Hedgehog Signal Transduction Research Articles

Broad-Minded Links Cell Cycle-Related Kinase to Cilia Assembly and Hedgehog Signal Transduction

Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. We show that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. We find that Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, we further show that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. We propose that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling.

Chemical and Biological Studies of Nakiterpiosin and Nakiterpiosinone

Nakiterpiosin and nakiterpiosinone are two related C-nor-D-homosteroids isolated from the sponge Terpios hoshinota that show promise as anticancer agents. We have previously described the asymmetric synthesis and revision of the relative configuration of nakiterpiosin. We now provide detailed information on the stereochemical analysis that supports our structure revision and the synthesis of the originally proposed and revised nakiterpiosin. In addition, we herein describe a refined approach for the synthesis of nakiterpiosin, the first synthesis of nakiterpiosinone, and preliminary mechanistic studies of nakiterpiosin's action in mammalian cells. Cells treated with nakiterpiosin exhibit compromised formation of the primary cilium, an organelle that functions as an assembly point for components of the Hedgehog signal transduction pathway. We provide evidence that the biological effects exhibited by nakiterpiosin are mechanistically distinct from those of well-established antimitotic agents such as taxol. Nakiterpiosin may be useful as an anticancer agent in those tumors resistant to existing antimitotic agents and those dependent on Hedgehog pathway responses for growth.

Zebrafish inositol polyphosphate kinases: New effectors of cilia and developmental signaling

We described here our recent findings that Ipk1 catalyzed production of IP6 regulates LR-axis specification (Sarmah et al., 2005) and that IP6 is an essential effector of ciliary beating and length maintenance in zebrafish (Sarmah et al., 2007). We have also uncovered a novel role for the IP kinase IP6k2 in craniofacial development, neural crest cell migration, and hedgehog signal transduction (B.S. an S.R.W., unpublished). Together, these findings place IP production as a key mediator for cellular signaling mechanisms that regulate vital cellular and developmental processes. How these and other IPs are integrated with cell-cell signaling networks during complex processes, such as, tissue morphogenesis and maintenance of cell fate and function? We propose that with its enormous resource and unique set of structural, functional, and sensory attributes, cilium provide a platform for executing IP-based signaling functions. Given the evolutionary conservation of the IP repertoire and pathways, the developmental and molecular events uncovered in our studies in the zebrafish system could be applicable in other vertebrates including humans. This unbiased approach of systematic identification of IP functions in cilia and development will aid in understanding of multiple disease pathologies including ciliopathies and dysmorphic syndromes.

Planar cell polarity effector gene Fuzzy regulates cilia formation and Hedgehog signal transduction in mouse

Precise planar cell polarity (PCP) is critical for the development of multiple organ systems in animals. A group of core-PCP proteins are recognized to play crucial roles in convergent extension and other PCP-related processes in mammals. However, the functions of another group of PCP-regulating proteins, the PCP-effector proteins, are yet to be fully studied. In this study, the generation and characterization of a mouse mutant for the PCP effector gene Fuzzy (Fuz) is reported. Fuz homozygous mutants are embryonically lethal, with multiple defects including neural tube defects, abnormal dorsal/ventral patterning of the spinal cord, and defective anterior/posterior patterning of the limb buds. Fuz mutants also exhibit abnormal Hedgehog (Hh) signaling and inefficient proteolytic processing of Gli3. Finally, a significant decrease in cilia was found in Fuz homozygous mutants. In conclusion, Fuz plays an important role in cilia formation, Hh signal transduction, and embryonic development in mammals.

A Quantification of Pathway Components Supports a Novel Model of Hedgehog Signal Transduction

The secreted protein Hedgehog (Hh) plays a critical instructional role during metazoan development. In Drosophila, Hh signaling is interpreted by a set of conserved, downstream effectors that differentially localize and interact to regulate the stability and activity of the transcription factor Cubitus interruptus. Two essential models that integrate genetic, cell biological, and biochemical information have been proposed to explain how these signaling components relate to one another within the cellular context. As the molar ratios of the signaling effectors required in each of these models are quite different, quantitating the cellular ratio of pathway components could distinguish these two models. Here, we address this important question using a set of purified protein standards to perform a quantitative analysis of Drosophila cell lysates for each downstream pathway component. We determine each component's steady-state concentration within a given cell, demonstrate the molar ratio of Hh signaling effectors differs more than two orders of magnitude and that this ratio is conserved in vivo. We find that the G-protein-coupled transmembrane protein Smoothened, an activating component, is present in limiting amounts, while a negative pathway regulator, Suppressor of Fused, is present in vast molar excess. Interestingly, despite large differences in the steady-state ratio, all downstream signaling components exist in an equimolar membrane-associated complex. We use these quantitative results to re-evaluate the current models of Hh signaling and now propose a novel model of signaling that accounts for the stoichiometric differences observed between various Hh pathway components.

Regulation of Hh signal transduction as Drosophila eye differentiation progresses

Differentiation of the Drosophila retina occurs as a morphogenetic furrow sweeps anteriorly across the eye imaginal disc, driven by Hedgehog secretion from photoreceptor precursors differentiating behind the furrow. A BTB protein, Roadkill, is expressed posterior to the furrow and targets the Hedgehog signal transduction component Cubitus interruptus for degradation by Cullin-3 and the proteosome. Clonal analysis and conditional mutant studies establish that roadkill transcription is activated by the EGF receptor and Ras pathway in most differentiating retinal cells, and by both EGF receptor/Ras and by Hedgehog signaling in cells that remain unspecified. These findings outline a circuit by which Hedgehog signal transduction is modified as Hedgehog signaling initiates retinal differentiation. A model is presented for regulation of the Cullin-3 and Cullin-1 pathways that modifies Hedgehog signaling as the morphogenetic furrow moves and the responses of retinal cells change.

Widening the mutation spectrum ofEVCandEVC2: ectopic expression of Weyer variants in NIH 3T3 fibroblasts disrupts hedgehog signaling

Autosomal recessive Ellis-van Creveld syndrome and autosomal dominant Weyer acrodental dysostosis are allelic conditions caused by mutations in EVC or EVC2. We performed a mutation screening study in 36 EvC cases and 3 cases of Weyer acrodental dysostosis, and identified pathogenic changes either in EVC or in EVC2 in all cases. We detected 40 independent EVC/EVC2 mutations of which 29 were novel changes in Ellis-van Creveld cases and 2 were novel mutations identified in Weyer pedigrees. Of interest one EvC patient had a T>G nucleotide substitution in intron 7 of EVC (c.940-150T>G), which creates a new donor splice site and results in the inclusion of a new exon. The T>G substitution is at nucleotide +5 of the novel 5' splice site. The three Weyer mutations occurred in the final exon of EVC2 (exon 22), suggesting that specific residues encoded by this exon are a key part of the protein. Using murine versions of EVC2 exon 22 mutations we demonstrate that the expression of a Weyer variant, but not the expression of a truncated protein that mimics an Ellis-van Creveld syndrome mutation, impairs Hedgehog signal transduction in NIH 3T3 cells in keeping with its dominant effect.

The Mammalian Cos2 Homolog Kif7 Plays an Essential Role in Modulating Hh Signal Transduction during Development

The Hedgehog (Hh) signaling pathway regulates development in animals ranging from flies to humans. Although its framework is conserved, differences in pathway components have been reported. A kinesin-like protein, Costal2 (Cos2), plays a central role in the Hh pathway in flies. Knockdown of a zebrafish homolog of Cos2, Kif7, results in ectopic Hh signaling, suggesting that Kif7 acts primarily as a negative regulator of Hh signal transduction. However, in vitro analysis of the function of mammalian Kif7 and the closely related Kif27 has led to the conclusion that neither protein has a role in Hh signaling. Using Kif7 knockout mice, we demonstrate that mouse Kif7, like its zebrafish and Drosophila homologs, plays a role in transducing the Hh signal. We show that Kif7 accumulates at the distal tip of the primary cilia in a Hh-dependent manner. We also demonstrate a requirement for Kif7 in the efficient localization of Gli3 to cilia in response to Hh and for the processing of Gli3 to its repressor form. These results suggest a role for Kif7 in coordinating Hh signal transduction at the tip of cilia and preventing Gli3 cleavage into a repressor form in the presence of Hh.

Smoothened antagonists: a promising new class of antitumor agents

Background: Hedgehog signaling is essential for the development of most metazoans. In recent years, evidence has accumulated showing that many human tumors aberrantly re-activate this developmental signaling pathway and that interfering with it may provide a new strategy for the development of novel anti-cancer therapeutics. Smoothened is a G-protein coupled receptor-like protein that is essentially involved in hedgehog signal transduction and small molecule antagonists of Smoothened have started to show antitumor activity in preclinical models and in clinical trials. Objective: We critically review the role of hedgehog signaling in normal development and in human malignancies, the available drug discovery tools and the classes of small molecule inhibitors that are in development. We further aim to address the potential impact that pathway antagonists may have on the treatment options of cancer patients. Methods: Literature, patents and clinical trial results from the past 5 years were analyzed. Conclusions: 1) A large body of evidence suggests a frequent reactivation of hedgehog signaling in human cancer. 2) Smoothened is an attractive, highly druggable target with extensive preclinical and initial clinical validation in basal cell carcinoma. Several promising novel classes of Smoothened antagonists have been discovered and are being developed as anticancer agents. 3) Our knowledge of the biology of hedgehog signaling in cancer is still very incomplete and significant efforts will be required to understand how to use the emerging novel agents in the clinic.

823 Activation of the Hedgehog Signal Transduction Pathway During the Development of Biliary Atresia in a Mouse Model

823 Activation of the Hedgehog Signal Transduction Pathway During the Development of Biliary Atresia in a Mouse Model

Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant or spontaneous disorder characterized by multiple cutaneous basal cell carcinomas, odontogenic keratocysts, skeletal anomalies and facial dysmorphology, including cleft lip and palate. Causative mutations for NBCCS occur in the PTCH1 gene on chromosome 9q22.3–q31, which encodes the principle receptor for the Hedgehog signalling pathway. We have investigated the molecular basis of craniofacial defects seen in NBCCS using a transgenic mouse model expressing Shh in basal epithelium under a Keratin-14 promoter. These mice have an absence of flat bones within the skull vault, hypertelorism, open-bite malocclusion, cleft palate and arrested tooth development. Significantly, increased Hedgehog signal transduction in these mice can influence cell fate within the craniofacial region. In medial edge epithelium of the palate, Shh activity prevents apoptosis and subsequent palatal shelf fusion. In contrast, high levels of Shh in odontogenic epithelium arrests tooth development at the bud stage, secondary to a lack of cell proliferation in this region. These findings illustrate the importance of appropriately regulated Hedgehog signalling during early craniofacial development and demonstrate that oro-facial clefting and hypodontia seen in NBCCS can occur as a direct consequence of increased Shh signal activity within embryonic epithelial tissues.

Intraflagellar transport/Hedgehog-related signaling components couple sensory cilium morphology and serotonin biosynthesis in Caenorhabditis elegans.

Intraflagellar transport in cilia has been proposed as a crucial mediator of Hedgehog signal transduction during embryonic pattern formation in both vertebrates and invertebrates. Here, we show that the Hh receptor Patched-related factor DAF-6 and intraflagellar transport modulate serotonin production in Caenorhabditis elegans animals, by remodeling the architecture of dendritic cilia of a pair of ADF serotonergic chemosensory neurons. Wild-type animals under aversive environment drastically reduce DAF-6 expression in glia-like cells surrounding the cilia of chemosensory neurons, resulting in cilium structural remodeling and upregulation of the serotonin-biosynthesis enzyme tryptophan hydroxylase tph-1 in the ADF neurons. These cellular and molecular modifications are reversed when the environment improves. Mutants of daf-6 or intraflagellar transport constitutively upregulate tph-1 expression. Epistasis analyses indicate that DAF-6/intraflagellar transport and the OCR-2/OSM-9 TRPV channel act in concert, regulating two layers of activation of tph-1 in the ADF neurons. The TRPV signaling turns on tph-1 expression under favorable and aversive conditions, whereas inactivation of DAF-6 by stress results in further upregulation of tph-1 independently of OCR-2/OSM-9 activity. Behavioral analyses suggest that serotonin facilitates larval animals resuming development when the environment improves. Our study revealed the cilium structure of serotonergic neurons as a trigger of regulated serotonin production, and demonstrated that a Hedgehog-related signaling component is dynamically regulated by environment and underscores neuroplasticity of serotonergic neurons in C. elegans under stress and stress recovery.

Ciliary Abnormalities Due to Defects in the Retrograde Transport Protein DYNC2H1 in Short-Rib Polydactyly Syndrome

The short-rib polydactyly (SRP) syndromes are a heterogeneous group of perinatal lethal skeletal disorders with polydactyly and multisystem organ abnormalities. Homozygosity by descent mapping in a consanguineous SRP family identified a genomic region that contained DYNC2H1, a cytoplasmic dynein involved in retrograde transport in the cilium. Affected individuals in the family were homozygous for an exon 12 missense mutation that predicted the amino acid substitution R587C. Compound heterozygosity for one missense and one null mutation was identified in two additional nonconsanguineous SRP families. Cultured chondrocytes from affected individuals showed morphologically abnormal, shortened cilia. In addition, the chondrocytes showed abnormal cytoskeletal microtubule architecture, implicating an altered microtubule network as part of the disease process. These findings establish SRP as a cilia disorder and demonstrate that DYNC2H1 is essential for skeletogenesis and growth.

17β-Estradiol inhibits chondrogenesis in the skull development of zebrafish embryos

17β-Estradiol (E2) plays important roles in the development and differentiation of the gonad and central nervous systems, but little is known regarding the effects of exogenous E2 on chondrogenesis in skeletal development. In the present study, we found that treatment with E2 1–5 days post-fertilization (dpf) at concentrations above 1.5 × 10 −5 M increased the mortality rate in zebrafish embryos. Morphological analysis showed that treatment with E2 1–5 dpf caused abnormal cartilage formation in a dose-dependent manner at concentrations above 5 × 10 −6 M. E2 1–5 dpf at 1.5 × 10 −5 M caused defects of the ethmoid plate, parallel cleft of the trabecular cartilage, and hypoplasia of Meckel's cartilage and the ceratohyal cartilage. The sensitivity of embryos to E2 depended on the developmental stage. In early chondrogenesis (1–2 dpf), the embryos were highly sensitive to E2, leading to hypoplasia of the cartilage. In situ hybridization studies showed that expression levels of patched1 ( ptc1) and patched2 ( ptc2) receptor mRNAs were markedly decreased by exposure to 2 × 10 −5 M E2 1–2 dpf. However, the expression levels of sonic hedgehog ( shh) and tiggywinkle hedgehog ( twhh) mRNAs were constant in the E2-treated embryos. In addition, the estrogen receptor antagonist ICI 182,780 did not completely abolish the effects of E2, suggesting that E2 may not inhibit chondrogenesis through its nuclear estrogen receptor. These results suggest that exposure to exogenous E2 possibly inhibits chondrogenesis via inhibition of the hedgehog (Hh) signal transduction system.

Hedgehog signal transduction by Smoothened: Pharmacologic evidence for a 2-step activation process

The Hedgehog (Hh) signaling pathway controls growth, cell fate decisions, and morphogenesis during development. Damage to Hh transduction machinery can lead to birth defects and cancer. The transmembrane protein Smoothened (Smo) relays the Hh signal and is an important drug target in cancer. Smo enrichment in primary cilia is thought to drive activation of target genes. Using small-molecule agonists and antagonists to dissect Smo function, we find that Smo enrichment in cilia is not sufficient for signaling and a distinct second step is required for full activation. This 2-step mechanism--localization followed by activation--has direct implications for the design and use of anticancer therapeutics targeted against Smo.

Lack of Evidence for Activation of the Hedgehog Pathway in Psoriasis

Recent reports have suggested that the hedgehog (Hh) pathway is activated in lesional psoriatic skin, and that treatment with the Hh pathway antagonist cyclopamine may lead to rapid resolution of the disease. To assess Hh pathway activity in psoriasis, we isolated RNA from lesional and uninvolved skin of 58 psoriatic patients, and from 63 normal control subjects, and subjected these samples to global gene expression profiling on Affymetrix HU133 Plus 2.0 gene arrays. We were especially interested in Hh target genes (PTCH1 and GLI1), whose expression is elevated in response to Hh signaling. The microarray data demonstrated downregulation of PTCH1 expression in uninvolved and lesional skin (1.1-fold and 2-fold, respectively; P<0.0001). Additionally GLI1 mRNA was downregulated in lesional skin (1.7 fold; P<0.05). No significant changes were observed between lesional and uninvolved skin for the Hh ligands or Smoothened. Quantitative PCR confirmed these findings. In situ hybridization for GLI1 and PTCH1 was positive in basal cell carcinoma tumor cells, but was negligible in uninvolved or lesional psoriatic skin. The absence of elevated Hh target gene expression in lesional psoriatic skin indicates that the Hh pathway is not activated in this disease, raising questions regarding the proposed use of Hh antagonists as antipsoriatic agents.

Sonic hedgehog ligand partners with caveolin-1 for intracellular transport

Prenatal alcohol exposure is the most common environmental factor leading to congenital birth defects in the United States. Although significant progress has been made in this field, the detailed molecular pathology of fetal alcohol syndrome (FAS) remains to be determined. Previously, we have shown that alcohol exposure perturbs hedgehog signal transduction in zebrafish embryos by inhibiting the post-translational cholesterol modification of Sonic hedgehog (Shh), leading to decreased levels of mature Shh ligand that is associated with the plasma membrane, and causing transient loss of Hh signaling, resulting in permanent FAS-related morphological abnormalities. In the present study, we further elucidate the mechanisms that regulate the intracellular transportation and secretion of Shh using the hepatic stellate cell line HSC8B. We have found that Shh is associated with caveolin-1 in the Golgi apparatus to form protein complexes and that these complexes are packaged as large punctuate structures (transport vesicles) that are transported to the plasma membrane in lipid raft microdomains. Alcohol exposure does not significantly interrupt translation of shh mRNA in endoplasmic reticulum (ER) or the trafficking of Shh from the ER to the Golgi apparatus. However, alcohol does prevent the entry of Shh into transport vesicles from the Golgi to the plasma membrane and specifically decreases the amount of caveolin-1/Shh complex found in lipid rafts, causing cytoplasmic accumulation of Shh and leading to a deficiency of Shh ligand secretion into the extracellular matrix.

Control of the Postmating Behavioral Switch in Drosophila Females by Internal Sensory Neurons

Mating induces changes in the receptivity and egg-laying behavior in Drosophila females, primarily due to a peptide pheromone called sex peptide which is transferred with the sperm into the female reproductive tract during copulation. Whereas sex peptide is generally believed to modulate fruitless-GAL4-expressing neurons in the central nervous system to produce behavioral changes, we found that six to eight sensory neurons on the reproductive tract labeled by both ppk-GAL4 and fruitless-GAL4 can sense sex peptide to control the induction of postmating behaviors. In these sensory neurons, sex peptide appears to act through Pertussis toxin-sensitive G proteins and suppression of protein kinase A activity to reduce synaptic output. Our results uncover a neuronal mechanism by which sex peptide exerts its control over reproductive behaviors in Drosophila females.

Hedgehog Signaling: Is Smo a G Protein-Coupled Receptor?

The Hedgehog signal transducer Smoothened is structurally similar to G protein-coupled receptors. Now there is direct evidence that Smoothened relies on heterotrimeric G proteins in order to transduce the Hedgehog signal.

PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation

The seven-transmembrane protein Smoothened (Smo) and Zn-finger transcription factor Ci/Gli are crucial components in Hedgehog (Hh) signal transduction that mediates a variety of processes in animal development. In Drosophila, multiple kinases have been identified to regulate Hh signaling by phosphorylating Smo and Ci; however, the phosphatase(s) involved remain obscured. Using an in vivo RNAi screen, we identified PP4 and PP2A as phosphatases that influence Hh signaling by regulating Smo and Ci, respectively. RNAi knockdown of PP4, but not of PP2A, elevates Smo phosphorylation and accumulation, leading to increased Hh signaling activity. Deletion of a PP4-interaction domain (amino acids 626-678) in Smo promotes Smo phosphorylation and signaling activity. We further find that PP4 regulates the Hh-induced Smo cell-surface accumulation. Mechanistically, we show that Hh downregulates Smo-PP4 interaction that is mediated by Cos2. We also provide evidence that PP2A is a Ci phosphatase. Inactivating PP2A regulatory subunit (Wdb) by RNAi or by loss-of-function mutation downregulates, whereas overexpressing regulatory subunit upregulates, the level and thus signaling activity of full-length Ci. Furthermore, we find that Wdb counteracts kinases to prevent Ci phosphorylation. Finally, we have obtained evidence that Wdb attenuates Ci processing probably by dephosphorylating Ci. Taken together, our results suggest that PP4 and PP2A are two phosphatases that act at different positions of the Hh signaling cascade.