RESUMO
The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.
Assuntos
Neoplasias Colorretais , Sistema Nervoso Entérico , Humanos , Transdução de Sinais/fisiologia , Diferenciação Celular/fisiologia , Sistema Nervoso Entérico/metabolismo , Movimento Celular/fisiologia , Neoplasias Colorretais/metabolismo , Crista Neural/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismoRESUMO
Gastrointestinal stromal tumor (GIST), the most common sarcoma, is mainly caused by an oncogenic mutation in the KIT receptor tyrosine kinase. Targeting KIT using tyrosine kinase inhibitors, such as imatinib and sunitinib, provides substantial benefit; however, in most patients, the disease will eventually progress due to KIT secondary mutations leading to treatment failure. Understanding how GIST cells initially adapt to KIT inhibition should guide the selection of appropriate therapies to overcome the emergence of resistance. Several mechanisms have been broadly implicated in the resistance to imatinib anti-tumoral effects, including the reactivation of MAPK signaling upon KIT/PDGFRA targeted inhibition. This study provides evidence that LImb eXpression 1 (LIX1), a protein we identified as a regulator of the Hippo transducers YAP1 and TAZ, is upregulated upon imatinib or sunitinib treatment. LIX1 silencing in GIST-T1 cells impaired imatinib-induced MAPK signaling reactivation and enhanced imatinib anti-tumor effect. Our findings identified LIX1 as a key regulator of the early adaptative response of GIST cells to targeted therapies.
Assuntos
Antineoplásicos , Tumores do Estroma Gastrointestinal , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Relacionadas à Autofagia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Sistema de Sinalização das MAP QuinasesRESUMO
YAP1 and TAZ are transcriptional co-activator proteins that play fundamental roles in many biological processes, from cell proliferation and cell lineage fate determination to tumorigenesis. We previously demonstrated that Limb Expression 1 (LIX1) regulates YAP1 and TAZ activity and controls digestive mesenchymal progenitor proliferation. However, LIX1 mode of action remains elusive. Here, we found that endogenous LIX1 is localized in mitochondria and is anchored to the outer mitochondrial membrane through S-palmitoylation of cysteine 84, a residue conserved in all LIX1 orthologs. LIX1 downregulation altered the mitochondrial ultrastructure, resulting in a significantly decreased respiration and attenuated production of mitochondrial reactive oxygen species (mtROS). Mechanistically, LIX1 knock-down impaired the stability of the mitochondrial proteins PHB2 and OPA1 that are found in complexes with mitochondrial-specific phospholipids and are required for cristae organization. Supplementation with unsaturated fatty acids counteracted the effects of LIX1 knock-down on mitochondrial morphology and ultrastructure and restored YAP1/TAZ signaling. Collectively, our data demonstrate that LIX1 is a key regulator of cristae organization, modulating mtROS level and subsequently regulating the signaling cascades that control fate commitment of digestive mesenchyme-derived cells.
Assuntos
Cisteína , Mitocôndrias , Cisteína/metabolismo , Mesoderma/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Gastrointestinal motor activity has been extensively studied in adults; however, only few studies have investigated fetal motor skills. It is unknown when the gastrointestinal tract starts to contract during the embryonic period and how this function evolves during development. Here, we adapted a non-invasive high-resolution echography technique combined with speckle tracking analysis to examine the gastrointestinal tract motor activity dynamics during chick embryo development. We provided the first recordings of fetal gastrointestinal motility in living embryos without anesthesia. We found that, although gastrointestinal contractions appear very early during development, they become synchronized only at the end of the fetal period. To validate this approach, we used various pharmacological inhibitors and BAPX1 gene overexpression in vivo. We found that the enteric nervous system determines the onset of the synchronized contractions in the stomach. Moreover, alteration of smooth muscle fiber organization led to an impairment of this functional activity. Altogether, our findings show that non-invasive high-resolution echography and speckle tracking analysis allows visualization and quantification of gastrointestinal motility during development and highlight the progressive acquisition of functional and coordinated gastrointestinal motility before birth.
Assuntos
Sistema Nervoso Entérico , Motilidade Gastrointestinal , Animais , Embrião de Galinha , Motilidade Gastrointestinal/fisiologia , Trato Gastrointestinal/diagnóstico por imagem , Miócitos de Músculo Liso , UltrassonografiaRESUMO
Smooth Muscle Cells (SMC) are unique amongst all muscle cells in their capacity to modulate their phenotype. Indeed, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate, switching between a quiescent contractile state and a highly proliferative and migratory phenotype, a quality often associated to SMC dysfunction. However, phenotypic plasticity remains poorly examined in the field of gastroenterology in particular in pathologies in which gut motor activity is impaired. Here, we assessed SMC status in biopsies of infants with chronic intestinal pseudo-obstruction (CIPO) syndrome, a life-threatening intestinal motility disorder. We showed that CIPO-SMCs harbour a decreased level of contractile markers. This phenotype is accompanied by an increase in Platelet-Derived Growth Factor Receptor-alpha (PDGFRA) expression. We showed that this modulation occurs without origin-related differences in CIPO circular and longitudinal-derived SMCs. As we characterized PDGFRA as a marker of digestive mesenchymal progenitors during embryogenesis, our results suggest a phenotypic switch of the CIPO-SMC towards an undifferentiated stage. The development of CIPO-SMC culture and the characterization of SMC phenotypic switch should enable us to design therapeutic approaches to promote SMC differentiation in CIPO.
Assuntos
Diferenciação Celular , Pseudo-Obstrução Intestinal/patologia , Contração Muscular , Miócitos de Músculo Liso/patologia , Fenótipo , Adolescente , Proliferação de Células , Células Cultivadas , Criança , Feminino , Humanos , Pseudo-Obstrução Intestinal/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Transdução de SinaisRESUMO
The first contractile waves in the developing embryonic gut are purely myogenic; they only involve smooth muscle. Here, we provide evidence for a transition from smooth muscle to interstitial cell of Cajal (ICC)-driven contractile waves in the developing chicken gut. In situ hybridization staining for anoctamin-1 (ANO1), a known ICC marker, shows that ICCs are already present throughout the gut, as from embryonic day (E)7. We devised a protocol to reveal ICC oscillatory and propagative calcium activity in embryonic gut whole mount and found that the first steady calcium oscillations in ICCs occur on (E14). We show that the activation of ICCs leads to an increase in contractile wave frequency, regularity, directionality, and velocity between E12 and E14. We finally demonstrate that application of the c-KIT antagonist imatinib mesylate in organ culture specifically depletes the ICC network and inhibits the transition to a regular rhythmic wave pattern. We compare our findings to existing results in the mouse and predict that a similar transition should take place in the human fetus between 12 and 14 wk of development. Together, our results point to an abrupt physiological transition from smooth muscle mesenchyme self-initiating waves to ICC-driven motility in the fetus and clarify the contribution of ICCs to the contractile wave pattern.NEW & NOTEWORTHY We reveal a sharp transition from smooth muscle to interstitial cell of Cajal (ICC)-driven motility in the chicken embryo, leading to higher-frequency, more rhythmic contractile waves. We predict the transition to happen between 12 and 14 embryonic wk in humans. We image for the first time the onset of ICC activity in an embryonic gut by calcium imaging. We show the first KIT and anoctamin-1 (ANO1) in situ hybridization micrographs in the embryonic chicken gut.
Assuntos
Motilidade Gastrointestinal/fisiologia , Células Intersticiais de Cajal/fisiologia , Intestinos/embriologia , Animais , Anoctamina-1/análise , Cálcio/metabolismo , Embrião de Galinha , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/fisiologia , Humanos , Células Intersticiais de Cajal/química , Intestinos/fisiologia , Camundongos , Contração Muscular/fisiologia , Músculo Liso/embriologia , Músculo Liso/fisiologia , Fatores de TempoRESUMO
Gastrointestinal stromal tumours (GISTs), the most common mesenchymal neoplasm of the gastrointestinal tract, result from deregulated proliferation of transformed KIT-positive interstitial cells of Cajal that share mesenchymal progenitors with smooth muscle cells. Despite the identification of selective KIT inhibitors, primary resistance and relapse remain a major concern. Moreover, most patients develop resistance partly through reactivation of KIT and its downstream signalling pathways. We previously identified the Limb Expression 1 (LIX1) gene as a unique marker of digestive mesenchyme immaturity. We also demonstrated that LIX1 regulates mesenchymal progenitor proliferation and differentiation by controlling the Hippo effector YAP1, which is constitutively activated in many sarcomas. Therefore, we wanted to determine LIX1 role in GIST development. We found that LIX1 is strongly up-regulated in GIST samples and this is associated with unfavourable prognosis. Moreover, LIX1 controls GIST cell proliferation in vitro and in vivo. Upon LIX1 inactivation in GIST cells, YAP1/TAZ activity is reduced, KIT (the GIST signature) is down-regulated, and cells acquire smooth muscle lineage features. Our data highlight LIX1 role in digestive mesenchyme-derived cell-fate decisions and identify this novel regulator as a target for drug design for GIST treatment by influencing its differentiation status.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Plasticidade Celular , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/patologia , Recidiva Local de Neoplasia/patologia , Fatores de Transcrição/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Proliferação de Células , Embrião de Galinha , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Taxa de Sobrevida , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The enteric nervous system (ENS) is a complex network constituted of neurons and glial cells that ensures the intrinsic innervation of the gastrointestinal tract. ENS cells originate from vagal and sacral neural crest cells that are initially located at the border of the neural tube. In birds, sacral neural crest cells (sNCCs) first give rise to an extramural ganglionated structure (the so-called Nerve of Remak [NoR]) and to the pelvic plexus. Later, sNCCs enter the colon mesenchyme to colonize and contribute to the intrinsic innervation of the caudal part of the gut. However, no specific sNCC marker has been described. Here, we report the expression pattern of prospero-related homeobox 1 (PROX1) in the developing chick colon. PROX1 is a homeobox domain transcription factor that plays a role in cell type specification in various tissues. Using in situ hybridization and immunofluorescence techniques, we showed that PROX1 is expressed in sNCCs localized in the NoR and in the pelvic plexus. Then, using real-time quantitative PCR we found that PROX1 displays a strong and highly dynamic expression pattern during NoR development. Moreover, we demonstrated using in vivo cell tracing, that sNCCs are the source of the PROX1-positive cells within the NoR. Our results indicate that PROX1 is the first marker that specifically identifies sNCCs. This might help to better identify the role of the different neural crest cell populations in distal gut innervation, and consequently to improve the diagnosis of diseases linked to incomplete ENS formation, such as Hirschsprung's disease.
Assuntos
Proteínas de Homeodomínio/metabolismo , Intestinos/inervação , Crista Neural/metabolismo , Animais , Biomarcadores/metabolismo , Embrião de Galinha , Sistema Nervoso Entérico/citologia , Crista Neural/citologiaRESUMO
During development, the gastrointestinal (GI) tract arises from a primary tube composed of mesoderm and endoderm. The mesoderm gives rise to the digestive mesenchyme, which in turn differentiates into multiple tissues, namely the submucosa, the interstitial cells of Cajal and the smooth muscle cells (SMCs). Concomitant with these early patterning events, the primitive GI tract is colonized by vagal enteric neural crest-derived cells (vENCDCs), a population of cells that gives rise to the enteric nervous system, the intrinsic innervation of the GI tract. Reciprocal neuro-mesenchymal interactions are essential for the coordinated development of GI musculature. The aim of this study is to examine and compare the kinetics of mesenchymal cell differentiation into SMCs along the anterior-posterior axis to the pattern of vENCDCs migration using whole-mount in situ hybridization and paraffin section immunofluorescence analyses on chick embryonic GI tracts from E4-Stage 23 to E7-Stages 30-31. We confirmed that gastric and pre-umbilical intestine mesenchyme differentiation into SMCs occurs after vENCDCs colonization. However, we found that colonic and post-umbilical intestine mesenchyme differentiation occurs before vENCDCs colonization. These findings suggest that regional-specific mechanisms are involved in the mesenchyme differentiation into SMCs along the GI anterior-posterior axis.
Assuntos
Colo/embriologia , Sistema Nervoso Entérico/embriologia , Mesoderma/embriologia , Músculo Liso/embriologia , Crista Neural/embriologia , Animais , Padronização Corporal , Diferenciação Celular , Embrião de Galinha , Colo/citologia , Colo/inervação , Intestinos/citologia , Intestinos/embriologia , Mesoderma/citologia , Estômago/citologia , Estômago/embriologiaRESUMO
BACKGROUND: Smooth muscle cell (SMC) plasticity maintains the balance between differentiated SMCs and proliferative mesenchymal progenitors, crucial for muscular tissue homeostasis. Studies on the development of mesenchymal progenitors into SMCs have proven useful in identifying molecular mechanisms involved in digestive musculature plasticity in physiological and pathological conditions. RESULTS: Here, we show that Limb Expression 1 (LIX1) molecularly defines the population of mesenchymal progenitors in the developing stomach. Using in vivo functional approaches in the chick embryo, we demonstrate that LIX1 is a key regulator of stomach SMC development. We show that LIX1 is required for stomach SMC determination to regulate the expression of the pro-proliferative gene YAP1 and mesenchymal cell proliferation. However, as stomach development proceeds, sustained LIX1 expression has a negative impact on further SMC differentiation and this is associated with a decrease in YAP1 activity. CONCLUSIONS: We demonstrate that expression of LIX1 must be tightly regulated to allow fine-tuning of the transcript levels and state of activation of the pro-proliferative transcriptional coactivator YAP1 to regulate proliferation rates of stomach mesenchymal progenitors and their differentiation. Our data highlight dual roles for LIX1 and YAP1 and provide new insights into the regulation of cell density-dependent proliferation, which is essential for the development and homeostasis of organs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular , Proliferação de Células , Células-Tronco Mesenquimais/citologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Embrião de Galinha , Regulação da Expressão Gênica , Inativação Gênica , Marcadores Genéticos , Miócitos de Músculo Liso/citologia , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Estômago/citologiaRESUMO
This review addresses the developmental roles of 2 GTPases of the Rho family, RhoV/Chp and RhoU/Wrch. These two GTPases form a distinct subfamily related to Rac and Cdc42 proteins and were detected in a screen for Rho members that are particularly expressed in the neural crest, an embryonic tissue peculiar to vertebrates. The neural crest represents a physiological model of normal epithelial to mesenchymal transition (EMT), in which epithelial cells at the border of neural and non-neural ectoderm differentiate, lose their intercellular connections and migrate throughout the embryo. We showed that RhoV, transiently induced by the canonical Wnt pathway, is required for the full differentiation of neural crest cells, while RhoU, induced later by the non-canonical Wnt pathway, is necessary for the migration process. These two GTPases, which are highly conserved across vertebrates, are thus tightly functionally linked to Wnt signaling, whose implication in embryonic development and cancer progression is well established. In the light of the recent literature, we discuss how RhoV and RhoU may achieve their physiological functions.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Crista Neural/embriologia , Via de Sinalização Wnt/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Ligação ao GTP/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration.
Assuntos
Mucosa Intestinal/embriologia , Mesoderma/embriologia , Comunicação Celular , Diferenciação Celular , Trato Gastrointestinal/citologia , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Miócitos de Músculo Liso/citologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
In vertebrates, the digestive tract develops from a uniform structure where reciprocal epithelial-mesenchymal interactions pattern this complex organ into regions with specific morphologies and functions. Concomitant with these early patterning events, the primitive GI tract is colonized by the vagal enteric neural crest cells (vENCCs), a population of cells that will give rise to the enteric nervous system (ENS), the intrinsic innervation of the GI tract. The influence of vENCCs on early patterning and differentiation of the GI tract has never been evaluated. In this study, we report that a crucial number of vENCCs is required for proper chick stomach development, patterning and differentiation. We show that reducing the number of vENCCs by performing vENCC ablations induces sustained activation of the BMP and Notch pathways in the stomach mesenchyme and impairs smooth muscle development. A reduction in vENCCs also leads to the transdifferentiation of the stomach into a stomach-intestinal mixed phenotype. In addition, sustained Notch signaling activity in the stomach mesenchyme phenocopies the defects observed in vENCC-ablated stomachs, indicating that inhibition of the Notch signaling pathway is essential for stomach patterning and differentiation. Finally, we report that a crucial number of vENCCs is also required for maintenance of stomach identity and differentiation through inhibition of the Notch signaling pathway. Altogether, our data reveal that, through the regulation of mesenchyme identity, vENCCs act as a new mediator in the mesenchymal-epithelial interactions that control stomach development.
Assuntos
Diferenciação Celular/fisiologia , Sistema Nervoso Entérico/embriologia , Morfogênese/fisiologia , Crista Neural/embriologia , Transdução de Sinais/fisiologia , Estômago/embriologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião de Galinha , Imunofluorescência , Hibridização In Situ , Músculo Liso/embriologia , Técnicas de Cultura de Órgãos , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estômago/inervaçãoRESUMO
The pacemaking activity of specialized tissues in the heart and gut results in lifelong rhythmic contractions. Here we describe a new syndrome characterized by Chronic Atrial and Intestinal Dysrhythmia, termed CAID syndrome, in 16 French Canadians and 1 Swede. We show that a single shared homozygous founder mutation in SGOL1, a component of the cohesin complex, causes CAID syndrome. Cultured dermal fibroblasts from affected individuals showed accelerated cell cycle progression, a higher rate of senescence and enhanced activation of TGF-ß signaling. Karyotypes showed the typical railroad appearance of a centromeric cohesion defect. Tissues derived from affected individuals displayed pathological changes in both the enteric nervous system and smooth muscle. Morpholino-induced knockdown of sgol1 in zebrafish recapitulated the abnormalities seen in humans with CAID syndrome. Our findings identify CAID syndrome as a novel generalized dysrhythmia, suggesting a new role for SGOL1 and the cohesin complex in mediating the integrity of human cardiac and gut rhythm.
Assuntos
Anormalidades Múltiplas/genética , Arritmias Cardíacas/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Enteropatias/genética , Contração Muscular/fisiologia , Transdução de Sinais/genética , Animais , Arritmias Cardíacas/patologia , Ciclo Celular/genética , Sistema Nervoso Entérico/patologia , Fibroblastos , Efeito Fundador , Trato Gastrointestinal/fisiopatologia , Técnicas de Silenciamento de Genes , Humanos , Enteropatias/fisiopatologia , Cariotipagem , Contração Muscular/genética , Músculo Liso Vascular/patologia , Mutação/genética , Quebeque , Síndrome , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra , CoesinasRESUMO
In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39-51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.
Assuntos
Miócitos de Músculo Liso/metabolismo , Proteínas de Ligação a RNA/química , Animais , Linhagem Celular , Células Cultivadas , Células HEK293 , Humanos , Leucina/química , Modelos Moleculares , Miócitos de Músculo Liso/citologia , Multimerização ProteicaRESUMO
Nonylphenol (NP) is an endocrine disruptor with harmful effects including feminization and carcinogenesis on various organisms. This substance is a degradation product of nonylphenol ethoxylates (NPEO) that is used in several industrial and agricultural processes. In this paper, we examined the assessment of NP exposure on chick embryo development, using a concentration consistent with the environmental concentrations of NP. With this aim, NP (between 0.1 and 50 µg/egg) was injected into the yolk of egg through a small needle hole in the shell. We report the effect of NP on chick reproductive system development although the effect we observed is lower than those observed by exposition to other endocrine disruptors. However, histological analysis highlighted a decrease of intraluminal seminiferous surface area in 64.12% of case (P=0.0086) and an heterogeneous organization of the renal tubules when 10 µg/egg were injected. Moreover, an impairment of liver development with an abnormal bile spillage was observed when higher concentration of NP was injected (50 µg/egg).
Assuntos
Aves/embriologia , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Sistema Urogenital/efeitos dos fármacos , Vísceras/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Embrião de Galinha , Disruptores Endócrinos/análise , Sistema Urogenital/embriologia , Vísceras/embriologia , Poluentes Químicos da Água/análiseRESUMO
Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.
Assuntos
Proteínas Aviárias/genética , Colo/metabolismo , Genes Homeobox , Moela das Aves/metabolismo , Fatores de Transcrição/genética , Animais , Proteínas Aviárias/metabolismo , Embrião de Galinha , Colo/citologia , Colo/embriologia , Mucosa Gástrica/embriologia , Mucosa Gástrica/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Moela das Aves/citologia , Moela das Aves/embriologia , Mucosa Intestinal/embriologia , Mucosa Intestinal/metabolismo , Miócitos de Músculo Liso/metabolismo , Especificidade de Órgãos , Piloro/citologia , Piloro/embriologia , Piloro/metabolismo , Reto/citologia , Reto/embriologia , Reto/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n=23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p<0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy.
Assuntos
Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Linhagem Celular Tumoral , Feminino , Trato Gastrointestinal/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/biossíntese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de SinaisRESUMO
BACKGROUND & AIMS: Gastrointestinal development requires regulated differentiation of visceral smooth muscle cells (SMCs) and their contractile activities; alterations in these processes might lead to gastrointestinal neuromuscular disorders. Gastrointestinal SMC development and remodeling involves post-transcriptional modification of messenger RNA. We investigated the function of the RNA-binding protein for multiple splicing 2 (RBPMS2) during normal development of visceral smooth muscle in chicken and expression of its transcript in human pathophysiological conditions. METHODS: We used avian replication-competent retroviral misexpression approaches to analyze the function of RBPMS2 in vivo and in primary cultures of chicken SMCs. We analyzed levels of RBPMS2 transcripts in colon samples from pediatric patients with Hirschsprung's disease and patients with chronic pseudo obstruction syndrome (CIPO) with megacystis. RESULTS: RBPMS2 was expressed strongly during the early stage of visceral SMC development and quickly down-regulated in differentiated and mature SMCs. Misexpression of RBPMS2 in differentiated visceral SMCs induced their dedifferentiation and reduced their contractility by up-regulating expression of Noggin, which reduced activity of bone morphogenetic protein. Visceral smooth muscles from pediatric patients with CIPO expressed high levels of RBPMS2 transcripts, compared with smooth muscle from patients without this disorder. CONCLUSIONS: Expression of RBPMS2 is present in visceral SMC precursors. Sustained expression of RBPMS2 inhibits the expression of markers of SMC differentiation by inhibiting bone morphogenetic protein activity, and stimulates SMC proliferation. RBPMS2 transcripts are up-regulated in patients with CIPO; alterations in RBPMS2 function might be involved in digestive motility disorders, particularly those characterized by the presence of muscular lesions (visceral myopathies).