Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21.

Type 1 diabetes (T1D) is a metabolic disease resulting from progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the majority of beta cells are lost in T1D, a small subset undergoes senescence, a stress response involving growth arrest, DNA damage response, and activation of a senescence-associated secretory phenotype (SASP). SASP in beta cells of the nonobese diabetic (NOD) mouse model of T1D and primary human islets is regulated at the level of transcription by bromodomain extra-terminal (BET) proteins, but the mechanisms remain unclear. To explore how SASP is transcriptionally regulated in beta cells, we used the NOD beta cell line NIT-1 to model beta cell SASP and identified binding partners of BET protein Brd4 and explored the role of the cyclin-dependent kinase inhibitor p21. Brd4 interacted with a variety of proteins in senescent NIT-1 cells including subunits of the Ino80 chromatin remodeling complex, which was expressed in beta cells during T1D progression in NOD mice and in human beta cells of control, autoantibody-positive, and T1D donors as determined from single-cell RNA-seq data. RNAi knockdown of p21 during senescence in NIT-1 cells did not significantly impact viability or SASP. Taken together, these results suggest that Brd4 interacts with several protein partners during senescence in NIT-1 cells, some of which may play roles in SASP gene activation and that p21 is dispensable for the SASP in this beta cell model.

Establishing evidence for immune surveillance of ß-cell senescence.

Cellular senescence is a programmed state of cell cycle arrest that involves a complex immunogenic secretome, eliciting immune surveillance and senescent cell clearance. Recent work has shown that a subpopulation of pancreatic ß-cells becomes senescent in the context of diabetes; however, it is not known whether these cells are normally subject to immune surveillance. In this opinion article, we advance the hypothesis that immune surveillance of ß-cells undergoing a senescence stress response normally limits their accumulation during aging and that the breakdown of these mechanisms is a driver of senescent ß-cell accumulation in diabetes. Elucidation and therapeutic activation of immune surveillance mechanisms in the pancreas holds promise for the improvement of approaches to target stressed senescent ß-cells in the treatment of diabetes.

Islet autoimmunity in human type 1 diabetes: initiation and progression from the perspective of the beta cell.

Type 1 diabetes results from the poorly understood process of islet autoimmunity, which ultimately leads to the loss of functional pancreatic beta cells. Mounting evidence supports the notion that the activation and evolution of islet autoimmunity in genetically susceptible people is contingent upon early life exposures affecting the islets, especially beta cells. Here, we review some of the recent advances and studies that highlight the roles of these changes as well as antigen presentation and stress response pathways in beta cells in the onset and propagation of the autoimmune process in type 1 diabetes. Future progress in this area holds promise for advancing islet- and beta cell-directed therapies that could be implemented in the early stages of the disease and could be combined with immunotherapies.

DNA damage to ß cells in culture recapitulates features of senescent ß cells that accumulate in type 1 diabetes.

OBJECTIVE: Type 1 Diabetes (T1D) is characterized by progressive loss of insulin-producing pancreatic ß cells as a result of autoimmune destruction. In addition to ß cell death, recent work has shown that subpopulations of ß cells acquire dysfunction during T1D. We previously reported that ß cells undergoing a DNA damage response (DDR) and senescence accumulate during the pathogenesis of T1D. However, the question of how senescence develops in ß cells has not been investigated. METHODS: Here, we tested the hypothesis that unrepaired DNA damage in the context of genetic susceptibility triggers ß cell senescence using culture models including the mouse NIT1 ß cell line derived from the T1D-susceptible nonobese diabetic (NOD) strain, human donor islets and EndoC ß cells. DNA damage was chemically induced using etoposide or bleomycin and cells or islets were analyzed by a combination of molecular assays for senescence phenotypes including Western blotting, qRT-PCR, Luminex assays, flow cytometry and histochemical staining. RNA-seq was carried out to profile global transcriptomic changes in human islets undergoing DDR and senescence. Insulin ELISAs were used to quantify glucose-stimulated insulin secretion from chemically-induced senescent human islets, EndoC ß cells and mouse ß cell lines in culture. RESULTS: Sub-lethal DNA damage in NIT1 cells led to several classical hallmarks of senescence including sustained DDR activation, growth arrest, enlarged flattened morphology and a senescence-associated secretory phenotype (SASP) resembling what occurs in primary ß cells during T1D in NOD mice. These phenotypes differed between NIT1 cells and the MIN6 ß cell line derived from a non-T1D susceptible mouse strain. RNA-seq analysis of DNA damage-induced senescence in human islets from two different donors revealed a p53 transcriptional program and upregulation of prosurvival and SASP genes, with inter-donor variability in this response. Inter-donor variability in human islets was also apparent in the extent of persistent DDR activation and SASP at the protein level. Notably, chemically induced DNA damage also led to DDR activation and senescent phenotypes in EndoC-ßH5 human ß cells, confirming that this response can occur directly in a human ß cell line. Finally, DNA damage led to different effects on glucose-stimulated insulin secretion in mouse ß cell lines as compared with human islets and EndoC ß cells. CONCLUSIONS: Taken together, these findings suggest that some of the phenotypes of senescent ß cells that accumulate during the development of T1D in the NOD mouse and humans can be modeled by chemically induced DNA damage to mouse ß cell lines, human islets and EndoC ß cells in culture. The differences between ß cells from different mouse strains and different human islet donors and EndoC ß cells highlights species differences and the role for genetic background in modifying the ß cell response to DNA damage and its effects on insulin secretion. These culture models will be useful tools to understand some of the mechanisms of ß cell senescence in T1D.