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The Rac2 GTPase contributes to Cathepsin H-mediated protection against cytokine-induced β-cell apoptosis

ABSTRACT: In type 1 diabetes (T1D), the pancreatic β-cells are specifically destroyed by the immune system. In this process, local release of pro-inflammatory cytokines, such as IL-1β and IFNγ, contribute to β-cell apoptosis. Genome-wide association studies have identified more than 60 risk loci for T1D, including chr15q25.1 with the candidate gene CTSH (cathepsin H). We previously showed that T1D-associated risk variants in CTSH affect the expression of CTSH and are associated with disease progression in children with newly diagnosed T1D.We further found that CTSH increases β-cell function and protects against cytokine-induced apoptosis. Using global gene expression analysis, the purpose of the present study was to identify the genes and mechanisms through which CTSH mediates its protective effects. Microarray analysis identified a total of 63 annotated genes differentially expressed between stable CTSH-overexpressing insulin-secreting INS-1 cells and control cells transfected with an empty vector after treatment with IL-1β and IFNγ for 0, 6 and 16 hours. Permutation test taking all time-points into consideration identified 10 genes differentially expressed between the CTSH-overexpressing cells and the control cells: Elmod1, Fam49a, Gas7, Gna15, Msrb3, Nox1, Ptgs1, Rac2, Scn7a and Ttn. Pathway analysis identified one significant pathway “Inflammation mediated by chemokine and cytokine signaling pathway” with the genes Gna15, Ptgs1 and Rac2. Since Gna15 and Rac2 were upregulated by CTSH overexpression, their expression was knocked down using small interfering RNAs (siRNAs) to evaluate their potential as mediators of the protective effect of CTSH. Knockdown of Rac2 abolished the protective effect of CTSH overexpression on cytokine-induced apoptosis.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE136643 | GEO | 2020/09/01

REPOSITORIES: GEO

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Project description:Purpose: In the early phases of T1D development exposure of β-cells to IFNγ, IL-1β, and TNFα is thought to induce β-cell dysfunction and the expression of chemokines and cytokines. These changes are mediated in part by post-translational histone modifications within cis-regulatory regions. The purpose of this study was to determine if prevention of these epigenetic changes can alter cytokine induced gene expression changes. Methods: Pancreatic islets were isolated from both male and female CD1 mice by collagenase digestion and were picked by hand. Hand-picked islets were cultured for four days with 1.6ng/ml IFNγ, 0.25ng/ml IL-1β and 0.16ng/ml TNFα (cytokine) or were left untreated (nocytokine). For RNA-seq islets were collected and processed for NGS using the Illumina TruSeq library prep kit. Chromatin immunoprecipitation (ChIP) was performed using 3μg of anti-H3K4me1 (Abcam) with islets from at least 10 adult ICR mice (8-10 weeks old) exposed to 1.6ng/ml IFNγ, 0.25ng/ml Il-1β and 0.16ng/ml TNFα for four days. DNA from 4 separate ChIP experiments was pooled for library construction and sequencing using the Illumina platform. Results: Comparison of gene expression levels between untreated and cytokine-treated islets resulted in the identification of 205 genes that showed an IFNγ, Il-1β, and TNFα-induced chromatin state change (from repressed or inactive to H3K4me1 enriched, i.e. the de novo regions), or increased H3K4me1 enrichment, that were associated with genes with up-regulated expression in response to IFNγ, Il-1β, and TNFα, and 111 genes with reduced H3K4me1 and down-regulated expression in response to IFNγ, Il-1β, and TNFα. Conclusions: Our study confirms that exposure of islets to pro-inflammatory cytokines induces the expression of chemokines and that these changes are the result of a switch from an inactive to an active chromatin state. Further we show that disruption of the trithorax group complex inhibits the upregulation of pro-inflammatory gene expression.

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The Rac2 GTPase contributes to Cathepsin H-mediated protection against cytokine-induced β-cell apoptosis

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