The Smoothened agonist SAG Modulates the Male and Female Peripheral Immune Systems Differently in an Immune Model of Central Nervous System Demyelination.

Both Hedgehog and androgen signaling pathways are known to promote myelin regeneration in the central nervous system. Remarkably, the combined administration of agonists of each pathway revealed their functional cooperation towards higher regeneration in demyelination models in males. Since multiple sclerosis, the most common demyelinating disease, predominates in women, and androgen effects were reported to diverge according to sex, it seemed essential to assess the existence of such cooperation in females. Here, we developed an intranasal formulation containing the Hedgehog signaling agonist SAG, either alone or in combination with testosterone. We show that SAG promotes myelin regeneration and presumably a pro-regenerative phenotype of microglia, thus mimicking the effects previously observed in males. However, unlike in males, the combined molecules failed to cooperate in the demyelinated females, as shown by the level of functional improvement observed. Consistent with this observation, SAG administered in the absence of testosterone amplified peripheral inflammation by presumably activating NK cells and thus counteracting a testosterone-induced reduction in Th17 cells when the molecules were combined. Altogether, the data uncover a sex-dependent effect of the Hedgehog signaling agonist SAG on the peripheral innate immune system that conditions its ability to cooperate or not with androgens in the context of demyelination.

Elucidation of Distinct Modular Assemblies of Smoothened Receptor by Bitopic Ligand Measurement.

Class F G protein-coupled receptors are characterized by a large extracellular domain (ECD) in addition to the common transmembrane domain (TMD) with seven α-helixes. For smoothened receptor (SMO), structural studies revealed dissected ECD and TMD, and their integrated assemblies. However, distinct assemblies were reported under different circumstances. Using an unbiased approach based on four series of cross-conjugated bitopic ligands, we explore the relationship between the active status and receptor assembly. Different activity dependency on the linker length for these bitopic ligands corroborates the various occurrences of SMO assembly. These results reveal a rigid "near" assembly for active SMO, which is in contrast to previous results. Conversely, inactive SMO adopts a free ECD, which would be remotely captured at "far" assembly by cholesterol. Altogether, we propose a mechanism of cholesterol flow-caused SMO activation involving an erection of ECD from far to near assembly.

Transcription factor POU4F2 promotes colorectal cancer cell migration and invasion through hedgehog-mediated epithelial-mesenchymal transition.

As a POU homeodomain transcription factor, POU4F2 has been implicated in regulating tumorigenic processes in various cancers. However, the role of POU4F2 in colorectal cancer (CRC) remains unclear. Here, we revealed that POU4F2 functions as a tumor promotor in CRC. Bioinformatics analysis in specimens from CRC patients and expression analysis in CRC cell lines showed that POU4F2 was upregulated at the mRNA and protein levels in CRC. Depletion of POU4F2 suppressed the metastatic phenotypes of CRC cells, including cell migration, invasion, and the expression of epithelial-mesenchymal transition (EMT) markers. Moreover, depletion of POU4F2 decreased the number of lung metastatic nodes in nude mice. Mechanistically, POU4F2 positively regulated the Hedgehog signaling pathway, as inferred from the downregulation of the expression of sonic Hedgehog homolog, patched 1, Smoothened, and GLI family zinc finger 1 in vitro and vivo following silencing of POU4F2. Furthermore, the SMO agonist SAG reversed the effects of POU4F2 knockdown in CRC. Functionally, POU4F2 contributed to the Hedgehog signaling-regulated activation of the EMT process and promotion of CRC cell migration and invasion. Collectively, these findings elucidated the role of POU4F2 as a tumor promotor in CRC through the regulation of Hedgehog signaling-mediated EMT and suggested that POU4F2 suppression might be a promising therapeutic target in inhibiting CRC metastasis.

Clobetasol promotes neuromuscular plasticity in mice after motoneuronal loss via sonic hedgehog signaling, immunomodulation and metabolic rebalancing.

Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.

A non-canonical Hedgehog pathway initiates ciliogenesis and autophagy.

Primary cilia function as critical signaling hubs whose absence leads to severe disorders collectively known as ciliopathies; our knowledge of ciliogenesis remains limited. We show that Smo induces ciliogenesis through two distinct yet essential noncanonical Hh pathways in several cell types, including neurons. Surprisingly, ligand activation of Smo induces autophagy via an LKB1-AMPK axis to remove the satellite pool of OFD1. This is required, but not sufficient, for ciliogenesis. Additionally, Smo activates the Gαi-LGN-NuMA-dynein axis, causing accumulation of a portion of OFD1 at centrioles in early ciliogenesis. Both pathways are critical for redistribution of BBS4 from satellites to centrioles, which is also mediated by OFD1 centriolar translocation. Notably, different Smo agonists, which activate Smo distinctly, activate one or the other of these pathways; only in combination they recapitulate the activity of Hh ligand. These studies provide new insight into physiological stimuli (Hh) that activate autophagy and promote ciliogenesis and introduce a novel role for the Gαi-LGN-NuMA-dynein complex in this process.

Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling.

Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO-Gi complexes bound to the synthetic SMO agonist (SAG) and to 24(S),25-epoxycholesterol (24(S),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMOD384R and SMOG111C/I496C, showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.

Structural insight into small molecule action on Frizzleds.

WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.

Molecular Pharmacology of Class F Receptor Activation.

The class Frizzled (FZD) or class F of G protein-coupled receptors consists of 10 FZD paralogues and Smoothened (SMO). FZDs coordinate wingless/Int-1 signaling and SMO mediates Hedgehog signaling. Class F receptor signaling is intrinsically important for embryonic development and its dysregulation leads to diseases, including diverse forms of tumors. With regard to the importance of class F signaling in human disease, these receptors provide an attractive target for therapeutics, exemplified by the use of SMO antagonists for the treatment of basal cell carcinoma. Here, we review recent structural insights in combination with a more detailed functional understanding of class F receptor activation, G protein coupling, conformation-based functional selectivity, and mechanistic details of activating cancer mutations, which will lay the basis for further development of class F-targeting small molecules for human therapy. SIGNIFICANCE STATEMENT: Stimulated by recent insights into the activation mechanisms of class F receptors from structural and functional analysis of Frizzled and Smoothened, we aim to summarize what we know about the molecular details of ligand binding, agonist-driven conformational changes, and class F receptor activation. A better understanding of receptor activation mechanisms will allow us to engage in structure- and mechanism-driven drug discovery with the potential to develop more isoform-selective and potentially pathway-selective drugs for human therapy.

Activation of sonic hedgehog signaling by a Smoothened agonist restores congenital defects in mouse models of endocrine-cerebro-osteodysplasia syndrome.

BACKGROUND: Endocrine-cerebro-osteodysplasia (ECO) syndrome is a genetic disorder associated with congenital defects of the endocrine, cerebral, and skeletal systems in humans. ECO syndrome is caused by mutations of the intestinal cell kinase (ICK) gene, which encodes a mitogen-activated protein (MAP) kinase-related kinase that plays a critical role in controlling the length of primary cilia. Lack of ICK function disrupts transduction of sonic hedgehog (SHH) signaling, which is important for development and homeostasis in humans and mice. Craniofacial structure abnormalities, such as cleft palate, are one of the most common defects observed in ECO syndrome patients, but the role of ICK in palatal development has not been studied. METHODS: Using Ick-mutant mice, we investigated the mechanisms by which ICK function loss causes cleft palate and examined pharmacological rescue of the congenital defects. FINDINGS: SHH signaling was compromised with abnormally elongated primary cilia in the developing palate of Ick-mutant mice. Cell proliferation was significantly decreased, resulting in failure of palatal outgrowth, although palatal adhesion and fusion occurred normally. We thus attempted to rescue the congenital palatal defects of Ick mutants by pharmacological activation of SHH signaling. Treatment of Ick-mutant mice with an agonist for Smoothened (SAG) rescued several congenital defects, including cleft palate. INTERPRETATIONS: The recovery of congenital defects by pharmacological intervention in the mouse models for ECO syndrome highlights prenatal SHH signaling modulation as a potential therapeutic measure to overcome congenital defects of ciliopathies.

Small Molecule-Based Strategy Promotes Nucleus Pulposus Specific Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are promising for regenerating degenerated intervertebral discs (IVDs), but the low efficiency of nucleus pulposus (NP)-specific differentiation limits their clinical applications. The Sonic hedgehog (Shh) signaling pathway is important in NP-specific differentiation of ADSCs, and Smoothened Agonist (SAG) is a highly specific and effective agonist of Shh signaling. In this study, we proposed a new differentiation strategy with the use of the small molecule SAG. The NP-specific differentiation and extracellular matrix (ECM) synthesis of ADSCs were measured in vitro , and the regenerative effects of SAG pretreated ADSCs in degenerated IVDs were verified in vivo . The results showed that the combination of SAG and transforming growth factor-ß3 (TGF-ß3) is able to increase the ECM synthesis of ADSCs. In addition, the gene and protein expression levels of NP-specific markers were increased by treatment with SAG and TGF-ß3. Furthermore, SAG pretreated ADSCs can also improve the disc height, water content, ECM content, and structure of degenerated IVDs in vivo . Our new differentiation scheme has high efficiency in inducing NP-specific differentiation of ADSCs and is promising for stem cell-based treatment of degenerated IVDs.

Smoothened stimulation by membrane sterols drives Hedgehog pathway activity.

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

Neuroprotective effects of a Smoothened receptor agonist against postoperative cognitive dysfunction by promoting autophagy in the dentate gyrus of aged rats.

Objectives: To investigate the effect of purmorphamine (PUR), a Shh co-receptor Smoothened (Smo) agonist, on postoperative cognitive dysfunction (POCD) rat models. Methods: Eighteen-month-old male Sprague-Dawley rats were subjected to intramedullary fixation of a tibial fracture with 7% chloral hydrate anesthesia to mimic human clinical surgery. PUR was administered via an intraperitoneal injection at a dose of 15mg/kg/day for 3 consecutive days at 6 h after surgery. The aged rats were sacrificed after performing a Morris water maze test 1, 3, and 7 days postoperatively to evaluate the expression of related proteins at the appointed time. Results: Compared to the POCD + vehicle group and sham + PUR group, the POCD + PUR group restored neurological deficit (P = 0.01). PUR administration induced upregulation of Shh expression on postoperative day 1 (P = 0.02), which continued on the third day (P = 0.008) but dropped by the 7th day (P = 0.03). Immunofluorescent analysis, similar to western blot analysis, showed a significant increase in the autophagy-marker LC3 (P = 0.006) as well as p62 degradation (P = 0.000) in the dentate gyrus of the aged rats (P = 0.000) after PUR treatment. Importantly, LC3 was mainly found in the presynaptic and postsynaptic membranes of the hippocampus. Conclusions: These results indicate a link between Shh and autophagy in the rat model of POCD, providing new insights into Shh signaling pathway-mediated mechanisms of neuroprotection and cognitive repair after POCD. It also provides a potential entry point for the development of clinical drugs.

Sonic hedgehog signaling is negatively regulated in reactive astrocytes after forebrain stab injury.

Following injury to the central nervous system, astrocytes perform critical and complex functions that both promote and antagonize neural repair. Understanding the molecular signaling pathways that coordinate their diverse functional properties is key to developing effective therapeutic strategies. In the healthy, adult CNS, Sonic hedgehog (Shh) signaling is active in mature, differentiated astrocytes. Shh has been shown to undergo injury-induced upregulation and promote neural repair. Here, we investigated whether Shh signaling mediates astrocyte response to injury. Surprisingly, we found that following an acute, focal injury, reactive astrocytes exhibit a pronounced reduction in Shh activity in a spatiotemporally-defined manner. Shh signaling is lost in reactive astrocytes at the lesion site, but persists in mild to moderately reactive astrocytes in distal tissues. Nevertheless, local pharmacological activation of the Shh pathway in astrocytes mitigates inflammation, consistent with a neuroprotective role for Shh signaling after injury. Interestingly, we find that Shh signaling is restored to baseline levels two weeks after injury, a time during which acute inflammation has largely subsided and lesions have matured. Taken together, these data suggest that endogenous Shh signaling in astrocytes is dynamically regulated in a context dependent manner. In addition, exogenous activation of the Shh pathway promotes neuroprotection mediated by reactive astrocytes.

Kruppel-Like Factor 4 represses osteoblast differentiation via ciliary Hedgehog signaling.

Primary cilia are appendages observed in most types of cells, and serve as cellular antennae for sensing environmental signals. Evidence is accumulating that correct ciliogenesis and ciliary functions are indispensable for normal skeletal development by regulating signaling pathways important for bone development. However, whether ciliogenesis is regulated by bone-related factors in osteoblasts is largely unknown. Here we show that Kruppel-Like Factor 4 (KLF4), which is known to repress osteoblast differentiation, supports the formation and maintenance of cilia in cultured osteoblasts; however, the length of the cilia observed in KLF4-induced cells were significantly shorter compared to the control cells. Basal Hedgehog signaling was repressed by KLF4. Significantly, activating Hedgehog signaling using a Smoothened agonist significantly rescued osteoblast mineralization and osteoblastic gene expressions. Global gene expression analysis showed that KLF4 induced number of genes including the nuclear receptor, Pregnane X receptor (PXR), and PXR repressed calvarial osteoblast mineralization and repressed Gli1 expression similar as the effect observed by inducing KLF4. Our results implicate that KLF4 plays important roles for maintaining osteoblasts in an immature state by repressing basal activation of the Hedgehog signaling.

Loss of pericyte smoothened activity in mice with genetic deficiency of leptin.

BACKGROUND: Obesity is associated with multiple diseases, but it is unclear how obesity promotes progressive tissue damage. Recovery from injury requires repair, an energy-expensive process that is coupled to energy availability at the cellular level. The satiety factor, leptin, is a key component of the sensor that matches cellular energy utilization to available energy supplies. Leptin deficiency signals energy depletion, whereas activating the Hedgehog pathway drives energy-consuming activities. Tissue repair is impaired in mice that are obese due to genetic leptin deficiency. Tissue repair is also blocked and obesity enhanced by inhibiting Hedgehog activity. We evaluated the hypothesis that loss of leptin silences Hedgehog signaling in pericytes, multipotent leptin-target cells that regulate a variety of responses that are often defective in obesity, including tissue repair and adipocyte differentiation. RESULTS: We found that pericytes from liver and white adipose tissue require leptin to maintain expression of the Hedgehog co-receptor, Smoothened, which controls the activities of Hedgehog-regulated Gli transcription factors that orchestrate gene expression programs that dictate pericyte fate. Smoothened suppression prevents liver pericytes from being reprogrammed into myofibroblasts, but stimulates adipose-derived pericytes to become white adipocytes. Progressive Hedgehog pathway decay promotes senescence in leptin-deficient liver pericytes, which, in turn, generate paracrine signals that cause neighboring hepatocytes to become fatty and less proliferative, enhancing vulnerability to liver damage. CONCLUSIONS: Leptin-responsive pericytes evaluate energy availability to inform tissue construction by modulating Hedgehog pathway activity and thus, are at the root of progressive obesity-related tissue pathology. Leptin deficiency inhibits Hedgehog signaling in pericytes to trigger a pericytopathy that promotes both adiposity and obesity-related tissue damage.

Hedgehog Signaling: From Basic Biology to Cancer Therapy.

The Hedgehog (HH) signaling pathway was discovered originally as a key pathway in embryonic patterning and development. Since its discovery, it has become increasingly clear that the HH pathway also plays important roles in a multitude of cancers. Therefore, HH signaling has emerged as a therapeutic target of interest for cancer therapy. In this review, we provide a brief overview of HH signaling and the key molecular players involved and offer an up-to-date summary of our current knowledge of endogenous and exogenous small molecules that modulate HH signaling. We discuss experiences and lessons learned from the decades-long efforts toward the development of cancer therapies targeting the HH pathway. Challenges to develop next-generation cancer therapies are highlighted.

Inhibition of smoothened decreases proliferation of synoviocytes in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLSs) contribute to synovial hyperplasia in rheumatoid arthritis (RA). Smoothened (Smo) is a key component of sonic hedgehog (Shh) signaling and contributes to tumor cell proliferation. The objective of this study was to investigate the role of Smo in RA synoviocyte proliferation. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist (GDC-0449) and small interfering RNA (siRNA) targeting the Smo gene in FLSs. Cell proliferation was quantified by using kit-8 assay and cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell cycle-related genes and proteins were detected by real-time PCR and western blot. FLSs treated with GDC-0449 or Smo-siRNA showed significantly decreased proliferation compared to controls (P < 0.05). Incubation with GDC-0449 or transfection with Smo-siRNA resulted in a significant increase of G1 phase cells compared to controls (P < 0.05). Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells (P < 0.05). Protein expression of cyclin D1 was also downregulated after Smo gene knockdown (P < 0.05). The results suggest that Shh signaling plays an important role in RA-FLSs proliferation in a Smo-dependent manner and may contribute to synovial hyperplasia. Targeting Shh signaling may help control joint damage in patients with RA.

Preaxial polydactyly following early gestational exposure to the smoothened agonist, SAG, in C57BL/6J mice.

BACKGROUND: While pharmacological activation of the Hedgehog (HH) signaling pathway may have therapeutic benefits for developmental and adult diseases, its teratogenic potential is of concern. The membrane molecule Smoothened (SMO) transduces HH signaling and can be acutely modulated by antagonists and agonists. The objective of the current experiments was to determine how maternal treatment with the Smo agonist, SAG, affects the developing limb. METHODS: Pregnant C57BL/6J mice received a single injection of SAG (15, 17, or 20 mg/kg, i.p.) or its vehicle on gestational day (GD) 9.25, the time of limb bud induction. Embryos were examined on GD 15 for gross dysmorphology and skeletal staining was performed to visualize the number and type of digits on the fore- and hindlimbs. Additionally, in situ hybridization was performed 4 hr after GD 9.25 SAG administration to determine SAG's effects on Gli1 and Gli2 mRNA expression. RESULTS: The most prevalent effect of SAG was the dose-dependent induction of pre-axial polydactyly; defects ranged from a broad thumb to the duplication of two finger-like digits on the preaxial side of the thumb. The highest SAG dose was effective in ca. 80% of the embryos and increased Gli1 and Gli2 mRNA expression in the limb bud, with Gli1 mRNA being the most upregulated. CONCLUSION: Preaxial polydactyly can be caused in the developing embryo by acute maternal administration of a Smo agonist that activates HH signaling. These results are consistent with the preaxial polydactyly induced in developmental disorders associated with mutations in HH signaling genes.Birth Defects Research 109:49-54, 2017. © 2016 Wiley Periodicals, Inc.

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling.

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.

Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling.

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.