Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells

Abstract

Inorganic phosphate (Pi) plays a critical role in diverse cellular functions. Among three classes of sodium/phosphate co-transporters (NPTs), two types have been identified in mammalian lung. The potential importance of Pi as a novel signaling molecule and pulmonary expression of NPTs with poor prognosis of diverse lung diseases including cancer have prompted us to begin to define the pathways by which Pi regulates nontumorigenic human bronchial epithelial cells. Pi activates Akt phosphorylation on Thr308 specifically, and activated signal transmits on the Raf/MEK/ERK signaling. Here, we report that Pi controls cell growth by activating ERK cascades and by facilitating the translocation of Mnk1 from cytosol into nucleus through an Akt-mediated MEK pathway. Sequentially, translocated Mnk1 increases eIF4E-BP1 phosphorylation. As a result, Pi stimulates cap-dependent protein translation. Such Akt-mediated signaling of inorganic phosphate may provide critical clues for treatment as well as prevention of diverse lung diseases.

Figure 1.

Concentration-dependent effects of inorganic phosphate (Pi) on NHBE cells. (A) NHBE cells were treated with various concentrations of Pi and incubated for 48 h, and cell viability was measured using MTT assay. The open bar represents the control/normal media containing 5.63 mM Pi, whereas filled bars represent different Pi concentrations. (B) Concentration-dependent NPT-IIa protein expression. NHBE cells were treated with increasing concentrations of Pi (0–20 mM) for 24 h and 25 μg of protein lysate was used for Western blot. Upper panels, Western blot; lower panel, densitometric analysis. (C) Concentration-dependent Akt phosphorylation on Thr308 and Ser473. NHBE cells were treated with increasing concentrations of Pi (0–20 mM) for 24 h and 25 μg of protein was used for Western blot. Upper panels, Western blot; lower panel, densitometric analysis. Open and filled bars represent degrees of phosphorylation of Akt at Thr308 and S473, respectively. Values are the means ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Time-dependent effects of Pi on NPT-IIa, Akt phosphorylation, and Raf/MEK/ERK signals. Cells were treated with 10 mM Pi for various time points, then lysed, and 25 μg of protein was used for Western blot. (A) Time-dependent NPT-IIa protein expression. Upper panel, Western blot; lower panel, densitometric analysis. (B) Time-dependent Akt phosphorylation on Thr308 and Ser473. Upper panels, Western blot; lower panel, Densitometric analysis. Open and filled circles represent degrees of phosphorylation of Akt at Thr308 and S473, respectively. (C) Time-dependent expressions of Raf/MEK/ERK signal proteins. (D) Time-dependent expression of eIF4E-BP1 protein in NHBE cells.

Figure 2.

Time-dependent effects of Pi on NPT-IIa, Akt phosphorylation, and Raf/MEK/ERK signals. Cells were treated with 10 mM Pi for various time points, then lysed, and 25 μg of protein was used for Western blot. (A) Time-dependent NPT-IIa protein expression. Upper panel, Western blot; lower panel, densitometric analysis. (B) Time-dependent Akt phosphorylation on Thr308 and Ser473. Upper panels, Western blot; lower panel, Densitometric analysis. Open and filled circles represent degrees of phosphorylation of Akt at Thr308 and S473, respectively. (C) Time-dependent expressions of Raf/MEK/ERK signal proteins. (D) Time-dependent expression of eIF4E-BP1 protein in NHBE cells.

Figure 3.

Effects of phosphate transport inhibitor on NPT-IIa, Akt phosphorylation, and cell growth. Cells were pretreated with Foscarnet phosphate transport inhibitor for 30 min, and then 10 mM of Pi was added for an additional 24 h. After 24 h, cells were lysed and 25 μg of protein was used for Western blot. (A) Changes of NPT-IIa protein (left panels) and Akt phosphorylation at Thr308 (right panels) were detected by Western blot. GAPDH and Akt1 were used as each corresponding internal standard. Upper panels, Western blot; lower panels, densitometric analysis. Values are the means ± SE of three independent experiments. **P < 0.01, ***P < 0.001 versus without Pi, ^##P < 0.01, ^###P < 0.001 versus without inhibitors. (B) Effects of various concentrations of Foscarnet on viability of cells treated with/without Pi measured by MTT assay. Three different numbers of cells grown in 96 wells (left: 5,000 cells/well; middle: 7,000 cells/well; right: 10,000 cells/well) were treated with increasing concentrations of Foscarnet (0–5 mM) in 10 mM of Pi. Based on above study, optimal concentration of Foscarnet (0.5 mM) as well as appropriate cell number (7,000 cells/well) were selected for further studies shown in panel C. ^$$P < 0.01, ^$$$P < 0.001 significantly increased compared with control, and ***P < 0.001 significantly decreased compared with control. (C) Effects of Pi on cell growth. Cells (7,000 cells/well) were grown in wells and treated with Pi in the presence or absence of 0.5 mM Foscarnet and observed under ×200 magnification. Pi caused the increase of cell size (middle panel) compared with control (left panel); however, Foscarnet treatment suppressed the cell growth significantly (right panel). The photograph is representative of three independent experiments. For all above studies, control cells were treated with 10 mM Na₂SO₄ to clarify the potential effect of salts. Note that the pattern of negative control was almost identical to that of control cells (data not shown).

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Figure 6.

Time-dependent effects of Pi on Mnk1 translocation between cytosol and nucleus. (A) Western blot analysis of phospho-Mnk1 between cytosol and nucleus. Cells were treated with 10 mM Pi for various time points, collected, then, separated into cytosol and nucleic fractions by Nuclear Extract Kit. Twenty-five micrograms of protein was used for Western blot of phospho-Mnk1. GAPDH and Histone H1 were used as controls for cytosol and nucleus, respectively. Upper panels, Western blot analysis; lower panel, densitometric analysis. (B) Fluorescence imaging of the distribution of phospho-Mnk1 between cytosol and nucleus. At different time points, cells were collected, fixed, and assayed by immunostaining for phospho-Mnk1 (green via FITC), and nuclei (blue via DAPI). Two representative time points are shown for better explanation. Scale bars = 10 μm.

Figure 6.

Time-dependent effects of Pi on Mnk1 translocation between cytosol and nucleus. (A) Western blot analysis of phospho-Mnk1 between cytosol and nucleus. Cells were treated with 10 mM Pi for various time points, collected, then, separated into cytosol and nucleic fractions by Nuclear Extract Kit. Twenty-five micrograms of protein was used for Western blot of phospho-Mnk1. GAPDH and Histone H1 were used as controls for cytosol and nucleus, respectively. Upper panels, Western blot analysis; lower panel, densitometric analysis. (B) Fluorescence imaging of the distribution of phospho-Mnk1 between cytosol and nucleus. At different time points, cells were collected, fixed, and assayed by immunostaining for phospho-Mnk1 (green via FITC), and nuclei (blue via DAPI). Two representative time points are shown for better explanation. Scale bars = 10 μm.

Figure 7.

Increase in nuclear phosphorylated Mnk1 in response to small changes in extracellular phosphate. NHBE cells were treated with 2 mM Pi for 5 d, then collected, and separated into total (A), cytosol (B), and nucleic (C) fractions by Nuclear Extract Kit. Twenty-five micrograms of protein was used for Western blot of phospho-Mnk1. GAPDH and Histone H1 were used as controls for cytosol and nucleus, respectively. Left panels, Western blot analysis; right panels, densitometric analysis.

Figure 8.

Schematic signal pathway by which Pi affects in NHBE cells. Based on our studies with the transport inhibitor, Foscarnet, Pi is transported into NHBE cells through a sodium-dependent phosphate transporter (NPT) and results in the activation of Akt by phosphorylation of Thr308. Such activated Akt phosphorylates Raf, MEK, ERK, then facilitates Mnk1 translocation from cytosol into nucleus resulting increased cap-dependent protein translation.