Abstract: Although both retinoic acid (RA) and L-ascorbic acid-2-phosphate trisodium salt (AscPNa) can promote the induction of mouse embryonic fibroblasts (MEF) into induced pluripotent stem cells (iPS), their coexistence Under the circumstances, the induction ability of both decreases instead. Zheng Hui’s team from the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences put forward the following hypothesis: RA and AscPNa may share some downstream signaling pathways, causing mutual “restraints.” Their study confirmed through RNA-seq that the downstream activated genes of RA and AscPNa during the reprogramming process are highly overlapped. In addition, RA can help L-ascorbic acid membrane (Asc) transfer by regulating Glut1/3, promoting and maintaining its long-term high-level oxidation state in the cell. On the other hand, AscPNa mainly down-regulates MET in the process of promoting it. Zeb1 and Twist1, thereby inhibiting the expression of Cyp26a1/b1, thereby maintaining intracellular RA levels. The two have different abilities and functions in somatic cell reprogramming. This study concluded that there is a certain positive feedback effect between RA and AscPNa (vitamins A and C) during induced reprogramming. The specific results are as follows.
The authors found that as long as AscPNa is present, Oct4-positive clones can appear in MEF cells under different culture conditions for about 12 days. However, AscPNa can inhibit the induction efficiency of RA (Figure 1B-D). Next, the authors performed RNA-seq on inducibly reprogrammed cells cultured with N2B27 medium for 6 days. Overlapping genes regulated by RA and AscPNa were identified from the differential genes. The up-regulated genes were mainly related to cell cycle, neurogenesis, and cell adhesion; the down-regulated genes were related to extracellular matrix, cell migration, and chemotaxis (Figure 1E-H).
The author speculated that a possible explanation is that RA and AscPNa conflict in the process of participating in the synthesis and degradation of Asc, so they followed the clues and found the SVCT and GLUT families, because they play a role in transporting and maintaining Asc. The results showed that the RARA elements in the promoter regions of Glut1 and Glut3 were activated on day 6 during RA-induced reprogramming, and their expression increased, and the corresponding intracellular levels of Asc also increased (Figure 2A-D). Next, the authors tested the effect of AscPNa on Glut1 and Glut3 in MEF cells during the early stage of drug induction (dehydroascorbic acid DHAA was also used for comparison, but it was found that DHAA was not as stable as AscPNa). The results showed that RA can maintain intracellular intracellular Asc levels and prevent the late intracellular Asc decline (Figure 2E-N). In summary, RA helps maintain intracellular Asc levels.
Conversely, what is the impact of AscPNa on RA? In the same way, RA is degraded by CYP26A1 and CYP26B1 in the body. Now under the action of AscPNa, these two enzymes are inhibited (Figure 3A-B). Then the author constructed a GFP reporter plasmid containing three RARA elements plus a TK promoter, and transfected it into MEF cells. When RA is treated, GFP will be expressed. When RA is removed, GFP expression will gradually decrease. When RA was removed and AscPNa was added at the same time, GFP was still expressed (Figure 3C-D). The same was true when the promoters of CYP26A1 and CYP26B1 were replaced (Figure 3E-F). Next, the author thought that a downstream transcription factor regulated by AscPNa could bind to these promoters to play a role. The author started from the perspective of EMT and used TGF-β and TGF-β inhibitors to also achieve the transcription of CYP26A1 and CYP26B1. The regulatory effect (Figure 3G-H), then the author followed the clues and thought of some transcription factors that are more important in the M state in EMT, such as ZEB1, TWIST1, etc., and predicted through Pscan software that they can indeed combine with the CYP26A1 and CYP26B1 promoters. and was verified (Figure 3I-K). It is concluded here that AscP Tosoh Matte Powder Na inhibits the expression of CYP26A1 and CYP26B1 by inducing MET, thereby causing the accumulation of RA in cells.
Finally, the author concluded as follows: During the iPS-induced reprogramming process, RA upregulates Glut1, Glut3, etc., maintaining high levels of intracellular Asc, thereby promoting reprogramming; while AscPNa causes MET to occur, thereby inhibiting the expression of CYP26A1 and CYP26B1. ultimately disrupting the degradation of RA. This regulatory mechanism is conserved in other cells and biological behaviors, so this molecular mechanism is of great significance.
