/3, which we found indeed involved in P. aeruginosa-dependent activation of IL-8 gene transcription by testing the effect of pharmacological inhibitors. In addition, we found a docking site of TMA into NF-κB by in silico analysis, whereas inhibition of the NF-κB/DNA interactions in vitro by EMSA was observed at high concentrations (10 mM TMA). To further understand whether NF-κB pathway should be considered a target of TMA, chromatin immunoprecipitation was performed, and we observed that TMA (100 nM) preincubated in whole living cells reduced the interaction of NF-κB with the promoter of IL-8 gene. These results suggest that TMA could inhibit IL-8 gene transcription mainly by intervening on driving the recruitment of activated transcription factors on IL-8 gene promoter, as demonstrated here for NF-κB. Although the complete understanding of the mechanism of action of TMA deserves further investigation, an activity of TMA on phosphorylating pathways was already demonstrated by our study. Finally, since psoralens have been shown to potentiate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport, TMA was tested and found to potentiate CFTR-dependent chloride efflux. In conclusion, TMA is a dual-acting compound reducing excessive IL-8 expression and potentiating CFTR function.

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

“…Here, we extended these studies and we found that both VX-809 and TMA, a molecule that both corrects and potentiates the F508del-CFTR-dependent chloride secretion (Favia et al, 2014;Tamanini et al, 2011), also restore the apical expression of phosphorylated ezrin and actin organization in polarized CFBE cell monolayers. Furthermore, in line with previous findings demonstrating that pharmacological molecules (correctors and potentiators) might increase the susceptibility of the R-domain of F508del CFTR to be phosphorylated (Pyle et al, 2011), we found that both correctors increase both the cAMP and the activated PKA compartmentalization in the sub-plasma-membrane region, allowing the consequent rescue of F508del-CFTR-dependent chloride efflux (Fig.…”

“…In this regard, it has been recently demonstrated that the known small-molecule corrector VX-809 enhances the stability and function on the cell surface of F508del CFTR (Eckford et al, 2014) and improves its interaction with NHERF1 (Arora et al, 2014;Loureiro et al, 2015). Indeed, we find here that both VX-809 ) and 4,6,4′-trimethylangelicin (TMA), a recently reported corrector and potentiator of F508del CFTR (Favia et al, 2014;Tamanini et al, 2011), involve both ezrin activation and actin cytoskeleton re-organization in their rescuing effect. Moreover, both correctors restore the sub-cortical compartmentalization of cAMP and activated PKA resulting in the rescue of the cAMP-PKA-dependent chloride secretion in both primary and secondary cystic fibrosis airway cells.…”

Immunomodulatory and Anti-inflammatory Activity in Vitro and in Vivo of a Novel Antimicrobial Candidate

“…With a view to possible application of SET-M33 in CF lung infections, we analyzed the inhibition of inflammatory cytokines in IB3–1 bronchial cells isolated from a CF patient (20). The effects of SET-M33 on cytokine production in CF cells were evaluated by Luminex technology (21), detecting and quantifying a panel of proteins excreted into the medium.…”

“…IB3–1 cells (ATCC), derived from a CF patient with a ΔF508/W1282X mutant genotype and immortalized with adeno12/SV40, were grown in LHC-8 supplemented with 5% FBS in the absence of gentamycin at 37 °C under 5% CO 2 (20). …”

CFTR and lung homeostasis

“…A study by Favia et al (26) follows this idea by identifying a compound that has both potentiator and corrector activities. Their initial screen of this compound showed that trimethylangelicin (TMA) reduced IL-8 transcription and potentiated CFTR function (71). Follow-up studies showed that nanomolar concentrations of TMA in primary human bronchial epithelial cells from ⌬F508 CFTR homozygous patients elevated CFTR-dependent chloride secretion to almost 40% of WT levels, suggesting that single-drug treatments are a possibility (26).…”