Isorhamnetin, a 3'-methoxylated flavonol, enhances the lysosomal proteolysis in J774.1 murine macrophages in a TFEB-independent manner.

ABSTRACT

Lysosome is the principal organelle for the ultimate degradation of cellular macromolecules, which are delivered through endocytosis, phagocytosis, and autophagy. The lysosomal functions have been found to be impaired by fatty foods and aging, and more importantly, the lysosomal dysfunction in macrophages has been reported as a risk of atherosclerosis development. In this study, we searched for dietary polyphenols which possess the activity for enhancing the lysosomal degradation in J774.1, a murine macrophage-like cell line. Screening test utilizing DQ-BSA digestion identified isorhamnetin (3'-O-methylquercetin) as an active compound. Interestingly, structural comparison to inactive flavonols revealed that the chemical structure of the B-ring moiety in isorhamnetin is the primary determinant of its lysosome-enhancing activity. Unexpectedly isorhamnetin failed to inhibit mTORC1-TFEB signaling, a master regulator of lysosomal biogenesis and function. Our data suggested that the other molecular mechanism might be critical for the regulation of lysosomes in macrophages.Abbreviations ANOVA analysis of variance; ApoE apolipoprotein E; ATP6V0D2 ATPase H+ transporting V0 subunit d2; BAF bafilomycin A1; BODIPY boron dipyrromethene; BSA bovine serum albumin; CTSD cathepsin D; CTSF cathepsin F; DMEM Dulbecco's modified eagle medium; DMSO dimethyl sulfoxide; EGCG epigallocatechin-3-gallate; FBS fetal bovine serum; GAPDH glyceraldehyde-3-phosphate dehydrogenase; HPLC high-performance liquid chromatography; LAMP1 lysosomal-associated membrane protein 1; LAMP2A lysosomal-associated membrane protein 2A; LC-MS/MS liquid chromatography tandem mass spectrometry; MITF microphthalmia-associated transcription factor; MRM multiple reaction monitoring; mTORC1 mechanistic target of rapamycin complex 1; PBS phosphate-buffered saline; PPARγ peroxisome proliferator-activated receptor γ; RT-qPCR reverse transcription quantitative polymerase chain reaction; SDS sodium dodecyl sulfate; SNARE soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TBS Tris-buffered saline; TFA trifluoroacetic acid; TFE3 transcription factor binding to IGHM enhancer 3; TFEB transcriptional factor EB; TFEC transcription factor EC; V-ATPase vacuolar-type proton ATPase.