RESUMEN
Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes.
Asunto(s)
Colesterol , Proteínas Hedgehog , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ligandos , Colesterol/metabolismo , Transducción de Señal , Esteroles/metabolismoRESUMEN
Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.
Asunto(s)
Cilios/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Optogenética/métodos , Transducción de Señal/fisiología , Pez Cebra/metabolismoRESUMEN
Understanding transcription factor navigation through the nucleus remains critical for developing targeted therapeutics. The GLI1 transcription factor must maintain maximal Hedgehog pathway output in basal cell carcinomas (BCCs), and we have previously shown that resistant BCCs increase GLI1 deacetylation through atypical protein kinase Cι/λ (aPKC) and HDAC1. Here we identify a lamina-associated polypeptide 2 (LAP2) isoform-dependent nuclear chaperoning system that regulates GLI1 movement between the nuclear lamina and nucleoplasm to achieve maximal activation. LAP2ß forms a two-site interaction with the GLI1 zinc-finger domain and acetylation site, stabilizing an acetylation-dependent reserve on the inner nuclear membrane (INM). By contrast, the nucleoplasmic LAP2α competes with LAP2ß for GLI1 while scaffolding HDAC1 to deacetylate the secondary binding site. aPKC functions to promote GLI1 association with LAP2α, promoting egress off the INM. GLI1 intranuclear trafficking by LAP2 isoforms represents a powerful signal amplifier in BCCs with implications for zinc finger-based signal transduction and therapeutics.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Células 3T3 , Animales , Carcinoma Basocelular/metabolismo , Línea Celular , Cromatina , Proteínas de Unión al ADN/fisiología , Células HEK293 , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/fisiología , Histona Desacetilasa 1/metabolismo , Humanos , Proteínas de la Membrana/fisiología , Ratones , Chaperonas Moleculares/metabolismo , Lámina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1/fisiología , Dedos de ZincRESUMEN
Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
Asunto(s)
Colesterol/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Microscopía por Crioelectrón , Dimerización , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Ratones , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Receptor Patched-1/química , Receptor Patched-1/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Transducción de SeñalRESUMEN
Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.
Asunto(s)
Adipogénesis , Proteínas Hedgehog/metabolismo , Músculo Esquelético/metabolismo , Transducción de Señal , Células Madre/metabolismo , Adipocitos/metabolismo , Animales , Cilios/metabolismo , Distrofina/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones , Desarrollo de Músculos , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Regeneración , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole-exome sequencing and DNA copy number analyses, and we analyzed 196 HCC cases by DNA methylation, RNA, miRNA, and proteomic expression also. DNA sequencing and mutation analysis identified significantly mutated genes, including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or downregulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.
Asunto(s)
Carcinoma Hepatocelular/genética , Genómica , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/virología , Metilación de ADN , Humanos , Isocitrato Deshidrogenasa/genética , Neoplasias Hepáticas/virología , MicroARNs/genética , MutaciónRESUMEN
The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas Hedgehog/metabolismo , Proteínas de la Membrana/metabolismo , Acilación , Aciltransferasas/genética , Aciltransferasas/ultraestructura , Regulación Alostérica , Animales , Células COS , Dominio Catalítico , Chlorocebus aethiops , Microscopía por Crioelectrón , Células HEK293 , Hemo/metabolismo , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/ultraestructura , Simulación de Dinámica Molecular , Palmitoil Coenzima A/metabolismo , Conformación Proteica , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.
Asunto(s)
Quinasas DyrK , Proteínas Hedgehog , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Transducción de Señal , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de Zinc , Animales , Proteína Gli3 con Dedos de Zinc/metabolismo , Proteína Gli3 con Dedos de Zinc/genética , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proliferación Celular , Cilios/metabolismo , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteínas Nucleares , Proteínas RepresorasRESUMEN
Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's.
Asunto(s)
Cilios , Neuronas Dopaminérgicas , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Neuroprotección , Enfermedad de Parkinson , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Cilios/metabolismo , Animales , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Humanos , Ratones , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Neuroprotección/genética , Mutación , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , MasculinoRESUMEN
The transmembrane proteins cdon and boc are implicated in regulating hedgehog signaling during vertebrate development. Recent work showing roles for these genes in axon guidance and neural crest cell migration suggest that cdon and boc may play additional functions in regulating directed cell movements. We use newly generated and existing mutants to investigate a role for cdon and boc in zebrafish neural crest cell migration. We find that single mutant embryos exhibit normal neural crest phenotypes, but that neural crest migration is strikingly disrupted in double cdon;boc mutant embryos. We further show that this migration phenotype is associated with defects in the differentiation of slow-twitch muscle cells, and the loss of a Col1a1a-containing extracellular matrix, suggesting that neural crest defects may be a secondary consequence to defects in mesoderm development. Combined, our data add to a growing literature showing that cdon and boc act synergistically to promote hedgehog signaling during vertebrate development, and suggest that the zebrafish can be used to study the function of hedgehog receptor paralogs.
Asunto(s)
Cresta Neural , Pez Cebra , Animales , Moléculas de Adhesión Celular/genética , Diferenciación Celular , Movimiento Celular/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Desarrollo de Músculos/genética , Mutación/genética , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
During vertebrate embryogenesis, hematopoietic stem and progenitor cell (HSPC) production through endothelial-to-hematopoietic transition requires suitable developmental signals, but how these signals are accurately regulated remains incompletely understood. Cytoplasmic polyadenylation, which is one of the posttranscriptional regulations, plays a crucial role in RNA metabolism. Here, we report that Cpeb1b-mediated cytoplasmic polyadenylation is important for HSPC specification by translational control of Hedgehog (Hh) signaling during zebrafish early development. Cpeb1b is highly expressed in notochord and its deficiency results in defective HSPC production. Mechanistically, Cpeb1b regulates hemogenic endothelium specification by the Hedgehog-Vegf-Notch axis. We demonstrate that the cytoplasmic polyadenylation element motif-dependent interaction between Cpeb1b and shha messenger RNA (mRNA) in the liquid-like condensates, which are induced by Pabpc1b phase separation, is required for cytoplasmic polyadenylation of shha mRNA. Intriguingly, the cytoplasmic polyadenylation regulates translation but not stability of shha mRNA, which further enhances the Shha protein level and Hh signal transduction. Taken together, our findings uncover the role of Cpeb1b-mediated cytoplasmic polyadenylation in HSPC development and provide insights into how posttranscriptional regulation can direct developmental signals with high fidelity to translate them into cell fate transition.
Asunto(s)
Poliadenilación , Pez Cebra , Animales , Pez Cebra/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Hedgehog/metabolismo , Hematopoyesis/genéticaRESUMEN
Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity.
Asunto(s)
Ciliopatías , Síndromes Orofaciodigitales , Cilios/genética , Cilios/metabolismo , Ciliopatías/genética , Proteínas Hedgehog/metabolismo , Humanos , Intrones/genética , Mutación/genética , Síndromes Orofaciodigitales/genética , Empalme del ARN/genética , Factores de Empalme de ARN/metabolismo , ARN Interferente Pequeño/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
During brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis crucially affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to the neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation at the expense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of hedgehog signaling in a smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of smoothened and an inhibitor of smoothened ameliorated the hyperproliferation of NPCs in the telencephalon. Thus, these findings support the idea that PAR3 has a crucial role in the transition of NPCs from the expansion phase to the neurogenic phase by restricting hedgehog signaling through the establishment of ciliary integrity.
Asunto(s)
Proteínas Hedgehog , Células-Madre Neurales , Células-Madre Neurales/fisiología , Neuronas , Neurogénesis , Transducción de Señal/fisiologíaRESUMEN
Diabetic nephropathy (DN) is the leading cause of end-stage renal disease globally. Currently, there are no effective drugs for the treatment of DN. Although several studies have reported the therapeutic potential of mesenchymal stem cells, the underlying mechanisms remain largely unknown. Here, we report that both human umbilical cord MSCs (UC-MSCs) and UC-MSC-derived exosomes (UC-MSC-exo) attenuate kidney damage, and inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis in streptozotocin-induced DN rats. Strikingly, the Hedgehog receptor, smoothened (SMO), was significantly upregulated in the kidney tissues of DN patients and rats, and positively correlated with EMT and renal fibrosis. UC-MSC and UC-MSC-exo treatment resulted in decrease of SMO expression. In vitro co-culture experiments revealed that UC-MSC-exo reduced EMT of tubular epithelial cells through inhibiting Hedgehog/SMO pathway. Collectively, UC-MSCs inhibit EMT and renal fibrosis by delivering exosomes and targeting Hedgehog/SMO signaling, suggesting that UC-MSCs and their exosomes are novel anti-fibrotic therapeutics for treating DN.
Asunto(s)
Diabetes Mellitus , Nefropatías Diabéticas , Exosomas , Células Madre Mesenquimatosas , Humanos , Ratas , Animales , Nefropatías Diabéticas/metabolismo , Exosomas/metabolismo , Receptor Smoothened , Proteínas Hedgehog/metabolismo , Fibrosis , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the Hhip gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.
Asunto(s)
Habénula , Enfermedades Pulmonares , Receptores Nicotínicos , Ratones , Animales , Nicotina/farmacología , Nicotina/metabolismo , Habénula/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Receptores Nicotínicos/metabolismo , Neuronas Colinérgicas/metabolismo , Enfermedades Pulmonares/metabolismoRESUMEN
The origin and diversification of appendage types is a central question in vertebrate evolution. Understanding the genetic mechanisms that underlie fin and limb development can reveal relationships between different appendages. Here we demonstrate, using chemical genetics, a mutually agonistic interaction between Fgf and Shh genes in the developing dorsal fin of the channel catfish, Ictalurus punctatus. We also find that Fgf8 and Shh orthologs are expressed in the apical ectodermal ridge and zone of polarizing activity, respectively, in the median fins of representatives from other major vertebrate lineages. These findings demonstrate the importance of this feedback loop in median fins and offer developmental evidence for a median fin-first scenario for vertebrate paired appendage origins.
Asunto(s)
Aletas de Animales/embriología , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Ictaluridae/embriología , Animales , Tipificación del Cuerpo/genética , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Ictaluridae/anatomía & histología , Ictaluridae/metabolismoRESUMEN
During Hedgehog signaling, the ciliary levels of Ptch1 and Smo are regulated by the pathway. At the basal state, Ptch1 localizes to cilia and prevents the ciliary accumulation and activation of Smo. Upon binding a Hedgehog ligand, Ptch1 exits cilia, relieving inhibition of Smo. Smo then concentrates in cilia, becomes activated and activates downstream signaling. Loss of the ubiquitin E3 ligase Arih2 elevates basal Hedgehog signaling, elevates the cellular level of Smo and increases basal levels of ciliary Smo. Mice express two isoforms of Arih2 with Arih2α found primarily in the nucleus and Arih2ß found on the cytoplasmic face of the endoplasmic reticulum (ER). Re-expression of ER-localized Arih2ß but not nuclear-localized Arih2α rescues the Arih2 mutant phenotypes. When Arih2 is defective, protein aggregates accumulate in the ER and the unfolded protein response is activated. Arih2ß appears to regulate the ER-associated degradation (ERAD) of Smo preventing excess and potentially misfolded Smo from reaching the cilium and interfering with pathway regulation.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Proteínas Hedgehog , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Ratones , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , UbiquitinaciónRESUMEN
Reactive oxygen species (ROS), originally classified as toxic molecules, have attracted increasing interest given their actions in cell signaling. Hydrogen peroxide (H2O2), the major ROS produced by cells, acts as a second messenger to modify redox-sensitive proteins or lipids. After caudal fin amputation, tight spatiotemporal regulation of ROS is required first for wound healing and later to initiate the regenerative program. However, the mechanisms carrying out this sustained ROS production and their integration with signaling pathways remain poorly understood. We focused on the early dialog between H2O2 and Sonic hedgehog (Shh) during zebrafish fin regeneration. We demonstrate that H2O2 controls Shh expression and that Shh in turn regulates the H2O2 level via a canonical pathway. Moreover, the means of this tight reciprocal control change during the successive phases of the regenerative program. Dysregulation of the Hedgehog pathway has been implicated in several developmental syndromes, diabetes and cancer. These data support the existence of an early positive crosstalk between Shh and H2O2 that might be more generally involved in various processes paving the way to improve regenerative processes, particularly in vertebrates.
Asunto(s)
Proteínas Hedgehog , Pez Cebra , Animales , Proteínas Hedgehog/metabolismo , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno , Cicatrización de Heridas , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Sonic hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage, which depends on Hedgehog signaling for its perinatal expansion. Whereas SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate whether the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as a function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream SHH pathway component Smoothened, whereas sensitivity to downstream components SUFU and GLI family proteins was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients.
Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Células-Madre Neurales , Animales , Proliferación Celular/fisiología , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Ratones , Células-Madre Neurales/metabolismoRESUMEN
The primary cilium is a hair-like projection that controls cell development and tissue homeostasis. Although accumulated studies identify the molecular link between cilia and cilia-related diseases, the underlying etiology of ciliopathies has not been fully understood. In this paper, we determine the function of Rab34, a small GTPase, as a key regulator for controlling ciliogenesis and type I collagen trafficking in craniofacial development. Mechanistically, Rab34 is required to form cilia that control osteogenic proliferation, survival, and differentiation via cilia-mediated Hedgehog signaling. In addition, Rab34 is indispensable for regulating type I collagen trafficking from the ER to the Golgi. These results demonstrate that Rab34 has both ciliary and non-ciliary functions to regulate osteogenesis. Our study highlights the critical function of Rab34, which may contribute to understanding the novel etiology of ciliopathies that are associated with the dysfunction of RAB34 in humans.