All-trans-retinoic acid-mediated cytoprotection in LLC-PK1 renal epithelial cells is coupled to p-ERK activation in a ROS-independent manner.

Although all-trans-retinoic acid (ATRA) provides protection against a variety of conditions in vivo, particularly ischemia, the molecular mechanisms underpinning these effects remain unclear. The present studies were designed to assess potential mechanisms by which ATRA affords cytoprotection against renal toxicants in LLC-PK1 cells. Pretreatment of LLC-PK1 cells with ATRA (25 µM) for 24 h afforded cytoprotection against oncotic cell death induced by p-aminophenol (PAP), 2-(glutathion-S-yl)hydroquinone (MGHQ), and iodoacetamide but not against apoptotic cell death induced by cisplatin. Inhibition of protein synthesis with cycloheximide blunted ATRA protection, indicating essential cell survival pathways must be engaged before toxicant exposure to provide cytoprotection. Interestingly, ATRA did not prevent the PAP-induced generation of reactive oxygen species (ROS) nor did it alter glutathione levels. Moreover, ATRA had no significant effect on Nrf2 protein expression, and the Nrf2 inducers sulforaphane and MG132 did not influence ATRA cytoprotection, suggesting cytoprotective pathways beyond those that influence ROS levels contribute to ATRA protection. In contrast, ATRA rapidly (15 min) induced levels of the cellular stress kinases p-ERK and p-AKT at concentrations of ATRA (10 and 25 µM) required for cytoprotection. Consistent with a role for p-ERK in ATRA-mediated cytoprotection, inhibition of p-ERK with PD98059 reduced the ability of ATRA to afford protection against PAP toxicity. Collectively, these data suggest that p-ERK and its downstream targets, independent of ROS and antioxidant signaling, are important contributors to the cytoprotective effects of ATRA against oncotic cell death.

Current Therapeutic Landscape and Safety Roadmap for Targeting the Aryl Hydrocarbon Receptor in Inflammatory Gastrointestinal Indications.

Target modulation of the AhR for inflammatory gastrointestinal (GI) conditions holds great promise but also the potential for safety liabilities both within and beyond the GI tract. The ubiquitous expression of the AhR across mammalian tissues coupled with its role in diverse signaling pathways makes development of a "clean" AhR therapeutically challenging. Ligand promiscuity and diversity in context-specific AhR activation further complicates targeting the AhR for drug development due to limitations surrounding clinical translatability. Despite these concerns, several approaches to target the AhR have been explored such as small molecules, microbials, PROTACs, and oligonucleotide-based approaches. These various chemical modalities are not without safety liabilities and require unique de-risking strategies to parse out toxicities. Collectively, these programs can benefit from in silico and in vitro methodologies that investigate specific AhR pathway activation and have the potential to implement thresholding parameters to categorize AhR ligands as "high" or "low" risk for sustained AhR activation. Exploration into transcriptomic signatures for AhR safety assessment, incorporation of physiologically-relevant in vitro model systems, and investigation into chronic activation of the AhR by structurally diverse ligands will help address gaps in our understanding regarding AhR-dependent toxicities. Here, we review the role of the AhR within the GI tract, novel therapeutic modality approaches to target the AhR, key AhR-dependent safety liabilities, and relevant strategies that can be implemented to address drug safety concerns. Together, this review discusses the emerging therapeutic landscape of modalities targeting the AhR for inflammatory GI indications and offers a safety roadmap for AhR drug development.

Hepatic triacylglycerol hydrolysis regulates peroxisome proliferator-activated receptor alpha activity.

Recent evidence suggests that fatty acids generated from intracellular triacylglycerol (TAG) hydrolysis may have important roles in intracellular signaling. This study was conducted to determine if fatty acids liberated from TAG hydrolysis regulate peroxisome proliferator-activated receptor alpha (PPARalpha). Primary rat hepatocyte cultures were treated with adenoviruses overexpressing adipose differentiation-related protein (ADRP) or adipose triacylglycerol lipase (ATGL) or treated with short interfering RNA (siRNA) targeted against ADRP. Subsequent effects on TAG metabolism and PPARalpha activity and target gene expression were determined. Overexpressing ADRP attenuated TAG hydrolysis, whereas siRNA-mediated knockdown of ADRP or ATGL overexpression resulted in enhanced TAG hydrolysis. Results from PPARalpha reporter activity assays demonstrated that decreasing TAG hydrolysis by ADRP overexpression resulted in a 35-60% reduction in reporter activity under basal conditions or in the presence of fatty acids. As expected, PPARalpha target genes were also decreased in response to ADRP overexpression. However, the PPARalpha ligand, WY-14643, was able to restore PPARalpha activity following ADRP overexpression. Despite its effects on PPARalpha, overexpressing ADRP did not affect PPARgamma activity. Enhancing TAG hydrolysis through ADRP knockdown or ATGL overexpression increased PPARalpha activity. These results indicate that TAG hydrolysis and the consequential release of fatty acids regulate PPARalpha activity.