RESUMEN
The glucocorticoid receptor (GR) is an important anti-cancer target in lymphoid cancers but has been understudied in solid tumors like lung cancer, although glucocorticoids are often given with chemotherapy regimens to mitigate side effects. Here, we identify a dexamethasone-GR mediated anti-cancer response in a subset of aggressive non-small cell lung cancers (NSCLCs) that harbor Serine/Threonine Kinase 11 (STK11/LKB1) mutations. High tumor expression of carbamoyl phosphate synthase 1 (CPS1) was strongly linked to the presence of LKB1 mutations, was the best predictor of NSCLC dexamethasone (DEX) sensitivity (p < 10-16) but was not mechanistically involved in DEX sensitivity. Subcutaneous, orthotopic and metastatic NSCLC xenografts, biomarker-selected, STK11/LKB1 mutant patient derived xenografts, and genetically engineered mouse models with KRAS/LKB1 mutant lung adenocarcinomas all showed marked in vivo anti-tumor responses with the glucocorticoid dexamethasone as a single agent or in combination with cisplatin. Mechanistically, GR activation triggers G1/S cell cycle arrest in LKB1 mutant NSCLCs by inducing the expression of the cyclin-dependent kinase inhibitor, CDKN1C/p57(Kip2). All findings were confirmed with functional genomic experiments including CRISPR knockouts and exogenous expression. Importantly, DEX-GR mediated cell cycle arrest did not interfere with NSCLC radiotherapy, or platinum response in vitro or with platinum response in vivo. While DEX induced LKB1 mutant NSCLCs in vitro exhibit markers of cellular senescence and demonstrate impaired migration, in vivo DEX treatment of a patient derived xenograft (PDX) STK11/LKB1 mutant model resulted in expression of apoptosis markers. These findings identify a previously unknown GR mediated therapeutic vulnerability in STK11/LKB1 mutant NSCLCs caused by induction of p57(Kip2) expression with both STK11 mutation and high expression of CPS1 as precision medicine biomarkers of this vulnerability.
RESUMEN
RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Neoplasias Pulmonares/metabolismo , Complejos Multiproteicos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Replicación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Estructura Molecular , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Tolerancia a RadiaciónRESUMEN
Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral/metabolismo , Metaboloma/fisiología , Biomarcadores de Tumor/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Regulación Neoplásica de la Expresión Génica/fisiología , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Redes y Vías Metabólicas/genética , Metabolómica/métodos , Neoplasias/metabolismoRESUMEN
Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K-mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448-59. ©2017 AACR.
Asunto(s)
Carcinoma de Células Escamosas/terapia , Neoplasias Pulmonares/terapia , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Transfección , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Although non-small cell lung cancer (NSCLC) patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/uso terapéutico , Carboplatino/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/uso terapéutico , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Taxoides/uso terapéutico , Aminopiridinas/efectos adversos , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzazepinas/efectos adversos , Benzazepinas/farmacología , Benzazepinas/uso terapéutico , Hidrocarburos Aromáticos con Puentes/farmacología , Carboplatino/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Hidrazonas/efectos adversos , Hidrazonas/farmacología , Hidrazonas/uso terapéutico , Histona Demetilasas con Dominio de Jumonji/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metilación , Ratones , Terapia Neoadyuvante , Pirimidinas/efectos adversos , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Taxoides/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Globally, an estimated 13 million preterm babies are born each year. These babies are at increased risk of infant mortality and life-long health complications. Interventions to prevent preterm birth (PTB) require an understanding of processes driving parturition. Prostaglandins (PGs) have diverse functions in parturition, including regulation of uterine contractility and tissue remodeling. Our studies on cervical remodeling in mice suggest that although local synthesis of PGs are not increased in term ripening, transcripts encoding PG-endoperoxide synthase 2 (Ptgs2) are induced in lipopolysaccharide (LPS)-mediated premature ripening. This study provides evidence for two distinct pathways of cervical ripening: one dependent on PGs derived from paracrine or endocrine sources and the other independent of PG actions. Cervical PG levels are increased in LPS-treated mice, a model of infection-mediated PTB, consistent with increases in PG synthesizing enzymes and reduction in PG-metabolizing enzymes. Administration of SC-236, a PTGS2 inhibitor, along with LPS attenuated cervical softening, consistent with the essential role of PGs in LPS-induced ripening. In contrast, during term and preterm ripening mediated by the antiprogestin, mifepristone, cervical PG levels, and expression of PG synthetic and catabolic enzymes did not change in a manner that supports a role for PGs. These findings in mice, supported by correlative studies in women, suggest PGs do not regulate all aspects of the parturition process. Additionally, it suggests a need to refocus current strategies toward developing therapies for the prevention of PTB that target early, pathway-specific processes rather than focusing on common late end point mediators of PTB.
Asunto(s)
Maduración Cervical/metabolismo , Lipopolisacáridos/metabolismo , Progestinas/metabolismo , Prostaglandinas/metabolismo , Animales , Cuello del Útero/efectos de los fármacos , Femenino , Citometría de Flujo , Regulación de la Expresión Génica , Ratones , Mifepristona/farmacología , Misoprostol/farmacología , Trabajo de Parto Prematuro , Embarazo , Preñez , Nacimiento Prematuro , Pirazoles/química , Esteroides/metabolismo , Sulfonamidas/química , Nacimiento a TérminoRESUMEN
Multiple sclerosis (MS) is thought to be a CD4+ T cell mediated autoimmune demyelinating disease of the central nervous system (CNS) that is rarely diagnosed during infancy. Cellular and molecular mechanisms that confer disease resistance in this age group are unknown. We tested the hypothesis that a differential composition of immune cells within the CNS modulates age-associated susceptibility to CNS autoimmune disease. C57BL/6 mice younger than eight weeks were resistant to experimental autoimmune encephalomyelitis (EAE) following active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35-55. Neonates also developed milder EAE after transfer of adult encephalitogenic T cells primed by adult or neonate antigen presenting cells (APC). There was a significant increase in CD45+ hematopoietic immune cells and CD45+ high side scatter granulocytes in the CNS of adults, but not in neonates. Within the CD45+ immune cell compartment of adults, the accumulation of CD4+ T cells, Gr-1+ and Gr-1- monocytes and CD11c+ dendritic cells (DC) was identified. A significantly greater percentage of CD19+ B cells in the adult CNS expressed MHC II than neonate CNS B cells. Only in the adult CNS could IFNγ transcripts be detected 10 days post immunization for EAE. IFNγ is highly expressed by adult donor CD4+ T cells that are adoptively transferred but not by transferred neonate donor cells. In contrast, IL-17 transcripts could not be detected in adult or neonate CNS in this EAE model, and neither adult nor neonate donor CD4+ T cells expressed IL-17 at the time of adoptive transfer.
Asunto(s)
Linfocitos B/patología , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/patología , Células TH1/patología , Traslado Adoptivo , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Proliferación Celular , Citometría de Flujo , Genes MHC Clase II/genética , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Glicoproteína Mielina-Oligodendrócito/metabolismo , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T Colaboradores-Inductores/fisiologíaRESUMEN
OBJECTIVE: We sought to determine if endothelial microparticles (EMPs), markers of endothelial damage, are associated with soluble fms-like tyrosine kinase 1 (sFlt1), soluble endoglin, and placental growth factor (PlGF) in women with preeclampsia. STUDY DESIGN: A prospective cohort study was conducted on 20 preeclamptic women and 20 controls. EMPs by flow cytometry, sFlt1, soluble endoglin, and PlGF were measured at time of enrollment, 48-hours postpartum, and 1-week postpartum. RESULTS: Preeclamptic CD31(+)/42(-), CD62E(+), and CD105(+) EMP levels were significantly elevated in preeclamptics vs controls at time of enrollment. The sFlt1:PlGF ratio was correlated with CD31(+)/42(-) and CD105(+) EMPs (r = 0.69 and r = 0.51, respectively) in preeclampsia. Levels of CD31(+)/42(-) EMPs remained elevated 1-week postpartum (P = .026). CONCLUSION: EMPs are elevated in preeclampsia. The correlation of EMPs and the sFlt1:PlGF ratio suggests that antiangiogenesis is related to apoptosis of the endothelia. Endothelial damage persists 1 week after delivery.
Asunto(s)
Micropartículas Derivadas de Células/fisiología , Células Endoteliales/fisiología , Periodo Posparto/sangre , Preeclampsia/sangre , Adulto , Antígenos CD/sangre , Estudios de Casos y Controles , Endoglina , Femenino , Citometría de Flujo , Humanos , Factor de Crecimiento Placentario , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Embarazo , Proteínas Gestacionales/sangre , Estudios Prospectivos , Receptores de Superficie Celular/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangreRESUMEN
In the current study, the mechanisms of premature cervical ripening in murine models of preterm birth resulting from infection or early progesterone withdrawal were compared with the process of term cervical ripening. Tissue morphology, weight, gene expression, and collagen content along with immune cell populations were evaluated. Premature ripening induced by the progesterone receptor antagonist mifepristone results from an acceleration of processes in place during term ripening as well as partial activation of proinflammatory and immunosuppressive processes observed during postpartum repair. In contrast to term or mifepristone-induced preterm ripening, premature ripening induced in an infection model occurs by a distinct mechanism which is dominated by an influx of neutrophils into the cervix, a robust proinflammatory response and increased expression of prostaglandin-cyclooxygenase-endoperoxide synthase 2, important in prostaglandin biosynthesis. Key findings from this study confirm that cervical ripening can be initiated by more than one mechanism and is not necessarily an acceleration of the physiologic process at term. These results will influence current strategies for identifying specific etiologies of preterm birth and developing subsequent therapies.
Asunto(s)
Maduración Cervical/fisiología , Nacimiento Prematuro , Animales , Maduración Cervical/efectos de los fármacos , Cuello del Útero/patología , Femenino , Regulación de la Expresión Génica/fisiología , Antagonistas de Hormonas/farmacología , Lipopolisacáridos/toxicidad , Ratones , Mifepristona/farmacología , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Appropriate and timely cervical remodeling is key for successful birth. Premature cervical opening can result in preterm birth which occurs in 12.5% of pregnancies. Research focused on the mechanisms of term and preterm cervical remodeling is essential to prevent prematurity. This review highlights recent findings that better define molecular processes driving progressive disorganization of the cervical extracellular matrix. This includes studies that redefine the role of immune cells and identify diverse functions of the cervical epithelia and hyaluronan in remodeling. New investigations proposing that infection-induced premature cervical remodeling is distinct from the normal process are presented. Recent advances in our understanding of term and preterm cervical remodeling provide new directions for investigation and compel investigators to reevaluate currently accepted models.
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Maduración Cervical/fisiología , Cuello del Útero/fisiología , Primer Periodo del Trabajo de Parto/fisiología , Parto/fisiología , Embarazo/fisiología , Animales , Cuello del Útero/anatomía & histología , Femenino , HumanosRESUMEN
Preterm birth occurs at a rate of 12.7% in the U.S. and is the primary cause of fetal morbidity in the first year of life as well as the cause of later health problems. Elucidation of mechanisms controlling cervical remodeling is critical for development of therapies to reduce the incidence of prematurity. The cervical extracellular matrix must be disorganized during labor to allow birth, followed by a rapid repair postpartum. Leukocytes infiltrate the cervix before and after birth and are proposed to regulate matrix remodeling during cervical ripening via release of proteolytic enzymes. In the current study, flow cytometry and cell sorting were used to determine the role of immune cells in cervical matrix remodeling before, during, and after parturition. Markers of myeloid cell differentiation and activation were assessed to define phenotype and function. Tissue monocytes and eosinophils increased in the cervix before birth in a progesterone-regulated fashion, whereas macrophage numbers were unchanged. Neutrophils increased in the postpartum period. Increased mRNA expression of Csfr1 and markers of alternatively activated M2 macrophages during labor or shortly postpartum suggest a function of M2 macrophages in postpartum tissue repair. Changes in cervical myeloid cell numbers are not reflected in the peripheral blood. These data along with our previous studies suggest that myeloid-derived cells do not orchestrate processes required for initiation of cervical ripening before birth. Additionally, macrophages with diverse phenotypes (M1 and M2) are present in the cervix and are most likely involved in the postpartum repair of tissue.
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Cuello del Útero/citología , Cuello del Útero/inmunología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Parto/inmunología , Parto/metabolismo , Animales , Animales Recién Nacidos , Movimiento Celular/inmunología , Cuello del Útero/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Femenino , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Recuento de Leucocitos , Activación de Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Monocitos/inmunología , Monocitos/metabolismo , Células Mieloides/citología , Embarazo , Factores de Tiempo , Cicatrización de Heridas/inmunologíaRESUMEN
Cervical remodeling during pregnancy and parturition is a single progressive process that can be loosely divided into four overlapping phases termed softening, ripening, dilation/labor, and post partum repair. Elucidating the molecular mechanisms that facilitate all phases of cervical remodeling is critical for an understanding of parturition and for identifying processes that are misregulated in preterm labor, a significant cause of perinatal morbidity. In the present study, biomechanical measurements indicate that softening was initiated between gestation days 10 and 12 of mouse pregnancy, and in contrast to cervical ripening on day 18, the softened cervix maintains tissue strength. Although preceded by increased collagen solubility, cervical softening is not characterized by significant increases in cell proliferation, tissue hydration or changes in the distribution of inflammatory cells. Gene expression studies reveal a potentially important role of cervical epithelia during softening and ripening in maintenance of an immunomucosal barrier that protects the stromal compartment during matrix remodeling. Expression of two genes involved in repair and protection of the epithelial permeability barrier in the gut (trefoil factor 1) and skin (serine protease inhibitor Kazal type 5) were increased during softening and/or ripening. Another gene whose function remains to be elucidated, purkinje cell protein 4, declines in expression as remodeling progressed. Collectively, these results indicate that cervical softening during pregnancy is a unique phase of the tissue remodeling process characterized by increased collagen solubility, maintenance of tissue strength, and upregulation of genes involved in mucosal protection.
Asunto(s)
Cuello del Útero/fisiología , Regulación de la Expresión Génica , Preñez/fisiología , Animales , Fenómenos Biomecánicos , Cuello del Útero/inmunología , Colágeno/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Edad Gestacional , Inmunohistoquímica , Leucocitos/citología , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resistencia a la TracciónRESUMEN
Cervical epithelia have numerous functions that include proliferation, differentiation, maintenance of fluid balance, protection from environmental hazards, and paracellular transport of solutes via tight junctions (TJs). Epithelial functions must be tightly regulated during pregnancy and parturition as the cervix undergoes extensive growth and remodeling. This study evaluated TJ proteins, as well as markers of epithelial cell differentiation in normal and cervical ripening defective mice to gain insights into how the permeability barrier is regulated during pregnancy and parturition. Although numerous TJ proteins are expressed in the nonpregnant cervix, claudins 1 and 2 are temporally regulated in pregnancy. Claudin 1 mRNA expression is increased, whereas claudin 2 expression declines. The cellular localization of claudin 1 shifts at the end of pregnancy (gestation d 18.75) to the plasma membrane in a lattice pattern, consistent with TJs in the apical cells. The timing of claudin 1-enriched TJs coincides with initiation of terminal differentiation of cervical squamous epithelia as evidenced by the increased expression of genes by differentiated epithelia late on gestation d 18. The cervical ripening defective steroid 5alpha-reductase type 1 deficient mouse, which has an elevated local progesterone concentration, also has aberrant claudin 1 and 2 expressions, fails to form claudin 1-enriched TJs, and lacks normal expression of genes involved in epithelial terminal differentiation. These data suggest that changes in permeability barrier properties during cervical ripening are, in part, negatively regulated by progesterone, and that dynamic changes in barrier properties of the cervix occur during pregnancy and parturition.
Asunto(s)
Diferenciación Celular , Maduración Cervical/fisiología , Cuello del Útero/citología , Células Epiteliales/citología , Parto/fisiología , Preñez , Uniones Estrechas/química , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Animales , Maduración Cervical/metabolismo , Cuello del Útero/metabolismo , Claudina-1 , Claudina-4 , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas/genética , Parto/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Embarazo , Uniones Estrechas/ultraestructura , Proteína de la Zonula Occludens-1RESUMEN
A greater understanding of the processes that regulate cervical remodeling during pregnancy, parturition, and the postpartum period is required to understand causes of preterm and posterm birth in which abnormal cervical function is the primary culprit. In the current study, gene expression patterns unique to cervical ripening as compared with cervical dilation and/or postpartum repair are identified in a mouse model. Genes differentially regulated from gestation day 15 to late day 18 reveal processes important for cervical ripening. Genes differentially regulated from late day 18 to 2 hours after birth reveal processes that could be important during cervical dilation or the postpartum recovery period. Based on expression patterns, cervical ripening requires a downregulation of collagen assembly genes; increased synthesis of glycosaminoglycans that disrupt the matrix, such as hyaluronan; increased metabolism of progesterone; and changes in epithelial barrier properties. The latter phases of dilation and postpartum recovery are associated with increased assembly of mature collagen, synthesis of matrix proteins that promote a dense connective tissue, activation of inflammatory responses, prostaglandin synthesis, and further changes in epithelial barrier properties and differentiation. Processes/gene expression required for cervical ripening are distinct from those important in latter phases of cervical remodeling and highlight the importance of timing of tissue collection for understanding the molecular mechanisms of cervical ripening.
Asunto(s)
Maduración Cervical/genética , Regulación de la Expresión Génica , Primer Periodo del Trabajo de Parto/genética , Periodo Posparto/genética , Preñez/genética , Cicatrización de Heridas/genética , Animales , Maduración Cervical/metabolismo , Matriz Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Edad Gestacional , Humanos , Primer Periodo del Trabajo de Parto/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Periodo Posparto/metabolismo , Embarazo , Preñez/metabolismo , Factores de TiempoRESUMEN
The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1-/-), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1-/- mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, and after birth. The effect of neutrophil depletion on parturition was tested. Tissue macrophages, myeloperoxidase activity, and expression of proinflammatory molecules are not increased within the cervix until after birth. Neutrophil numbers do not change after birth and neutrophil depletion before term has no effect on timing or success of parturition. These results suggest that cervical ripening does not require neutrophils. Moreover, neutrophil activation and a general inflammatory response are not initiated within the cervix until shortly after parturition. The timing of inflammatory cell migration and activation in pregnant cervix suggest a role for these cells in postpartum remodeling of the cervix rather than in the initiation of cervical ripening at parturition.
Asunto(s)
Biomarcadores/metabolismo , Maduración Cervical/fisiología , Inflamación/metabolismo , Activación Neutrófila , Neutrófilos/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Animales , Cuello del Útero/citología , Cuello del Útero/metabolismo , Eosinófilos/citología , Eosinófilos/fisiología , Membranas Extraembrionarias/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Macrófagos/fisiología , Ratones , Ratones Endogámicos , Ratones Mutantes , Peroxidasa/metabolismo , Periodo Posparto , EmbarazoRESUMEN
The molecular mechanisms controlling the initiation of parturition remain largely undefined. We report a new animal model in which parturition does not occur. A line of mice expressing a human apolipoprotein B (APOB) gene fail to deliver their young if the transgene is present in homozygous (Tg/Tg), but not in heterozygous (Tg/Wt), form. Cloning and mapping of the transgene insertion locus indicate that 10 copies of the 80-kilobase APOB genomic fragment inserted into mouse chromosome 6 result in a small, 390-base pair deletion of mouse genomic DNA. Nine other lines expressing the transgene have normal labor, suggesting that transgene insertion in this mutant line disrupted a mouse gene crucial for successful parturition. The pathophysiology of parturition failure in these animals was defined using physiological, endocrinological, and morphological techniques. Results indicate that luteolysis occurs in Tg/Tg mice but is delayed by 1 day. Delivery did not occur in mutant mice at term after spontaneous luteolysis or even after removal of progesterone action by ovariectomy or antiprogestin treatment. Biomechanical and functional studies of the uterus and cervix revealed that the primary cause of failed parturition in Tg/Tg mice was not inadequate uterine contractions of labor but, rather, a rigid, inelastic cervix at term that was abnormally rich in neutrophils and tissue monocytes. Characterization of the transgene insertional mutant, Tg/Tg, indicates that progesterone withdrawal is insufficient to complete parturition in the presence of inadequate cervical ripening at term.
Asunto(s)
Cuello del Útero/fisiopatología , Cromosomas , Ratones Transgénicos/genética , Parto/genética , 20-alfa-Dihidroprogesterona/metabolismo , Animales , Cuello del Útero/metabolismo , Femenino , Perfilación de la Expresión Génica , Homocigoto , Ratones , Oxitocina/metabolismo , Parto/fisiología , Embarazo , Progesterona/metabolismo , Transducción de Señal , Transcripción Genética , Contracción Uterina/genéticaRESUMEN
A high-virulence clone (HVC) was proposed as causing much of the morbidity and mortality when a collection of group B Streptococcus (GBS) isolates was examined by multi-locus enzyme electrophoresis. HVC isolates could be further distinguished by their inability to grow at 40 degrees C, and a temperature-sensitive aldolase was identified as responsible for this characteristic. In the present study, the HVC was sought in a collection of 57 GBS isolates by hybridization with a probe containing a putative aldolase gene on genomic DNA restriction enzyme digests. Isolates were initially classified as HVC or non-HVC by their inability to grow at 40 degrees C. Three serotype III invasive isolates had the HVC control restriction/hybridization pattern. They were also unable to grow at 40 degrees C. The remaining 11 invasive and all carrier isolates showed a pattern identical to that of the non-HVC control. These results provide additional support for the existence of a highly virulent clonal group among serotype III isolates and suggest that hybridization with a probe containing the aldolase gene on DNA restriction enzyme digests can be an alternative method for identifying highly virulent isolates.