Interoception refers to the representation of the internal world, and includes the processes by which an organism senses, interprets, integrates, and regulates signals from within itself.The brain communicates with internal organs via the peripheral nervous system and non-neuronal systems.

The G protein-coupled receptor Smoothened (Smo) is the signal transducer of the developmentally- and therapeutically-relevant Hedgehog pathway. Although recent structural analyses have advanced our understanding of Smo biology, a number of questions remain. Chief among them are the identity of its natural ligand, the regulatory processes controlling its activation, and the mechanisms by which it signals to downstream effectors. This review will discuss recent discoveries from multiple model systems that have set the stage for solving these mysteries. We focus on the roles of distinct Smo functional domains, post-translational modifications and trafficking, and conclude by discussing their contributions to signal output.

The mammalian unfolded protein response (UPR) is propagated by three ER-resident transmembrane proteins, each of which initiates a signaling cascade that ultimately culminates in production of a transcriptional activator. The UPR was originally characterized as a pathway for upregulating ER chaperones, and a comprehensive body of subsequent work has shown that protein synthesis, folding, oxidation, trafficking, and degradation are all transcriptionally enhanced by the UPR. However, the global reach of the UPR extends to genes involved in diverse physiological processes having seemingly little to do with ER protein folding, and this includes a substantial number of mRNAs that are suppressed by stress rather than stimulated. Through multiple non-canonical mechanisms emanating from each of the UPR pathways, the cell dynamically regulates transcription and mRNA degradation. Here we highlight these mechanisms and their increasingly appreciated impact on physiological processes.

Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC). Although these conditions also lead to integrated stress response (ISR) or unfolded protein response (UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

The unfolded protein response (UPR) responds to disruption of endoplasmic reticulum (ER) function by initiating signaling cascades that ultimately culminate in extensive transcriptional regulation. Classically, this regulation includes genes encoding ER chaperones, ER-associated degradation factors, and others involved in secretory protein folding and processing, and is carried out by the transcriptional activators that are produced as a consequence of UPR activation. However, up to half of the mRNAs regulated by ER stress are downregulated rather than upregulated, and the mechanisms linking ER stress and UPR activation to mRNA suppression are poorly understood. To begin to address this issue, we used a “bottom-up” approach to study the metabolic gene regulatory network controlled by the UPR in the liver, because ER stress in the liver leads to lipid accumulation, and fatty liver disease is the most common liver disease in the western world. qRT-PCR profiling of mouse liver mRNAs during ER stress revealed that suppression of the transcriptional regulators C/EBPα, PPARα, and PGC-1α preceded lipid accumulation, and was then followed by suppression of mRNAs encoding key enzymes involved in fatty acid oxidation and lipoprotein biogenesis and transport. Mice lacking the ER stress sensor ATF6α, which experience persistent ER stress and profound lipid accumulation during challenge, were then used as the basis for a functional genomics approach that allowed genes to be grouped into distinct expression profiles. This clustering predicted that ER stress would suppress the activity of the metabolic transcriptional regulator HNF4α—a finding subsequently confirmed by chromatin immunopreciptation at the Cebpa and Pgc1a promoters. Our results establish a framework for hepatic gene regulation during ER stress and suggest that HNF4α occupies the apex of that framework. They also provide a unique resource for the community to further explore the temporal regulation of gene expression during ER stress in vivo.

Summary The G protein-coupled receptor Smoothened (Smo) is the signal transducer of the Sonic Hedgehog (Shh) pathway. Smo signals through G protein-dependent and independent routes, with G protein-independent canonical signaling to Gli effectors requiring Smo accumulation in the primary cilium. Mechanisms controlling Smo activation and trafficking are not yet clear, but likely entail small-molecule binding to pockets in its extracellular cysteine-rich domain (CRD) and/or transmembrane bundle. Herein we demonstrate cytosolic phospholipase cPLA2α is activated through Gβγ downstream of Smo to release arachidonic acid. Arachidonic acid binds Smo and synergizes with CRD-binding agonist, promoting Smo ciliary trafficking and high-level signaling. Chemical or genetic cPLA2α inhibition dampens Smo signaling to Gli, revealing an unexpected contribution of G protein-dependent signaling to canonical pathway activity. Arachidonic acid displaces the Smo transmembrane domain inhibitor cyclopamine to rescue CRD agonist-induced signaling, suggesting arachidonic acid may target the transmembrane bundle to allosterically enhance signaling by CRD agonist-bound Smo.

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.