RESUMEN
In 1999, The American Journal of Pathology published an article entitled "Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry," by Maniotis and colleagues, which ignited a spirited debate for several years and earned distinction as a citation classic. Tumor cell vasculogenic mimicry (VM) refers to the plasticity of aggressive cancer cells forming de novo vascular networks, which thereby contribute to perfusion of rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with the constitutional endothelial-lined vasculature. The tumor cells capable of VM share a plastic, transendothelial phenotype, which may be induced by hypoxia. Since VM was introduced as a novel paradigm for melanoma tumor perfusion, many studies have contributed new findings illuminating the underlying molecular pathways supporting VM in a variety of tumors, including carcinomas, sarcomas, glioblastomas, astrocytomas, and melanomas. Facilitating the functional plasticity of tumor cell VM are key proteins associated with vascular, stem cell, and hypoxia-related signaling pathways, each deserving serious consideration as potential therapeutic targets and diagnostic indicators of the aggressive, metastatic phenotype.
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Imitación Molecular , Neoplasias/irrigación sanguínea , Neoplasias/terapia , Investigación Biomédica Traslacional , Animales , Humanos , Metástasis de la Neoplasia , Neoplasias/patología , Células Madre Neoplásicas/patología , Transducción de Señal , Microambiente TumoralRESUMEN
INTRODUCTION: The re-emergence of the tumour growth factor-beta (TGF-beta)-related embryonic morphogen Nodal has recently been reported in several different human cancers. In this study, we examined the expression of Nodal in a series of benign and malignant human breast tissues to determine the clinical significance of this expression and whether Nodal could represent a potential therapeutic target in breast cancer. METHODS: Tissue sections from 431 therapeutically naive patients diagnosed with benign or malignant breast disease were stained for Nodal by immunohistochemistry and analysed in a blinded manner. The degree of Nodal staining was subsequently correlated with available clinical data, such as diagnoses and disease stage. These tissue findings were further explored in breast cancer cell lines MDA-MB-231 and MDA-MB-468 treated with a Nodal blocking antibody to determine biological effects for target validation. RESULTS: A variable degree of Nodal staining was detected in all samples. The intensity of Nodal staining was significantly greater in undifferentiated, advanced stage, invasive breast cancer compared with benign breast disease or early stage breast cancer. Treatment of human breast cancer cells in vitro with Nodal blocking antibody significantly reduced proliferation and colony-forming ability in soft agar, concomitant with increased apoptosis. CONCLUSIONS: These data suggest a potential role for Nodal as a biomarker for disease progression and a promising target for anti-Nodal therapy in breast cancer.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Proteína Nodal/metabolismo , Adulto , Anciano , Anticuerpos Bloqueadores/inmunología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Proteína Nodal/inmunología , PronósticoRESUMEN
BACKGROUND: FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos. RESULTS: We find that pharmacological inhibition of FGFR activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of E-cadherin. E-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. FGFR inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. This includes members of the FGF, NOTCH, EPH, PDGF, and canonical and non-canonical WNT pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. SNAI2 expression in the primitive streak and mesoderm is not altered by FGFR inhibition, but is downregulated only in the preingression epiblast region with no significant effect on E-cadherin. Furthermore, over expression of SNAIL has no discernable effect on E-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. FGFR activity modulates distinct downstream pathways including RAS/MAPK and PI3K/AKT. Pharmacological inhibition of MEK or AKT indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. FGFR activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including RHOA, the non-canonical WNT pathway, PDGF signalling and the cell adhesion protein N-cadherin. CONCLUSIONS: In chicken embryos, FGF signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in E-cadherin expression or protein localization. The positive and negative effects on large groups of genes by pharmacological inhibition of FGF signalling, including major signalling pathways and transcription factor families, indicates that the FGF pathway is a focal point of regulation during gastrulation in chicken.
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Cadherinas/genética , Movimiento Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Primitiva/metabolismo , Proteínas ras/metabolismo , Animales , Western Blotting , Cadherinas/metabolismo , Embrión de Pollo , Electroporación , Factores de Crecimiento de Fibroblastos/genética , Gastrulación , Expresión Génica , Hibridación in Situ , Análisis por Micromatrices , Proteínas Quinasas Activadas por Mitógenos/genética , Fosfatidilinositol 3-Quinasas/genética , Reacción en Cadena de la Polimerasa , Línea Primitiva/embriología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas ras/genéticaRESUMEN
As the frequency of melanoma diagnosis increases, current treatment strategies are still struggling to significantly impact patient survival. Some promise has been shown in treating certain melanomas by targeting activated signaling pathways resulting from specific mutations in proteins, such as BRAF and NRAS. Recently, the identification of embryonic signaling pathways in melanoma has helped us better understand certain biological characteristics, such as cellular heterogeneity and phenotypic plasticity, and has provided novel insight pertinent to diagnosis and therapy. For instance, our studies have shown that the TGF-ß family member, Nodal, is expressed in melanoma and is responsible, at least in part, for tumor cell plasticity and aggressiveness. Since the majority of normal adult tissues do not express Nodal, we reason that this embryonic morphogen could be used to identify and target aggressive melanoma cells. We have also identified that molecular cross-talk between the Notch and Nodal pathways may represent a mechanism responsible for the overexpression of Nodal in melanoma. Further exploitation of the relationship between embryonic signaling pathways and cancer pathogenesis could lead to novel approaches for diagnosis and therapy in cancers, such as melanoma.
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Embrión de Mamíferos/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/metabolismo , Proteína Nodal/metabolismo , Fenotipo , Transducción de Señal/fisiología , Línea Celular Tumoral , Embrión de Mamíferos/metabolismo , Humanos , Melanoma/diagnóstico , Melanoma/terapia , Modelos Biológicos , Receptores Notch/metabolismo , Células Madre/citologíaRESUMEN
Activation of the ErbB family of receptor tyrosine kinases via cognate Epidermal Growth Factor (EGF)-like peptide ligands constitutes a major group of related signaling pathways that control proliferation, survival, angiogenesis and metastasis of breast cancer. In this respect, clinical trials with various ErbB receptor blocking antibodies and specific tyrosine kinase inhibitors have proven to be partially efficacious in the treatment of this heterogeneous disease. Induction of an embryonic program of epithelial-to-mesenchymal transition (EMT) in breast cancer, whereupon epithelial tumor cells convert to a more mesenchymal-like phenotype, facilitates the migration, intravasation, and extravasation of tumor cells during metastasis. Breast cancers which exhibit properties of EMT are highly aggressive and resistant to therapy. Activation of ErbB signaling can regulate EMT-associated invasion and migration in normal and malignant mammary epithelial cells, as well as modulating discrete stages of mammary gland development. The purpose of this review is to summarize current information regarding the role of ErbB signaling in aspects of EMT that influence epithelial cell plasticity during mammary gland development and tumorigenesis. How this information may contribute to the improvement of therapeutic approaches in breast cancer will also be addressed.
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Neoplasias de la Mama/fisiopatología , Transdiferenciación Celular , Factor de Crecimiento Epidérmico/fisiología , Glándulas Mamarias Humanas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal/fisiología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Desdiferenciación Celular , Diferenciación Celular , Desarrollo Embrionario , Células Epiteliales/metabolismo , Femenino , Humanos , Ligandos , Glándulas Mamarias Animales/embriología , Glándulas Mamarias Animales/fisiología , Glándulas Mamarias Animales/fisiopatología , Glándulas Mamarias Humanas/embriología , Glándulas Mamarias Humanas/fisiopatología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/fisiopatología , Células Madre Mesenquimatosas/metabolismo , Invasividad Neoplásica , Metástasis de la NeoplasiaRESUMEN
Knowledge of the molecular mechanisms regulating cell ingression, epithelial-mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.
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Movimiento Celular/fisiología , Gastrulación , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Proteínas Aviares , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/química , Miocitos Cardíacos/química , Distribución TisularRESUMEN
Cardiac malformations constitute the most common birth defects, of which heart septal and valve defects are the most frequent forms diagnosed in infancy. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of hyaluronan (HA), cardiac jelly, and endocardial cushions, a loss of vascular integrity, and death at embryonic day 9.5. Despite the requirements for Has2 and its product, HA, in the developing heart, little is known about the normal processing and removal of HA during development. Cell culture studies show that HA obtains new bioactivity after depolymerization into small oligosaccharides. We previously showed reduction in Has2 expression and diminished presence of HA at later stages of heart development as tissue remodeling formed the leaflets of the cardiac valves. Here we show that small oligosaccharide forms of HA (o-HA) act antagonistically to developmental epithelial-to-mesenchymal transformation (EMT), which is required to generate the progenitor cells that populate the endocardial cushions. We further show that o-HA induces vascular endothelial growth factor (VEGF), which acts as a negative regulator of EMT. This is the first report illustrating a functional link between oligosaccharide HA and VEGF. Collectively, our data indicate that following endocardial cell EMT, native HA is likely processed to o-HA, which stimulates VEGF activity to attenuate cardiac developmental EMT.
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Inducción Embrionaria , Células Endoteliales/citología , Epitelio/embriología , Corazón/embriología , Ácido Hialurónico/farmacología , Mesodermo/citología , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Células Cultivadas , Embrión de Pollo , Embrión de Mamíferos , Regulación de la Expresión Génica , Corazón/crecimiento & desarrollo , Ácido Hialurónico/metabolismo , Ratones , Oligosacáridos/farmacologíaRESUMEN
UNLABELLED: Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. Despite a complete response in fewer than 5% of patients, the chemotherapeutic agent dacarbazine (DTIC) remains the reference drug after almost 40 years. More recently, FDA-approved drugs have shown promise but patient outcome remains modest, predominantly due to drug resistance. As such, combinatorial targeting has received increased attention, and will advance with the identification of new molecular targets. One attractive target for improving melanoma therapy is the growth factor Nodal, whose normal expression is largely restricted to embryonic development, but is reactivated in metastatic melanoma. In this study, we sought to determine how Nodal-positive human melanoma cells respond to DTIC treatment and to ascertain whether targeting Nodal in combination with DTIC would be more effective than monotherapy. A single treatment with DTIC inhibited cell growth but did not induce apoptosis. Rather than reducing Nodal expression, DTIC increased the size of the Nodal-positive subpopulation, an observation coincident with increased cellular invasion. Importantly, clinical tissue specimens from patients with melanomas refractory to DTIC therapy stained positive for Nodal expression, both in pre- and post-DTIC tumors, underscoring the value of targeting Nodal. In vitro, anti-Nodal antibodies alone had some adverse effects on proliferation and apoptosis, but combining DTIC treatment with anti-Nodal antibodies decreased cell growth and increased apoptosis synergistically, at concentrations incapable of producing meaningful effects as monotherapy. IMPLICATIONS: Targeting Nodal in combination with DTIC therapy holds promise for the treatment of metastatic melanoma.
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Antibióticos Antineoplásicos/farmacología , Dacarbazina/farmacología , Melanoma/tratamiento farmacológico , Proteína Nodal/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanoma/metabolismo , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Proteína Nodal/inmunología , Neoplasias Cutáneas/metabolismoRESUMEN
Cripto-1 (CR-1) protein function differs according to cellular or extracellular expression. In this study, we explore the significance of cell surface CR-1 expression in human melanoma cells. Cell surface CR-1-expressing human melanoma cells (CR1-CS+) were selected by fluorescence-activated cell sorting (FACS) and grown in vitro and in vivo in nude mice to study their growth characteristics. The CR1-CS+ melanoma cells were found to express increased levels of Oct4, MDR-1 and activated c-Src compared with cells lacking this subpopulation (CR1-CS-) or unsorted cells, used as control. CR1-CS+ show reduced proliferation rates and diminished spherical colony formation compared with control cells when cultured in vitro. Orthotopic injections of CR1-CS+ in nude mice formed slow growing tumors with histologic variability across different areas of the CR1-CS+ xenografts. CR-1-expressing cells from first generation CR1-CS+ tumors showed significantly increased tumor-forming rate and aggressiveness following subsequent transplants in nude mice. These data demonstrate that within a heterogeneous melanoma cell population there resides a slow proliferating, cell surface CR-1-expressing subpopulation capable of giving rise to a fast growing, aggressive progeny that may contribute to disease recurrence and progression.
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Proteínas Ligadas a GPI/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanoma/metabolismo , Melanoma/patología , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Ratones , Ratones Desnudos , Transducción de Señal/genéticaRESUMEN
Nodal is a TGF-ß-related embryonic morphogen that is expressed in multiple human cancers. Detection of Nodal expression in these tissues can be challenging if issues related to Nodal transcription and protein processing are not considered. Here, we discuss certain characteristics related to Nodal expression and function and how these can facilitate acquisition and interpretation of expression data, contributing to our understanding of the potential role of Nodal in human cancer. We also discuss how Nodal could be exploited clinically as a novel biomarker for cancer progression and therapeutic target.
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Neoplasias , Proteína Nodal/fisiología , HumanosRESUMEN
Tumor cell vasculogenic mimicry (VM) describes the functional plasticity of aggressive cancer cells forming de novo vascular networks, thereby providing a perfusion pathway for rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with endothelial-lined vasculature. The underlying induction of VM seems to be related to hypoxia, which may also promote the plastic, transendothelial phenotype of tumor cells capable of VM. Since its introduction in 1999 as a novel paradigm for melanoma tumor perfusion, many studies have contributed new insights into the underlying molecular pathways supporting VM in a variety of tumors, including melanoma, glioblastoma, carcinomas, and sarcomas. In particular, critical VM-modulating genes are associated with vascular (VE-cadherin, EphA2, VEGF receptor 1), embryonic and/or stem cell (Nodal, Notch4), and hypoxia-related (hypoxia-inducible factor, Twist1) signaling pathways. Each of these pathways warrants serious scrutiny as potential therapeutic, vascular targets, and diagnostic indicators of plasticity, drug resistance, and the aggressive metastatic phenotype.
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Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica , Transducción de Señal , Animales , Hipoxia de la Célula , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Melanoma/irrigación sanguínea , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Neoplasias/metabolismo , Neoplasias/terapiaRESUMEN
Metastatic melanoma is an aggressive skin cancer associated with poor prognosis. The reactivation of the embryonic morphogen Nodal in metastatic melanoma has previously been shown to regulate the aggressive behavior of these tumor cells. During the establishment of left-right asymmetry in early vertebrate development, Nodal expression is specifically regulated by a Notch signaling pathway. We hypothesize that a similar relationship between Notch and Nodal may be reestablished in melanoma. In this study, we investigate whether cross talk between the Notch and Nodal pathways can explain the reactivation of Nodal in aggressive metastatic melanoma cells. We show a molecular link between Notch and Nodal signaling in the aggressive melanoma cell line MV3 via the activity of an RBPJ-dependent Nodal enhancer element. We show a precise correlation between Notch4 and Nodal expression in multiple aggressive cell lines but not poorly aggressive cell lines. Surprisingly, Notch4 is specifically required for expression of Nodal in aggressive cells and plays a vital role both in the balance of cell growth and in the regulation of the aggressive phenotype. In addition, Notch4 function in vasculogenic mimicry and anchorage-independent growth in vitro is due in part to Notch4 regulation of Nodal. This study identifies an important role for cross talk between Notch4 and Nodal in metastatic melanoma, placing Notch4 upstream of Nodal, and offers a potential molecular target for melanoma therapy.
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Melanoma/metabolismo , Proteína Nodal/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Notch/metabolismo , Apoptosis/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Humanos , Melanoma/irrigación sanguínea , Melanoma/genética , Melanoma/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Proteína Nodal/biosíntesis , Proteína Nodal/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/biosíntesis , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Receptor Cross-Talk , Receptor Notch4 , Receptores Notch/antagonistas & inhibidores , Receptores Notch/biosíntesis , Transducción de Señal , TransfecciónRESUMEN
Aggressive tumor cells express a plastic, multipotent phenotype similar to embryonic stem cells. However, the absence of major regulatory checkpoints in these tumor cells allows aberrant activation of embryonic signaling pathways, which seems to contribute to their plastic phenotype. Emerging evidence showing the molecular cross-talk between two major stem cell signaling pathways Nodal and Notch suggests a promising therapeutic strategy that could target aggressive tumor cells on the basis of their unique plasticity, and provide new insights into the mechanisms underlying the re-emergence of developmental signaling pathways during tumor progression.
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Neoplasias/metabolismo , Neoplasias/patología , Proteína Nodal/metabolismo , Receptores Notch/metabolismo , Animales , Humanos , Transducción de SeñalRESUMEN
Myocardin, a serum response factor cofactor, plays an important role in regulating heart and smooth muscle development. To investigate myocardin function during early stages of heart development, we isolated the chicken orthologue of myocardin and characterized its expression between Hamburger and Hamilton stages 3 and 15. At stage 4, myocardin transcripts are detected in the lateral and extraembryonic mesoderm, become progressively localized to the precardiac mesoderm and the differentiated myocardium and are also seen in smooth muscle cells of the developing vascular plexus. Surprisingly, myocardin expression within the developing chicken embryo precedes that of the homeodomain transcription factor Nkx2.5. Embryonic dissection studies demonstrate that signals from the endoderm are required for myocardin expression within the precardiac mesoderm. However, unlike Nkx2.5, myocardin expression is not regulated by bone morphogenetic protein (BMP) signaling. These results suggest that initial expression of myocardin in the precardiac mesoderm is regulated by a signaling pathway that is parallel to, and independent of, Nkx2.5 expression.
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Proteínas Morfogenéticas Óseas/fisiología , Endodermo/metabolismo , Corazón/embriología , Proteínas Nucleares/genética , Transducción de Señal/fisiología , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas/genética , Células CHO , Proteínas Portadoras/genética , Embrión de Pollo , Cricetinae , Cricetulus , Endodermo/embriología , Hibridación in Situ , Mesodermo/embriología , Mesodermo/metabolismo , Datos de Secuencia Molecular , Desarrollo de Músculos/genética , Desarrollo de Músculos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
The 11th Annual Weinstein Cardiovascular Development Conference was held May 19-22, 2005 at the Westward Look Resort and Conference Center in Tucson, Arizona. The Westward Look was the site of the 6th Weinstein Meeting in 1999, and this year, 330 basic research scientists and research clinicians returned to Tucson for 3 days of meetings, Mariachis, and margaritas. The meeting was hosted by the cardiovascular research group at the University of Arizona and offered flavors of the desert southwest that included record temperatures, the Skopopelli conference logo modified from the Kokopelli of Native American mythology (Fig. 1), and liberal use of a cattle prod to encourage speaker timeliness.
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Sistema Cardiovascular/embriología , Animales , Distinciones y Premios , Sistema Cardiovascular/diagnóstico por imagen , Diferenciación Celular/fisiología , Vasos Coronarios/embriología , Femenino , Válvulas Cardíacas/embriología , Humanos , Masculino , Microscopía Confocal , Miocardio/citología , Miocardio/metabolismo , Cresta Neural/embriología , Cresta Neural/fisiología , Pericardio/embriología , RadiografíaRESUMEN
Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.