Abstract
The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.
Highlights
Macroautophagy or autophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long lived proteins and organelles [1,2,3]
The increase in proteolysis induced by C2-Cer was essentially due to the augmentation in the fraction of protein degradation sensitive to 3-MA (Fig. 1A). This result suggested that C2-Cer stimulates autophagy in HT-29 cells. This conclusion was supported by morphometric analysis showing that the fractional volume occupied by autophagic vacuoles is significantly increased in cells treated with C2-Cer and incubated either in nutrient-free medium or in complete medium (Fig. 1B)
Ceramide Interferes with the Class I PI3K Signaling Pathway to Stimulate Autophagy in HT-29 Cells—C2-Cer is able to stimulate the accumulation of autophagic vacuoles, whereas HT-29 cells were cultured in complete medium (Fig. 1B, b) suggesting that ceramide can relieve the inhibitory effect induced by growth factors
Summary
Macroautophagy or autophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long lived proteins and organelles [1,2,3]. Ceramide reverts the inhibition of the class I PI3K signaling pathway on autophagy by interfering with the IL-13-dependent activation of protein kinase B (PKB) [34] and stimulates the expression of beclin 1. This conclusion was supported by morphometric analysis showing that the fractional volume occupied by autophagic vacuoles is significantly increased in cells treated with C2-Cer and incubated either in nutrient-free medium or in complete medium (Fig. 1B).
Sign-in/Register to view highlights & summary 
Published Version (
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