TGFβ-induced PI 3 kinase-dependent Mnk-1 activation is necessary for Ser-209 phosphorylation of eIF4E and mesangial cell hypertrophy

Abstract

Transforming growth factorβ (TGFβ)-induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time-dependently stimulated eIF4E phosphorylation at Ser-209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen-activated and extracellular signal-regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ-induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser-209 is mediated by Mnk-1 (mitogen-activated protein kinase signal-integrating kinase-1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk-1 in response to TGFβ. Dominant negative Mnk-1 significantly inhibited TGFβ-stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E-4EBP-1 complex, decreased TGFβ-stimulated phosphorylation of eIF4E without any effect on Mnk-1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP-1-eIF4E complex for Mnk-1-mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ-induced noncanonical signaling circuit involving PI 3 kinase-dependent Mnk-1-mediated phosphorylation of eIF4E at Ser-209 is required to facilitate mesangial cell hypertrophy.

Fig. 1

TGFβ increases phosphorylation of eIF4E and mesangial cell hypertrophy in Erk1/2-dependent manner. A, B: Serum-starved (48 h) mesangial cells were incubated with 2 ng/ml TGFβ for the indicated times. The cell lysates were immunoblotted with phospho-eIF4E, eIF4E, phospho-Erk1/2, Erk1/2 (part A), and phospho-MEK and MEK (part B) antibodies. C: Mesangial cells were treated with 5 μM U0126 for 1 h prior to incubation with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with the indicated antibodies. D, E: Serum-starved (24 h) mesangial cells were incubated with 5 μM of U0126 for last 1 h. TGFβ was added for 24 h. Protein synthesis (part D) and hypertrophy (part E) were determined as described (Das et al., 2008a). In part E, cell hypertrophy was determined as increase in the ratio of total protein to cell number as described (Das et al., 2008a). In part D,^*P < 0.01 versus control; ^**P < 0.05 versus TGFβ-stimulated. In part E,^*P < 0.05 versus control; ^**P < 0.05 versus TGFβ-stimulated. F, G: Mesangial cells were transfected with dominant negative Myc Erk2. Transfected cells were serum starved for 24 h and then incubated with 2 ng/ml TGFβ for 24 h. Protein synthesis (part F) and hypertrophy (part G) were determined as described above. Mean ±SE of triplicate measurements is shown. For part F, ^*P < 0.05 versus control; ^**P < 0.05 versus TGFβ-stimulated. For part G, ^*P < 0.01 versus control; ^**P < 0.05 versus TGFβ-stimulated. Bottom parts show expression of Myc Erk2 and actin in one representative samples.

Fig. 2

PI 3 kinase regulates Erk1/2 dependent phosphorylation of eIF4E. Forty hours serum-starved mesangial cells were treated with 25 μM Ly294002 prior to incubation with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with antibodies against phospho-MEK, MEK (part A), phospho-Erk1/2, Erk1/2 (part B), and phospho-eIF4E, eIF4E (part C).

Fig. 3

PI 3 kinase regulates Raf-1 phosphorylation in the absence of Akt-mediated Erk1/2 and eIF4E phosphorylation. A: Mesangial cells were serum-starved for 48 h and treated with 25 μM Ly294002 for 1 h followed by incubation with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with phospho-Raf-1 (Ser-259) and Raf-1 antibodies. B–E: Mesangial cells were infected with adenovirus vector expressing dominant negative Akt (Ad DN Akt) for 24 h in serum-free medium. The infected cells were further serum-starved for 24 h and incubated with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with indicated antibodies.

Fig. 4

Erk1/2 and PI 3 kinase regulate TGFβ-stimulated phosphorylation of Mnk-1. A: Forty-eight hours serum-starved mesangial cells were incubated with 2 ng/ml TGFβ for the indicated times. The cell lysates were immunoblotted with phospho-Mnk-1 and Mnk-1 antibodies. B,D: Mesangial cells were incubated with 5 μMU0126 (part B) and 25 μMLy294002 (part D) for 1 h. The cells were then incubated with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with phospho-Mnk-1 and Mnk-1 antibodies. Part C: Mesangial cells were transfected with empty vector or dominant negative Myc-tagged Erk2 (DN Myc Erk2). The serum-starved cells were then incubated with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with phospho-Mnk-1, Myc, Mnk-1 antibodies as indicated.

Fig. 5

Requirement of Mnk-1 for TGFβ-induced protein synthesis and hypertrophy. Mesangial cells were transfected with dominant negative Mnk-1 plasmid or empty vector (control) and serum-starved for 24 h. To these cells2 ng/ml TGFβ was added for 24 h. Protein synthesis (part A) and hypertrophy (part B) were measured as described (Das et al., 2008a). The bottom parts show expression of Mnk-1 and actin in representative samples.

Fig. 6

Dissociation of eIF4E-4EBP-1 complex is necessary for eIF4E phosphorylation. A, E: Serum-starved mesangial cells were treated with 25 nM rapamycin prior to incubation with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with phospho-eIF4E, phospho-S6 kinase, eIF4E, S6 kinase (part A), phospho-Mnk-1, and Mnk-1 (part E) antibodies as indicated. B: Lysates of mesangial cells treated with rapamycin and TGFβ similar to as described in parts A and E were immunoprecipitated with 4EBP-1 antibody followed by immunoblotting with eIF4E antibody. C, D, F, G: Mesangial cells were transfected with vector expressing scrambled RNA or shRNA against raptor (shRaptor 1, parts C and F) (shRaptor 2, parts D and G). The transfected cells were serum-starved for 48 hand incubated with 2 ng/ml TGFβ for 15 min. The cell lysates were immunoblotted with phospho-eIF4E, phospho-S6 kinase, eIF4E, S6 kinase, phospho-Mnk-1, Mnk-1, raptor, and actin antibodies as indicated.

Fig. 7

Phosphorylation of eIF4E is required for protein synthesis and hypertrophy. A, B: Mesangial cells were transfected with eIF4E S209D phospho-mimetic mutant. The transfected cells serum-starved for 24 h and then incubated with 2 ng/ml TGFβ for 24 h. Protein synthesis (part A) and hypertrophy (part B) were measured as described in Figure 2. Mean ±SE of triplicate measurements is shown. In part A, ^*P < 0.001 versus control; ^**P < 0.05 versus control. In part B, ^*P < 0.05 versus control; ^**P < 0.01 versus control. C, D: Mesangial cells were transfected with eIF4E S209A phospho-deficient mutant. The transfected cells were starved and incubated with 2 ng/ml TGFβ as described above. Protein synthesis (part C) and hypertrophy (part D) were measured as described in Figure 2. Mean ±SE of triplicate measurements is shown. In part C, ^*P < 0.01 versus control; ^**P < 0.01 versus TGFβ-stimulated. In part D, ^*P < 0.001 versus control; ^**P < 0.05 versus TGFβ-stimulated.

Fig. 8

Schematic summarizing our results.