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Macrophage migration inhibitory factor governs endothelial cell sensitivity to LPS-induced apoptosis
Rachel L Damico1, Alan Chesley, Laura Johnston
1Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, MD 21224, USA.
Abstract:
Human endothelial cells (EC) are typically resistant to the apoptotic effects of stimuli associated with lung disease. The determinants of this resistance remain incompletely understood. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by human pulmonary artery EC (HPAEC). Its expression increases in response to various death-inducing stimuli, including lipopolysaccharide (LPS). We show here that silencing MIF expression by RNA interference (MIF siRNA) dramatically reduces MIF mRNA expression and the LPS-induced increase in MIF protein levels, thereby sensitizing HPAECs to LPS-induced cell death. Addition of recombinant human MIF (rhMIF) protein prevents the death-sensitizing effect of MIF siRNA. A common mediator of apoptosis resistance in ECs is the death effector domain (DED)-containing protein, FLIP (FLICE-like inhibitory protein). We show that LPS induces a transcription-independent increase in the short isoform of FLIP (FLIP(s)). This increase is blocked by MIF siRNA but restored with the addition of recombinant MIF protein (rHMIF). While FLIP(s) siRNA also sensitizes HPAECs to LPS-induced death, the addition of rhMIF does not affect this sensitization, placing MIF upstream of FLIP(s) in preventing HPAEC death. These studies demonstrate that MIF is an endogenous pro-survival factor in HPAECs and identify a novel mechanism for its role in apoptosis resistance through the regulation of FLIP(s). These results show that MIF can protect vascular endothelial cells from inflammation-associated cell damage.
Insights
Macrophage migration inhibitory factor (MIF) protects human pulmonary artery endothelial cells (HPAECs) from cell death. MIF regulates the FLICE-like inhibitory protein (FLIP), preventing apoptosis during inflammation.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Human endothelial cells (ECs) exhibit resistance to apoptosis, a crucial factor in lung disease pathogenesis.
- The molecular mechanisms underlying EC apoptosis resistance are not fully understood.
- Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is produced by ECs and its expression is upregulated by death-inducing stimuli like lipopolysaccharide (LPS).
Purpose of the Study:
- To investigate the role of MIF in EC apoptosis resistance.
- To elucidate the molecular pathway through which MIF confers protection against apoptosis.
- To determine if MIF regulates the expression of apoptosis-related proteins like FLIP.
Main Methods:
- Utilized RNA interference (siRNA) to silence MIF expression in human pulmonary artery ECs (HPAECs).
- Assessed the impact of MIF silencing on HPAEC sensitivity to LPS-induced cell death.
- Investigated the effect of recombinant human MIF (rhMIF) on cell survival and apoptosis.
- Examined the regulation of FLICE-like inhibitory protein (FLIP) expression by MIF using Western blotting and siRNA.
- Determined the position of MIF in the apoptotic pathway relative to FLIP.
Main Results:
- Silencing MIF expression significantly sensitized HPAECs to LPS-induced apoptosis.
- Addition of rhMIF reversed the cell death-sensitizing effect of MIF siRNA.
- LPS induced an increase in the short isoform of FLIP (FLIP(s)), which was blocked by MIF siRNA but restored by rhMIF.
- FLIP(s) siRNA also sensitized HPAECs to LPS-induced death, but rhMIF did not alter this sensitization.
- These findings place MIF upstream of FLIP(s) in the pathway preventing HPAEC death.
Conclusions:
- MIF acts as an endogenous pro-survival factor in HPAECs.
- MIF protects vascular endothelial cells from inflammation-associated cell damage by regulating FLIP(s) expression.
- This study identifies a novel mechanism of MIF-mediated apoptosis resistance in ECs.
