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Hedgehogs (Hhs) are key signaling regulators of stem cell maintenance and tissue patterning in embryos, and activating mutations in the pathway that increase Gli transcriptional activity are causal in a diversity of cancers. Here, we report that phosphoinositide 3-kinase (PI3-kinase)-dependent Akt activation is essential for Sonic Hedgehog (Shh) signaling in the specification of neuronal fates in chicken neural explants, chondrogenic differentiation of 10T1/2 cells, and Gli activation in NIH 3T3 cells. Stimulation of PI3-kinase/Akt by insulin-like growth factor I potentiates Gli activation induced by low levels of Shh; however, insulin-like growth factor I alone is insufficient to induce Gli-dependent transcription. Protein kinase A (PKA) and glycogen synthase kinase 3β sequentially phosphorylate Gli2 at multiple sites, identified by mutagenesis, thus resulting in a reduction of its transcriptional activity. Gli2 mutant proteins in which the major PKA and glycogen synthase kinase 3β phosphorylation sites were mutated to alanine remain fully transcriptionally active; however, PKA-mutant Gli2 functions independently of Akt signaling, indicating that Akt positively regulates Shh signaling by controlling PKA-mediated Gli inactivation. Our findings provide a basis for the synergistic role of PI3-kinase/Akt in Hh signaling in embryonic development and Hh-dependent tumors.

Hedgehog (Hh) proteins regulate the development of a wide range of metazoan embryonic and adult structures, and disruption of Hh signaling pathways results in various human diseases. Here, we provide a comprehensive review of the signaling pathways regulated by Hh, consolidating data from a diverse array of organisms in a variety of scientific disciplines. Similar to the elucidation of many other signaling pathways, our knowledge of Hh signaling developed in a sequential manner centered on its earliest discoveries. Thus, our knowledge of Hh signaling has for the most part focused on elucidating the mechanism by which Hh regulates the Gli family of transcription factors, the so-called “canonical” Hh signaling pathway. However, in the past few years, numerous studies have shown that Hh proteins can also signal through Gli-independent mechanisms collectively referred to as “noncanonical” signaling pathways. Noncanonical Hh signaling is itself subdivided into two distinct signaling modules: (i) those not requiring Smoothened (Smo) and (ii) those downstream of Smo that do not require Gli transcription factors. Thus, Hh signaling is now proposed to occur through a variety of distinct context-dependent signaling modules that have the ability to crosstalk with one another to form an interacting, dynamic Hh signaling network.

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The Hedgehog (Hh) pathway orchestrates developmental and homeostatic angiogenesis. the three Hh isoforms--Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh)--signal through patched-1 (ptCH1) and Smoothened (SMo), to activate the Gli transcription factors with a characteristic rank of potency (Shh >> Ihh > Dhh). To dissect the mechanisms through which Hh proteins promote angiogenesis, we analyzed processes inherent to vessel formation in endothelial cells. We found that none of the Hh ligands were able to induce Gli-target genes in human umbilical vein (HUVeC) or human cardiac microvascular endothelial cells (HMVeC), suggesting that endothelial cells do not respond to Hh through the canonical pathway. However, our results show that the three Hh proteins promote endothelial cell tubulogenesis in 3D cultures in a SMo- and Gi protein-dependent manner. Consistent with the required cytoskeletal re-arrangement for tubulogenesis, Shh, Ihh and Dhh all stimulated the small GTPase RhoA and the formation of actin stress fibers. This effect, which was mediated by SMO, Gi proteins and Rac1, defines a new non-canonical Hh pathway. In addition to regulating the actin cytoskeleton, the Hh ligands promoted survival through inhibition of the pro-apoptotic effect of PTCH1 in a SMO-independent manner. Altogether, our results support the existence of Gli-independent Hh responses in endothelial cells that regulate tubulogenesis and apoptosis. The identification of novel non-canonical responses elicited by Hh proteins in endothelial cells highlights the complexity of the Hh signaling pathway and reveals striking differences in ligand strength for transcriptional and non-transcriptional responses

One third of all lethal cancers are associated with excessive activation of the Hedgehog (HH) pathway by mutations of its signaling components or by increased responsiveness of cells to the HH ligand. HH signaling through the GLI transcription factors leads to increased cell proliferation by up-regulation of the extracellular regulated kinase (ERK) pathway and by expression of S phase cyclins. In this study, we have tested the hypothesis that the HH pathway can integrate ERK signaling to modulate the activity of GLI. Using NIH 3T3 cells, we show that phorbol esters, acting through protein kinase C-D (PKCD) and mitogen-activated protein/extracellular signal-regulated kinase-1 (MEK-1), fully stimulate the transcriptional activity of endogenous and overexpressed GLI proteins, as assessed by GLI-luciferase reporter assays, and induce the expression of endogenous GLI1 and PTCH-1 target genes, as assessed by reverse transcription-PCR. Moreover, activation of GLI elicited by Sonic Hedgehog also requires PKCD and MEK-1 function. Remarkably, coexpression of activated MEK-1 and GLI1 or GLI2 induced a 10-fold synergistic increase in GLI-luciferase activity that was totally blocked by PD98059. The NH 2 -terminal region of GLI1 (amino acids 1-130) is required for sensing the ERK pathway, as deletion of this domain produces active GLI1 protein with greatly reduced response to activation by MEK-1. Basic fibroblast growth factor activation of the ERK pathway also stimulated GLI1 activity through its NH 2 -terminal domain. Our results identify PKCD and MEK-1 as essential, positive regulators of GLI-mediated HH signaling. Furthermore, our findings suggest that tumors with deregulated HH and ERK synergize to stimulate cell proliferation pathways. (Cancer Res 2006; 66(2): 839-45)

Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a Gi-coupled receptor contrasts with the lack of an apparently consistent requirement for Gi in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-Gi coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for Gi in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a Gi 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-Gi coupling in non-canonical Hh signaling.

The notion of noncanonical hedgehog (Hh) signaling in mammals has started to receive support from numerous observations. By noncanonical, we refer to all those cellular and tissue responses to any of the Hh isoforms that are independent of transcriptional changes mediated by the Gli family of transcription factors. In this chapter, we discuss the most recent findings that suggest that Patched1 can regulate cell proliferation and apoptosis independently of Smoothened (Smo) and Gli and the reports that Smo modulates actin cytoskeleton-dependent processes such as fibroblast migration, endothelial cell tubulogenesis, axonal extension, and neurite formation by diverse mechanisms that exclude any involvement of Gli-dependent transcription. We also acknowledge the existence of less stronger evidence of noncanonical signaling in Drosophila.