mTOR transcriptionally and post-transcriptionally regulates Npm1 gene expression to contribute to enhanced proliferation in cells with Pten inactivation

Abstract

The mammalian target of rapamycin (mTOR) plays essential roles in the regulation of growth-related processes such as protein synthesis, cell sizing and metabolism in both normal and pathological growing conditions. These functions of mTOR are thought to be largely a consequence of its cytoplasmic activity in regulating translation rate, but accumulating data highlight supplementary role(s) for this serine/threonine kinase within the nucleus. Indeed, the nuclear activities of mTOR are currently associated with the control of protein biosynthetic capacity through its ability to regulate the expression of gene products involved in the control of ribosomal biogenesis and proliferation. Using primary murine embryo fibroblasts (MEFs), we observed that cells with overactive mTOR signaling displayed higher abundance for the growth-associated Npm1 protein, in what represents a novel mechanism of Npm1 gene regulation. We show that Npm1 gene expression is dependent on mTOR as demonstrated by treatment of wild-type and Pten inactivated MEFs cultured with rapamycin or by transient transfections of small interfering RNA directed against mTOR. In accordance, the mTOR kinase localizes to the Npm1 promoter gene in vivo and it enhances the activity of a human NPM1-luciferase reporter gene providing an opportunity for direct control. Interestingly, rapamycin did not dislodge mTOR from the Npm1 promoter but rather strongly destabilized the Npm1 transcript by increasing its turnover. Using a prostate-specific Pten-deleted mouse model of cancer, Npm1 mRNA levels were found up-regulated and sensitive to rapamycin. Finally, we also showed that Npm1 is required to promote mTOR-dependent cell proliferation. We therefore proposed a model whereby mTOR is closely involved in the transcriptional and posttranscriptional regulation of Npm1 gene expression with implications in development and diseases including cancer.

Keywords: Npm1; cell proliferation; gene expression; mTOR; prostate cancer.

Figure 1.

Npm1 mRNA and protein levels are enhanced in MEF cells inactivated for Pten. (A) Thirty µg of total protein extracts from wild-type (WT) and Pten knockout (KO) mouse embryonic fibroblasts were separated by SDS-PAGE and immunoblotted with specific antibodies as indicated (n = 3). (B) RT-qPCR analysis of Npm1 mRNA accumulation in WT and Pten KO MEFs (normalized to 1.0 relative to 36b4 mRNA gene for control WT MEFs). Bar graphs show the mean level of three independent experiments (±SEM) and a student's test was performed to assess statistical significance. **p < 0.01.

Figure 2.

Inhibition of mTOR signaling reduces Npm1 expression in MEFs. (A) Cultured wild-type (WT) and Pten knockout (KO) cells were treated for 24 hours in the absence or the presence of 20 nM rapamycin. Cell extracts were prepared and aliquots containing 30 µg of total proteins were resolved by SDS-PAGE for proteins visualization by Western blotting. (B) Cells were incubated as above except that rapamycin was added to the medium for 4 hours. Npm1 and mTOR expression levels were then assayed by RT-qPCR from mRNA isolated from WT and KO MEFs for Pten and levels were normalized by comparison to the 36b4 mRNA. Bar graphs show the mean levels of at least three independent experiments (±SEM) of qPCR-amplified Npm1 (normalized to 1.0 for control untreated WT MEF cells). (C) Western blot analysis of total Npm1 and mTOR in MEF cells treated with a pool of siRNA (50 nM) for 48 hours against mTOR. β-Actin levels are shown as a loading control. (D) RT-qPCR analysis of Npm1 expression after depletion of mTOR. Errors bars represent mean ± SEM normalized to WT untreated cells (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3.

mTOR binding and activation of Npm1 promoter. (A) Endogenous mTOR is associated with Npm1 promoter region in a rapamycin-independent manner. ChIP assays were conducted using a mTOR antibody in WT and Pten KO MEFs in the absence or the presence of 20 nM rapamycin for 4 hours. Binding of mTOR to Npm1 promoter and Pol-III transcribed tRNA^Arg/Tyr were determined with PCR primer sets described in Material and Methods. Rabbit IgG were used as negative control. (B) Npm1 is a transcriptional target of mTOR. HeLa (left) and MEF (middle) cell lines were co-transfected for 48 hours with 200 ng of hNPM1-luc plasmid encoding the luciferase under the control of the human NPM1 promoter region and increasing amounts of a pCMV-Flag mTOR vector (100, 200 and 500 ng). The luciferase activity measured in WT MEF cells transfected with hNPM1-luc or CMV-luc was normalized to 1 and a relative fold-induced luciferase activity was then calculated for Pten KO MEFs in the same experimental conditions (right). Error bars represent ± SEM (n = 3; asterisk, p < 0.05; two asterisks, p < 0.01; three asterisks, p < 0.001).

Figure 4.

mTOR signaling inhibition by rapamycin increase Npm1 mRNA turnover. Total mRNA from early log phase growing MEFs were used for qPCR analysis of Npm1 expression. Cells were treated with actinomycin D (5µg/ml), rapamycin (20 nM), or rapamycin plus actinomycin D as indicated. Cells were harvested after 10 h and total mRNA was extracted for Npm1 expression evaluation. The mRNA level in WT MEFs alternatively treated with vehicle was considered as 100%. Data are representative of 3 independent experiments and expressed as fold change relative to WT vehicle-treated MEFs, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and bar graphs represent mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5.

Npm1 overexpression in mouse prostate tumors induced by Pten loss is sensitive to chronic mTOR inhibition by rapamycin. (A) Homozygous Pten deletion increases Npm1 abundance in the mouse dorsolateral prostate gland lobe. Protein lysates were prepared from three month-old animals and were probed with the indicated antibodies by western immunoblotting. (B) Chronic mTOR inhibition decreases the abundance of Npm1 in Pten-null prostate cancer. Three-month old Pten conditional knockout mice were chronically administrated with either vehicle or rapamyscin (10 mg/kg/day). After 4 days, protein levels in the dorsolateral compartment of the Pten-null prostate lobe were separated by SDS-PAGE and assayed by western blotting using the indicated antibodies. Representative blots are derived from 2 different animals per group. Compared Pten knockout mice treated with vehicle (lanes 1 and 2) with rapamycin-treated animals lanes 3 and 4).

Figure 6.

Pten deletion-induced proliferation in MEFs with hyperactive mTOR requires Npm1. (A) Exponentially growing MEFs were transfected with specific Npm1 or control GFP (Ctrl) siRNAs. 24 hours after transfection with 50 nM Npm1 and Ctrl GFP siRNAs, whole-cell extracts were prepared and protein gel blot was performed using antibodies against Npm1 and β-Actin for loading control. (B) Npm1 depletion reverses Pten-loss induced hyper-proliferation of MEFs with hyperactive mTOR signaling. Wild-type and inactivated MEFs for Pten were plated at a respective density of 17,5 × 10³ and 7,5 × 10³ cell per well into a 96-wells plate and were transfected 1 day later with either Npm1 and Ctrl GFP siRNAs. After 24 hours, medium was changed and the cell proliferation was assayed by using the BrdU reagent according to the Material and Methods. (C) Npm1 silencing decreased the upregulated Cyclin D1 protein level induced by Pten inactivation. MEFs deleted for Pten were transfected as above and total protein lysate of equal numbers of cells were subjected to Western blot analysis with antibodies to cyclin D1, cyclin D2, cyclin E, and cyclin B1. β-Actin levels are shown as a loading control. (n = 3) *p < 0.05.