Inflammatory stress signaling via NF-kB alters accessible cholesterol to upregulate SREBP2 transcriptional activity in endothelial cells
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Evaluation Summary:
This study shows a direct link between inflammation and cholesterol metabolism in endothelial cells. Specifically, the authors show a pathway by which the major inflammatory factor, NF kappa B, activates a gene called STARD10, which, in turn, leads to the activation of the cholesterol biosynthetic pathway. The study, therefore, provides important insights into the inter-relationship between cholesterol metabolism and inflammation at the molecular level.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
There is a growing appreciation that a tight relationship exists between cholesterol homeostasis and immunity in leukocytes; however, this relationship has not been deeply explored in the vascular endothelium. Endothelial cells (ECs) rapidly respond to extrinsic signals, such as tissue damage or microbial infection, by upregulating factors to activate and recruit circulating leukocytes to the site of injury and aberrant activation of ECs leads to inflammatory based diseases, such as multiple sclerosis and atherosclerosis. Here, we studied the role of cholesterol and a key transcription regulator of cholesterol homeostasis, SREBP2, in the EC responses to inflammatory stress. Treatment of primary human ECs with pro-inflammatory cytokines upregulated SREBP2 cleavage and cholesterol biosynthetic gene expression within the late phase of the acute inflammatory response. Furthermore, SREBP2 activation was dependent on NF-κB DNA binding and canonical SCAP-SREBP2 processing. Mechanistically, inflammatory activation of SREBP was mediated by a reduction in accessible cholesterol, leading to heightened sterol sensing and downstream SREBP2 cleavage. Detailed analysis of NF-κB inducible genes that may impact sterol sensing resulted in the identification of a novel RELA -inducible target, STARD10 , that mediates accessible cholesterol homeostasis in ECs. Thus, this study provides an in-depth characterization of the relationship between cholesterol homeostasis and the acute inflammatory response in EC.
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Evaluation Summary:
This study shows a direct link between inflammation and cholesterol metabolism in endothelial cells. Specifically, the authors show a pathway by which the major inflammatory factor, NF kappa B, activates a gene called STARD10, which, in turn, leads to the activation of the cholesterol biosynthetic pathway. The study, therefore, provides important insights into the inter-relationship between cholesterol metabolism and inflammation at the molecular level.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The manuscript by Folwer et al examines the mechanism whereby endothelial cells respond to inflammatory stimuli. Using primary Human Vascular Endothelial Cells (HUVEC) as their model, they find that upon treatment with TNF the induction and repression of hundreds of genes at both 4 and 10 hours. They found by IPA the expected inflammatory pathway and unexpectedly found that the cholesterol biosynthetic pathway was also a prominent pathway upregulated. Upon deletion of RELA, the genes that were significantly downregulated were associated with SREBP2, suggesting that TNF somehow activates SREBP2 in HUVEC. Therefore, the authors focused on understanding the mechanism of SREBP2 activation in HUVEC cells by NF-Kappa-B.
They examined TNF-induced SREBP2 cleavage over 24 hours and found that cleaved SREBP2 peaked at …
Reviewer #1 (Public Review):
The manuscript by Folwer et al examines the mechanism whereby endothelial cells respond to inflammatory stimuli. Using primary Human Vascular Endothelial Cells (HUVEC) as their model, they find that upon treatment with TNF the induction and repression of hundreds of genes at both 4 and 10 hours. They found by IPA the expected inflammatory pathway and unexpectedly found that the cholesterol biosynthetic pathway was also a prominent pathway upregulated. Upon deletion of RELA, the genes that were significantly downregulated were associated with SREBP2, suggesting that TNF somehow activates SREBP2 in HUVEC. Therefore, the authors focused on understanding the mechanism of SREBP2 activation in HUVEC cells by NF-Kappa-B.
They examined TNF-induced SREBP2 cleavage over 24 hours and found that cleaved SREBP2 peaked at 10 hours. Over the same time course, they interrogated both NF-Kappa-B and SREBP2 targets and found that the expression of the NF-Kappa-B targets proceeds the SREBP2 targets, suggesting that NF-Kappa-B is somehow activating the SREBP2 pathway. Consistent with this hypothesis are studies with IKK inhibitor (which prevents NF-Kappa-B activation) and RELA knockdown that show a reduction in SREBP2 cleavage, and the inhibition of SREBP2 gene targets involved in cholesterol biosynthesis.
A series of SREBP2-processing inhibitors were used to define that the cleavage of SREBP2 by TNF-NF-Kappa-B activation was mediated by the canonical processing pathway. They then posited that TNF treatment affected the cellular cholesterol levels to activate SREPB cleavage. Whereas TNF did not change total cholesterol, they did find a change in the amount of cholesterol in the membrane both in HUVECs and in vivo by labeling the membranes with a fluorescently-labeled cholesterol-binding protein. This prompted them to look at the genes regulated by TNF-NF-Kappa-B that might be responsible for a reduction in accessible cholesterol and they focused on the lipid transporter STARD10. Interrogation of publicly available ChIP-seq of RELA from TNF-treated HUVEC cells indicates occupancy at the promoter, suggesting STARD10 is a direct RELA target. Depletion of STARD10 inhibited TNF-induced expression of cholesterol biosynthesis genes and reduced the TNF-stimulated SREBP2 cleavage and LDLR protein abundance.
Overall the data are consistent with the conclusion that NF-Kappa-B induction of STARD10 reduces cholesterol at the membrane and activates SREBP2 cleavage (model is presented in Figure 7). This illuminates the mechanism of regulation of the inflammatory response in endothelial cells. A few controls are missing and some additional analyses should be included to strengthen an already strong study.
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Reviewer #2 (Public Review):
This paper by Fowler and colleagues has examined the little-known relationship between cholesterol metabolism, its master regulator SREBP2, and inflammatory signalling/responses in endothelial cells. Here, they show that pro-inflammatory cytokines including TNF and IL-1 induce a transcriptional response via NF-kB that alters cholesterol homeostasis in the endothelium, which then activates SREBP2 processing. This, in turn, compensates for the altered flux of cholesterol between the plasma membrane and endoplasmic reticulum. They identified a novel NF-kB inducible gene, STARD10, that bridges TNF activation to changes in bioavailable cholesterol and SREBP2 activation. Taken together, these data support the growing evidence for a fundamental and biologically relevant relationship between cholesterol and …
Reviewer #2 (Public Review):
This paper by Fowler and colleagues has examined the little-known relationship between cholesterol metabolism, its master regulator SREBP2, and inflammatory signalling/responses in endothelial cells. Here, they show that pro-inflammatory cytokines including TNF and IL-1 induce a transcriptional response via NF-kB that alters cholesterol homeostasis in the endothelium, which then activates SREBP2 processing. This, in turn, compensates for the altered flux of cholesterol between the plasma membrane and endoplasmic reticulum. They identified a novel NF-kB inducible gene, STARD10, that bridges TNF activation to changes in bioavailable cholesterol and SREBP2 activation. Taken together, these data support the growing evidence for a fundamental and biologically relevant relationship between cholesterol and inflammation. The experiments that were carried out to highlight this link are multipronged in strategy, appropriate for the experimental question being asked are robust.
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