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Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development.
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Enfermedad de la Válvula Aórtica Bicúspide , Enfermedades de las Válvulas Cardíacas , Humanos , Ratones , Animales , Enfermedades de las Válvulas Cardíacas/genética , Válvula Aórtica/anomalías , Enfermedad de la Válvula Aórtica Bicúspide/metabolismo , Perfilación de la Expresión Génica , Transcriptoma/genéticaRESUMEN
G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium "paradox" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled ß2-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.
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Músculo Liso , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Músculo Liso/metabolismo , Músculo Liso/efectos de los fármacos , Fosforilación , Relajación Muscular/efectos de los fármacos , Histamina/metabolismo , Histamina/farmacología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Isoproterenol/farmacología , Calcio/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Gusto/fisiología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Transducción de Señal , Células CultivadasRESUMEN
Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes, dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes, and RNA sequencing shows significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Mitocondrias/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Reproducción/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP , Animales , Transporte Biológico/fisiología , Caenorhabditis elegans/metabolismo , Hemo/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
Finasteride is commonly prescribed to treat benign prostate hyperplasia and male-pattern baldness in cis men and, more recently, trans individuals. However, the effect of finasteride on cardiovascular disease remains elusive. We evaluated the role of finasteride on atherosclerosis using low-density lipoprotein (LDL) receptor-deficient (Ldlr-/-) mice. Next, we examined the relevance to humans by analyzing the data deposited between 2009 and 2016 in the National Health and Nutrition Examination Survey. We show that finasteride reduces total plasma cholesterol and delays the development of atherosclerosis in Ldlr-/- mice. Finasteride reduced monocytosis, monocyte recruitment to the lesion, macrophage lesion content, and necrotic core area, the latter of which is an indicator of plaque vulnerability in humans. RNA sequencing analysis revealed a downregulation of inflammatory pathways and an upregulation of bile acid metabolism, oxidative phosphorylation, and cholesterol pathways in the liver of mice taking finasteride. Men reporting the use of finasteride showed lower plasma levels of cholesterol and LDL-cholesterol than those not taking the drug. Our data unveil finasteride as a potential treatment to delay cardiovascular disease in people by improving the plasma lipid profile.
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Aterosclerosis , Enfermedades Cardiovasculares , Humanos , Masculino , Animales , Ratones , Finasterida/farmacología , Finasterida/uso terapéutico , Encuestas Nutricionales , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/metabolismo , Colesterol/metabolismo , Receptores de LDL/genética , Ratones NoqueadosRESUMEN
Retinoid X receptors (RXRs) are nuclear transcription factors that partner with other nuclear receptors to regulate numerous physiological processes. Although RXR represents a valid therapeutic target, only a few RXR-specific ligands (rexinoids) have been identified, in part due to the lack of clarity on how rexinoids selectively modulate RXR response. Previously, we showed that rexinoid UAB30 potentiates all-trans-retinoic acid (ATRA) signaling in human keratinocytes, in part by stimulating ATRA biosynthesis. Here, we examined the mechanism of action of next-generation rexinoids UAB110 and UAB111 that are more potent in vitro than UAB30 and the FDA-approved Targretin. Both UAB110 and UAB111 enhanced ATRA signaling in human organotypic epithelium at a 50-fold lower concentration than UAB30. This was consistent with the 2- to 5- fold greater increase in ATRA in organotypic epidermis treated with UAB110/111 versus UAB30. Furthermore, at 0.2 µM, UAB110/111 increased the expression of ATRA genes up to 16-fold stronger than Targretin. The less toxic and more potent UAB110 also induced more changes in differential gene expression than Targretin. Additionally, our hydrogen deuterium exchange mass spectrometry analysis showed that both ligands reduced the dynamics of the ligand-binding pocket but also induced unique dynamic responses that were indicative of higher affinity binding relative to UAB30, especially for Helix 3. UAB110 binding also showed increased dynamics towards the dimer interface through the Helix 8 and Helix 9 regions. These data suggest that UAB110 and UAB111 are potent activators of RXR-RAR signaling pathways but accomplish activation through different molecular responses to ligand binding.
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Tetrahidronaftalenos , Tretinoina , Humanos , Receptores X Retinoide/metabolismo , Bexaroteno , Ligandos , Tetrahidronaftalenos/farmacología , Tretinoina/farmacología , Tretinoina/metabolismo , Epidermis/metabolismoRESUMEN
All paired sensory organs arise from a common precursor domain called the pre-placodal region (PPR). In Xenopus, Zic1 non-cell autonomously regulates PPR formation by activating retinoic acid (RA) production. Here, we have identified two Zic1 targets, the RA catabolizing enzyme Cyp26c1 and the transcription factor Pitx2c, expressed in the vicinity of the PPR as being crucially required for maintaining low RA levels in a spatially restricted, PPR-adjacent domain. Morpholino- or CRISPR/Cas9-mediated Cyp26c1 knockdown abrogated PPR gene expression, yielding defective cranial placodes. Direct measurement of RA levels revealed that this is mediated by a mechanism involving excess RA accumulation. Furthermore, we show that pitx2c is activated by RA and required for Cyp26c1 expression in a domain-specific manner through induction of FGF8. We propose that Zic1 anteriorly establishes a program of RA containment and regulation through activation of Cyp26c1 and Pitx2c that cooperates to promote PPR specification in a spatially restricted domain.
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Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Homeodominio/metabolismo , Organogénesis , Factores de Transcripción/metabolismo , Tretinoina/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Tipificación del Cuerpo/genética , Sistema Enzimático del Citocromo P-450/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Inmunohistoquímica , Modelos Biológicos , Organogénesis/genética , Fenotipo , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Entering pregnancy with a history of adversity, including adverse childhood experiences and racial discrimination stress, is a predictor of negative maternal and fetal health outcomes. Little is known about the biological mechanisms by which preconception adverse experiences are stored and impact future offspring health outcomes. In our maternal preconception stress (MPS) model, female mice underwent chronic stress from postnatal days 28-70 and were mated 2 weeks post-stress. Maternal preconception stress dams blunted the pregnancy-induced shift in the circulating extracellular vesicle proteome and reduced glucose tolerance at mid-gestation, suggesting a shift in pregnancy adaptation. To investigate MPS effects at the maternal:fetal interface, we probed the mid-gestation placental, uterine, and fetal brain tissue transcriptome. Male and female placentas differentially regulated expression of genes involved in growth and metabolic signaling in response to gestation in an MPS dam. We also report novel offspring sex- and MPS-specific responses in the uterine tissue apposing these placentas. In the fetal compartment, MPS female offspring reduced expression of neurodevelopmental genes. Using a ribosome-tagging transgenic approach we detected a dramatic increase in genes involved in chromatin regulation in a PVN-enriched neuronal population in females at PN21. While MPS had an additive effect on high-fat-diet (HFD)-induced weight gain in male offspring, both MPS and HFD were necessary to induce significant weight gain in female offspring. These data highlight the preconception period as a determinant of maternal health in pregnancy and provides novel insights into mechanisms by which maternal stress history impacts offspring developmental programming.
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Placenta , Aumento de Peso , Humanos , Embarazo , Ratones , Femenino , Masculino , Animales , Placenta/metabolismo , Feto/metabolismo , Transducción de Señal , Dieta Alta en Grasa/efectos adversosRESUMEN
Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.
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Meninges/metabolismo , Neurogénesis , Neuronas/citología , Tretinoina/metabolismo , Animales , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Técnicas In Vitro , Ratones , Prosencéfalo/citología , Prosencéfalo/metabolismoRESUMEN
OBJECTIVE: Electronic cigarette (e-cigarette) use among adults in the United States continues to rise. Particularly concerning is the impact of e-cigarette aerosol inhalation on the oral mucosa. Aerosols are derived from a heated e-liquid base of propylene glycol/glycerin (PG/G) often mixed with nicotine and chemical flavors. Of note, harmful and potentially harmful constituents (HPHCs), including metals and volatile organic compounds, have been detected in e-cigarette aerosols. It remains unknown, however, whether aerosols exclusively derived from e-liquid PG/G are detrimental to oral keratinocytes. The present study analyzed toxicological outcomes in normal oral keratinocytes exposed to model nicotine-free, unflavored PG/G e-liquid aerosols. MATERIALS AND METHODS: Cell viability/cytotoxicity, genotoxicity, and immunoblotting assays were conducted in NOKSI, a gingiva-derived oral keratinocyte cell line, following exposure to model e-liquid aerosols or non-aerosolized controls. The HPHC acrolein, reported to form DNA adducts in the buccal mucosa from e-cigarette users, was also used in similar assays. RESULTS: PG/G e-liquid aerosol extracts significantly enhanced cytotoxic and DNA damaging responses in NOKSI cells when compared to non-aerosolized e-liquid treatment. Acrolein treatment led to similar results. CONCLUSIONS: The aerosolization process of PG/G e-liquid is a critical determinant of marked cytotoxic and genotoxic stimuli in oral keratinocytes.
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The gasotransmitter hydrogen sulfide (H2S) is thought to be involved in the post-translational modification of cysteine residues to produce reactive persulfides. A persulfide-specific chemoselective proteomics approach with mammalian cells has identified a broad range of zinc finger (ZF) proteins as targets of persulfidation. Parallel studies with isolated ZFs show that persulfidation is mediated by ZnII, O2, and H2S, with intermediates involving oxygen- and sulfur-based radicals detected by mass spectrometry and optical spectroscopies. A small molecule ZnII complex exhibits analogous reactivity with H2S and O2, giving a persulfidated product. These data show that ZnII is not just a biological structural element, but also plays a critical role in mediating H2S-dependent persulfidation. ZF persulfidation appears to be a general post-translational modification and a possible conduit for H2S signaling. This work has implications for our understanding of H2S-mediated signaling and the regulation of ZFs in cellular physiology and development.
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Sulfuro de Hidrógeno , Proteómica , Sulfuros , Dedos de Zinc , Zinc , Sulfuro de Hidrógeno/química , Sulfuro de Hidrógeno/metabolismo , Zinc/química , Humanos , Sulfuros/química , Procesamiento Proteico-PostraduccionalRESUMEN
Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.
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ERK1/2 (extracellular signal-regulated kinases 1 and 2) regulate the activity of various transcription factors that contribute to asthma pathogenesis. Although an attractive drug target, broadly inhibiting ERK1/2 is challenging because of unwanted cellular toxicities. We have identified small molecule inhibitors with a benzenesulfonate scaffold that selectively inhibit ERK1/2-mediated activation of AP-1 (activator protein-1). Herein, we describe the findings of targeting ERK1/2-mediated substrate-specific signaling with the small molecule inhibitor SF-3-030 in a murine model of house dust mite (HDM)-induced asthma. In 8- to 10-week-old BALB/c mice, allergic asthma was established by repeated intranasal HDM (25 µg/mouse) instillation for 3 weeks (5 days/week). A subgroup of mice was prophylactically dosed with 10 mg/kg SF-3-030/DMSO intranasally 30 minutes before the HDM challenge. Following the dosing schedule, mice were evaluated for alterations in airway mechanics, inflammation, and markers of airway remodeling. SF-3-030 treatment significantly attenuated HDM-induced elevation of distinct inflammatory cell types and cytokine concentrations in BAL and IgE concentrations in the lungs. Histopathological analysis of lung tissue sections revealed diminished HDM-induced pleocellular peribronchial inflammation, mucus cell metaplasia, collagen accumulation, thickening of airway smooth muscle mass, and expression of markers of cell proliferation (Ki-67 and cyclin D1) in mice treated with SF-3-030. Furthermore, SF-3-030 treatment attenuated HDM-induced airway hyperresponsiveness in mice. Finally, mechanistic studies using transcriptome and proteome analyses suggest inhibition of HDM-induced genes involved in inflammation, cell proliferation, and tissue remodeling by SF-3-030. These preclinical findings demonstrate that function-selective inhibition of ERK1/2 signaling mitigates multiple features of asthma in a murine model.
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Asma , Animales , Ratones , Asma/metabolismo , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inflamación/metabolismo , Pulmón/patología , Ratones Endogámicos BALB C , PyroglyphidaeRESUMEN
The main active metabolite of Vitamin A, all-trans retinoic acid (RA), is required for proper cellular function and tissue organization. Heart development has a well-defined requirement for RA, but there is limited research on the role of RA in the adult heart. Homeostasis of RA includes regulation of membrane receptors, chaperones, enzymes, and nuclear receptors. Cellular retinol-binding protein, type 1 (CRBP1), encoded by retinol-binding protein, type 1 (Rbp1), regulates RA homeostasis by delivering vitamin A to enzymes for RA synthesis and protecting it from non-specific oxidation. In this work, a multi-omics approach was used to characterize the effect of CRBP1 loss using the Rbp1-/- mouse. Retinoid homeostasis was disrupted in Rbp1-/- mouse heart tissue, as seen by a 33% and 24% decrease in RA levels in the left and right ventricles, respectively, compared to wild-type mice (WT). To further inform on the effect of disrupted RA homeostasis, we conducted high-throughput targeted metabolomics. A total of 222 metabolite and metabolite combinations were analyzed, with 33 having differential abundance between Rbp1-/- and WT hearts. Additionally, we performed global proteome profiling to further characterize the impact of CRBP1 loss in adult mouse hearts. More than 2606 unique proteins were identified, with 340 proteins having differential expression between Rbp1-/- and WT hearts. Pathway analysis performed on metabolomic and proteomic data revealed pathways related to cellular metabolism and cardiac metabolism were the most disrupted in Rbp1-/- mice. Together, these studies characterize the effect of CRBP1 loss and reduced RA in the adult heart.
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Retinoides , Vitamina A , Animales , Homeostasis , Ratones , Proteómica , Retinoides/metabolismo , Proteínas de Unión al Retinol , Proteínas Celulares de Unión al Retinol/genética , Proteínas Celulares de Unión al Retinol/metabolismo , Tretinoina/metabolismo , Vitamina A/metabolismoRESUMEN
TLR7 (Toll-like receptor 7), the sensor for single-stranded RNA, contributes to systemic inflammation and mortality in murine polymicrobial sepsis. Recent studies show that extracellular miR-146a-5p serves as a TLR7 ligand and plays an important role in regulating host innate immunity. However, the role of miR-146a-5p and TLR7 signaling in pulmonary inflammation, endothelial activation, and sepsis-associated acute respiratory distress syndrome remains unclear. Here, we show that intratracheal administration of exogenous miR-146a-5p in mice evokes lung inflammation, activates endothelium, and increases endothelial permeability via TLR7-dependent mechanisms. TLR7 deficiency attenuates pulmonary barrier dysfunction and reduces lung inflammatory response in a murine sepsis model. Moreover, the impact of miR-146a-5p-TLR7 signaling on endothelial activation appears to be a secondary effect because TLR7 is undetectable in the human pulmonary artery and microvascular endothelial cells (ECs), which show no response to direct miR-146a-5p treatment in vitro. Both conditioned media of miR-146a-5p-treated macrophages (MÏ) and septic sera of wild-type mice induce a marked EC barrier disruption in vitro, whereas MÏ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the MÏ conditioned media and septic sera identify TNFα (tumor necrosis factor α) as the main downstream effector of miR-146a-5p-TLR7 signaling responsible for the EC barrier dysfunction, which is further supported by neutralizing anti-TNFα antibody intervention. Together, these data demonstrate that TLR7 activation elicits pulmonary inflammation and endothelial barrier disruption by sensing extracellular miR-146a-5p and contributes to sepsis-associated acute respiratory distress syndrome.
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Glicoproteínas de Membrana , MicroARNs , Síndrome de Dificultad Respiratoria , Sepsis , Receptor Toll-Like 7 , Animales , Medios de Cultivo Condicionados , Células Endoteliales/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Síndrome de Dificultad Respiratoria/inmunología , Sepsis/complicaciones , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismoRESUMEN
In mammals, all-trans retinoic acid (ATRA) is instrumental to spermatogenesis. It is synthesized by two retinaldehyde dehydrogenases (RALDH) present in both Sertoli cells (SCs) and germ cells (GCs). In order to determine the relative contributions of each source of ATRA, we have generated mice lacking all RALDH activities in the seminiferous epithelium (SE). We show that both the SC- and GC-derived sources of ATRA cooperate to initiate and propagate spermatogenetic waves at puberty. In adults, they exert redundant functions and, against all expectations, the GC-derived source does not perform any specific roles despite contributing to two-thirds of the total amount of ATRA present in the testis. The production from SCs is sufficient to maintain the periodic expression of genes in SCs, as well and the cycle and wave of the SE, which account for the steady production of spermatozoa. The production from SCs is also specifically required for spermiation. Importantly, our study shows that spermatogonia differentiation depends upon the ATRA synthesized by RALDH inside the SE, whereas initiation of meiosis and expression of STRA8 by spermatocytes can occur without ATRA.
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Epitelio Seminífero/metabolismo , Células de Sertoli/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Tretinoina/metabolismo , Animales , Femenino , Masculino , Meiosis/fisiología , Ratones , Ratones Transgénicos , Epitelio Seminífero/citología , Células de Sertoli/citología , Espermatocitos/citología , Espermatogonias/citologíaRESUMEN
Cancer stem cells (CSCs) virtually present in all tumors albeit in small numbers are primarily responsible for driving cancer progression, metastasis, drug resistance, and recurrence. Prostate cancer (PCa) is the second most frequent cancer in men worldwide, and castration resistant prostate cancer (CRPC) remains a major challenge despite the tremendous advancements in medicine. Currently, none of the available treatment options are effective in treating CRPC. We earlier reported that VNPP433-3ß, the lead next-generation galeterone analog is effective in treating preclinical in vivo models of CRPC. In this study using RNA-seq, cytological, and biochemical methods, we report that VNPP433-3ß inhibits prostate CSCs by targeting key pathways critical to stemness and epithelial-mesenchymal transition. VNPP433-3ß inhibits CSCs in PCa, presumably by degrading the androgen receptor (AR) thereby decreasing the AR-mediated transcription of several stem cell markers including BMI1 and KLF4. Transcriptome analyses by RNA-seq, Ingenuity Pathway Analysis, and Gene Set Enrichment Analysis demonstrate that VNPP433-3ß inhibits transcription of several genes and functional pathways critical to the prostate CSCs thereby inhibiting CSCs in PCa besides targeting the bulk of the tumor.
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Neoplasias de la Próstata Resistentes a la Castración , Androstadienos , Bencimidazoles , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Perfilación de la Expresión Génica , Humanos , Masculino , Células Madre Neoplásicas/patología , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Vitamin A deficiency has been shown to exacerbate allergic asthma. Previous studies have postulated that retinoic acid (RA), an active metabolite of vitamin A and high-affinity ligand for RA receptor (RAR), is reduced in airway inflammatory condition and contributes to multiple features of asthma including airway hyperresponsiveness and excessive accumulation of airway smooth muscle (ASM) cells. In this study, we directly quantified RA and examined the molecular basis for reduced RA levels and RA-mediated signaling in lungs and ASM cells obtained from asthmatic donors and in lungs from allergen-challenged mice. Levels of RA and retinol were significantly lower in lung tissues from asthmatic donors and house dust mite (HDM)-challenged mice compared to non-asthmatic human lungs and PBS-challenged mice, respectively. Quantification of mRNA and protein expression revealed dysregulation in the first step of RA biosynthesis consistent with reduced RA including decreased protein expression of retinol dehydrogenase (RDH)-10 and increased protein expression of RDH11 and dehydrogenase/reductase (DHRS)-4 in asthmatic lung. Proteomic profiling of non-asthmatic and asthmatic lungs also showed significant changes in the protein expression of AP-1 targets consistent with increased AP-1 activity. Further, basal RA levels and RA biosynthetic capabilities were decreased in asthmatic human ASM cells. Treatment of human ASM cells with all-trans RA (ATRA) or the RARγ-specific agonist (CD1530) resulted in the inhibition of mitogen-induced cell proliferation and AP-1-dependent transcription. These data suggest that RA metabolism is decreased in asthmatic lung and that enhancing RAR signaling using ATRA or RARγ agonists may mitigate airway remodeling associated with asthma.
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Remodelación de las Vías Aéreas (Respiratorias) , Asma/patología , Hipersensibilidad Respiratoria/patología , Tretinoina/metabolismo , Adulto , Alérgenos/toxicidad , Animales , Asma/etiología , Asma/metabolismo , Estudios de Casos y Controles , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Receptores de Ácido Retinoico/agonistas , Hipersensibilidad Respiratoria/etiología , Hipersensibilidad Respiratoria/metabolismo , Receptor de Ácido Retinoico gammaRESUMEN
BACKGROUND: The serotonin transporter (SERT) mRNA was previously reported to be a quantitative and pathophysiology-based biomarker of heavy drinking in 5HTTLPR:LL genotype-carriers treated with ondansetron. Here, we validated the potential use of SERT mRNA for quantitative prediction of recent alcohol consumption (in the absence of treatment) and compared it with the known biomarkers ethyl glucuronide (EtG) and ethyl sulfate (EtS). METHODS: Binge drinking men and women of European ancestry aged 21 to 65 years were enrolled in a 12-day, in-patient, randomized, double-blind, crossover study, where they were administered three beverage doses (placebo, 0.5 g/kg [0.4 g/kg] ethanol, and 1 g/kg [0.9 g/kg] ethanol for men [women]) individually in three 4-day periods (experiments), separated by minimum 7-day washout period. Diet, sleep, and physical activity were controlled throughout the inpatient experiments. Twenty-nine participants were randomized to receive beverage doses counterbalancing the sequence of treatment and gender within subgroups stratified by SERT genotypes 5HTTLPR:LL+rs25531:AA (LA LA ) versus 5HTTLPR:LS/SS. Peripheral venous blood was collected daily for (1) quantification of SERT mRNA (the primary outcome measure) using qRT-PCR and (2) plasma EtG and EtS levels using tandem mass-spectrometry. RESULTS: The association between administered beverage dose and SERT mRNA from completers of at least one 4-day experiment (N = 18) assessed by a linear mixed model was not statistically significant. Significant positive associations were found with beverage dose and plasma EtG, EtS and EtG/EtS ratio (ß = 5.8, SE = 1.2, p < 0.0001; ß = 1.3, SE = 0.6, p = 0.023; and ß = 3.0, SE = 0.7, p < 0.0001, respectively; the C-statistics for discriminating outcomes were 0.97, 0.8, and 0.92, respectively). Additionally, we observed a sequence effect with a greater placebo effect on SERT mRNA when it was administered during the first experiment (p = 0.0009), but not on EtG/EtS measures. CONCLUSION: The findings do not validate the use of SERT as a biomarker of heavy drinking. Larger and more innovative studies addressing the effects of placebo, race, gender, and response to treatment with serotonergic agents are needed to fully assess the utility of SERT as a biomarker of heavy and binge drinking.
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Consumo Excesivo de Bebidas Alcohólicas , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Femenino , Humanos , Masculino , Consumo de Bebidas Alcohólicas/genética , Biomarcadores , Estudios Cruzados , Etanol , Glucuronatos/análisis , Ondansetrón , ARN Mensajero/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Ésteres del Ácido Sulfúrico/análisis , Adulto Joven , Adulto , Persona de Mediana Edad , AncianoRESUMEN
PURPOSE: Despite no broad, direct evidence in humans, there is a potential concern that surfactants alter active or passive drug intestinal permeation to modulate oral drug absorption. The purpose of this study was to investigate the impact of the surfactant polysorbate 80 on active and passive intestinal drug absorption in humans. METHODS: The human (n = 12) pharmacokinetics (PK) of three probe substrates of intestinal absorption, valacyclovir, chenodeoxycholic acid (CDCA), and enalaprilat, were assessed. Endogenous bile acid levels were assessed as a secondary measure of transporter and microbiota impact. RESULTS: Polysorbate 80 did not inhibit peptide transporter 1 (PepT1)- or apical sodium bile acid transporter (ASBT)-mediated PK of valacyclovir and CDCA, respectively. Polysorbate 80 did not increase enalaprilat absorption. Modest increases in unconjugated secondary bile acid Cmax ratios suggest a potential alteration of the in vivo intestinal microbiota by polysorbate 80. CONCLUSIONS: Polysorbate 80 did not alter intestinal membrane fluidity or cause intestinal membrane disruption. This finding supports regulatory relief of excipient restrictions for Biopharmaceutics Classification System-based biowaivers.
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Enalaprilato , Polisorbatos , Ácidos y Sales Biliares , Enalaprilato/farmacología , Excipientes/farmacología , Humanos , Absorción Intestinal , Permeabilidad , Tensoactivos/farmacología , Valaciclovir/farmacologíaRESUMEN
The shorter synthesis of a novel poly(propylene imine) (PPI) dendron that can be quantitatively conjugated in good yields in a modular fashion to various modified Michael acceptors is reported herein. The focal point of the PPI dendron was coupled to an ester-linked thioctic acid-modified spacer to allow for an improved scalable synthesis and to allow attachment to other suitable systems, such as nanoparticle surfaces. The two modified Michael acceptors reported here are an acyl hydrazine Michael acceptor as well as an azide Michael acceptor. The acyl hydrazine modified third generation PPI dendron was further conjugated to doxorubicin (DOX) as a model system to test acid-sensitive drug delivery. The PPI-DOX conjugate displayed fast release of DOX at pH 4.5 while remaining stable at pH 7.4 and the PPI-DOX conjugate showed low in vitro cytotoxicity against PC3 prostate cancer cells. This modular platform represents a powerful dendronized system for incorporation onto nanoparticles or other systems to allow for multifunctional drug delivery.