L-arginine deprivation regulates cyclin D3 mRNA stability in human T cells by controlling HuR expression

Abstract

Myeloid-derived suppressor cells are a major mechanism of tumor-induced immune suppression in cancer. Arginase I-producing myeloid-derived suppressor cells deplete l-arginine (L-Arg) from the microenvironment, which arrests T cells in the G(0)-G(1) phase of the cell cycle. This cell cycle arrest correlated with an inability to increase cyclin D3 expression resulting from a decreased mRNA stability and an impaired translation. We sought to determine the mechanisms leading to a decreased cyclin D3 mRNA stability in activated T cells cultured in medium deprived of L-Arg. Results show that cyclin D3 mRNA instability induced by L-Arg deprivation is dependent on response elements found in its 3'-untranslated region (UTR). RNA-binding protein HuR was found to be increased in T cells cultured in medium with L-Arg and bound to the 3'-untranslated region of cyclin D3 mRNA in vitro and endogenously in activated T cells. Silencing of HuR expression significantly impaired cyclin D3 mRNA stability. L-Arg deprivation inhibited the expression of HuR through a global arrest in de novo protein synthesis, but it did not affect its mRNA expression. This alteration is dependent on the expression of the amino acid starvation sensor general control nonderepressible 2 kinase. These data contribute to an understanding of a central mechanism by which diseases characterized by increased arginase I production may cause T cell dysfunction.

FIGURE 1

Cyclin D3 3′-UTR is responsible for the low cyclin D3 mRNA stability found in L-Arg–deprived T cells. A, Similar expression of cyclin D3 in selected clones of EBV cells transfected with cyclin D3 ORF or cyclin D3 cDNA plasmids. B, EBV cells stably transfected with cyclin D3 ORF or cyclin D3 cDNA plasmids were cultured for 12 h in medium containing or deprived of L-Arg, after which actinomycin D was added, and cyclin D3 mRNA half-life was tested by Northern blot. Values are from three similar experiments.

FIGURE 2

HuR binds to the 3′-UTR of cyclin D3 mRNA only in T cells cultured in the presence L-Arg. A, RNA-binding shift assay, where cell extracts from T cells cultured in medium with and without L-Arg for 24 and 48 h were mixed with an in vitro-transcribed radiolabeled 3′-UTR cyclin D3 mRNA. Specificity of the reaction was determined by adding an excess (15 or 150 pg) of unlabeled in vitro-transcribed cyclin D3 3′-UTR mRNA (related) or GAPDH mRNA (unrelated) to the protein extracts of activated T cells cultured with L-Arg and radiolabeled 3′-UTR cyclin D3 mRNA. B, Expression of HuR, AUF1, and TTP in whole-cell extracts from T cells cultured for 48 h in medium with or without L-Arg by Western blot. C, Blocking Abs against TTP and HuR were added before the mix with the in vitro-transcribed mRNA. D, Activated T cells were cultured in the presence or the absence of L-Arg for 48 h, after which mRNA was isolated from cells (input), cytoplasmic protein extracts (Cyto), and after immunoprecipitation of HuR or TTP. The mRNA was then tested for the expression of cyclin D3 mRNA using RT-PCR. Values are from three similar experiments.

FIGURE 3

L-Arg deprivation impairs HuR expression in activated T cells. Representative experiment from three similar experiments showing the cytoplasmic (A) and nuclear (B) expression of HuR in activated T cells cultured with and without L-Arg by Western blot.

FIGURE 4

HuR silencing impairs cyclin D3 mRNA stability and expression. A, Primary T cells were transfected by electroporation with FAM-labeled siRNA against HuR or nonhomologous sequences (Control siRNA). FAM-positive cells were sorted, activated for 24 h in medium with or without L-Arg, and tested for cyclin mRNA stability using Northern blot. B, After 24 h of activation, transfected T cells were tested for the expression of HuR and cyclin D3 by Western blot. The experiment was repeated a minimum of three times, with similar results having been obtained.

FIGURE 5

L-Arg deprivation inhibits HuR expression by impairing global translation. A, Detection of HuR mRNA levels using real-time PCR after culture of cells in medium with or without L-Arg for 24 and 48 h. B, Pulse-chase analysis for HuR in activated T cells cultured in the presence or absence of L-Arg for 48 h. C and D, HuR mRNA and total mRNA levels were tested in polysomes isolated using sucrose gradients from activated T cells cultured in the presence or absence of L-Arg for 48 h. The experiment was repeated a minimum of three times, with similar results having been obtained.

FIGURE 6

GCN2 mediates changes in cyclin D3 mRNA stability caused by L-Arg deprivation. A, Detection of phospho-eIF2α and phospho-GCN2 in T cells cultured in medium with or without L-Arg. B, GCN2 mRNA levels in activated T cells cultured for 24 h in the presence or starvation of L-Arg. HuR expression (C) and cyclin D3 mRNA stability (D) were tested in activated T cells from GCN2 knockout and wild-type mice, cultured with or without L-Arg, by Western blot and Northern blot, respectively. The experiments were repeated three times, with similar results having been obtained.