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NRF2-independent iron-triggered signaling via MAPK p38-ETS1 regulates LSEC Bmp6 mRNA expression [I]

ABSTRACT: BMP6 is an iron-sensing cytokine produced by liver sinusoidal endothelial cells (LSECs). The transcription factor NRF2 was proposed to induce Bmp6 expression in response to iron-triggered oxidative stress, and NRF2 knock-out mice were shown not capable of regulating Bmp6 in response to massive diet-imposed iron overload. However, LSEC Bmp6 expression levels in the hemochromatosis mouse model reflect hepatocytic, not endothelial iron content. Hence, it is not fully resolved how iron signals translate into alterations of Bmp6 mRNA levels. To further explore the mechanisms of Bmp6 regulation, we employed female mice aged 10-11 months that are hallmarked by hepatocytic, but not LSEC iron accumulation and decreased transferrin saturation, hinting at the absence of systemic iron overload. We found that LSECs of aged mice exhibit increased Bmp6 levels as compared to young controls, but do not show oxidative stress or a transcriptional signature characteristic of activated NFR2-mediated signaling in FACS-sorted LSECs. We further observed that primary murine LSECs derived from both wild-type and NRF2 knock-out mice induce Bmp6 in response to acute iron exposure. By analyzing RNA sequencing data of FACS-sorted LSECs from aged versus young mice, as well as upon acute injections of iron citrate in young mice, we identified ETS1 as a candidate transcription factor involved in Bmp6 regulation. By conducting siRNA-mediated knock-down and small-molecule treatments in primary LSECs, we show that Bmp6 transcription is regulated in an NRF2-independent manner via ETS1, which is activated downstream of p38 MAP kinase-mediated signaling. This knowledge expands the understanding of Bmp6 transcriptional control in both iron-triggered oxidative stress and under the conditions uncoupled to LSECs reactive oxygen species levels.

ORGANISM(S): Mus musculus

PROVIDER: GSE235692 | GEO | 2024/01/25

REPOSITORIES: GEO

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Project description:Liver sinusoidal endothelial cells (LSECs) are specialized fenestrated scavenger endothelial cells involved in the elimination of modified plasma proteins and tissue turnover waste macromolecules from blood. LSECs also participate in liver immune responses. A challenge when studying LSEC biology is the rapid loss of the in vivo cell phenotype in culture. In this study, we have examined biological processes and pathways affected during early-stage primary culture of rat LSECs and checked for cell responses to the pro-inflammatory cytokine interleukin (IL)-1 and the anti-inflammatory drug dexamethasone. Methods: LSECs from male Sprague Dawley rats were cultured on type I collagen in a 5% oxygen atmosphere in DMEM with serum-free supplements for 2 and 24 h. Quantitative proteomics using tandem mass tag technology were used to examine proteins in cells and supernatants. Validation was done with ELISA and multiplex immunobead assays, cell ultrastructure was examined by scanning electron microscopy, and scavenger function by quantitative endocytosis assays. Results: LSECs cultured for 24 h showed a characteristic pro-inflammatory phenotype both in the presence and absence of IL-1, displaying upregulation of cellular responses to cytokines and interferon-, cell-cell adhesion, and glycolysis, and downregulation of membrane/endocytosis receptors (MCAM, STAB1, STAB2, LYVE1, CLEC4G) and proteins involved in pyruvate metabolism, citric acid cycle, fatty acid elongation, amino acid metabolism, and oxidation-reduction processes. Dexamethasone improved LSEC viability in culture and repressed the culture-induced stimulation of glycolysis, inflammatory and immune regulatory pathways, and secretion of pro-inflammatory cytokines while increasing IL-10 secretion compared to time-matched control. The number of sieve plates in LSECs was reduced at 24 h both in the presence and absence of dexamethasone but the dexamethasone-treated cells showed a more quiescent phenotype. Conclusion: Rat LSECs become activated towards a pro-inflammatory phenotype during early culture. Dexamethasone represses LSEC activation and improves cell viability.

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NRF2-independent iron-triggered signaling via MAPK p38-ETS1 regulates LSEC Bmp6 mRNA expression [I]

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