RESUMEN
The transcription factor forkhead/winged-helix domain proteins Foxp1 and Foxp2 have previously been studied in mouse retina, where they are expressed in retinal ganglion cells named F-mini and F-midi. Here we show that both transcription factors are expressed by small subpopulations (on average less than 10%) of retinal ganglion cells in the retina of the marmoset monkey (Callithrix jacchus). The morphology of Foxp1- and Foxp2-expressing cells was revealed by intracellular DiI injections of immunofluorescent cells. Foxp1- and Foxp2-expressing cells comprised multiple types of wide-field ganglion cells, including broad thorny cells, narrow thorny cells, and tufted cells. The large majority of Foxp2-expressing cells were identified as tufted cells. Tufted cells stratify broadly in the middle of the inner plexiform layer. They resemble broad thorny cells but their proximal dendrites are bare of branches and the distal dendrites branch frequently forming dense dendritic tufts. Double labeling with calretinin, a previously established marker for broad thorny and narrow thorny cells, showed that only a small proportion of ganglion cells co-expressed calretinin and Foxp1 or Foxp2 supporting the idea that the two markers are differentially expressed in retinal ganglion cells of marmoset retina.
Asunto(s)
Callithrix , Factores de Transcripción Forkhead , Células Ganglionares de la Retina , Animales , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/biosíntesis , Células Ganglionares de la Retina/metabolismo , Masculino , Femenino , Retina/metabolismo , Retina/citologíaRESUMEN
PURPOSE: Immunodysregulation, Polyendocrinopathy, Enteropathy, and X-linked syndrome (IPEX), caused by pathogenic FOXP3 variants, is a rare autoimmune disorder with diverse clinical features, including early-onset diabetes, eczema, and enteropathy. Atypical cases show milder symptoms and unique signs, requiring different treatments. Therefore, there are ambiguities in the accurate diagnosis and management of IPEX. We sought to present clinical, genetic, and immunological assessments of 12 IPEX patients with long-term follow-up to facilitate the diagnosis and management of the disease. METHODS: Clinical findings and treatment options of the patients were collected over time. Lymphocyte subpopulations, protein expressions, regulatory T (Treg) and circulating T follicular helper (cTFH) cells, and T-cell proliferation were analyzed. RESULTS: Predominant presentations included autoimmunity (91.6%), failure to thrive (66.7%), and eczema (58.3%). There were four classical and eight atypical IPEX individuals. Allergic manifestations were more common in atypical patients. Notably, chronic diarrhea demonstrated heightened severity compared to other manifestations. Four patients (33.3%) demonstrated eosinophilia, and nine (75%) showed high serum IgE levels. Most patients exhibited normal percentages of Treg cells with reduced CD25, FOXP3, and CTLA-4 expressions, corrected after hematopoietic stem cell transplantation (HSCT). Compared to healthy controls, the TH2-like skewing accompanied by reduced TH17-like responses was observed in cTFH and Treg cells of patients. Overall, nine patients (75%) received immunosuppressants (ISs), and six (50%) underwent HSCT, which was the only treatment revealing sustained control. Sirolimus was used in six patients and showed better control than other ISs. CONCLUSIONS: The first cohort from Turkey with long-term follow-up results, comparing typical and atypical cases, provides insights into the outcomes of different therapeutic modalities and T- cell subtype changes in IPEX syndrome.
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Factores de Transcripción Forkhead , Enfermedades Genéticas Ligadas al Cromosoma X , Linfocitos T Reguladores , Humanos , Turquía , Masculino , Preescolar , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Linfocitos T Reguladores/inmunología , Lactante , Femenino , Niño , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/congénito , Enfermedades del Sistema Inmune/diagnóstico , Enfermedades del Sistema Inmune/genética , Enfermedades del Sistema Inmune/terapia , Enfermedades del Sistema Inmune/congénito , Autoinmunidad , Adolescente , DiarreaRESUMEN
Based on the joint analysis of multi-omic data and the biological experiments, we demonstrate that FOXF1 inhibits invasion and metastasis of lung adenocarcinoma cells and enhances anti-tumor immunity via regulating MFAP4/FAK signal axis in this study. The levels of FOXF1 and MFAP4 are significantly down-regulated in LUAD, and the increased levels of two genes can improve the clinical prognosis of LUAD patients. Fluorescein reporter gene determination, chromatin immunoprecipitation and gene co-expression analysis indicate that MFAP4 level is positively regulated by transcription factor FOXF1. The function enrichment analysis shows that the levels of FOXF1 and MFAP4 are closely associated with an enrichment of tumor metastasis signatures. FOXF1 can inhibit the migration and invasion of LAUD cells by transcriptionally activating MFAP4 expression. And the overexpression of FOXF1/MFAP4 can reduce focal adhesion kinase (FAK) phosphorylation, while their knockdown result in the opposite effects. The increased levels of FOXF1/MFAP4 enhance the antitumor immunity by increasing the infiltration of dendritic cells and CD4+ T cells, and the interactions between LUAD cells and immune cells, and activating multiple anti-tumor immunity-related pathways. In conclusion, our study reveals the potential function of FOXF1/MFAP4/FAK signal axis in inhibiting metastasis of LUAD cells and modulating anti-tumor immunity of LUAD patients.
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Adenocarcinoma del Pulmón , Factores de Transcripción Forkhead , Neoplasias Pulmonares , Invasividad Neoplásica , Transducción de Señal , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Humanos , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Movimiento Celular , Ratones , Animales , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismoRESUMEN
Our goal is to improve the outcomes of cancer immunotherapy by targeting FOXP3+ T-regulatory (Treg) cells with a next generation of antisense oligonucleotides (ASO), termed FOXP3 AUMsilence ASO. We performed in vitro experiments with human healthy donor PBMC and clinical samples from patients with lung cancer, mesothelioma and melanoma, and tested our approach in vivo using ASO FOXP3 in syngeneic murine cancer models and in humanized mice. ASO FOXP3 had no effects on cell viability or cell division, did not affect expression of other FOXP members, but decreased expression of FOXP3 mRNA in PBMC by 54.9% and in cancer samples by 64.7%, with corresponding 41.0% (PBMC) and 60.0% (cancer) decreases of Treg numbers (all p<0.0001). Hence, intratumoral Treg were more sensitive to the effects of ASO FOXP3 than peripheral blood Tregs. Isolated human Treg, incubated with ASO FOXP3 for 3.5 hours, had significantly impaired suppressive function (66.4%) versus Scramble control. In murine studies, we observed a significant inhibition of tumor growth, while 13.6% (MC38) to 22% (TC1) of tumors were completely resorbed, in conjunction with ~50% decrease of Foxp3 mRNA by qPCR and decreased numbers of intratumoral Tregs. In addition, there were no changes in FOXP3 mRNA expression or in the numbers of Tregs in draining lymph nodes and in spleens of tumor bearing mice, confirming that intratumoral Treg had enhanced sensitivity to ASO FOXP3 in vivo compared to other Treg populations. ASO FOXP3 Treg targeting in vivo and in vitro was accompanied by significant downregulation of multiple exhaustion markers, and by increased expression of perforin and granzyme-B by intratumoral T cells. To conclude, we report that targeting the key Treg transcription factor FOXP3, with ASO FOXP3, has a powerful anti-tumoral effect and enhances T cell response in vitro and in vivo.
Asunto(s)
Factores de Transcripción Forkhead , Oligonucleótidos Antisentido , Linfocitos T Reguladores , Animales , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Humanos , Ratones , Femenino , Neoplasias/inmunología , Neoplasias/terapia , Línea Celular Tumoral , Ratones Endogámicos C57BL , Inmunoterapia/métodosRESUMEN
BACKGROUND: Currently, creating more effector T cells and augmenting their functions is a focal point in pancreatic ductal adenocarcinoma (PDAC) treatment research. T cell immunoglobulin domain and mucin domain molecule 4 (TIM-4), known for promoting cancer progression in various malignancies, is implicated in the suppressive immune microenvironment of tumors. Analyzing of the role of TIM-4 in the immune regulation of PDAC can offer novel insights for immune therapy. METHODS: We analyzed the TIM-4 expression in tumor specimens from PDAC patients. Meanwhile, multiple fluorescent immunohistochemical staining was used to study the distribution characteristics of TIM-4, and through tissue microarrays, we explored its correlation with patient prognosis. The influence of TIM-4 overexpression on cell function was analyzed using RNA-seq. Flow cytometry and ELISA were used for verification. Finally, the relationship between TIM-4 and T lymphocytes was analyzed by tissue microarray, and the impacts of TIM-4 on T cell subsets were observed by cell coculture technology and a mouse pancreatic cancer in situ model. RESULTS: In PDAC, TIM-4 is mainly expressed in tumor cells and negatively correlated with patient prognosis. TIM-4 influences the differentiation of Treg by inhibiting IL-6 secretion in pancreatic cancer cells and facilitates the proliferation of pancreatic cancer in mice. Additionally, the mechanism may be through the CD8+ effector T cells (CD8+Tc). CONCLUSION: TIM-4 has the potential to be an immunotherapeutic target or to improve the efficacy of chemotherapy for PDAC.
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Carcinoma Ductal Pancreático , Factores de Transcripción Forkhead , Interleucina-6 , Neoplasias Pancreáticas , Linfocitos T Reguladores , Microambiente Tumoral , Humanos , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Microambiente Tumoral/inmunología , Interleucina-6/metabolismo , Animales , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Ratones , Factores de Transcripción Forkhead/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Línea Celular Tumoral , Pronóstico , Femenino , Masculino , Proliferación Celular , Proteínas de la MembranaRESUMEN
lncRNA can regulate tumorigenesis development and distant metastasis of colorectal cancer (CRC). However, the detailed molecular mechanisms are still largely unknown. Using RNA-sequencing data, RT-qPCR, and FISH assay, we found that HIF1A-AS2 was upregulated in CRC tissues and associated with poor prognosis. Functional experiments were performed to determine the roles of HIF1A-AS2 in tumor progression and we found that HIF1A-AS2 can promote the proliferation, metastasis, and aerobic glycolysis of CRC cells. Mechanistically, HIF1A-AS2 can promote FOXC1 expression by sponging miR-141-3p. SP1 can transcriptionally activate HIF1A-AS2. Further, HIF1A-AS2 can be packaged into exosomes and promote the malignant phenotype of recipient tumor cells. Taken together, we discovered that SP1-induced HIF1A-AS2 can promote the metabolic reprogramming and progression of CRC via miR-141-3p/FOXC1 axis. HIF1A-AS2 is a promising diagnostic marker and treatment target in CRC.
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Neoplasias Colorrectales , Progresión de la Enfermedad , Factores de Transcripción Forkhead , Regulación Neoplásica de la Expresión Génica , MicroARNs , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Animales , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Línea Celular Tumoral , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ratones , Ratones Desnudos , Proliferación Celular/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/genética , Glucólisis/genética , Ratones Endogámicos BALB C , Masculino , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Movimiento Celular/genética , Reprogramación MetabólicaRESUMEN
Introduction: The role of zinc (Zn) in tumor development and immune modulation has always been paradoxical. This study redefines our understanding of the impact of Zn on cancer progression and therapeutic strategies. Methods: We investigated the effects of dietary Zn levels on tumor progression and immune responses. This included examining the impact of both high and deficient dietary Zn, as well as Zn chelation, on tumor growth and immune cell populations. Specifically, we analyzed the frequency of Foxp3+ regulatory T-cells (Tregs) and identified the role of FOXO1 in Zn-mediated effects on Tregs. Additionally, we explored the therapeutic potential of clioquinol (CQ) in enhancing α-PD-1 immunotherapy responses, particularly in melanoma. Results: Our findings show that high dietary Zn promotes tumor progression by fostering a protumorigenic environment mediated by T cells. Increased Zn intake was found to facilitate tumor progression by increasing Foxp3+ Treg frequency. In contrast, deficiency in dietary Zn and chelation of tissue Zn emerged as potent drivers of antitumor immunity. We pinpointed FOXO1 as the master regulator governing the influence of Zn on Tregs. Discussion: These results reveal a novel mechanistic insight into how Zn influences tumor progression and immune regulation. The identification of FOXO1 as a key regulator opens new avenues for understanding the role of Zn in cancer biology. Furthermore, we introduce a promising therapeutic approach by showing that administering clioquinol (CQ) significantly enhances α-PD-1 immunotherapy response, particularly in melanoma. These revelations transform our comprehension of the multifaceted role of Zn in tumorigenesis and immune regulation, highlighting innovative possibilities for cancer therapy.
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Factores de Transcripción Forkhead , Linfocitos T Reguladores , Zinc , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Animales , Zinc/metabolismo , Factores de Transcripción Forkhead/metabolismo , Ratones , Clioquinol/farmacología , Ratones Endogámicos C57BL , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Melanoma Experimental/inmunología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Melanoma/inmunología , Melanoma/tratamiento farmacológico , Humanos , Línea Celular Tumoral , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de los fármacos , Inmunoterapia/métodos , FemeninoRESUMEN
Glycation Stress (GS), induced by advanced glycation end-products (AGEs), significantly impacts aging processes. This study introduces a new model of GS of Caenorhabditis elegans by feeding them Escherichia coli OP50 cultured in a glucose-enriched medium, which better simulates human dietary glycation compared to previous single protein-glucose cross-linking methods. Utilizing WormCNN, a deep learning model, we assessed the health status and calculated the Healthy Aging Index (HAI) of worms with or without GS. Our results demonstrated accelerated aging in the GS group, evidenced by increased autofluorescence and altered gene expression of key aging regulators, daf-2 and daf-16. Additionally, we observed elevated pharyngeal pumping rates in AGEs-fed worms, suggesting an addictive response similar to human dietary patterns. This study highlights the profound effects of GS on worm aging and underscores the critical role of computer vision in accurately assessing health status and aiding in the establishment of disease models. The findings provide insights into glycation-induced aging and offer a comprehensive approach to studying the effects of dietary glycation on aging processes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Productos Finales de Glicación Avanzada , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Productos Finales de Glicación Avanzada/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Envejecimiento Saludable/metabolismo , Envejecimiento/metabolismo , Estrés Fisiológico , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Glicosilación , Glucosa/metabolismo , Modelos Animales de Enfermedad , Receptor de InsulinaRESUMEN
Regulatory T (Treg) cells play a central role in immune homeostasis. Forkhead box P3 (Foxp3), a hallmark molecule in Treg cells, is a vital transcription factor for their development and function. Studies have shown that degradation of the Foxp3 could provide therapeutic benefits in achieving effective anti-tumor immunity. In this study, we designed three PROTAC molecules, P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, based on a 15-mer peptide inhibitor of Foxp3 (P60), and explored their potential in regulating Foxp3 expression and function. Our data show that, among these molecules, P60-L3-VHL can inhibit the expression and nuclear localization of Foxp3 in HEK 293 T and HeLa cells, respectively. Meanwhile, use of proteasome inhibitor in P60-L3-VHL treated cells revealed an increased Foxp3 expression, indicating that P60-L3-VHL mediates the inhibition of Foxp3 through its degradation in the proteasome pathway. We further substantiate that P60-L3-VHL reduces the differentiation and Foxp3 expression in the in-vitro activated Treg cells. Overall, our findings suggest that P60-L3-VHL inhibits the differentiation of Treg cells by degrading the Foxp3, which may have potential implications in cancer immunotherapy.
Asunto(s)
Factores de Transcripción Forkhead , Proteolisis , Humanos , Factores de Transcripción Forkhead/metabolismo , Proteolisis/efectos de los fármacos , Células HEK293 , Células HeLa , Linfocitos T Reguladores/efectos de los fármacos , Relación Estructura-Actividad , Estructura Molecular , Descubrimiento de Drogas , Relación Dosis-Respuesta a Droga , Complejo de la Endopetidasa Proteasomal/metabolismo , Quimera Dirigida a la ProteólisisRESUMEN
Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103- counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.
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Antígenos CD , Diferenciación Celular , Factores de Transcripción Forkhead , Cadenas alfa de Integrinas , Linfocitos T Reguladores , Humanos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Antígenos CD/metabolismo , Antígenos CD/inmunología , Cadenas alfa de Integrinas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/inmunología , Fenotipo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Tolerancia Inmunológica , Receptores Inmunológicos/metabolismo , Proteómica/métodos , Receptores CXCR3/metabolismo , Proteína del Gen 3 de Activación de Linfocitos , Células CultivadasRESUMEN
Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.
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Oogénesis , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/fisiología , Oogénesis/fisiología , Oogénesis/genética , Femenino , Masculino , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Células Germinativas/fisiología , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Espermatogénesis/fisiología , Espermatogénesis/genética , Oocitos/fisiologíaRESUMEN
Ovarian development was traditionally recognized as a "default" sexual outcome and therefore received much less scientific attention than testis development. In turtles with temperature-dependent sex determination (TSD), how the female pathway is initiated to induce ovary development remains unknown. In this study, we have found that phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3) and Foxl2 exhibit temperature-dependent sexually dimorphic patterns and tempo-spatial coexpression in early embryos of the red-eared slider turtle (Trachemys scripta elegans). Inhibition of pSTAT3 at a female-producing temperature of 31 °C induces 64.7% female-to-male sex reversal, whereas activation of pSTAT3 at a male-producing temperature of 26 °C triggers 75.6% male-to-female sex reversal. In addition, pSTAT3 directly binds to the locus of the female sex-determining gene Foxl2 and promotes Foxl2 transcription. Overexpression or knockdown of Foxl2 can rescue the sex reversal induced by inhibition or activation of pSTAT3. This study has established a direct genetic link between warm temperature-induced STAT3 phosphorylation and female pathway initiation in a TSD system, highlighting the critical role of pSTAT3 in the cross talk between female and male pathways.
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Factor de Transcripción STAT3 , Procesos de Determinación del Sexo , Temperatura , Tortugas , Animales , Femenino , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Masculino , Fosforilación , Tortugas/metabolismo , Tortugas/genética , Tortugas/embriología , Ovario/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Proteína Forkhead Box L2/metabolismo , Proteína Forkhead Box L2/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
BACKGROUND: Foxn1-/- deficient mice are a rare model of regenerative skin wound healing among mammals. In wounded skin, the transcription factor Foxn1 interacting with hypoxia-regulated factors affects re-epithelialization, epithelial-mesenchymal transition (EMT) and dermal white adipose tissue (dWAT) reestablishment and is thus a factor regulating scar-forming/reparative healing. Here, we hypothesized that transcriptional crosstalk between Foxn1 and Hif-1α controls the switch from scarless (regenerative) to scar-present (reparative) skin wound healing. To verify this hypothesis, we examined (i) the effect of hypoxia/normoxia and Foxn1 signalling on the proteomic signature of Foxn1-/- (regenerative) dermal fibroblasts (DFs) and then (ii) explored the effect of Hif-1α or Foxn1/Hif-1α introduced by a lentiviral (LV) delivery vector to injured skin of regenerative Foxn1-/- mice with particular attention to the remodelling phase of healing. RESULTS: We showed that hypoxic conditions and Foxn1 stimulation modified the proteome of Foxn1-/- DFs. Hypoxic conditions upregulated DF protein profiles, particularly those related to extracellular matrix (ECM) composition: plasminogen activator inhibitor-1 (Pai-1), Sdc4, Plod2, Plod1, Lox, Loxl2, Itga2, Vldlr, Ftl1, Vegfa, Hmox1, Fth1, and F3. We found that Pai-1 was stimulated by hypoxic conditions in regenerative Foxn1-/- DFs but was released by DFs to the culture media exclusively upon hypoxia and Foxn1 stimulation. We also found higher levels of Pai-1 protein in DFs isolated from Foxn1+/+ mice (reparative/scar-forming) than in DFs isolated from Foxn1-/- (regenerative/scarless) mice and triggered by injury increase in Foxn1 and Pai-1 protein in the skin of mice with active Foxn1 (Foxn1+/+ mice). Then, we demonstrated that the introduction of Foxn1 and Hif-1α via lentiviral injection into the wounded skin of regenerative Foxn1-/- mice activates reparative/scar-forming healing by increasing the wounded skin area and decreasing hyaluronic acid deposition and the collagen type III to I ratio. We also identified a stimulatory effect of LV-Foxn1 + LV-Hif-1α injection in the wounded skin of Foxn1-/- mice on Pai-1 protein levels. CONCLUSIONS: The present data highlight the effect of hypoxia and Foxn1 on the protein profile and functionality of regenerative Foxn1-/- DFs and demonstrate that the introduction of Foxn1 and Hif-1α into the wounded skin of regenerative Foxn1-/- mice activates reparative/scar-forming healing.
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Cicatriz , Fibroblastos , Factores de Transcripción Forkhead , Cicatrización de Heridas , Animales , Cicatrización de Heridas/fisiología , Cicatrización de Heridas/genética , Fibroblastos/metabolismo , Ratones , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Cicatriz/metabolismo , Piel/metabolismo , Piel/lesiones , Ratones Noqueados , Proteoma/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteómica/métodos , Hipoxia/metabolismoRESUMEN
Naïve pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naïve pluripotency-specific transcription factors (TFs). Upon exiting naïve pluripotency, embryonic stem cells (ESCs) transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage choice. However, the mechanisms underlying disengagement from the naïve GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localisation of FoxO TFs in naïve ESCs. PTEN-mediated reduction of AKT activity upon exit from naïve pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for the activation of the formative pluripotency-specific GRN. Our work uncovers a pivotal role for FoxO TFs in establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition.
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Redes Reguladoras de Genes , Células Madre Pluripotentes , Animales , Ratones , Células Madre Pluripotentes/metabolismo , Diferenciación Celular/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Fosforilación , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
OBJECTIVE: The TGF-ß superfamily member activin, a dimer of the gene products of INHBA and/or INHBB, has been implicated in immune cell maturation and recruitment, but its immune impact within epithelial ovarian cancer (EOC) is not well characterized. We sought to explore differences in activin (INHBA/ Inhibin-ßA and INHBB/ Inhibin-ßB) between malignant and ovarian tissues at the RNA and protein level and assess the relationship between activin and immune cells in EOC. METHODS: Publicly available RNA sequencing data were accessed from GEO (#GSE143897) with normalization and quantification performed via DESeq2. Immune gene expression profile was further explored within the TCGA-OV cohort derived from The Cancer Genome Atlas (TCGA). Immunohistochemical analysis was performed to evaluate activin A and T-cell markers CD8 and FoxP3 at the protein level. ELISA to activin-A was used to assess levels in the ascites of advanced EOC patients. Kaplan-Meier curves were generated to visualize survival outcomes. RESULTS: Gene expression levels of components of the activin signaling pathway were elevated within EOC when compared to a benign cohort, with differences in activin type I/II receptor gene profiles identified. Additionally, INHBA gene expression was linked to lymphocytic immune markers in EOC samples. Immunohistochemistry analysis revealed a positive correlation of CD8 and FOXP3 staining with activin A at the protein level in both primary and metastatic epithelial ovarian cancer samples. Furthermore, Activin-A (inhibin-ßA) is significantly elevated in EOC patient ascites. CONCLUSION: INHBA expression is elevated within EOC, correlating with worse survival, with activin protein levels correlating with specific immune infiltration. Our findings suggest that activin-A may play a role in suppressing anti-tumor immunity in EOC, highlighting its potential as a therapeutic target.
Asunto(s)
Activinas , Carcinoma Epitelial de Ovario , Subunidades beta de Inhibinas , Neoplasias Ováricas , Humanos , Femenino , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/inmunología , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/mortalidad , Neoplasias Ováricas/patología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/genética , Subunidades beta de Inhibinas/genética , Subunidades beta de Inhibinas/metabolismo , Activinas/metabolismo , Activinas/genética , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Persona de Mediana Edad , Regulación Neoplásica de la Expresión Génica , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genéticaRESUMEN
The aim of this study was to elucidate the therapeutic effect of simvastatin on experimental autoimmune encephalomyelitis (EAE) by regulating the balance between Th17 and Treg cells in mice. C57BL/6 mice were randomly divided into four groups: normal group, EAE group, simvastatin (2 and 10 mg/kg) group, and AG490 group (with AG490 serving as the positive control). Neurological function scores of mice were assessed daily. The four groups received treatments of normal saline, normal saline, and simvastatin (2 and 10 mg/kg), respectively. In the AG490 group, mice were injected intraperitoneally with AG490 (1 mg) every other day, and treatment was halted after 3 weeks. The spinal cord was stained with hematoxylin and eosin (H&E), and immunohistochemical staining for retinoic acid receptor-related orphan receptor γ(RORγ) and Foxp3 (Foxp3) was performed. Spleen samples were taken for Th17 and Treg analysis using flow cytometry. The levels of interleukin-17 and transforming growth factor-ß (TGF-ß) were detected using enzyme-linked immunosorbent assay (ELISA). In the simvastatin and AG490 groups, recovery from neurological impairment was earlier compared to the EAE group, and the symptoms were notably improved. Both simvastatin and AG490 reduced focal inflammation, decreased RORγ-positive cell infiltration, and significantly increased the number of FOXP3-positive cells. The number of Th17 cells and the level of IL-17 in the spleen were decreased in the simvastatin and AG490 treatment groups, while the number of Treg cells and TGF-ß levels were significantly increased across all treatment groups. Simvastatin exhibits anti-inflammatory and immunomodulatory effects, potentially alleviating symptoms of neurological dysfunction of EAE. Regulating the balance between Th17 and Treg may represent a therapeutic mechanism for simvastatin in treating EAE.
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Encefalomielitis Autoinmune Experimental , Ratones Endogámicos C57BL , Simvastatina , Linfocitos T Reguladores , Células Th17 , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Células Th17/inmunología , Células Th17/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Simvastatina/farmacología , Simvastatina/administración & dosificación , Ratones , Femenino , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Interleucina-17/metabolismo , Factores de Transcripción Forkhead/metabolismo , Médula Espinal/inmunología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Regulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma. METHODS: We performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings. RESULTS: High CD25+, FOXP3+, and CD25+FOXP3+CD45RA- stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA+ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25+ or CD25+FOXP3+CD45RA- stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25+ : CD8+ cell count or CD25+FOXP3+CD45RA- : CD8+ cell count correlated with longer TFI (p = 0.021 and 0.021). CONCLUSION: The current observations suggest that the balance between CD25+ or CD25+FOXP3+CD45RA- cells and CD8+ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25+FOXP3+CD45RA- effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas.
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Biomarcadores de Tumor , Neoplasias Endometriales , Factores de Transcripción Forkhead , Subunidad alfa del Receptor de Interleucina-2 , Antígenos Comunes de Leucocito , Linfocitos T Reguladores , Humanos , Femenino , Neoplasias Endometriales/inmunología , Neoplasias Endometriales/patología , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/genética , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Pronóstico , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Biomarcadores de Tumor/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Persona de Mediana Edad , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Anciano , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Adulto , Estimación de Kaplan-Meier , Antígeno CTLA-4/metabolismo , Anciano de 80 o más AñosRESUMEN
The microenvironment within solid tumors often becomes acidic due to various factors associated with abnormal metabolism and cellular activities, including increased lactate production as a result of dysregulated tumor glycolysis. Recently, we have identified multiple tumor microenvironment (TME) factors that potentiate regulatory T (Treg) cell function in evading anti-tumor immunosurveillance. Despite the strong correlation between lactate and acidity, the potential roles of acidity in intratumoral Treg cell adaptation and underlying molecular mechanisms have gone largely unstudied. In this study, we demonstrate that acidity significantly enhances immunosuppressive functions of nTreg cells, but not iTreg cells, without altering the expression of either FoxP3 or the cell surface receptors CD25, CTLA4, or GITR in these cells. Surprisingly, the addition of lactate, often considered a major contributor to increased acidity of the TME, completely abolished the acidity-induced enhancement of nTreg suppressive functions. Consistently, metabolic flux analyses showed elevated basal mitochondrial respiratory capacity and ATP-coupled respiration in acidity-treated nTreg cells without altering glycolytic capacity. Genome-wide transcriptome and metabolomics analyses revealed alterations in multiple metabolic pathways, particularly the one-carbon folate metabolism pathway, with reduced SAM, folate, and glutathione, in nTreg cells exposed to low pH conditions. Addition of a one-carbon metabolic contributor, formate, diminished the acidity-induced enhancement in nTreg cell suppressive functions, but neither SAM nor glutathione could reverse the phenotype. Remarkably, in vitro transient treatment of nTreg cells resulted in sustained enhancement of their functions, as evidenced by more vigorous tumor growth observed in mice adoptively receiving acidity-treated nTreg cells. Further analysis of intratumoral infiltrated T cells confirmed a significant reduction in CD8+ T cell frequency and their granzyme B production. In summary, our study elucidates how acidity-mediated metabolic reprogramming leads to sustained Treg-mediated tumor immune evasion.
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Linfocitos T Reguladores , Escape del Tumor , Microambiente Tumoral , Linfocitos T Reguladores/inmunología , Animales , Microambiente Tumoral/inmunología , Escape del Tumor/inmunología , Ratones , Ácido Láctico/metabolismo , Concentración de Iones de Hidrógeno , Ratones Endogámicos C57BL , Factores de Transcripción Forkhead/metabolismo , Glucólisis/efectos de los fármacos , Neoplasias/inmunología , Línea Celular Tumoral , Humanos , FemeninoRESUMEN
BACKGROUND: Zoledronic acid (ZOL) is a type of bisphosphonate with good therapeutic effects on orthopaedic diseases. However, the pharmacological functions of ZOL on steroid-induced avascular necrosis of femoral head (SANFH) and the underlying mechanism remain unclear, which deserve further research. METHODS: SANFH models both in vivo and in vitro were established by dexamethasone (Dex) stimulation. Osteoclastogenesis was examined by TRAP staining. Immunofluorescence was employed to examine autophagy marker (LC3) level. Cell apoptosis was analyzed by TUNEL staining. The interaction between Foxhead box D3 protein (FOXD3) and Annexin A2 (ANXA2) promoter was analyzed using ChIP and dual luciferase reporter gene assays. RESULTS: Dex aggravated osteoclastogenesis and induced osteoclast differentiation and autophagy in vitro, which was abrogated by ZOL treatment. PI3K inhibitor LY294002 abolished the inhibitory effect of ZOL on Dex-induced osteoclast differentiation and autophagy. FOXD3 overexpression neutralized the downregulation effects of ZOL on Dex-induced osteoclasts by transcriptionally activating ANXA2. ANXA2 knockdown reversed the effect of FOXD3 overexpression on ZOL-mediated biological effects in Dex-treated osteoclasts. In addition, ZOL improved SANFH symptoms in rats. CONCLUSION: ZOL alleviated SANFH through regulating FOXD3 mediated ANXA2 transcriptional activity and then promoting PI3K/AKT/mTOR pathway, revealing that FOXD3 might be a target for ZOL in SANFH treatment.
Asunto(s)
Anexina A2 , Autofagia , Necrosis de la Cabeza Femoral , Factores de Transcripción Forkhead , Activación Transcripcional , Ácido Zoledrónico , Animales , Necrosis de la Cabeza Femoral/inducido químicamente , Necrosis de la Cabeza Femoral/patología , Necrosis de la Cabeza Femoral/genética , Necrosis de la Cabeza Femoral/tratamiento farmacológico , Ácido Zoledrónico/farmacología , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Anexina A2/metabolismo , Anexina A2/genética , Masculino , Activación Transcripcional/efectos de los fármacos , Dexametasona/farmacología , Dexametasona/efectos adversos , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Diferenciación Celular/efectos de los fármacos , Ratones , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Apoptosis/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld-Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes.