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. 2008 Sep 1;321(1):188-96.
doi: 10.1016/j.ydbio.2008.06.014. Epub 2008 Jun 18.

The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

Wee-Chuang Low  1 , Chengbing Wang, Yong Pan, Xin-Yun Huang, James K Chen, Baolin Wang
Affiliations

The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

Wee-Chuang Low et al. Dev Biol. .

Abstract

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Galpha(i) proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Galpha(i) protein activity appears to significantly reduce Hh pathway activity in Ptc(-/-) mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Galpha(i) protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Galpha(i) protein, Galpha(i2)QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Galpha(i) activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.

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Figures

Figure 1
Figure 1
Smo activates G proteins in vitro. HEK293 cells were separately transfected with either an empty vector, Smo or SmoM2. Two days later, membranes were isolated from the cells and [35S]GTPγS binding was performed in the presence of the indicated chemicals. The results presented were obtained from three independent experiments.
Figure 2
Figure 2
Smo and SmoM2 induced Gli-dependent luciferase activity was inhibited by pertussis toxin (PTX), cholera toxin (CTX), forskolin (FSK), and cyclopamine (CPM). (A) ADP-ribosylation assay of Shh-light cells pre-incubated with or without PTX. This 32P-labeled band (approximately 41 kD) presumably corresponds to a mixture of Gαi2 and Gαi3 (Castro et al., 2001; Xu and Barbieri, 1995). See Materials and methods for details of the assay. (B) The reporter assay results. Shh-light cells, which contained a stably integrated Gli-dependent firefly luciferase reporter and a Renillar luciferase reporter control, were infected with a retrovirus containing Smo and SmoM2 cDNAs. After being treated with the indicated drugs for 36–48 hrs, the cells were lysed, and firefly and Renilla luciferase activities were measured. The results shown were obtained from three independent experiments.
Figure 3
Figure 3
Hh signaling in Shh-light cells was inhibited by PTX, CTX, FSK, and CPM. (A) The Gli-dependent luciferase reporter assay. Shh-light cells were treated with either ShhN protein or SAG together with the indicated agents,; otherwise, the experiment was performed as that in figure 2. (B) Semi-quantitative RT-PCR of Gli1 and Ptc. Total RNA was isolated from Shh-light cells treated with pharmacological agents as indicated above the panel and subjected to semi-quantitative RT-PCR using Gli1, Ptc, and GAPDH (control) specific primers. Histogram shows the relative intensity of bands.
Figure 4
Figure 4
PTX treatment reduced Hh signaling in Ptc−/− MEFs. (A) PTX treatment had little effect on the Ptc-driven lacZ reporter activity in Ptc−/− mutant cells. The β-galactosidase activity in Ptc−/− MEFs was measured, following incubation with SAG, PTX, CTX, FSK, or CPM overnight or for 36–48 hours. The β-galactosidase activity was normalized to the protein amount of cell lysates and derived from three independent experiments. (B) Semi-quantitative RT-PCR of Gli1 and GAPDH (control) in Ptc−/− MEFs treated with the indicated chemicals. Histogram shows the relative intensity of bands. (C) In vitro ADP-ribosylation assay of Ptc−/− MEFs preincubated with or without PTX. See Materials and methods and figure 2A legend for details of the assay.
Figure 5
Figure 5
PTX treatment failed to derepress Shh- or SAG-induced suppression of Gli3 processing. (A) Immunoblots show the full-length and processed forms of Gli3 (Gli3-190, Gli3-83, Gli3-75) in chick limb bud micromass cells (left panel), E11.5 mouse embryonic fibroblasts (MEFs, middle panel), and Ptc−/− MEFs (right panel). Cells were incubated overnight with the medium containing the indicated components above the panels. (B) Histograms show the ratio of the Gli3-83 repressor to Gli3-190 activator from panel A. (C) In vitro ADP-ribosylation assay of chick limb bud micromasses preincubated with or without ShhN and/or PTX. See Materials and methods and figure 2A legend for details of the assay.
Figure 6
Figure 6
Misexpression of the active SmoM2, not active Gαi2QL, ectopically activated the expression of ventral neural transcription factors and suppressed a dorsal neural marker. The neural tubes of HH stage 12–14 embryos were electroporated with SmoM2 or Gαi2QL together with GFP expression constructs, and assayed 24h later for the expression of the indicated transcription factors (red) and GFP expression (green). Missexpression of the active SmoM2 resulted in an ectopic expression of Nkx2.2 (a V3 progenitor marker) (Cs), Nkx6.1 (a marker for V3, MNs, and V2 progenitors)(Ds), and MNR2 (a MN marker)(Gs), but it suppressed the expression of the dorsal marker Pax7 (indicated by an arrow in H’). Note that the loss of Pax7 expression corresponded to the GFP expression (compare H with H’ and H”). Missexpression of Gαi2QL failed to do so (As, Bs, Es, and Fs). (Is and Js) Immunostaining showing coexpression of GFP and Gαi2QL or SmoM2 (detected using myc antibody).
Figure 7
Figure 7
Overexpression of PTX catalytic S1 subunit did not inhibit the Shh-regulated specification of neuronal cell types in chick neural tube. Chick neural tubes (see materials and methods for details) were electroporated with PTX-S1 wt or mutant (not shown) together with GFP expression constructs and assayed 24 h later for the expression of the molecular markers (red) and GFP (green) by immunofluorescence (As-Fs). See figure 6 legend for molecular markers. (G) Autoradiogram of the in vitro ADP-ribosylation assay. Expression of wt PTX-S1 reduced the labeling of Gαi proteins by 32P-NAD compared to mutant PTX-S1.
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References

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