RESUMEN
While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFß and TGFß-mediated fibroblast activation, indicating that TGFß-receptor loss on cancer cells increased TGFß bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFß responsiveness of cancer cells can affect the TME.
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Neoplasias , Microambiente Tumoral , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genómica , Ratones , Neoplasias/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Integrin αvß8 binds with exquisite specificity to latent transforming growth factor-ß (L-TGF-ß). This binding is essential for activating L-TGF-ß presented by a variety of cell types. Inhibiting αvß8-mediated TGF-ß activation blocks immunosuppressive regulatory T cell differentiation, which is a potential therapeutic strategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvß8 ectodomain and its intact natural ligand, L-TGF-ß, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvß8 binding specificity and TGF-ß activation. Our studies reveal a mechanism of TGF-ß activation where mature TGF-ß signals within the confines of L-TGF-ß and the release and diffusion of TGF-ß are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvß8-mediated L-TGF-ß activation.
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Microscopía por Crioelectrón/métodos , Integrinas/química , Integrinas/metabolismo , Proteínas de Unión a TGF-beta Latente/química , Proteínas de Unión a TGF-beta Latente/metabolismo , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Anticuerpos/inmunología , Sitios de Unión , Bronquios/citología , Células CHO , Cricetulus , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Integrinas/inmunología , Activación de Linfocitos , Masculino , Visón , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Linfocitos T Reguladores/inmunologíaRESUMEN
Transforming growth factor ß (TGF-ß) directly acts on naive, effector, and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-ß signaling. TGF-ß availability is altered by infections and cancer; however, the dose-dependent effects of TGF-ß on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-ß signals interact to shape the functional outcome of Tmem reactivation. We found that TGF-ß could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or proinflammatory signals. In contrast, even high doses of TGF-ß had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 Tmem may not always receive TGF-ß signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-ß signals is of importance. We found that exposure to TGF-ß before or after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-ß altered ~10% of the regulatory elements induced by reactivation and also elicited transcriptional changes indicative of broadly modulated functional properties. We confirmed some changes on the protein level and found that TGF-ß-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-ß is not simply suppressing CD8 Tmem but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner.
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Células T de Memoria , Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta/genética , Linfocitos T CD8-positivos/metabolismo , Transducción de Señal , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Regulators of G protein signaling (RGS) proteins constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase-activating protein activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and airway smooth muscle contraction, whereas RGS4 KO mice unexpectedly have decreased AHR because of increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here, we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GTPase-activating protein activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3K and inhibited PI3K-dependent PGE2 secretion elicited by transforming growth factor beta in airway epithelial cells. Together, these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.
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Asma , Proteínas RGS , Animales , Humanos , Ratones , Asma/metabolismo , Asma/genética , Asma/patología , Broncoconstricción/genética , Dinoprostona/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Hipersensibilidad Respiratoria/genética , Hipersensibilidad Respiratoria/patología , Proteínas RGS/metabolismo , Proteínas RGS/genética , Línea CelularRESUMEN
Transforming growth factor beta (TGF-ß) signaling is essential for a balanced immune response by mediating the development and function of regulatory T cells (Tregs) and suppressing autoreactive T cells. Disruption of this balance can result in autoimmune diseases, including multiple sclerosis (MS). MicroRNAs (miRNAs) targeting TGF-ß signaling have been shown to be upregulated in naïve CD4 T cells in MS patients, resulting in a limited in vitro generation of human Tregs. Utilizing the murine model experimental autoimmune encephalomyelitis, we show that perinatal administration of miRNAs, which target the TGF-ß signaling pathway, enhanced susceptibility to central nervous system (CNS) autoimmunity. Neonatal mice administered with these miRNAs further exhibited reduced Treg frequencies with a loss in T cell receptor repertoire diversity following the induction of experimental autoimmune encephalomyelitis in adulthood. Exacerbated CNS autoimmunity as a result of miRNA overexpression in CD4 T cells was accompanied by enhanced Th1 and Th17 cell frequencies. These findings demonstrate that increased levels of TGF-ß-associated miRNAs impede the development of a diverse Treg population, leading to enhanced effector cell activity, and contributing to an increased susceptibility to CNS autoimmunity. Thus, TGF-ß-targeting miRNAs could be a risk factor for MS, and recovering optimal TGF-ß signaling may restore immune homeostasis in MS patients.
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Autoinmunidad , Sistema Nervioso Central , Encefalomielitis Autoinmune Experimental , MicroARNs , Esclerosis Múltiple , Transducción de Señal , Linfocitos T Reguladores , Células Th17 , Factor de Crecimiento Transformador beta , MicroARNs/genética , MicroARNs/inmunología , Animales , Linfocitos T Reguladores/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/genética , Factor de Crecimiento Transformador beta/metabolismo , Ratones , Transducción de Señal/inmunología , Autoinmunidad/inmunología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/genética , Humanos , Sistema Nervioso Central/inmunología , Células Th17/inmunología , Ratones Endogámicos C57BL , Células TH1/inmunología , Diferenciación Celular/inmunología , FemeninoRESUMEN
Cancer immunotherapies have rapidly changed the therapeutic landscape for cancer. Nevertheless, most of the patients show innate or acquired resistance to these therapies. Studies conducted in recent years have highlighted an emerging role of cancer-associated fibroblasts (CAFs) in immune regulation that shapes the tumor immune microenvironment (TIME) and influences response to cancer immunotherapies. In this review, we outline recent advances in the understanding of phenotypic and functional heterogeneity of CAFs. We will focus on emerging roles of CAFs in shaping the TIME, especially under a framework of tumor immunity continuum, and discuss current and future CAF-targeting therapeutic strategies in particular in the context of optimizing the success of immunotherapies.
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Fibroblastos Asociados al Cáncer , Neoplasias , Humanos , Inmunoterapia , Neoplasias/terapia , Microambiente TumoralRESUMEN
RECK is a candidate tumor suppressor gene isolated as a gene that induces flat reversion in a cell line transformed by the KRAS oncogene. Since RECK knockout mice die in utero, they are not suitable for studying the effects of RECK on tumor formation. In this study, we found an increased incidence of spontaneous pulmonary adenomas in mice with reduced RECK expression (RECK-Hypo mice). To evaluate the effects of RECK expressed by either tumor cells or host cells on tumor growth, we established a tumorigenic cell line (MKER) from the kidney of a C57BL/6 mouse and performed syngeneic transplantation experiments. Our results indicate that when RECK expression is low in host cells, transplanted MKER cells grow faster and kill the animal more rapidly. Since RECK is required for the formation of proper fibrillin fibers that serve as a tissue reservoir for precursors of TGFß-family cytokines, we assessed the levels of TGFß1 in the peripheral blood. We found a significant increase in TGFß1 in RECK-Hypo mice compared to wild-type mice. We also found that the proportion of FOXP3-positive regulatory T (Treg) cells among splenocytes was higher in RECK-Hypo mice compared to the control mice. Furthermore, the number of FOXP3-positive cells in spontaneous hematopoietic neoplasms in the lungs as well as tumors that formed after MKER transplantation was significantly higher in RECK-Hypo mice compared to the control mice. These findings indicate that RECK-mediated tumor suppression involves a non-cell-autonomous mechanism and that possible roles of TGFß1 and Treg cells in such a mechanism warrant further study.
RESUMEN
BACKGROUND: Activins are novel therapeutic targets in pulmonary arterial hypertension (PAH). We therefore studied whether key members of the activin pathway could be used as PAH biomarkers. METHODS: Serum levels of activin A, activin B, α-subunit of inhibin A and B proteins, and the antagonists follistatin and follistatin-like 3 (FSTL3) were measured in controls and in patients with newly diagnosed idiopathic, heritable, or anorexigen-associated PAH (n=80) at baseline and 3 to 4 months after treatment initiation. The primary outcome was death or lung transplantation. Expression patterns of the inhibin subunits, follistatin, FSTL3, Bambi, Cripto, and the activin receptors type I (ALK), type II (ACTRII), and betaglycan were analyzed in PAH and control lung tissues. RESULTS: Death or lung transplantation occurred in 26 of 80 patients (32.5%) over a median follow-up of 69 (interquartile range, 50-81) months. Both baseline (hazard ratio, 1.001 [95% CI, 1.000-1.001]; P=0.037 and 1.263 [95% CI, 1.049-1.520]; P=0.014, respectively) and follow-up (hazard ratio, 1.003 [95% CI, 1.001-1.005]; P=0.001 and 1.365 [95% CI, 1.185-1.573]; P<0.001, respectively) serum levels of activin A and FSTL3 were associated with transplant-free survival in a model adjusted for age and sex. Thresholds determined by receiver operating characteristic analyses were 393 pg/mL for activin A and 16.6 ng/mL for FSTL3. When adjusted with New York Heart Association functional class, 6-minute walk distance, and N-terminal pro-B-type natriuretic peptide, the hazard ratios for transplant-free survival for baseline activin A <393 pg/mL and FSTL3 <16.6 ng/mL were, respectively, 0.14 (95% CI, 0.03-0.61; P=0.009) and 0.17 (95% CI, 0.06-0.45; P<0.001), and for follow-up measures, 0.23 (95% CI, 0.07-0.78; P=0.019) and 0.27 (95% CI, 0.09-0.78, P=0.015), respectively. Prognostic values of activin A and FSTL3 were confirmed in an independent external validation cohort. Histological analyses showed a nuclear accumulation of the phosphorylated form of Smad2/3, higher immunoreactivities for ACTRIIB, ALK2, ALK4, ALK5, ALK7, Cripto, and FSTL3 in vascular endothelial and smooth muscle layers, and lower immunostaining for inhibin-α and follistatin. CONCLUSIONS: These findings offer new insights into the activin signaling system in PAH and show that activin A and FSTL3 are prognostic biomarkers for PAH.
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Folistatina , Hipertensión Arterial Pulmonar , Humanos , Folistatina/metabolismo , Inhibinas/metabolismo , Activinas/metabolismo , Pulmón/metabolismoRESUMEN
Importin 8, encoded by IPO8, is a ubiquitously expressed member of the importin-ß protein family that translocates cargo molecules such as proteins, RNAs, and ribonucleoprotein complexes into the nucleus in a RanGTP-dependent manner. Current knowledge of the cargoes of importin 8 is limited, but TGF-ß signaling components such as SMAD1-4 have been suggested to be among them. Here, we report that bi-allelic loss-of-function variants in IPO8 cause a syndromic form of thoracic aortic aneurysm (TAA) with clinical overlap with Loeys-Dietz and Shprintzen-Goldberg syndromes. Seven individuals from six unrelated families showed a consistent phenotype with early-onset TAA, motor developmental delay, connective tissue findings, and craniofacial dysmorphic features. A C57BL/6N Ipo8 knockout mouse model recapitulates TAA development from 8-12 weeks onward in both sexes but most prominently shows ascending aorta dilatation with a propensity for dissection in males. Compliance assays suggest augmented passive stiffness of the ascending aorta in male Ipo8-/- mice throughout life. Immunohistological investigation of mutant aortic walls reveals elastic fiber disorganization and fragmentation along with a signature of increased TGF-ß signaling, as evidenced by nuclear pSmad2 accumulation. RT-qPCR assays of the aortic wall in male Ipo8-/- mice demonstrate decreased Smad6/7 and increased Mmp2 and Ccn2 (Ctgf) expression, reinforcing a role for dysregulation of the TGF-ß signaling pathway in TAA development. Because importin 8 is the most downstream TGF-ß-related effector implicated in TAA pathogenesis so far, it offers opportunities for future mechanistic studies and represents a candidate drug target for TAA.
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Aneurisma de la Aorta Torácica/etiología , Mutación con Pérdida de Función , Pérdida de Heterocigocidad , Fenotipo , beta Carioferinas/genética , Adulto , Animales , Aneurisma de la Aorta Torácica/metabolismo , Aneurisma de la Aorta Torácica/patología , Niño , Preescolar , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linaje , Transducción de Señal , Síndrome , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Adulto Joven , beta Carioferinas/metabolismoRESUMEN
The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-ß) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-ß-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-ß1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-ß1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.
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Nefropatías Diabéticas , Factor de Crecimiento Transformador beta1 , Humanos , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Transición Epitelial-Mesenquimal , Nefropatías Diabéticas/metabolismo , Fibrosis , Técnicas de Cultivo de Célula , Células Epiteliales/metabolismoRESUMEN
The purpose of this study was to evaluate transforming growth factor beta (TGFß) isoform localization in rabbit corneas with spontaneous persistent epithelial defects (PEDs) after photorefractive keratectomy (PRK). Four cryofixed corneas from a previously reported series of PEDs in rabbits that had PRK were evaluated with triplex immunohistochemistry (IHC) for TGFß3, myofibroblast marker alpha-smooth muscle actin (α-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFß1, TGFß2, or TGFß3 (each with α-SMA and vimentin) using isoform-specific antibodies. All three TGFß isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFß isoform co-localized with α-SMA of myofibroblasts, which could be TGFß isoform autocrine production by myofibroblasts or TGFß-1, -2, and -3 binding to these myofibroblasts.
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Queratectomía Fotorrefractiva , Animales , Conejos , Vimentina/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Sustancia Propia/metabolismo , Córnea/metabolismo , Isoformas de Proteínas/metabolismo , Actinas/metabolismoRESUMEN
BACKGROUND: Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-ß pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. METHODS: Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-ß pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. RESULTS: We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. CONCLUSIONS: Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-ß signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.
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Vesículas Extracelulares , MicroARNs , Humanos , Femenino , Bovinos , Animales , Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , MicroARNs/genética , Oviductos/metabolismo , Vesículas Extracelulares/metabolismo , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Osteoporosis is a genetic disease caused by the imbalance between osteoblast-led bone formation and osteoclast-induced bone resorption. However, further gene-related pathogenesis remains to be elucidated. METHODS: The aberrant expressed genes in osteoporosis was identified by analyzing the microarray profile GSE100609. Serum samples of patients with osteoporosis and normal group were collected, and the mRNA expression of candidate genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mouse cranial osteoblast MC3T3-E1 cells were treated with dexamethasone (DEX) to mimic osteoporosis in vitro. Alizarin Red staining and alkaline phosphatase (ALP) staining methods were combined to measure matrix mineralization deposition of MC3T3-E1 cells. Meanwhile, the expression of osteogenesis related genes including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix, and bone morphogenetic protein 2 (BMP2) were evaluated by qRT-PCR and western blotting methods. Then the effects of candidate genes on regulating impede bone loss caused by ovariectomy (OVX) in mice were studied. RESULTS: Cyclin A1 (CCNA1) was found to be significantly upregulated in serum of osteoporosis patients and the osteoporosis model cells, which was in line with the bioinformatic analysis. The osteogenic differentiation ability of MC3T3-E1 cells was inhibited by DEX treatment, which was manifested by decreased Alizarin Red staining intensity, ALP staining intensity, and expression levels of ALP, OCN, OPN, Osterix, and BMP2. The effects of CCNA1 inhibition on regulating osteogenesis were opposite to that of DEX. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that genes negatively associated with CCNA1 were enriched in the TGF-beta signaling pathway. Inhibitor of TGF-beta signaling pathway partly reversed osteogenesis induced by suppressed CCNA1. Furthermore, suppressed CCNA1 relieved bone mass of OVX mice in vivo. CONCLUSION: Downregulation of CCNA1 could activate TGF-beta signaling pathway and promote bone formation, thus playing a role in treatment of osteoporosis.
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Antraquinonas , Osteoporosis , Factor de Crecimiento Transformador beta , Animales , Femenino , Humanos , Ratones , Fosfatasa Alcalina/metabolismo , Diferenciación Celular , Ciclina A1/metabolismo , Osteoblastos/metabolismo , Osteogénesis , Osteoporosis/inducido químicamente , Factor de Crecimiento Transformador beta/metabolismo , Factores de Crecimiento Transformadores/efectos adversos , Factores de Crecimiento Transformadores/metabolismoRESUMEN
Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.
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Rabdomiosarcoma Alveolar , Rabdomiosarcoma , Humanos , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patología , Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta1 , Factores de Transcripción Paired Box/genética , Transición Epitelial-Mesenquimal , Rabdomiosarcoma/genética , Proteínas de Fusión Oncogénica/genéticaRESUMEN
Cnidarians are fascinating creatures at the base of metazoan evolution possessing an almost unlimited regeneration capacity that has attracted the interest of researchers, from Abraham Trembley's discovery of regeneration to the present. They share a simple body plan and a high morphogenetic plasticity that has led to a broad spectrum of life cycles. With molecular genomics it became unequivocally clear that Cnidaria are the sister group of the Bilateria and how similar their molecular toolkit is to that of more complex animals. This has renewed interest in these simple animals, which have had an important role in the establishment of fundamental concepts for developmental biologists from the beginning. This review focuses on our current understanding of signaling centers (organizers) and morphogenetic gradients in cnidarians and how they relate to the emergence of the bilaterian body axes. The data are largely based on the cnidarian models Hydra and Nematostella and are supported by new studies on forms with a complete cnidarian life cycle, such as the medusozoans Aurelia and Clytia. Molecular studies on cnidarian development have revealed the existence of an ancient Wnt signaling center at the site of gastrulation, which was instrumental for the formation of a primary body axis and can be traced back to the common ancestor of bilaterian and non-bilaterian animals. New molecular data also suggest that the molecular vectors for the dorso-ventral and left-right body axis in bilaterians, Bmp and Nodal signaling, respectively, were already present but had different fates in the two clades. The close link of developmental processes in bilaterians and cnidarians but also their distinct differences make cnidarians an indispensable model for tackling fundamental questions in developmental biology from patterning, regeneration and other recent molecular approaches to theoretical concepts.
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Tipificación del Cuerpo , Anémonas de Mar , Animales , Tipificación del Cuerpo/genética , Biología Evolutiva , Evolución Molecular , Vía de Señalización Wnt/genéticaRESUMEN
Insulin signaling in blood vessels primarily functions to stimulate angiogenesis and maintain vascular homeostasis through the canonical PI3K and MAPK signaling pathways. However, angiogenesis is a complex process coordinated by multiple other signaling events. Here, we report a distinct crosstalk between the insulin receptor and endoglin/activin receptor-like kinase 1 (ALK1), an endothelial cell-specific TGF-ß receptor complex essential for angiogenesis. While the endoglin-ALK1 complex normally binds to TGF-ß or bone morphogenetic protein 9 (BMP9) to promote gene regulation via transcription factors Smad1/5, we show that insulin drives insulin receptor oligomerization with endoglin-ALK1 at the cell surface to trigger rapid Smad1/5 activation. Through quantitative proteomic analysis, we identify ependymin-related protein 1 (EPDR1) as a major Smad1/5 gene target induced by insulin but not by TGF-ß or BMP9. We found endothelial EPDR1 expression is minimal at the basal state but is markedly enhanced upon prolonged insulin treatment to promote cell migration and formation of capillary tubules. Conversely, we demonstrate EPDR1 depletion strongly abrogates these angiogenic effects, indicating that EPDR1 is a crucial mediator of insulin-induced angiogenesis. Taken together, these results suggest important therapeutic implications for EPDR1 and the TGF-ß pathways in pathologic angiogenesis during hyperinsulinemia and insulin resistance.
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Endoglina , Factor 2 de Diferenciación de Crecimiento , Insulina , Neovascularización Patológica , Proteínas del Tejido Nervioso , Receptores de Factores de Crecimiento Transformadores beta , Animales , Humanos , Ratones , Receptores de Activinas Tipo II/metabolismo , Chlorocebus aethiops , Células COS , Endoglina/genética , Endoglina/metabolismo , Factor 2 de Diferenciación de Crecimiento/genética , Insulina/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfatidilinositol 3-Quinasas , Proteómica , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
KIN17 DNA and RNA binding protein (Kin17) is involved in the regulation of tumorigenesis of diverse human cancers. However, its role in the cancer progression and metastasis in hepatocellular carcinoma (HCC) remains largely unknown. Bioinformatics and immunohistochemistry staining were used to investigate the expression pattern of KIN17 and its prognostic value in HCC patients. The transwell, wound-healing assay was employed to determine the effects of KIN17 on migration and invasion of HCC cells in vitro. The tail veins model was employed to determine the effects of KIN17 on lung metastasis in vivo. The biological mechanisms involved in cell migration and invasion regulated by KIN17 were determined with Western blot analysis method. KIN17 expression was significantly increased in HCC tissues compared with adjacent normal tissues, with particularly higher in portal vein tumor thrombus and intrahepatic metastasis tissues. Patients with higher KIN17 expression experienced poor overall and disease free survival. KIN17 knockdown in HuH7 and HepG2 cells significantly reduced cell migration and invasion abilities, whereas its overexpression promoted migration and invasion in MHCC-97L and HepG2 cells in vitro and in vivo. In HuH7 and HepG2 cells, KIN17 knockdown inhibited the TGF-ß/Smad2 pathway. In contrast, KIN17 overexpression stimulated TGF-ß/Smad2 pathway in MHCC-97L and HepG2 cells, along with the genes involved in the epithelial-mesenchymal transition. These findings suggest that KIN17 promotes migration and invasion in HCC cells by stimulating the TGF-ß/Smad2 pathway. KIN17 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Proteína Smad2/genética , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Limosilactobacillusmucosae (LM) exerts anti-inflammatory and health-promoting effects. However, its role in the modulation of gut serotonin or 5-hydroxytryptamine (5-HT) metabolism and 5-HT receptors (HTRs) in inflammation requires further investigation. OBJECTIVES: We compared LM with Lactobacillus amylovorus (LA) for the regulation of 5-HT, HTRs, inflammatory mediators, and their correlations in the colon of mice with experimental colitis. METHODS: Male C57BL/6 mice were randomly assigned to 6 groups: control (Con), LM, LA, dextran sodium sulfate (DSS), and DSS with pre-administration of LM (+LM) or LA (+LA). After 7 d of DSS treatment, mice were killed to analyze the expression of inflammatory mediators, HTRs, and concentrations of 5-HT and microbial metabolites in the colon. RESULTS: LM was more effective than LA in alleviating DSS-induced colonic inflammation. Compared with mice in the DSS group, mice receiving DSS + LM or DSS + LA treatment had lower (P < 0.05) colonic mRNA expression of proinflammatory cytokines. DSS + LM treatment had lower mRNA expression of Il1b, Tnfa, and Ccl3, an abundance of p-STAT3, and greater expression of Tgfb2 and Htr4 in the colon (P < 0.05). The expression of inflammatory mediators (including Tgfb-1) was positively correlated (P < 0.05) with 5-HT and Htr2a and negatively correlated (P < 0.05) with Htr4. However, the expression of Tgfb-2 showed reversed correlations with the 5-HT and HTRs described above. Patterns for these correlations were different for LM and LA. Mice receiving the DSS + LM treatment had greater (P < 0.05) concentrations of acetate and valerate and lower (P < 0.05) concentrations of indole-3-acetic acid in the cecal and colonic contents. CONCLUSIONS: LM showed greater efficacy than LA in alleviating DSS-induced colonic inflammation. The coordinated regulation of transforming growth factor-ß subtypes and serotonin receptors in the colon may be one of the most important mechanisms underlying the probiotic effects of lactobacilli in gut inflammation.
Asunto(s)
Colitis , Serotonina , Masculino , Animales , Ratones , Serotonina/metabolismo , Lactobacillus acidophilus/metabolismo , Regulación hacia Arriba , Ratones Endogámicos C57BL , Colitis/inducido químicamente , Colitis/prevención & control , Colitis/metabolismo , Colon/metabolismo , Inflamación/metabolismo , ARN Mensajero/metabolismo , Factores de Crecimiento Transformadores/efectos adversos , Factores de Crecimiento Transformadores/metabolismo , Sulfato de Dextran/toxicidad , Modelos Animales de EnfermedadRESUMEN
Alkali burns are one of the most common injuries used in corneal wound healing studies. Investigators have used different conditions to produce corneal alkali injuries that have varied in sodium hydroxide concentration, application methods, and duration of exposure. A critical factor in the subsequent corneal healing responses, including myofibroblast generation and fibrosis localization, is whether, or not, Descemet's membrane and the endothelium are injured during the initial exposure. After exposures that produce injuries confined to the epithelium and stroma, anterior stromal myofibroblasts and fibrosis are typical, with sparing of the posterior stroma. However, if there is also injury to Descemet's membrane and the endothelium, then myofibroblast generation and fibrosis is noted full corneal thickness, with predilection to the most anterior and most posterior stroma and a tendency for relative sparring of the central stroma that is likely related to the availability of TGF beta from the tears, epithelium, and the aqueous humor. A method is described where a 5 mm diameter circle of Whatman #1 filter paper wetted with only 30 µL of alkali solution is applied for 15 s prior to profuse irrigation in rabbit corneas. When 0.6N, or lower, NaOH is used, then the injury, myofibroblasts, and fibrosis generation are limited to the epithelium and stroma. Use of 0.75N NaOH triggers injury to Descemet's membrane and the corneal endothelium with fibrosis throughout the stroma, but rare corneal neovascularization (CNV) and persistent epithelial defects (PED). Use of 1N NaOH with this method produces greater stromal fibrosis and increased likelihood that CNV and PED will occur in individual corneas.
Asunto(s)
Quemaduras Químicas , Lesiones de la Cornea , Quemaduras Oculares , Animales , Conejos , Sustancia Propia/patología , Álcalis/toxicidad , Quemaduras Químicas/patología , Hidróxido de Sodio/toxicidad , Córnea/patología , Lesiones de la Cornea/patología , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/patología , Fibrosis , Estándares de ReferenciaRESUMEN
The purpose of this study was to evaluate the localization of TGF beta-3 in situ in unwounded rabbit corneas and corneas that had epithelial-stromal injuries produced by photorefractive keratectomy (PRK) in rabbits and to evaluate the in vitro effects of TGF beta-3 compared to TGF beta-1 on alpha-smooth muscle actin (α-SMA) protein expression and myofibroblast development in corneal fibroblasts. Forty-eight New Zealand white rabbits underwent either -3 diopter (D) or -9D PRK and were studied from one to eight weeks (four corneas in each group at each time point) after surgery with immunohistochemistry for TGF beta-3, laminin alpha-5, and alpha-smooth muscle actin (α-SMA). Rabbit corneal fibroblasts were treated with activated TGF beta-1 and/or TGF beta-3 at different concentrations and duration of exposure and studied with immunocytochemistry for myofibroblast development and the expression of α-SMA using Jess automated Western blotting. TGF beta-3 was detected at high levels in the stroma of unwounded corneas and corneas at one to eight weeks after -3D or -9D PRK, as well as in the epithelium and epithelial basement membrane (EBM). No difference was noted between corneas that healed with and without myofibroblast-mediated fibrosis, although TGF beta-3 was commonly associated with myofibroblasts. TGF beta-3 effects on corneal fibroblasts in vitro were similar to TGF beta-1 in stimulating transition to α-SMA-positive myofibroblasts and promoting α-SMA protein expression. The corneal stromal localization pattern of TGF beta-3 protein in unwounded corneas and corneas after epithelial-stromal injury was found to be higher and different from TGF beta-1 and TGF beta-2 reported in previous studies. TGF beta-3 had similar effects to TGF beta-1 in driving myofibroblast development and α-SMA expression in corneal fibroblasts cultured in medium with 1% fetal bovine serum.