Inhibition of Sphingolipid Synthesis as a Phenotype-Modifying Therapy in Cystic Fibrosis

Alessandra Mingione, Michele Dei Cas, Fabiola Bonezzi, Anna Caretti, Marco Piccoli, Luigi Anastasia, Riccardo Ghidoni, Rita Paroni, Paola Signorelli

Research output: Contribution to journalArticlepeer-review

Abstract

BACKGROUND/AIMS: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells.

METHODS: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned.

RESULTS: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin.

CONCLUSION: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF.

Original languageEnglish
Pages (from-to)110-125
Number of pages16
JournalCellular Physiology and Biochemistry
Volume54
Issue number1
DOIs
Publication statusPublished - Jan 31 2020

Keywords

  • Autophagy/drug effects
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics
  • Cell Line
  • Cholesterol/analysis
  • Chromatography, High Pressure Liquid
  • Cystic Fibrosis/metabolism
  • Fatty Acids, Monounsaturated/pharmacology
  • Forkhead Box Protein O1/genetics
  • Humans
  • Lipid Metabolism/drug effects
  • Mass Spectrometry
  • PPAR gamma/genetics
  • RNA Interference
  • RNA, Small Interfering/metabolism
  • Serine C-Palmitoyltransferase/antagonists & inhibitors
  • Sphingolipids/analysis
  • Sphingomyelins/analysis

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