These studies have determined the cellular amounts of the SL of RAW264.7 cells from the first detectable intermediate (sphinganine) through Cer and its initial metabolites (SM, GlcCer, etc.) and established how KLA, a TLR4 receptor agonist, alters the amounts of many of these compounds. Through the use of an inhibitor of de novo SL biosynthesis and stable isotope labeling, we found that both control and KLA-stimulated RAW264.7 cells are actively engaged in de novo SL biosynthesis; however, soon after KLA treatment, the cells stop dividing, resulting in an increase in the cell size and SL content.
Most of the changes in SL were due to de novo
biosynthesis and are reminiscent of the induction of SL biosynthesis in liver (20
) and a number of extrahepatic tissues (21
). Increases in Cer are an interesting exception because it was also derived from another source, most likely the turnover of cellular SM, which is also in agreement with previously noted activation of sphingomyelinase and production of Cer in LPS-treated cells (22
). It is intriguing that the amount of Cer that can be attributed to de novo
biosynthesis when the cells are incubated with [13
C]palmitate is similar to the total Cer in experiments without stable isotope labeling, and when cells are treated with ISP1 in addition to KLA (, , and ). This suggests that the cells have mechanism(s) to maintain some form of “Cer homeostasis” by coordination of the formation and removal of this compound to/from multiple sources.
We think that one of the most important findings of these studies is that activation of RAW264.7 cells not only increased the cellular SL content but also cell size (~24%), surface area (~53%), and the production of the intracellular membrane vacuoles termed autophagosomes. Furthermore, de novo
SL biosynthesis was shown to be necessary for autophagosome formation, which, to the best of our knowledge, is the first demonstration of the requirement for SL for a “normal” biological process for induction of autophagy, although exogenous addition of SL, or their endogenous accumulation when SL metabolism has been disrupted, has been noted to induce autophagy in other cell types (45
). Consistent with a requirement for de novo
SL biosynthesis for autophagy, we found that CHO-LYB cells are refractory to induction of autophagy by fenretinide until a functional SPT1 was reintroduced into the cells (supplemental Fig. S5
). Therefore, SL may play an essential role in many, if not all, forms of autophagy.
It is not yet known with certainty if the de novo
biosynthesized SL(s) required for autophagy is (are) (DH)Cer per se
, although the anti-Cer antibody co-localization studies suggest that this might be the case. This raises the possibility that the production of (DH)Cer and possibly other SL (66
) by de novo
biosynthesis in the ER might be a driving force for formation of the autophagosomal vacuole, in what has been referred to as the “membrane extension” step (68
) that occurs after many of the associated autophagosomal proteins have been recruited. If this process requires participation by the ER, this might explain why elevation of Cer by SL turnover did not appear to be adequate for induction of autophagy by KLA in RAW264.7 cells, which is also consistent with the previous observation that treatment of HT29 cells with bacterial sphingomyelinase to generate Cer had no effect on autophagy induction (46
). Furthermore, because the enzymes for de novo
Cer biosynthesis reside in the ER (56
), it is conceivable that they might be recruited into autophagosomes and, perhaps, continue to produce SL there. However, a preliminary experiment using antibodies against the first two enzymes in de novo
SL biosynthesis, serine palmitoyltransferase subunit 1 (SPT1, also called “LCB1”) and 3-ketosphinganine reductase, found no evidence that the enzymatic machinery for de novo
SL biosynthesis is recruited to the autophagosome. Therefore, the idea of how ceramide becomes associated with the autophagosome is an interesting area for continued study.
There are additional reasons to suspect that Cer might play a role in autophagy. First, Cer has been identified as a mediator in the ER-localized dissociation of the Beclin-Bcl-2 complex, which facilitates autophagosomal vesicle nucleation (47
). Second, activation of macrophages invokes ER stress (71
), which is one of the up-regulators of autophagy (72
) and might involve SL as intermediaries or modulators (73
). Third, Cer has been shown to inhibit the activation of the Akt/mammalian target of rapamycin cascade resulting in the induction of autophagy (46
), and the microarray analysis by the LIPID MAPS Consortium suggests that KLA decreases mammalian target of rapamycin mRNA; therefore, KLA might induce autophagy through repression of the Akt/mammalian target of rapamycin signaling pathway at multiple levels. All of the above might be inter-related or mean that there are multiple roles for SL in autophagy, as has often proven to be the case in other biological processes.
There is a strong likelihood that additional SL (66
) and other lipid categories (e.g.
) are involved in autophagy and that the categories differ among organisms. In yeast, sterol glycoside appears to be critical for membrane extension (68
), which suggests that future studies should explore if, by “lipid homology,” Cer-monohexoses (GlcCer and/or GalCer) might play a role in induction of autophagy in mammalian cells. Balances among lipid categories might also be important, as illustrated by the induction of autophagy by cholesterol overloading (74
). Considering how many lipid categories are changed by KLA in RAW264.7 cells, there are many other possibilities to be explored. The studies described in this study have, at least, mapped the changes in SL metabolism in activated RAW264.7 cells and demonstrated a direct link between de novo
SL biosynthesis and autophagy, one of the important cell behaviors of cells of the innate immune system.