Human PROM1 Stable Cell Line - CHO-K1

Prominin-1 (Prom1) is a transmembrane glycoprotein expressed in stem cell lineages and recently implicated in cancer stem cell survival. Mutations in the Prom1 gene have been shown to disrupt photoreceptor disc morphogenesis and cause autosomal dominant Stargardt-like macular dystrophy (STGD4). Here, researchers aimed to investigate the role of Prom1 in metabolically active cells of the retinal pigment epithelium (RPE). Studies have shown that Prom1 is primarily present in the cytoplasm of the RPE. Stress signals and physiological aging significantly increase autophagy, with upregulation of Prom1 expression. Knockout of Prom1 enhances mTORC1 and mTORC2 signaling, reduces trafficking of autophagosomes to lysosomes, increases p62 accumulation, and inhibits autophagy sites induced by autophagy activators. Conversely, ectopic overexpression of Prom1 inhibits the activity of mTORC1 and mTORC2 and enhances autophagic flux. Through interaction with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a novel cytoplasmic role for Prom1 in RPE function. These results suggest that Prom1 plays a key role in autophagy regulation by inhibiting upstream mTOR signaling and is also a component of the macromolecular scaffold engaged by p62 and HDAC6.

Here, researchers verified whether autophagic flux is enhanced by Prom1 overexpression. Control ARPE-19 cells and wild-type cells were cultured in normal growth medium (FBS) or amino acid starvation medium (EBSS) in the presence or absence of BAF. Consistent with the data shown in Figure 1A, BAF increased the accumulation of LC3-II in control cells. EBSS alone reduced LC3-I and partially increased LC3-II, while in control cells, EBSS+BAF reduced LC3-I but significantly increased LC3-II expression. In Prom1 overexpressing WT cells, BAF alone increased LC3-II to levels higher than those in control ARPE-19 cells. Interestingly, EBSS reduced LC3-I and increased LC3-II levels in wild-type cells, while BAF further increased LC3-II levels in EBSS-treated wild-type cells. Since LC3-II levels increase rapidly in wild-type (WT) cells, the researchers used the LC3-II/actin ratio and the p62/actin ratio to measure autophagy in wild-type cells. Under normal growth conditions, wild-type cells had an increased LC3-II/actin ratio and decreased p62/actin ratio, Akt, and S6 Rp phosphorylation levels, indicating that autophagy was constitutively and sustainably activated. BAF treatment enhanced this response (Figure 1C, 1D). EBSS treatment in wild-type cells further increased the LC3-II/actin ratio and decreased the expression of p62, p-Akt, and p-S6 Rp, confirming the enhancement of autophagy. The enhanced response was observed in wild-type cells treated with EBSS+BAF (Figure 1C, 1D), indicating that Prom1 overexpression enhances autophagic flux under BAF and amino acid starvation conditions.

Figure 1. Overexpression of Prom1 increases autophagic flux through inhibition of mTORC1 and mTORC2 in the RPE. (Bhattacharya S, et al., 2017)