Abstract
As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf) uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-β-cyclodextrine up regulated Tf uptake at greater intensity in the CD133high situation than in the CD133low situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post-transciptional effects. Taken together, these data extend our knowledge of the function of CD133 and underline the interest of further exploring the CD133-Tf-iron network.
Highlights
Following the use of new monoclonal antibodies raised against neuroepithelial and hematopoietic stem cells, CD133, known in humans and rodents as Prominin-1, was first isolated and cloned in 1997 [1,2,3]
Alternative internalization pathways have been described depending on cell types and differentiation status, TfAlexa 488, Dx-FITC and cholera toxin subunit B (CTB)-FITC were used as prototype markers of receptor mediated endocytosis [34], fluid phase endocytosis [24,35] and caveolae dependent endocytosis [23,36], respectively
CD133 knockdown had no impact on intracellular accumulation of DxFITC (Figure 1B) or on CTB-FITC (Figure 1C), cellular uptake of Tf-Alexa 488 was significantly amplified in CD133low-Caco-2 cells whatever the concentration tested (Figure 1D)
Summary
Following the use of new monoclonal antibodies raised against neuroepithelial and hematopoietic stem cells, CD133, known in humans and rodents as Prominin-1, was first isolated and cloned in 1997 [1,2,3]. CD133 is a five-domain transmembrane protein, composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites and a short C-terminal intracellular tail that can be alternatively spliced [4] or phosphorylated [5]. It has been shown that a truncated CD133, which is not transported to cell membrane, leads to human retinal degeneration [6]. Underlining this important observation, analysis of a generation of CD133-deficient mice revealed that, while expressed very early during retinal development, CD133 acted as a key regulator of disk morphogenesis and that loss of CD133 caused photoreceptor degeneration and blindness [7]. Other investigations revealed that CD133 is linked to cell metabolism as a glucose responsive gene in myotubes [11], as well as providing evidence for bioenergetic stress [12] and of non-exposure to high oxygen tension in gliomas (Bourseau-Guilmain et al, submitted)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
