All-trans retinoic acid induces lipophagy through reducing Rubicon in Hepa1c1c7 cells.

All-trans retinoic acid (atRA), a metabolite of vitamin A, reduces hepatic lipid accumulation in liver steatosis model animals. Lipophagy, a new lipolysis pathway, degrades a lipid droplet (LD) via autophagy in adipose tissue and the liver. We recently found that atRA induces lipophagy in adipocytes. However, it remains unclear whether atRA induces lipophagy in hepatocytes. In this study, we investigated the effects of atRA on lipophagy in Hepa1c1c7 cells and the liver of mice fed a high-fat diet (HFD). Firstly, we confirmed that atRA induced autophagy in Hepa1c1c7 cells by Western blotting and the GFP-LC3-mCherry probe. Next, we evaluated the lipolysis in fatty Hepa1c1c7 cells treated with the knockdown of Atg5, an essential gene in autophagy induction. Atg5-knockdown partly suppressed the atRA-induced lipolysis in fatty Hepa1c1c7 cells. We also found that atRA reduced the protein, but not mRNA, expression of Rubicon, a negative regulator of autophagy, in Hepa1c1c7 cells and the liver of HFD-fed mice. Rubicon-knockdown partly inhibited the atRA-induced lipolysis in fatty Hepa1c1c7 cells. In addition, atRA reduced hepatic Rubicon expression in young mice, but the effect of atRA on it diminished in aged mice. Lastly, we investigated the mechanism underlying reduced Rubicon protein expression by atRA in hepatocytes. A protein synthesis inhibitor, but not proteasome or lysosomal inhibitors, significantly blocked the reduction of Rubicon protein expression by atRA in Hepa1c1c7 cells. These results suggest that atRA may promote lipophagy in fatty hepatocytes by reducing hepatic Rubicon expression via inhibiting protein synthesis. (243/250 words).

Modulations of the mTORC2-GATA3 axis by an isorhamnetin activated endosomal-lysosomal system of the J774.1 macrophage-like cell line.

The endosomal-lysosomal system represents a crucial degradation pathway for various extracellular substances, and its dysfunction is linked to cardiovascular and neurodegenerative diseases. This degradation process involves multiple steps: (1) the uptake of extracellular molecules, (2) transport of cargos to lysosomes, and (3) digestion by lysosomal enzymes. While cellular uptake and lysosomal function are reportedly regulated by the mTORC1-TFEB axis, the key regulatory signal for cargo transport remains unclear. Notably, our previous study discovered that isorhamnetin, a dietary flavonoid, enhances endosomal-lysosomal proteolysis in the J774.1 cell line independently of the mTORC1-TFEB axis. This finding suggests the involvement of another signal in the mechanism of isorhamnetin. This study analyzes the molecular mechanism of isorhamnetin using transcriptome analysis and reveals that the transcription factor GATA3 plays a critical role in enhanced endosomal-lysosomal degradation. Our data also demonstrate that mTORC2 regulates GATA3 nuclear translocation, and the mTORC2-GATA3 axis alters endosomal formation and maturation, facilitating the efficient transport of cargos to lysosomes. This study suggests that the mTORC2-GATA3 axis might be a novel target for the degradation of abnormal substances.

Expression, phosphorylation, and mRNA-binding of heterogeneous nuclear ribonucleoprotein K in Xenopus oocytes, eggs, and early embryos.

Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the K homology domain-containing proteins, is expressed in Xenopus immature oocytes, unfertilized eggs, and early embryos. Fertilization or egg activation treatment involving upregulation of the egg tyrosine kinase Src promotes a rapid and transient tyrosine phosphorylation of hnRNP K. HnRNP K is also phosphorylated on serine/threonine residues in unfertilized eggs, dephosphorylated after fertilization, and re-phosphorylated during the premitotic phase of early embryogenesis. In vitro, Src and mitogen-activated protein kinase (MAPK) were capable of phosphorylating hnRNP K on tyrosine and serine/threonine residues, respectively. In support of this, pretreatment of oocytes, eggs, or embryos with inhibitors for Src (PP2) and MAPK (U0126) blocked effectively the phosphorylation of hnRNP K. We also identify some maternal mRNAs that coimmunoprecipitate with hnRNP K in unfertilized eggs. Specific binding of these mRNAs to hnRNP K was verified by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, real-time PCR analyses revealed a subset of the mRNAs whose binding to hnRNP K might be up or downregulated in activated eggs. In vitro binding assay with the use of poly U monopolymeric RNA-coupled beads demonstrated that the RNA-binding property of hnRNP K is negatively regulated by tyrosine phosphorylation and positively or neutrally regulated by serine/threonine phosphorylation. Taken together, it is attractive to suggest that hnRNP K is in association with certain pools of maternal mRNAs whose translational activation are modulated by the Src/MAPK phosphorylation of hnRNP K during oocyte-egg-embryo transition.