Brewed Coffee and Its Components Act Through Orphan Nuclear Receptor 4A1 (NR4A1)

Background/Objective: Coffee is the most highly consumed beverage worldwide, and coffee drinkers exhibit decreased mortality and protection from aging-related diseases. This study investigates the role of orphan nuclear receptor 4A1 (NR4A1) in mediating the effects of brewed coffee and the major polyphenolic and polyhydroxy compounds in brewed coffee and also in determining their binding to NR4A1. Methods: The interactions of brewed coffee and several of the major individual compounds in brewed coffee with the ligand-binding domain of NR4A1 were determined using a fluorescent binding assay. For specific compounds, binding was also carried out by surface plasmon resonance, and molecular docking studies were also performed. NR4A1-responsive Rh30 cancer cells were used as models to determine NR4A1-dependent transactivation, cell growth inhibition and inhibition of specific gene products, and in some studies, knockdown of NR4A1 by RNA interference was also determined. Inhibition of lipopolysaccharide-induced IkBα by key polyphenolics was also investigated in RAW264.7 macrophages. Results: Brewed coffee and several polyphenolics, including caffeic acid, ferulic acid, chlorogenic acid, p-coumaric acid, several cinnamic acid derivatives, kahweol, and cafestrol, bound NR4A1 in binding assays, and most Kd values were <10 µM. Brewed coffee and the major polyphenolics inhibited growth of NR4A1-responsive Rh30 cells, and this was attenuated in NR4A1-deficient Rh30 cells. These same compounds also exhibited NR4A1-dependent effects on transactivation and gene product responses in Rh30 and RAW264.7 macrophages and exhibited inverse NR4A1 agonist activity. In contrast, the NR4A1-dependent activity of caffeine and quinic acid was highly variable, suggesting that they are selective NR4A1 ligands. Conclusions: The results of this study demonstrate that brewed coffee and its major polyphenolics and polyhydroxy constituents are NR4A1 ligands and that NR4A1 may play an important role in the health-protective effects of coffee. These results, coupled with recent studies, indicate that NR4A1 and its ligands may play an important role in diet and health.