JUND regulates pancreatic β cell survival during metabolic stress

Abstract

Objective In type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in β cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions. Methods Translating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment. Results TRAP-seq in a PDX1-deficiency model of β cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in β cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of β cell dysfunction, consistent with a maladaptive role for JUND in islets. Conclusions A translation-centric approach uncovered JUND as a stress-responsive factor in β cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.

Metabolic stress activates an ERK/hnRNPK/DDX3X pathway in pancreatic β cells

“…We previously used Translating Ribosome Affinity Purification followed by RNA-seq (TRAP-seq) to search for mRNAs with altered ribosome binding density in a PDX1-deficiency model of β cell dysfunction [14]. Depletion of PDX1 is a useful model to screen for factors with critical roles in β cells given that it is a human diabetes gene that shapes β cell susceptibility to stress [26], [27].…”

“…Depletion of PDX1 is a useful model to screen for factors with critical roles in β cells given that it is a human diabetes gene that shapes β cell susceptibility to stress [26], [27]. Indeed, this approach led us to uncover a subset of mRNAs regulated at the level of ribosome binding, including the mRNA encoding JUND, a transcription factor that promotes β cell apoptosis via regulation of pro-oxidant and pro-inflammatory genes [14]. In this study, we build on these findings by investigating the factors controlling JUND mRNA translation with the goal of uncovering novel pathways influencing β cell adaptation to stress conditions.…”

“…First, we used a bioinformatic approach to search for sequence motifs in our TRAP-seq data set, which included 53 genes with increased ribosome occupancy and 57 genes with decreased ribosome occupancy during PDX1-deficiency [14]. Specifically, we hypothesized that the genes with altered ribosome occupancy may share a common regulatory element in their untranslated regions (UTRs) that imparts translational regulation via RBP binding.…”

“…Interestingly, this factor acts downstream of several signaling pathways, allowing for the integration of extracellular cues with changes in gene expression [10], [11], [12], [13]; however, the role of hnRNPK in β cells is unknown. By focusing on the post-transcriptional regulation of the mRNA encoding JUND, a transcription factor with links to β cell redox homeostasis and apoptosis [14], we elucidate a novel ERK/hnRNPK/DDX3X axis that is activated in islets during metabolic stress.…”

Antioxidant Activity of Parijoto Fruit Extract at Various Temperature of Food Processing

“…Oksidasi pada LDL menyebabkan plak yang memicu penyakit jantung (Ellulu et al, 2016). Stres oksidatif menyebabkan disfungsi dan kerusakan sel β pancreas, sehingga produksi insulin akan terganggu dan menyebabkan diabetes (Good et al, 2019). Upaya untuk mencegah terjadinya oksidasi yang merusak dalam tubuh dengan meningkatkan konsumsi antioksidan (Bacchetti et al, 2019).…”