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. 2011 Jun 3;286(22):19589-96.
doi: 10.1074/jbc.M110.197111. Epub 2011 Apr 7.

Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration

Ariel H Polizio  1 , Pilar Chinchilla, Xiaole Chen, Sangbumn Kim, David R Manning, Natalia A Riobo
Affiliations

Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration

Ariel H Polizio et al. J Biol Chem. .

Abstract

Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a G(i)-coupled receptor contrasts with the lack of an apparently consistent requirement for G(i) in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-G(i) coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for G(i) in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a G(i) protein- and PI3K-dependent mechanism, and that these are required for cell migration. These responses were independent of transcription by Gli and of the C-terminal domain of SMO, as we show using a combination of molecular and genetic tools. Our findings provide a mechanistic model for fibroblast migration in response to Shh and underscore the role of SMO-G(i) coupling in non-canonical Hh signaling.

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Figures

FIGURE 1.
FIGURE 1.
Shh promotes fibroblast migration through SMO. A, migration of Ptc1+/− mouse embryonic fibroblasts during 8 h after scratching a monolayer in the absence of serum. Vehicle (control) or 2.5 μg/ml Shh were added at t = 0 h. Photographs are representative of n = 6 experiments. B, quantification of Ptc1+/− migration without any stimulus (control), 2.5 μg/ml Shh (Shh), or 5 μm purmorphamine (PUR). Cells were pretreated with DMSO (black bars) or 0.5 μm KAAD-cyclopamine (KAAD) (white bars) (n = 6; *, p < 0.001). C, comparison of spontaneous migration of Ptc1+/− and Ptc1−/− MEFs in the absence or presence of 0.5 μm KAAD. D, quantification of basal Ptc1+/− and Ptc1−/− MEFs migration, the latter in the absence or presence of 0.5 μm KAAD (n = 6; *, p = 0.023). Representative images (E) and quantification (F) of wound healing assays using SMO−/− MEFs treated with 5 μm purmorphamine or 0.5 μm KAAD (n = 6).
FIGURE 2.
FIGURE 2.
SMO-dependent migration requires Gi and PI3K signaling. A, serum-starved Ptc1+/− MEFs were stimulated for 15 min with 5 μm purmorphamine (PUR) or vehicle (control) in the absence or presence of 0.5 μm KAAD-cyclopamine. Whole cell lysates were separated by SDS-PAGE and blotted for P-Akt (Ser-473) and total Akt. Densitometry values of P-Akt (Ser-473) normalized to total Akt represent the mean ± S.E. of three experiments. *, p < 0.05. B, quantification of wound healing assays of Ptc1+/− MEFs pretreated with 100 ng/ml pertussis toxin or 15 μm LY294002 and stimulated with 5 μm purmorphamine or vehicle (DMSO) (n = 6; *, p < 0.001) C–E, representative experiment of P-Akt (Ser-473) and P-PDK1 (Ser-241) levels in serum-starved Ptc1+/− MEFs (C), SMO−/− MEFs (D), and Ptc1−/− MEFs (E) treated overnight with 100 ng/ml PTX or 0.5 μm KAAD-cyclopamine (KAAD) (n = 3). F, migration of Ptc1−/− MEFs (gray bars) or SMO−/− MEFs (black bars) in scratch-wound healing assays (t = 8 h) preincubated with 0.5 μm KAAD-cyclopamine (KAAD), 100 ng/ml PTX, or 15 μm LY294002 (LY). (n = 4–6; *, p < 0.05; ¶, p < 0.01).
FIGURE 3.
FIGURE 3.
Shh stimulates RhoA and Rac1 small GTPases. A, representative Rac1 pulldown assay of serum-starved NIH 3T3 cells stimulated for 0–15 min with 2.5 μg/ml Shh. B, densitometry values of three independent time course Rac1 pulldown experiments. *, p < 0.05. C, representative RhoA pulldown assay of serum starved NIH 3T3 cells stimulated for 0–15 min with 2.5 μg/ml Shh. D, densitometry values of three independent time-course RhoA pulldown experiments; *, p < 0.05; ¶, p < 0.01. E, quantification of wound healing assays of NIH 3T3 cells co-transfected with GFP and pAX142 (empty vector), pAX142-RhoN19, or pAX142-RacN17 and stimulated at t = 0 with 2.5 μg/ml Shh or vehicle. Values represent the fold increase of GFP+ cells that migrate into the wound. *, p < 0.05. F, representative images of migration of cells expressing pAX142, pAX142-RhoN19, or pAX142-RacN17 after 8 h in the presence of Shh. The white line shows the scratch border at t = 0. Note that green cells expressing pAX142-RhoN19 or pAX142-RacN17 are incapable of moving into the wound.
FIGURE 4.
FIGURE 4.
Constitutive activation of SMO in Ptc1−/− MEFs leads to high basal RhoA and Rac1 activity. A, Ptc1+/− cells were serum-starved for 24 h and stimulated with 2.5 μg/ml Shh or vehicle for 10 min. Ptc1−/− MEFs were serum-starved for 24 h and incubated during the last 45 min with vehicle or 0.5 μm KAAD-cyclopamine. The basal and stimulated levels of GTP-RhoA were determined by pulldown assays as described under “Experimental Procedures.” B, GTP-Rac1 levels were evaluated by pulldown assays in conditions identical to A. Quantification of fractional RhoA (C) and Rac1 (D) GTP loading by densitometry (n = 3; *, p < 0.05).
FIGURE 5.
FIGURE 5.
Shh stimulates RhoA and Rac1 in a SMO-, Gi-, and PI3K-dependent manner. A, representative RhoA pulldown assay of serum starved NIH 3T3 cells stimulated for 10 min with 2.5 μg/ml Shh and pretreated with vehicle, 100 ng/ml PTX, 0.5 μm KAAD-cyclopamine (KAAD), or with 15 μm LY294002. B, representative Rac1 pulldown assay of serum-starved NIH 3T3 cells stimulated for 5 min with 2.5 μg/ml Shh and pretreated as in A. C, densitometry values of three independent RhoA pulldown experiments. *, p < 0.001. D, densitometry values of three independent Rac1 pulldown experiments. *, p < 0.05.
FIGURE 6.
FIGURE 6.
Rac1 activity is required for full RhoA activation by Shh. A, RhoA pulldown assay of Ptc1+/− MEFs transduced with control GFP-AdV or Myc-RacN17-AdV (MOI = 150) and stimulated with 2.5 μg/ml Shh or vehicle (control). Densitometry values of a representative experiment are shown below the gel. In the bottom gel, expression of RacN17 was confirmed by Western blot. B, Rac1 pulldown assay of Ptc1+/− MEFs transduced with control GFP-AdV or HA-RhoN19-AdV (MOI = 150) and stimulated with 2.5 μg/ml Shh or vehicle. Densitometry values of a representative experiment are shown below the gel. Expression of RhoN19 was confirmed by Western blot to the epitope tag (bottom gel).
FIGURE 7.
FIGURE 7.
Pertussis toxin does not affect Gli-dependent transcription in MEFs. A, expression of gli1, ptc1, and the housekeeping gene S15 was analyzed by semiquantitative RT-PCR. Ptc1+/− MEFs were grown until fully confluent, pretreated with 100 ng/ml PTX, 0.5 μm KAAD-cyclopamine, or 15 μm LY294002 and then stimulated with 2.5 μg/ml Shh, 5 μm purmorphamine, or vehicle for 24 h in DMEM with 0.5% FBS. B, effect of PTX treatment in Ptc1+/− cells was also quantified by real-time PCR. C, effect of PTX, KAAD-cyclopamine, and LY294002 on the basal expression level of gli1 in Ptc1−/− and SMO−/− cells. D, expression of Gαz in membranes isolated from Ptc1+/− MEFs or platelets (positive control) by Western blot.
FIGURE 8.
FIGURE 8.
SMO-mediated Rac1 activation is independent of Gli transcriptional activity. A, Ptc1+/− MEFs were transduced with control GFP-AdV or Gli3R-GFP-AdV (MOI = 150). Following 24 h of serum starvation, cells were stimulated with 5 μm purmorphamine (PUR) or vehicle (DMSO) for 5 min, and GTP-Rac1 levels were determined by pulldown assays. Expression of the transgenes was confirmed by Western blot against GFP. B, quantification of GTP-Rac1 activation by purmorphamine in the presence of Gli3R or GFP (n = 3). *, p < 0.05. C, quantification of wound healing assays of Ptc1+/− MEFs transduced with GFP-AdV or Gli3R-GFP-AdV and stimulated at t = 0 with 2.5 μg/ml Shh or vehicle. Values represent the fold increase of GFP+ cells that migrate into the wound; *, p < 0.05. D, representative images of migration of cells expressing GFP or Gli3R-GFP after 8 h in the presence of Shh. The white line shows the scratch border at t = 0.
FIGURE 9.
FIGURE 9.
SMO promotes monomeric GTPase activation and cell migration independently of its capacity to induce gli1. A, upper gels, expression of SMO variants in membranes prepared from SMO−/− MEFs stably transfected with empty pcDNA3.1vector (pcDNA), SMO-M2 (SMO), SMO-M2-CLD (SMO-CLD), or SMO-M2-ΔC (SMO-ΔC) was assessed by Western blot with anti-SMO E5 mAb. Full-length SMO variants run at ∼90 kDa, whereas SMO-ΔC runs at ∼55 kDa. The asterisk marks a cross-reactive band. Bottom gels, expression of gli1 and S15 in the same cell lines was determined by semiquantitative RT-PCR after 24 h of serum starvation at high cell density. B, densitometry values of Rac1 activation of the indicated cell lines after 5 min of stimulation with 5 μm purmorphamine (n = 3; *, p < 0.05; ¶, p < 0.01). C, densitometry values of RhoA activation of the indicated cell lines after 5 min of stimulation with 5 μm purmorphamine (n = 3; *, p < 0.05). D, purmorphamine-induced migration (fold compared with DMSO) of SMO−/− fibroblasts containing empty vector or the three SMO-M2 variants (n = 3). #, p < 0.01; *, p < 0.05.
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