Circadian Clock Regulation of Hepatic Lipid Metabolism by Modulation of m6A mRNA Methylation.

Transcriptional regulation of circadian rhythms is essential for lipid metabolic homeostasis, disruptions of which can lead to metabolic diseases. Whether N6-methyladenosine (m6A) mRNA methylation impacts circadian regulation of lipid metabolism is unclear. Here, we show m6A mRNA methylation oscillations in murine liver depend upon a functional circadian clock. Hepatic deletion of Bmal1 increases m6A mRNA methylation, particularly of PPaRα. Inhibition of m6A methylation via knockdown of m6A methyltransferase METTL3 decreases PPaRα m6A abundance and increases PPaRα mRNA lifetime and expression, reducing lipid accumulation in cells in vitro. Mechanistically, YTHDF2 binds to PPaRα to mediate its mRNA stability to regulate lipid metabolism. Induction of reactive oxygen species both in vitro and in vivo increases PPaRα transcript m6A levels, revealing a possible mechanism for circadian disruption on m6A mRNA methylation. These data show that m6A RNA methylation is important for circadian regulation of downstream genes and lipid metabolism, impacting metabolic outcomes.

Distinct roles of intracellular heat shock protein 70 in maintaining gastrointestinal homeostasis.

The inducible heat shock protein 70 (Hsp70) is both cytoprotective and immunomodulatory, potentially accounting for its critical role in maintaining gastrointestinal homeostasis. When levels are reduced in conditions like inflammatory bowel diseases (IBD), loss of function contributes to the severity and chronicity of these diseases, although through which cell types and mechanisms remains unclear. Here, the role of Hsp70-mediated intestinal epithelial protection and immune regulation in experimental colitis was examined by using a villin promoter-driven Hsp70 transgene in the 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models and in IL-10/Hsp70 double knockout (IL10-/-/Hsp70-/-) mice. In addition, Hsp70-mediated IL-10 production and immune protection were investigated using a CD45RBhigh transfer model and measuring colonic and immune cell cytokine expression during colitis. We found that the epithelial-specific expression of Hsp70 transgene attenuated DSS-induced colitis in Hsp70-/- mice by protecting tight junctions (TJ) and their interaction with the TJ-associated protein ZO-1. In the TNBS colitis and CD45RBhigh model, Hsp70 carried out its intracellular anti-inflammatory function by maintaining IL-10 production. Impaired ERK phosphorylation, but not p38 or JNK phosphorylation pathways, was associated with decreased IL-10 production in Hsp70-deficient cells. Together, these actions can be leveraged in the context of cellular specificity to develop complementary strategies that can lead to reduction in mucosal injury and immune activation in colonic colitis development. NEW & NOTEWORTHY Using four different experimental colitis models, we filled an important gap in knowledge by defining essential roles of intracellular heat shock protein 70 in different cell types in maintaining intestinal integrity and immune regulation. These findings are relevant to human inflammatory bowel diseases and represent potential avenues for developing therapeutic strategies, not only to counter the destructive processes of inflammation but also to promote tissue healing and prevent complications frequently associated with chronic intestinal inflammation.

Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation.

The intracellular protein HMGB1 is released from cells and acts as a damage-associated molecular pattern molecule during many diseases, including inflammatory bowel disease (IBD); however, the intracellular function of HMGB1 during inflammation is poorly understood. Here, we demonstrated that cytosolic HMGB1 regulates apoptosis by protecting the autophagy proteins beclin 1 and ATG5 from calpain-mediated cleavage during inflammation. Colitis in mice with an intestinal epithelial cell-specific Hmgb1 deletion and patients with IBD were both characterized by increased calpain activation, beclin 1 and ATG5 cleavage, and intestinal epithelial cell (IEC) death compared with controls. In vitro cleavage assays and studies of enteroids verified that HMGB1 protects beclin 1 and ATG5 from calpain-mediated cleavage events that generate proapoptotic protein fragments. Together, our results indicate that HMGB1 is essential for mitigating the extent and severity of inflammation-associated cellular injury by controlling the switch between the proautophagic and proapoptotic functions of beclin 1 and ATG5 during inflammation. Moreover, these studies demonstrate that HMGB1 is pivotal for reducing tissue injury in IBD and other complex inflammatory disorders.