Overlap in signaling between constitutively active forms of Smoothened and the α subunit of the heterotrimeric G protein G13
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE111669 | GEO | 2018/05/05
REPOSITORIES: GEO
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Project description:Overlap in signaling between constitutively active forms of Smoothened and the α subunit of the heterotrimeric G protein G13
| PRJNA437832 | ENA
Project description:The Hedgehog family of morphogens has long been known to utilize, through the 7-transmembrane protein Smoothened (Smo), the heterotrimeric G protein Gi in both canonical and noncanonical forms of signaling. Other G proteins, while not specifically utilized by Smo, may nonetheless provide access to some of the events controlled by it. We reported several years ago that the G protein G13 activates one or more forms of the Gli family of transcription factors. While the Gli transcription factors are well known targets for Smo, the uncertain mechanism of activation by G13 and the identity of the targeted Gli(s) limited predictions as to the extent to which G13 might mimic Smo's actions. We evaluate here the potential for overlap in G13 and Smo signaling using C3H10T1/2 and 3T3-L1 cells as models of osteogenesis and adipogenesis, respectively. We find in C3H10T1/2 cells that a constitutively active form of Gα13 (Gα13QL) increases Gli1 mRNA, as does a constitutively active form of Smo (SmoA1). We find as well that Gα13QL induces alkaline phosphatase activity, a marker of osteogenesis, albeit the induction is far less substantial than that achieved by SmoA1. In 3T3-L1 cells both Gα13QL and SmoA1 markedly suppress adipogenic differentiation as determined by triglyceride accumulation. RNA sequencing reveals that Gα13QL and SmoA1 regulate many of the same genes but that quantitative and qualitative differences exist. Differences also exist, we find, between SmoA1 and purmorphamine, an agonist for Smo. Therefore, while comparisons of constitutively active proteins are informative, extrapolations to the setting of agonists require care.
| S-EPMC5953476 | biostudies-literature
Project description:Purpose: Analysis of the differential gene expression of C3H10T1/2 cells undergoing Hedgehog (Hh) induced osteoblast differentiation compared to unstimulated (DMSO treated) cells and investigation of the differential gene expression caused by Picoberin, a highly potent inhibitor of Hh induced osteoblast differentiation (Comparison of purmorphamin + Picoberin treated C3H10T1/2 cells with purmorphamin + DMSO treated cells) Methods: 80 % confluent C3H10T1/2 cells were treated either with 1.5 µM purmorphamine + DMSO, only DMSO, 1.5 µM purmorphamine + 1 nM Picoberin, 1.5 µM purmorphamine +100 nM Picoberin or DMSO + 100 nM Picoberin for 24h, 48h, 96h or 7 days. Results: Picoberin inhibits osteoblast differentiation
2022-11-30 | GSE197970 | GEO
Project description:RNA sequencing is used to compare genes regulated by a constitutively active form of Smoothened, a protein critical to Hedgehog signaling, and a constitutively active form of the alpha subunit of the heterotrimeric G proteins G13, which shares some of the primary actions of Smoothened.
2018-05-05 | GSE98841 | GEO
Project description:The mechanisms by which the activation of Smoothened (Smo), a protein essential to the actions of the Hedgehog family of secreted proteins, is translated into signals that converge on the Gli transcription factors are not fully understood. The seven-transmembrane structure of Smo has long implied the utilization of heterotrimeric GTP-binding regulatory proteins (G proteins); however, evidence in this regard has been indirect and contradictory. In the current study we evaluated the capacity of mammalian Smo to couple to G proteins directly. We found that Smo, by virtue of what appears to be constitutive activity, activates all members of the G(i) family but does not activate members of the G(s), G(q), and G(12) families. The activation is suppressed by cyclopamine and other inhibitors of Hedgehog signaling and is enhanced by the Smo agonist purmorphamine. Activation of G(i) by Smo is essential in the activation of Gli in fibroblasts, because disruption of coupling to G(i) with pertussis toxin inhibits the activation of Gli by Sonic hedgehog and a constitutively active form of Smo (SmoM2). However, G(i) does not provide a sufficient signal because a truncated form of Smo, although capable of activating G(i), does not effect activation of Gli. Rescue of pertussis toxin-inhibited activation of Gli by Sonic hedgehog can be achieved with a constitutively active Galpha(i)-subunit. The data suggest that Smo is in fact the source of two signals relevant to the activation of Gli: one involving G(i) and the other involving events at Smo's C-tail independent of G(i).
| S-EPMC1567926 | biostudies-literature
Project description:The Hedgehog (Hh) pathway is essential for tissue homeostasis, and misregulation of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase 2 (GRK2)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking vertebrate Hh signal transduction at the membrane to transcription factor activation in the nucleus. In contrast to existing models, our work shows that this aspect of Hh signaling is fundamentally similar between species. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades.
2021-04-25 | PXD019346 | Pride
Project description:Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. Smoothened (SMO), the primary effector of Hh signaling, is expressed ectopically in human breast cancer, as well as in other cancers. Constitutive activation of SMO in mouse mammary glands leads to paracrine stimulation of proliferation, as well as hyperplasia. In canonical signaling, SMO functions via GLI transcription factor activation. However, recent data from Drosophila and mammalian cell lines indicate that SMO can function non-canonically as a G-protein coupled receptor (GPCR) by coupling to heterotrimeric G proteins, particularly those in the pertussis toxin (PTX)-sensitive G-alpha-i (Gai) class. Whether SMO functions as a GPCR in mammalian tissues in vivo is not known. Using genetically modified mouse models, we demonstrate here that SMO-induced stimulation of proliferation is PTX sensitive, and requires Gai2, but not Gai1 or Gai3. Our findings provide evidence for a non-canonical GPCR function of activated SMO in vivo, a finding that may have clinical significance given that most SMO-targeted agents were selected based largely on their ability to block canonical GLI-mediated transcription. Primary mammary epithelial cell RNA was deep-sequenced from mT-mG/SmoM2;MMTV-Cre (EGFP), mT-mG/SmoM2;MMTV-Cre (tdTomato), and mT-mG/SmoM2;+ cells to examine the effects of SmoM2 overexpression in the mammary gland.
2015-09-08 | E-GEOD-70210 | biostudies-arrayexpress
Project description:Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. Smoothened (SMO), the primary effector of Hh signaling, is expressed ectopically in human breast cancer, as well as in other cancers. Constitutive activation of SMO in mouse mammary glands leads to paracrine stimulation of proliferation, as well as hyperplasia. In canonical signaling, SMO functions via GLI transcription factor activation. However, recent data from Drosophila and mammalian cell lines indicate that SMO can function non-canonically as a G-protein coupled receptor (GPCR) by coupling to heterotrimeric G proteins, particularly those in the pertussis toxin (PTX)-sensitive G-alpha-i (Gai) class. Whether SMO functions as a GPCR in mammalian tissues in vivo is not known. Using genetically modified mouse models, we demonstrate here that SMO-induced stimulation of proliferation is PTX sensitive, and requires Gai2, but not Gai1 or Gai3. Our findings provide evidence for a non-canonical GPCR function of activated SMO in vivo, a finding that may have clinical significance given that most SMO-targeted agents were selected based largely on their ability to block canonical GLI-mediated transcription.
2015-09-08 | GSE70210 | GEO
Project description:Although the tubule is the epicenter of damage, kidney fibroblast is increasingly appreciated in governing the prognosis of acute kidney injury (AKI). Smoothened (Smo), a heptahelical transmembrane protein, carries a hedgehog signal to mediate the communications between kidney fibroblasts and tubular epithelium through an undetermined mechanism in AKI. Integrating in vivo, ex vivo, and in vitro approaches with systems biology, we report that Smo-specific ablation in fibroblast preserved kidney function and mitigated tubular cell apoptosis or inflammation after AKI. Loss of Smo in fibroblasts enhanced the activity of perivascular mesenchymal cells. Global proteomics revealed extracellular matrix is a key cellular compartment where significant proteins are distributed, and Nidogen-1 is a prominent basement membrane glycoprotein highly expressed in fibroblast-Smo specific ablation kidneys. Co-immunoprecipitation showed that Smo binds Nidogen-1. Phosphoproteomics identified wnt signaling pathway, the AKI protector, was activated in fibroblast Smo deletion kidneys. In vitro and ex vivo, Nidogen-1 induced wnts and repressed tubular cell apoptosis. Our results suggest that fibroblast-derived Smo dictates AKI fate through cell-matrix-cell interactions and provides open-access data resources to understand AKI pathogenesis better.
2021-12-23 | MSV000088609 | MassIVE
Project description:Aberrant activation of Hedgehog pathway is responsible for initiation and maintenance of various cancers, including medulloblastoma (MB), basal cell carcinoma (BCC), and other solid and hematological tumors. Therefore, targeting Hh pathway represents promising therapeutic prospects for Hh-driven cancers. In recent years, tremendous efforts have been dedicated to the discovery of Hh pathway inhibitor. While the majority of Hh pathway inhibitors target the upstream membrane protein Smoothened (SMO). Here, we performed Next Generation Sequencing to reveal the target genes of Hh pathway by treating mouse SHH-subtype medulloblastoma cells (SmoWT) with SMO inhibitor (GDC0449) or DMSO.
2021-07-08 | GSE171689 | GEO