The immunosuppressant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation

Abstract

RAFT1/FRAP/mTOR is a key regulator of cell growth and division and the mammalian target of rapamycin, an immunosuppressive and anticancer drug. Rapamycin deprivation and nutrient deprivation have similar effects on the activity of S6 kinase 1 (S6K1) and 4E-BP1, two downstream effectors of RAFT1, but the relationship between nutrient- and rapamycin-sensitive pathways is unknown. Using transcriptional profiling, we show that, in human BJAB B-lymphoma cells and murine CTLL-2 T lymphocytes, rapamycin treatment affects the expression of many genes involved in nutrient and protein metabolism. The rapamycin-induced transcriptional profile is distinct from those induced by glucose, glutamine, or leucine deprivation but is most similar to that induced by amino acid deprivation. In particular, rapamycin treatment and amino acid deprivation up-regulate genes involved in nutrient catabolism and energy production and down-regulate genes participating in lipid and nucleotide synthesis and in protein synthesis, turnover, and folding. Surprisingly, however, rapamycin had effects opposite from those of amino acid starvation on the expression of a large group of genes involved in the synthesis, transport, and use of amino acids. Supported by measurements of nutrient use, the data suggest that RAFT1 is an energy and nutrient sensor and that rapamycin mimics a signal generated by the starvation of amino acids but that the signal is unlikely to be the absence of amino acids themselves. These observations underscore the importance of metabolism in controlling lymphocyte proliferation and offer a novel explanation for immunosuppression by rapamycin.

FIG. 1.

Rapamycin-sensitive gene expression in human BJAB cells. (A) At all time points rapamycin eliminates the phosphorylation of threonine 389 of S6K1. EtOH, ethyl alcohol. (B) Representation of k means cluster analysis of 543 genes regulated at 0, 15, 30, 60, and 120 min and 12 and 24 h after addition of 20 nM rapamycin. Genes are represented vertically, and experimental conditions (i.e., rapamycin time course) are displayed horizontally. Fold changes are indicated colorimetrically. (C) Quantitative RT-PCR analysis confirms rapamycin-induced gene expression changes detected using microarrays. Expression changes in four genes (BCKAD E1 alpha in cluster C, ASS in cluster D, FAS in cluster G, and SREBP-1 in cluster H) were determined in RNA samples prepared independently from those used for microarray analysis. The standard deviation for each time point was calculated from the triplicate samples in RT-PCR.

FIG. 2.

Amino acid and glucose deprivation regulates unique sets of genes. (A) Proliferation of BJAB cells over a 24-h period is similarly affected by deprivation of glutamine, leucine, or glucose and by rapamycin treatment. Cells were cultured in media containing indicated concentrations of nutrients or rapamycin. (B) Comparison of transcriptional profiles induced in response to deprivation of glutamine, leucine, or glucose. Only genes affected at 12 or 24 h were used for the comparison. Glutamine deprivation affects 71 and 82% of the genes up- and down-regulated, respectively, by leucine deprivation. Fold changes are indicated colorimetrically, individual genes are represented vertically, and experimental conditions (i.e., time course of deprivation for individual nutrients) are displayed horizontally. (C) A core set of genes regulated by both glutamine and glucose deprivation. Unigene numbers are used to represent genes with unknown function. The gene expression changes for the 24-h time point are shown.

FIG. 3.

Global comparison of gene expression changes induced by rapamycin treatment and by deprivation for individual nutrient. The sets of genes affected at 12 or 24 h after rapamycin treatment and deprivation for nutrients significantly overlap. Rapamycin shares 34, 44, and 44% of up-regulated genes as well as 58, 69, and 31% of genes down-regulated by glutamine, leucine, and glucose deprivation, respectively.

FIG. 4.

Effects of rapamycin treatment on nutrient metabolism and cell proliferation in murine CTLL-2 T lymphocytes. All nutrient measurements were performed over a 48-h period in triplicate as described in Materials and Methods. (A) Cells treated with rapamycin increased the usage of glutamine by 54% (P < 0.01). (B) Cells treated with rapamycin increased the production of ammonia when cultured in media with normal (2 mM) or low (0.1 mM) concentrations of glutamine by 44% (P < 0.01) or 20% (P < 0.05), respectively. (C) Cells treated with rapamycin increased the consumption of glucose by 19% (P < 0.05) and also increased lactate production by 31% (P < 0.01). (D) CTLL-2 cells (starting at 10⁴/ml) were grown in control medium with or without rapamycin (20 nM), medium with 0.1 mM glutamine with or without rapamycin (20 nM), or medium with 0.3 mM glucose with or without rapamycin (20 nM) for 72 h. Triplicate samples were counted every 24 h.