The G12 family of heterotrimeric G proteins and Rho GTPase mediate Sonic hedgehog signalling

Abstract

Sonic hedgehog (Shh) is a secreted morphogen crucial for cell fate decision, cellular proliferation, and patterning during vertebrate development. The intracellular Shh signalling is transduced by Smoothened (Smo), a seven-transmembrane spanning protein that belongs to the G-protein coupled receptor family. Among four families of Gα α α α subunits, Gα α α α i has been thought to be responsible for transducing Shh signalling, while several lines of evidence indicated that other signalling pathways may be involved. We found that the G12 family of heterotrimeric G proteins and the small GTPase RhoA are involved in Shh/Smo-mediated cellular responses, including stimulation of target gene promoter and inhibition of neurite outgrowth of neuroblastoma cells. We also found that the G12/RhoA pathway is responsible for Smo-induced nuclear import of GLI3 which is thought to transduce Shh signals to nucleus. Furthermore, misexpression of a G12-specific GTPase-activating protein in rat neural tubes leads to pertubation of motor neurone and interneurone development, mimicking the effects of decreased Shh signalling. These results show that Shh signalling is mediated in part by activating G12 family coupled signalling pathways. The participation of RhoA, a pivotal molecular switch in many signal transduction pathways, may help explain how Shh can trigger a variety of cellular responses.

GLI1 facilitates the migration and invasion of pancreatic cancer cells through MUC5AC-mediated attenuation of E-cadherin

“…GBS1mut and GBS2mut, mutants for putative GBSs (GBS1 and GBS2) in the MUC5AC promoter, were generated using the GeneTailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). Luciferase reporter assays were carried out using a Dual-Glo Luciferase Assay System (Promega) as described previously (Kasai et al, 2004(Kasai et al, , 2008. The following Stealth RNAi siRNAs (Invitrogen) were used to knock down GLI1 and GLI2: for GLI1 (siGLI1), 5 0 -AAUGCAAGGUCCCUC GUCCAAGCUG-3 0 , 5 0 -CAGCUUGGACGAGGGACCUUG CAUU-3 0 and for GLI2 (siGLI2), 5 0 -CAUAGAUGACCAC CUCAGCCUCCUG-3 0 , 5 0 -CAGGAGGCUGAGGUGGUCA UCUAUG-3 0 .…”

“…The following duplexes were used as controls: siControl-1, 5 0 -CGUACGCG GAAUACAACGATT-3 0 and 5 0 -UCGUUGUAUUCCGCG UACGTT-3 0 and siControl-2, 5 0 -CGCGAAACGAACGAAU AAATT-3 0 and 5 0 -UUUAUUCGUUCGUUUCGCGTT-3 0 . Immunoblot and semi-quantitative RT-PCR analyses were performed as described previously (Kasai et al, 2004(Kasai et al, , 2008. RNAs isolated from normal human tissues (the trachea, stomach, small and large intestine, gallbladder, pancreas and prostate: BioChain, Hayward, CA, USA) were combined, reverse-transcribed either with or without reverse transcriptase…”

“…Cells were then used for immunofluorescence staining as reported previously (Kasai et al, 2004(Kasai et al, , 2008. Z-stack confocal fluorescence images were obtained using an LSM710 fluorescence confocal microscope (Carl Zeiss MicroImaging, Goettingen, Germany) under a 40 Â water immersion lens.…”

The cell adhesion molecule CHL1 interacts with patched-1 to regulate apoptosis during postnatal cerebellar development

“…Since PTCH1 also mediates hedgehog-triggered non-canonical Gli-independent signaling, such as activation of RhoA via a SMO-dependent pathway (Kasai et al, 2004;Riobo et al, 2006;Lipinski et al, 2008;Jenkins, 2009;Yam et al, 2009;Polizio et al, 2011a,b;Shen et al, 2013), we asked whether CHL1 triggers RhoA activation. In comparison to untreated neurons, application of CHL1-Fc or SAG enhanced the levels of activated RhoA as determined by pulldown assay and ELISA experiments (Fig.…”

Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways fromDrosophilato vertebrates

“…However, only limited evidence indicates that Smo directly couples with G proteins (DeCamp et al, 2000;Kasai et al, 2004) and it is unclear whether there is a ligand that regulates Smo activity. Sequence comparisons among vertebrate and invertebrate Smo proteins show that the TM region of Smo is relatively conserved across species, while significant divergence exists in the cytoplasmic C-terminal tails (Fig.…”