Crosstalk between VEGFR2 and muscarinic receptors regulates the mTOR pathway in serum starved SK-N-SH human neuroblastoma cells

Abstract

Muscarinic acetylcholine receptors (mAchRs) are guanosine nucleotide-binding protein (G protein) coupled receptors that crosstalk with receptor tyrosine kinases (RTKs) to signal mitogenic pathways. In particular, mAchRs are known to couple with RTKs for several growth factors to activate the mammalian target of rapamycin (mTOR)/Akt pathway, a regulator of protein synthesis. The RTK for the vascular endothelial growth factor (VEGF), VEGFR2, can signal protein synthesis but whether it cooperates with mAchRs to mediate mTOR activation has not been demonstrated. Using serum starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of the mTOR substrate p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2 dependent manner. Treatments with carbachol increased VEGFR2 phosphorylation, suggesting that mAchRs stimulate VEGFR2 transactivation to enhance mTOR signaling. Inhibitor studies revealed that phosphatidylinositol 3 kinase resides upstream from S6K, S6 and Akt phosphorylation while protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, okadaic acid elicited a far greater increase in phosphorylation, implicating phosphatase 2A as a critical determinant of their function. Finally, pilocarpine but not carbachol induced a time and dose dependent cell death that was associated with caspase activation and oxidative stress but independent of S6K and S6 activation through VEGFR2. Accordingly, our findings suggest that mAchRs crosstalk with VEGFR2 to enhance mTOR activity but signal divergent effects on survival through alternate mechanisms.

Figure 1

Carbachol and VEGF induce activation of mTORC1 targets with different effects on Akt phosphorylation in serum-deprived SK-N-SH cells. (A) SK-N-SH cells were serum deprived for 48 hours as described in Materials and Methods. Cells were treated without and with 1mM carbachol during the final 30 minutes of serum deprivation as indicated. Cell lysates were analyzed by immunoblotting as described in Materials and Methods. Blots were probed with antibodies that specifically recognize the phosphorylation of S6K on Thr389 and Akt on Thr308. (B) Cells were treated with 10ng/ml VEGF for 15 minutes and analyzed for phosphorylation of S6K on Thr389, S6 on Ser235/236 and Akt on Thr308 and Ser473. Blots in (A) and (B) were stripped and reprobed with antigen specific antibodies to detect total protein levels for use as loading controls. Data for each time frame were quantified as the fold difference of normalized phosphorylation relative to the untreated control. Results are typical of the phosphorylation levels observed in 3 independent experiments.

Figure 2

VEGFR2 signals VEGF and carbachol mediated S6K and S6 phosphorylation. (A) Serum deprived cells were pretreated for 2 hours with 10 μM SU1498 followed by stimulation with either 10 ng/ml VEGF or 1mM carbachol as indicated. Immunoblotting was carried out as described in Figure 1 for detection of total and phosphorylated S6K and S6. (B) Cells were transfected with either scrambled (nontargeting) or VEGFR2 siRNA and then serum deprived for 48 hours. During the final 15 minutes of serum deprivation, cells were treated with 1mM carbachol for 15 minutes. Immunoblotting and the quantification of phosphorylation were carried out as described for Figure 1. (C) VEGFR2 was immunoprecipitated from lysates of serum starved cells treated with either 10ng/ml VEGF or 1mM carbachol for 15 minutes. Immunoprecipitates were analyzed by immunoblotting and probed with antigen specific antibodies that detect VEGFR2 phosphorylation on TYR951 and total protein.

Fig 3

PKC positively regulates S6K and S6 phosphorylation and negatively regulates Akt activation. (A) Serum deprived cells were treated for 2 hours with 10 μM SU1498 or 1 μM GFX followed by 15 minute incubations with either 10 ng/ml VEGF (VE) or 1 mM carbachol (Carb) alone or in combination as indicated. Immunoblotting and data quantification for total and phosphorylated S6K, S6 and Akt were performed as described for Figure 1. (B, C) Cells were treated with inhibitors as described in (A) followed by a 15 minute exposure to 1 mM pilocarpine. Immunoblots were probed for total and phosphorylated S6K and S6 (B) and Akt (C).

Fig 3

PKC positively regulates S6K and S6 phosphorylation and negatively regulates Akt activation. (A) Serum deprived cells were treated for 2 hours with 10 μM SU1498 or 1 μM GFX followed by 15 minute incubations with either 10 ng/ml VEGF (VE) or 1 mM carbachol (Carb) alone or in combination as indicated. Immunoblotting and data quantification for total and phosphorylated S6K, S6 and Akt were performed as described for Figure 1. (B, C) Cells were treated with inhibitors as described in (A) followed by a 15 minute exposure to 1 mM pilocarpine. Immunoblots were probed for total and phosphorylated S6K and S6 (B) and Akt (C).

Fig 4

PI3K is upstream from TORC1 and Akt. Serum starved cells were treated with either 1 μM GFX or 20 μM LY294002 (LY) for 2 hours or 1 μM rapamycin (Rap) for 48 hours either alone or in combination. Lysates were then analyzed by immunoblotting for detection of total and phosphorylated S6 and Akt as described for Figure 1.

Fig 5

PP2A regulates the phosphorylation levels of S6K, S6 and Akt. (A) Serum starved cells were treated with either 400 nM OA or 50 μM Na₃VO₄ for 1 hour followed by a 15 minute exposure to 10ng/ml VEGF or 1mM carbachol as indicated. Cell lysates were analyzed by immunoblotting for detection of total and phosphorylated S6K, S6 and Akt followed by data quantification as described for Figure 1. (B) Cells were treated with 400 nM OA in combination with 1μM rapamycin (Rap) or 20 μM LY294002 (LY) as indicated and analyzed by immunoblotting for phosphorylated S6K, S6 and Akt as in (A).

Fig 5

PP2A regulates the phosphorylation levels of S6K, S6 and Akt. (A) Serum starved cells were treated with either 400 nM OA or 50 μM Na₃VO₄ for 1 hour followed by a 15 minute exposure to 10ng/ml VEGF or 1mM carbachol as indicated. Cell lysates were analyzed by immunoblotting for detection of total and phosphorylated S6K, S6 and Akt followed by data quantification as described for Figure 1. (B) Cells were treated with 400 nM OA in combination with 1μM rapamycin (Rap) or 20 μM LY294002 (LY) as indicated and analyzed by immunoblotting for phosphorylated S6K, S6 and Akt as in (A).

Fig 6

Pilocarpine, unlike carbachol, induces a dose and time dependent cell death that correlates with elevated TORC1 target activation. (A) Serum starved cells were treated with increasing concentrations of carbachol or pilocarpine as indicated for the final 8, 20 or 24 hours of the 48 hour incubation without serum. (A) Cells were assayed for viability as described in Materials and Methods. Survival was expressed as the percent viability relative to the vehicle treated control (100%) ± S.E.M from at least three independent experiments. Asterisks indicate significant differences between pilocarpine treatments at 20 and 24 hours versus the carbachol treatments at 8, 20 and 24 hours (***P < 0.001). (B) Cells were incubated for 24 hour with 1 mM carbachol or pilocarpine as in (A) and analyzed by immunoblotting for detection of phosphorylated S6K, S6 and Akt, phosphorylation of PKCα/βII on Thr638/641 and the total protein levels of S6K, S6 and Akt and PKCα. Total PKCα levels were quantified and plotted relative to the untreated control.

Fig 7

The cell death induced by pilocarpine involves oxidative stress and caspase cleavage. Serum deprived cells were treated for 24 hour with 1 mM pilocarpine alone or with 5 mM N-acetyl cysteine (Nac) or Nac in combination with 10 μM SU1498. (A) Cell viability and caspase 3/7 activation was measured as described in Materials and Methods. Results represent the percent cell viability or caspase activity (units/μg protein) relative to the vehicle treated control ± S.E.M from at least three independent experiments. The asterisk indicates a significant difference between pilocarpine treatments with Nac versus pilocarpine alone (*P < 0.05; ***P < 0.001). (B) Immunoblots from cells treated as in (A) were analyzed for detection of total and phosphorylated S6K and S6 as described for Figure 1.

Fig 8

Model of crosstalk between mAchR and VEGFR2 signaling. Agonists for mAchRs carbachol (carb) and pilocarpine (pilo) stimulate VEGFR2 activation by VEGF to enhance the phosphorylation of the TORC1 targets S6K and the ribosomal protein S6. PKC influences phosphorylation by positively regulating TORC1 targets and negatively regulating Akt activation at the PI3K site Thr308 and the TORC2 site, Ser473. Dephosphorylation by the phosphatase PP2A serves as an additional mode of regulation for S6K, S6 and Akt activation.