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1.
Genes Dev ; 38(11-12): 569-582, 2024 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-38997156

RESUMEN

Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHigh cells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHigh cells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of "cell cycling" and "oxidative stress response" pathways in TertHigh cells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHigh cells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.


Asunto(s)
Homeostasis , Regeneración , Telomerasa , Telomerasa/metabolismo , Telomerasa/genética , Animales , Homeostasis/genética , Homeostasis/efectos de la radiación , Ratones , Regeneración/efectos de la radiación , Regeneración/genética , Humanos , Glándulas Salivales/efectos de la radiación , Glándulas Salivales/metabolismo , Glándulas Salivales/citología , Proliferación Celular/efectos de la radiación , Proliferación Celular/genética , Supervivencia Celular/efectos de la radiación , Supervivencia Celular/genética , Glándula Submandibular/efectos de la radiación , Glándula Submandibular/metabolismo , Células Madre/efectos de la radiación , Células Madre/metabolismo , Células Madre/citología , Radioterapia/efectos adversos , Especies Reactivas de Oxígeno/metabolismo , Células Cultivadas
2.
Nat Rev Mol Cell Biol ; 24(9): 668-687, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-36932157

RESUMEN

The Hedgehog signalling pathway has crucial roles in embryonic tissue patterning, postembryonic tissue regeneration, and cancer, yet aspects of Hedgehog signal transmission and reception have until recently remained unclear. Biochemical and structural studies surprisingly reveal a central role for lipids in Hedgehog signalling. The signal - Hedgehog protein - is modified by cholesterol and palmitate during its biogenesis, thereby necessitating specialized proteins such as the transporter Dispatched and several lipid-binding carriers for cellular export and receptor engagement. Additional lipid transactions mediate response to the Hedgehog signal, including sterol activation of the transducer Smoothened. Access of sterols to Smoothened is regulated by the apparent sterol transporter and Hedgehog receptor Patched, whose activity is blocked by Hedgehog binding. Alongside these lipid-centric mechanisms and their relevance to pharmacological pathway modulation, we discuss emerging roles of Hedgehog pathway activity in stem cells or their cellular niches, with translational implications for regeneration and restoration of injured or diseased tissues.


Asunto(s)
Proteínas Hedgehog , Transducción de Señal , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Transducción de Señal/fisiología , Colesterol/metabolismo , Esteroles/química , Esteroles/metabolismo
3.
Sci Adv ; 8(45): eabm3548, 2022 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-36351009

RESUMEN

Metastasis is responsible for most breast cancer-related deaths; however, identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage-traced cells integrate into the vasculature and have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by tumor necrosis factor-α and interleukin-6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.


Asunto(s)
Neoplasias de la Mama , Neoplasias Pulmonares , Humanos , Ratones , Animales , Femenino , Neoplasias de la Mama/patología , Neoplasias Pulmonares/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo
4.
Nature ; 599(7884): 320-324, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34707294

RESUMEN

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters1,2 and to H+-driven transporters of the RND family3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na+ gradient across the plasma membrane. The transmembrane channel and Na+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na+ ions. DISP1-NNN and variants that disrupt single Na+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na+-site occupancies, which suggests a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na+ flux powers their conformationally driven activities.


Asunto(s)
Microscopía por Crioelectrón , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Sodio/metabolismo , Animales , Sitios de Unión , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas Hedgehog/ultraestructura , Lípidos de la Membrana/química , Lípidos de la Membrana/aislamiento & purificación , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/ultraestructura , Ratones , Modelos Moleculares , Mutación
5.
Proc Natl Acad Sci U S A ; 117(46): 28838-28846, 2020 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-33139559

RESUMEN

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.


Asunto(s)
Receptor Patched-1/agonistas , Receptor Patched-1/metabolismo , Receptor Patched-1/ultraestructura , Animales , Microscopía por Crioelectrón/métodos , Proteínas Hedgehog/metabolismo , Humanos , Receptores Patched/metabolismo , Transducción de Señal/fisiología , Anticuerpos de Dominio Único/farmacología
6.
Nature ; 571(7764): 284-288, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31263273

RESUMEN

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.


Asunto(s)
Membrana Celular/química , Proteínas Hedgehog/agonistas , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/agonistas , Receptor Smoothened/metabolismo , Esteroles/farmacología , Animales , Sitios de Unión , Técnicas Biosensibles , Dominio Catalítico/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacología , Proteínas Hedgehog/metabolismo , Ligandos , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Receptor Patched-1/antagonistas & inhibidores , Receptor Patched-1/metabolismo , Conformación Proteica , Estabilidad Proteica , Anticuerpos de Cadena Única/inmunología , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/química , Esteroles/química , Esteroles/metabolismo , Proteínas de Xenopus/química
7.
Cell ; 175(5): 1352-1364.e14, 2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30415841

RESUMEN

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 Señal
8.
Mol Cell ; 72(2): 316-327.e5, 2018 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-30340023

RESUMEN

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11ß2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.


Asunto(s)
Cilios/efectos de los fármacos , Cilios/metabolismo , Oxiesteroles/farmacología , Animales , Línea Celular , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Células 3T3 NIH , Transducción de Señal/efectos de los fármacos
9.
Proc Natl Acad Sci U S A ; 115(2): E200-E209, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29279401

RESUMEN

How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog (Shh), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh-expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration.


Asunto(s)
Epitelio/metabolismo , Proteínas Hedgehog/metabolismo , Papilas Gustativas/metabolismo , Lengua/metabolismo , Animales , Epitelio/crecimiento & desarrollo , Epitelio/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Organogénesis/genética , Regeneración/genética , Transducción de Señal/genética , Gusto/genética , Papilas Gustativas/citología , Papilas Gustativas/crecimiento & desarrollo , Factores de Tiempo , Lengua/citología , Lengua/crecimiento & desarrollo
10.
Proc Natl Acad Sci U S A ; 114(52): E11141-E11150, 2017 12 26.
Artículo en Inglés | MEDLINE | ID: mdl-29229834

RESUMEN

Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na+ to regulate Smoothened in our assays, raising the possibility that a Na+ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na+ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.


Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal/fisiología , Receptor Smoothened/metabolismo , Sodio/metabolismo , Animales , Membrana Celular/genética , Colesterol/genética , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Dominios Proteicos , Células Sf9 , Receptor Smoothened/genética , Spodoptera
11.
Nat Commun ; 8(1): 1275, 2017 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-29097673

RESUMEN

The Drosophila Hedgehog receptor functions to regulate the essential downstream pathway component, Smoothened, and to limit the range of signaling by sequestering Hedgehog protein signal within imaginal disc epithelium. Hedgehog receptor function requires both Patched and Ihog activity, the latter interchangeably encoded by interference hedgehog (ihog) or brother of ihog (boi). Here we show that Patched and Ihog activity are mutually required for receptor endocytosis and degradation, triggered by Hedgehog protein binding, and causing reduced levels of Ihog/Boi proteins in a stripe of cells at the anterior/posterior compartment boundary of the wing imaginal disc. This Ihog spatial discontinuity may contribute to classically defined cell segregation and lineage restriction at the anterior/posterior wing disc compartment boundary, as suggested by our observations that Ihog activity mediates aggregation of otherwise non-adherent cultured cells and that loss of Ihog activity disrupts wing disc cell segregation, even with downstream genetic rescue of Hedgehog signal response.


Asunto(s)
Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Proteínas Hedgehog/genética , Discos Imaginales/crecimiento & desarrollo , Glicoproteínas de Membrana/genética , Receptores de Superficie Celular/genética , Alas de Animales/crecimiento & desarrollo , Animales , Tipificación del Cuerpo , Drosophila/embriología , Drosophila/genética , Endocitosis/genética , Regulación del Desarrollo de la Expresión Génica , Transducción de Señal , Receptor Smoothened/genética
12.
Cancer Cell ; 32(4): 404-410, 2017 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-29017054

RESUMEN

Stromal restraint of cancer growth and progression-emerging as a widespread phenomenon in epithelial cancers such as bladder, pancreas, colon, and prostate-appears rooted in stromal cell niche activity. During normal tissue repair, stromal niche signals, often Hedgehog-induced, promote epithelial stem cell differentiation as well as self-renewal, thus specifying a regenerating epithelial pattern. In the case of cancerous tissue, stromal cell-derived differentiation signals in particular may provide a brake on malignant growth. Understanding and therapeutic harnessing of the role of stroma in cancer restraint may hinge on our knowledge of the signaling programs elaborated by the stromal niche.


Asunto(s)
Regeneración/fisiología , Nicho de Células Madre/fisiología , Células Madre/fisiología , Células del Estroma/fisiología , Neoplasias de la Mama/etiología , Neoplasias de la Mama/patología , Diferenciación Celular , Femenino , Proteínas Hedgehog/fisiología , Humanos , Masculino , Neoplasias Pancreáticas/etiología , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/patología , Neoplasias de la Vejiga Urinaria/etiología , Neoplasias de la Vejiga Urinaria/patología
13.
Science ; 356(6335)2017 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-28280246

RESUMEN

The stem cell niche is a complex local signaling microenvironment that sustains stem cell activity during organ maintenance and regeneration. The mammary gland niche must support its associated stem cells while also responding to systemic hormonal regulation that triggers pubertal changes. We find that Gli2, the major Hedgehog pathway transcriptional effector, acts within mouse mammary stromal cells to direct a hormone-responsive niche signaling program by activating expression of factors that regulate epithelial stem cells as well as receptors for the mammatrophic hormones estrogen and growth hormone. Whereas prior studies implicate stem cell defects in human disease, this work shows that niche dysfunction may also cause disease, with possible relevance for human disorders and in particular the breast growth pathogenesis associated with combined pituitary hormone deficiency.


Asunto(s)
Hormona del Crecimiento/metabolismo , Proteínas Hedgehog/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Nicho de Células Madre/genética , Proteína Gli2 con Dedos de Zinc/fisiología , Animales , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Estrógenos/metabolismo , Femenino , Expresión Génica , Hormona del Crecimiento/sangre , Hormona del Crecimiento/deficiencia , Proteínas Hedgehog/genética , Factor II del Crecimiento Similar a la Insulina/genética , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Ratones , Prolactina/metabolismo , Maduración Sexual/genética , Transducción de Señal/genética , Células del Estroma/metabolismo , Proteínas Wnt/genética , Proteína Gli2 con Dedos de Zinc/genética
14.
Sci Transl Med ; 9(372)2017 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-28077677

RESUMEN

Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.


Asunto(s)
Fracturas del Fémur/tratamiento farmacológico , Curación de Fractura/efectos de los fármacos , Proteínas Hedgehog/farmacología , Células Madre Mesenquimatosas/citología , Nicho de Células Madre , Animales , Huesos/patología , Proliferación Celular , Separación Celular , Diabetes Mellitus Experimental/patología , Femenino , Citometría de Flujo , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Osteogénesis , Transducción de Señal
15.
Proc Natl Acad Sci U S A ; 113(47): E7545-E7553, 2016 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-27815529

RESUMEN

Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.


Asunto(s)
Colitis/metabolismo , Sulfato de Dextran/efectos adversos , Proteínas Hedgehog/metabolismo , Interleucina-10/metabolismo , Transducción de Señal , Animales , Antígenos CD4/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Factores de Transcripción Forkhead/metabolismo , Proteínas Hedgehog/efectos de los fármacos , Humanos , Ratones , Mutación , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T Reguladores/metabolismo , Proteína con Dedos de Zinc GLI1/genética
16.
Cell ; 166(2): 451-467, 2016 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-27419872

RESUMEN

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.


Asunto(s)
Mesodermo/citología , Transducción de Señal , Proteínas Morfogenéticas Óseas/metabolismo , Huesos/citología , Huesos/metabolismo , Corazón/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Humanos , Mesodermo/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismo , Línea Primitiva/citología , Línea Primitiva/metabolismo , Análisis de la Célula Individual , Somitos/metabolismo , Células Madre , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismo
17.
Proc Natl Acad Sci U S A ; 113(21)2016 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-27162362

RESUMEN

Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith-Lemli-Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-ß-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3ß,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith-Lemli-Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.


Asunto(s)
Deshidrocolesteroles/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Receptor Smoothened/metabolismo , Animales , Chlamydomonas reinhardtii/química , Deshidrocolesteroles/química , Deshidrocolesteroles/farmacología , Flagelos/química , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Ratones , Células 3T3 NIH , Síndrome de Smith-Lemli-Opitz/genética , Síndrome de Smith-Lemli-Opitz/metabolismo , Receptor Smoothened/genética , Alcaloides de Veratrum/farmacología , beta-Ciclodextrinas/farmacología
18.
Elife ; 52016 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-26974344

RESUMEN

A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. Here we show that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control.


Asunto(s)
Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Alas de Animales/embriología , Alas de Animales/crecimiento & desarrollo , Animales , Antenas de Artrópodos/embriología , Antenas de Artrópodos/crecimiento & desarrollo , Drosophila/metabolismo , Extremidades/embriología , Extremidades/crecimiento & desarrollo , Organogénesis , Alas de Animales/metabolismo
19.
JAMA Dermatol ; 152(4): 452-6, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26765315

RESUMEN

IMPORTANCE: Tumor resistance is an emerging problem for Smoothened (SMO) inhibitor-treated metastatic basal cell carcinoma (BCC). Arsenic trioxide and itraconazole antagonize the hedgehog (HH) pathway at sites distinct from those treated by SMO inhibitors. OBJECTIVE: To determine whether administration of intravenous arsenic trioxide and oral itraconazole in patients with metastatic BCC is associated with a reduction in GLI1 messenger RNA expression in tumor and/or normal skin biopsy samples. DESIGN, SETTING, AND PARTICIPANTS: Five men with metastatic BCC who experienced relapse after SMO inhibitor treatment underwent intravenous arsenic trioxide treatment for 5 days, every 28 days, and oral itraconazole treatment on days 6 to 28. Data were collected from April 10 to November 14, 2013. Follow-up was completed on October 3, 2015, and data were analyzed from June 5 to October 6, 2015. MAIN OUTCOMES AND MEASURES: The primary outcome was the change in messenger RNA levels of the GLI family zinc finger 1 (GLI1) gene (HH-pathway target gene) in biopsy specimens of normal skin or BCC before and after treatment. Secondary objectives were evaluation of tumor response and tolerability. RESULTS: Of the 5 patients (mean [SD] age, 52 [9] years; age range, 43-62 years), 3 completed 3 cycles of treatment and 2 discontinued treatment early owing to disease progression or adverse events. Adverse effects included grade 2 transaminitis and grade 4 leukopenia with a grade 3 infection. Overall, arsenic trioxide and itraconazole reduced GLI1 messenger RNA levels by 75% from baseline (P < .001). The best overall response after 3 treatment cycles was stable disease in 3 patients. CONCLUSIONS AND RELEVANCE: Targeting the HH pathway with sequential arsenic trioxide and itraconazole treatment is a feasible treatment for metastatic BCC. Although some patients experienced stable disease for 3 months, none had tumor shrinkage, which may be owing to transient GLI1 suppression with sequential dosing. Continuous dosing may be required to fully inhibit the HH pathway and achieve clinical response.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Basocelular/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Factores de Transcripción/genética , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Trióxido de Arsénico , Arsenicales/administración & dosificación , Carcinoma Basocelular/patología , Resistencia a Antineoplásicos , Estudios de Seguimiento , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Itraconazol/administración & dosificación , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Óxidos/administración & dosificación , ARN Mensajero/metabolismo , Neoplasias Cutáneas/patología , Resultado del Tratamiento , Proteína con Dedos de Zinc GLI1
20.
PLoS One ; 10(8): e0135804, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26271100

RESUMEN

Suppressor of fused (Su(fu)/Sufu), one of the most conserved components of the Hedgehog (Hh) signaling pathway, binds Ci/Gli transcription factors and impedes activation of target gene expression. In Drosophila, the Su(fu) mutation has a minimal phenotype, and we show here that Ci transcriptional activity in large part is regulated independently of Su(fu) by other pathway components. Mutant mice lacking Sufu in contrast show excessive pathway activity and die as embryos with patterning defects. Here we show that in cultured cells Hh stimulation can augment transcriptional activity of a Gli2 variant lacking Sufu interaction and, surprisingly, that regulation of Hh pathway targets is nearly normal in the neural tube of Sufu-/- mutant embryos that also lack Gli1 function. Some degree of Hh-induced transcriptional activation of Ci/Gli thus can occur independently of Sufu in both flies and mammals. We further note that Sufu loss can also reduce Hh induction of high-threshold neural tube fates, such as floor plate, suggesting a possible positive pathway role for Sufu.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas Hedgehog/genética , Cinesinas/genética , Cinesinas/metabolismo , Ratones , Ratones Mutantes , Proteínas Oncogénicas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Transactivadores/genética , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1
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