Endothelin-1 couples betaPix to p66Shc: role of betaPix in cell proliferation through FOXO3a phosphorylation and p27kip1 down-regulation independently of Akt

Abstract

The phosphorylation of forkhead transcription factor FOXO3a by Akt is critical regulator of cell proliferation induced by serum. We show that endothelin-1 (ET-1) stimulation of primary human mesangial cells (HMCs) induces betaPix and p66Shc up-regulation, resulting in the formation of the betaPix/p66Shc complex. In transformed HMCs, ET-1 induces a biphasic phosphorylation of p66Shc and FOXO3a. The second phase leads to p27(kip1) down-regulation independently of Akt. Depletion of betaPix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27(kip1) down-regulation induced by ET-1. Depletion of either betaPix or p66Shc inhibits ET-1-induced cell proliferation. The expression of beta(1)Pix induces FOXO3a phosphorylation through activation of Rac1, ERK1/2, and p66Shc. Using either p66Shc- or Akt-depleted cells; we show that beta(1)Pix-induced FOXO3a phosphorylation requires p66Shc but not Akt. beta(1)Pix-induced p27(kip1) down-regulation was blocked by U0126 but not by wortmannin. Endogenous betaPix and FOXO3a are constitutively associated with endogenous p66Shc. FOXO3a and p66Shc binding requires beta(1)Pix homodimerization. Expression of beta(1)Pix homodimerization deficient mutant abrogates beta(1)Pix-induced p27(kip1) down-regulation and cell proliferation. Our results identify p66Shc and FOXO3a as novel partners of beta(1)Pix and represent the first direct evidence of beta(1)Pix in cell proliferation via Erk/p66Shc-dependent and Akt-independent mechanisms.

Figure 1.

Endothelin-1 induces βPix and p66Shc up-regulation in primary human mesangial cells. (A) Cells were serum-starved for 24 h before endothelin-1 (100 nM) was added for the indicated times. Equal amounts of proteins were probed by immunoblotting with anti-βPix, anti-SHC, or anti-actin antibodies. (B) Cells were serum-starved for 24 h before ET-1 (100 nM) was added for 24 h. Cell lysates were immunoprecipitated with anti-p66Shc antibody and the membranes were probed with the indicated antibodies.

Figure 2.

p66Shc binding requires in β₁Pix homodimerization. (A) Endogenous p66Shc was immunoprecipitated with anti-p66Shc antibody, and associated endogenous βPix was detected by anti-βPix antibody. (B) Myc-tagged β₁Pix or its mutants β₁PixΔDH, β₁PixΔPH, or β₁PixΔERD were expressed in HMCs, and anti-p66Shc antibody was used for immunoprecipitation. (C) Cell lysates expressing Myc-tagged β₁Pix, β₁PixΔ(605-608), β₁PixΔ(603-608), or β₁PixΔ(602-611) were immunoprecipitated with p66Shc antibody followed by immunoblotting as above. (D) Cells were transfected with Myc-β₁Pix, Flag-β₁Pix, combination of both, or Flag-β₁Pix and Myc-β₁PixΔ(603-608), and then cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting as indicated. These results are representative of four independent experiments.

Figure 3.

Kinetics of ET-1 stimulation on ERK1/2, p66Shc, FOXO3a phosphorylation, and p27^kip1 down-regulation. HMCs were serum-starved for 48 h and then stimulated with ET-1 (100 nM) for 0, 15 min, 4 h, or 24 h. Cells lysates were probed by immunoblotting using specific antibodies as indicated. These results are representative of three independent experiments.

Figure 4.

Effect of βPix depletion on ET-1 signaling. (A) Cells were transfected with βPix siRNA (50 nM) for 24 h in complete RPMI and serum-starved for an additional 24 h. Cells were then stimulated with ET-1 (100 nM) for the indicated times, and cell lysates were immunoblotted with different antibodies as indicated. (B) Cells were transfected with 5 nM of βPix siRNA, p66Shc siRNA, or negative control siRNA for 24 h and serum-starved for an additional 24 h before addition of ET-1 (100 nM) for 24 h. [³H]thymidine incorporation was performed as described in Materials and Methods. Values represent the mean ± SEM from four independent experiments performed in triplicates. Symbols above a column indicate a statistical comparison between the indicated sample and nonstimulated cells (NS). *p < 0.05.

Figure 5.

β₁Pix-induced p66Shc phosphorylation on serine 36 is mediated by Rac1 and ERK1/2 activation. (A) HMCs were transiently transfected with empty vector or Myc-tagged β₁Pix. Four hours after transfection cells were treated with vehicle, U0126 (10 μM), or wortmannin (100 nM) for 20 h. Endogenous p66Shc was immunoprecipitated from cell lysates followed by immunoblotting with anti-phospho-S36p66Shc or anti-p66Shc antibodies. Portions of the cell lysates were immunoblotted with anti-phospho-ERK1/2 or anti-ERK1/2 antibodies. (B) Cell lysates overexpressing β₁Pix or β₁Pix mutants were subjected to GST-Pak pulldown. Rac1-GTP bound to GST-Pak (top) and Rac1 in total lysates (middle) were detected by immunoblotting with anti-Rac1. The quantitative analysis of Rac1-GTP (active) was obtained by densitometry (bottom panel). (C) Lysates from cells expressing β₁Pix, β₁PixSH3m, β₁PixDHm, or β₁Pix(SH3m, DHm) were analyzed using anti-phospho-Erk1/2 or anti-Erk1/2 antibodies. (D) Myc-tagged β₁Pix was expressed alone or with Flag-tagged p66Shc or Flag-tagged p66ShcS36A. Cell lysates were immunoprecipitated with Flag antibody followed by Western blotting analysis as indicated. Anti-Flag and anti-myc antibodies show the expression levels of Flag-tagged p66Shc and Myc-tagged β₁Pix, respectively (bottom). Results are representative of at least three independent experiments.

Figure 6.

β₁Pix induces p27^kip1 down-regulation through ERK/p66Shc/FOXO3a pathway. (A) Cells transfected with β₁Pix were either left untreated or treated with U0126 (10 μM) or wortmannin (100 nM) 4 h after transfection. Cell lysates were then analyzed by Western blotting as indicated using phospho-Akt, phospho-FOXO3a, or p27^kip1 specific antibodies. FOXO3a phosphorylation was quantified by densitometry (bottom panel). (B) Nontransfected cells were serum-starved overnight before stimulation with 10% serum in the presence or absence of wortmannin (100 nM). Cell lysates were analyzed by Western blotting as indicated above. Similar results were obtained from three independent experiments.

Figure 7.

Serum-induced FOXO3a phosphorylation is Akt-dependent and p66Shc-independent. Cells were transfected with 100 nM of each Akt siRNA (A) or 100 nM of p66Shc siRNA or pSUPERp66Shc shRNA (8 μg; B) for 24 h. The cells were serum-starved for an additional 24 h before stimulation with 10% of serum for 20 min. The cell lysates were blotted as indicated. These results are representative of three independent experiments.

Figure 8.

β₁Pix-induced FOXO3a phosphorylation is p66Shc-dependent and Akt-independent. Cells were transfected with 100 nM of each Akt siRNA (A) or 100 nM of p66Shc siRNA or pSUPERp66Shc shRNA (8 μg) (B) for 24 h before transfection with empty vector or β₁Pix for another 24 h. The cell lysates were blotted as indicated. These results are representative of three independent experiments.

Figure 9.

FOXO3a binding, like p66Shc, requires β₁Pix homodimerization. (A) Lysates from cells expressing β₁Pix or β₁PixΔ(603-608) were immunoprecipitated with anti-myc antibody followed by Western blotting with anti-phospho-FOXO3a or anti- FOXO3a. (B) Lysates from nontransfected cells were immunoprecipitated with anti-p66Shc or IgG control followed by immunoblotting with anti-phospho-FOXO3a or anti-FOXO3a.

Figure 10.

β₁Pix/p66Shc/FOXO3a is required for p27^kip1 down-regulation and cell proliferation. (A) Lysates from cells expressing β₁Pix or β₁PixΔ(603-608) were analyzed by Western blot using anti-p27^kip1. (B) Cells were transfected with empty vector, β₁Pix, or β₁PixΔ(603-608) for 48 h. [³H]thymidine incorporation was performed as described in Materials and Methods. Values represent the mean ± SEM from four independent experiments performed in triplicates. Symbols above a column indicate a statistical comparison between the indicated sample and cells transfected with β₁Pix. *p < 0.05. (C) Cells expressing β₁Pix or β₁PixΔ(603–608) were trypsinized, fixed in 70% ethanol, and stained with propidium iodide. Cells were analyzed with a flow cytometer for the distribution of cell cycle. The experiment shown is representative of four independent experiments performed in triplicates.

Figure 11.

p66Shc is necessary for β₁Pix-induced cell proliferation. Cells were transfected with p66Shc siRNA, pSUPERp66Shc shRNA, or control siRNA for 24 h before transfection with empty vector or β₁Pix for another 24 h. [³H]thymidine incorporation was performed as described in Materials and Methods. Values represent the mean ± SEM from three independent experiments performed in triplicate. Symbols above a column indicate a statistical comparison between the indicated sample and cells transfected with β₁Pix. *p < 0.05.

Figure 12.

Schematic representation of proposed ET-1 signaling through βPix/p66Shc/FOXO3a complex in glomerular mesangial cells. Endothelin-1 (ET-1), acting via its G-protein–coupled receptor, induces expression of βPix and p66Shc, leading to the formation of βPix/p66Shc complex, which then recruit cytoplasmic FOXO3a to form a trimeric signaling complex. βPix expression, through sequential activation of ERK and p66Shc results in FOXO3a phosphorylation via Akt-independent pathway. Akt-dependent FOXO3a phosphorylation in the nucleus is also shown.