The Hedgehog network includes canonical and noncanonical signaling modules important for development, homeostasis, and disease.

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.

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 Gα _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 .

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 rearrangement 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.

Signalling by Hh (Hedgehog) proteins is among the most actively studied receptor-mediated phenomena relevant to development and post-embryonic homoeostatic events. The impact of signalling by the Hh proteins is profound, and work pertaining to the presentation of these proteins and the pathways engaged by them continues to yield unique insights into basic aspects of morphogenic signalling. We review here the mechanisms of signalling relevant to the actions of Hh proteins in vertebrates. We emphasize findings within the past several years on the recognition of, in particular, Sonic hedgehog by target cells, pathways of transduction employed by the seven-pass transmembrane protein Smoothened and end points of action, as manifest in the regulation of the Gli transcription factors. Topics of extended interest are those regarding the employment of heterotrimeric G-proteins and G-protein-coupled receptor kinases by Smoothened. We also address the pathways, insofar as known, linking Smoothened to the expression and stability of Gli1, Gli2 and Gli3. The mechanisms by which Hh proteins signal have few, if any, parallels. It is becoming clear in vertebrates, however, that several facets of signalling are shared in common with other venues of signalling. The challenge in understanding both the actions of Hh proteins and the overlapping forms of regulation will be in understanding, in molecular terms, both common and divergent signalling events.