RESUMEN

RNA sequencing (RNA-Seq) has become a widely adopted technique for studying gene expression. However, conventional RNA-Seq analyses rely on gene expression (GE) values that aggregate all the transcripts produced under a single gene identifier, overlooking the complexity of transcript variants arising from different transcription start sites or alternative splicing. Transcript variants may encode proteins with diverse functional domains, or noncoding RNAs. This study explored the implications of neglecting transcript variants in RNA-Seq analyses. Among the 1334 transcription factor (TF) genes expressed in mouse embryonic stem (ES) or trophoblast stem (TS) cells, 652 were differentially expressed in TS cells based on GE values (365 upregulated and 287 downregulated, ≥absolute 2-fold changes, false discovery rate (FDR) p-value ≤ 0.05). The 365 upregulated genes expressed 883 transcript variants. Further transcript expression (TE) based analyses identified only 174 (<20%) of the 883 transcripts to be upregulated. The remaining 709 transcripts were either downregulated or showed no significant changes. Meanwhile, the 287 downregulated genes expressed 856 transcript variants and only 153 (<20%) of the 856 transcripts were downregulated. The other 703 transcripts were either upregulated or showed no significant change. Additionally, the 682 insignificant TF genes (GE values < absolute 2-fold changes and/or FDR p-values > 0.05) between ES and TS cells expressed 2215 transcript variants. These included 477 (>21%) differentially expressed transcripts (276 upregulated and 201 downregulated, ≥absolute 2-fold changes, FDR p-value ≤ 0.05). Hence, GE based RNA-Seq analyses do not represent accurate expression levels due to divergent transcripts expression from the same gene. Our findings show that by including transcript variants in RNA-Seq analyses, we can generate a precise understanding of a gene's functional and regulatory landscape; ignoring the variants may result in an erroneous interpretation.

Asunto(s)

Perfilación de la Expresión Génica , Transcriptoma , Animales , Ratones , Transcriptoma/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Trofoblastos/metabolismo , Análisis de Secuencia de ARN , Empalme Alternativo/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo

RESUMEN

Increased activation of ovarian primordial follicles in Erß knockout (ErßKO) rats becomes evident as early as postnatal day 8.5. To identify the ERß-regulated genes that may control ovarian primordial follicle activation, we analyzed the transcriptome profiles of ErßKO rat ovaries collected on postnatal days 4.5, 6.5, and 8.5. Compared to wildtype ovaries, ErßKO ovaries displayed dramatic downregulation of Indian hedgehog (Ihh) expression. IHH-regulated genes, including Hhip, Gli1, and Ptch1, were also downregulated in ErßKO ovaries. This was associated with a downregulation of steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1. The expression of Ihh remained very low in ErßKO ovaries despite the high levels of Gdf9 and Bmp15, which are known upregulators of Ihh expression in the granulosa cells of activated ovarian follicles. Strikingly, the downregulation of the Ihh gene in ErßKO ovaries began to disappear on postnatal day 16.5 and recovered on postnatal day 21.5. In rat ovaries, the first wave of primordial follicles is rapidly activated after their formation, whereas the second wave of primordial follicles remains dormant in the ovarian cortex and slowly starts activating after postnatal day 12.5. We localized the expression of Ihh mRNA in postnatal day 8.5 wildtype rat ovaries but not in the age-matched ErßKO ovaries. In postnatal day 21.5 ErßKO rat ovaries, we detected Ihh mRNA mainly in the activated follicles in the ovaries' peripheral regions. Our findings indicate that the expression of Ihh in the granulosa cells of the activated first wave of ovarian follicles depends on ERß.

Asunto(s)

Receptor beta de Estrógeno , Proteínas Hedgehog , Animales , Femenino , Ratas , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Folículo Ovárico/metabolismo , Ovario/metabolismo , ARN Mensajero/metabolismo

RESUMEN

Loss of ERß increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERß in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erß knockout (Erßnull) mouse ovaries, and their transcriptomes were compared with those of control Erßfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erßnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERß has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erßnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erßfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erß-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erßnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erßnull PrFs were downregulated. Such deregulation of genes in Erßnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erßnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERß either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.

Asunto(s)

Receptor beta de Estrógeno , Folículo Ovárico , Femenino , Ratones , Animales , Receptor beta de Estrógeno/metabolismo , Folículo Ovárico/metabolismo , Ovario/metabolismo , Regulación de la Expresión Génica , Transcriptoma , Ratones Noqueados

RESUMEN

Kisspeptins (KPs) secreted from the hypothalamic KP neurons act on KP receptors (KPRs) in gonadotropin (GPN) releasing hormone (GnRH) neurons to produce GnRH. GnRH acts on pituitary gonadotrophs to induce secretion of GPNs, namely follicle stimulating hormone (FSH) and luteinizing hormone (LH), which are essential for ovarian follicle development, oocyte maturation and ovulation. Thus, hypothalamic KPs regulate oocyte maturation indirectly through GPNs. KPs and KPRs are also expressed in the ovarian follicles across species. Recent studies demonstrated that intraovarian KPs also act directly on the KPRs expressed in oocytes to promote oocyte maturation and ovulation. In this review article, we have summarized published reports on the role of hypothalamic and ovarian KP-signaling in oocyte maturation. Gonadal steroid hormones regulate KP secretion from hypothalamic KP neurons, which in turn induces GPN secretion from the hypothalamic-pituitary (HP) axis. On the other hand, GPNs secreted from the HP axis act on the granulosa cells (GCs) and upregulate the expression of ovarian KPs. While KPs are expressed predominantly in the GCs, the KPRs are in the oocytes. Expression of KPs in the ovaries increases with the progression of the estrous cycle and peaks during the preovulatory GPN surge. Intrafollicular KP levels in the ovaries rise with the advancement of developmental stages. Moreover, loss of KPRs in oocytes in mice leads to failure of oocyte maturation and ovulation similar to that of premature ovarian insufficiency (POI). These findings suggest that GC-derived KPs may act on the KPRs in oocytes during their preovulatory maturation. In addition to the intraovarian role of KP-signaling in oocyte maturation, in vivo, a direct role of KP has been identified during in vitro maturation of sheep, porcine, and rat oocytes. KP-stimulation of rat oocytes, in vitro, resulted in Ca2+ release and activation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1 and 2. In vitro treatment of rat or porcine oocytes with KPs upregulated messenger RNA levels of the factors that favor oocyte maturation. In clinical trials, human KP-54 has also been administered successfully to patients undergoing assisted reproductive technologies (ARTs) for increasing oocyte maturation. Exogenous KPs can induce GPN secretion from hypothalamus; however, the possibility of direct KP action on the oocytes cannot be excluded. Understanding the direct in vivo and in vitro roles of KP-signaling in oocyte maturation will help in developing novel KP-based ARTs.

Asunto(s)

Kisspeptinas , Oogénesis , Animales , Femenino , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Kisspeptinas/metabolismo , Hormona Luteinizante/metabolismo , Ratones , Oocitos/fisiología , Ratas , Ovinos , Porcinos

RESUMEN

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca2+) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1lKO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1lKO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca2+ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca2+ influx by TRPC2, Dot1lKO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca2+ influx. Such heightened Ca2+ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.

Asunto(s)

Calcio , Eritropoyetina , N-Metiltransferasa de Histona-Lisina , Animales , Calcio/metabolismo , Calcio de la Dieta , Epoetina alfa , Células Precursoras Eritroides/metabolismo , Eritropoyesis , Eritropoyetina/metabolismo , Eritropoyetina/farmacología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Ratones , Receptores de Eritropoyetina/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6

RESUMEN

DOT1L is essential for embryonic hematopoiesis but the precise mechanisms of its action remain unclear. The only recognized function of DOT1L is histone H3 lysine 79 (H3K79) methylation, which has been implicated in both transcriptional activation and repression. We observed that deletion of the mouse Dot1L gene (Dot1L-KO) or selective mutation of its methyltransferase domain (Dot1L-MM) can differentially affect early embryonic erythropoiesis. However, both mutations result in embryonic lethality by mid-gestation and growth of hematopoietic progenitor cells (HPCs) is similarly affected in extensively self-renewing erythroblast (ESRE) cultures established from yolk sac cells. To understand DOT1L-mediated gene regulation and to clarify the role of H3K79 methylation, we analyzed whole transcriptomes of wildtype and Dot1L-mutant ESRE cells. We observed that more than 80% of the differentially expressed genes (DEGs) were upregulated in the mutant ESRE cells either lacking the DOT1L protein or the DOT1L methyltransferase activity. However, approximately 45% of the DEGs were unique to either mutant group, indicating that DOT1L possesses both methyltransferase-dependent and -independent gene regulatory functions. Analyses of Gene Ontology and signaling pathways for the DEGs were consistent, with DEGs that were found to be common or unique to either mutant group. Genes related to proliferation of HPCs were primarily impacted in Dot1L-KO cells, while genes related to HPC development were affected in the Dot1L-MM cells. A subset of genes related to differentiation of HPCs were affected in both mutant groups of ESREs. Our findings suggest that DOT1L primarily acts to repress gene expression in HPCs, and this function can be independent of its methyltransferase activity.

RESUMEN

SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages that emerge during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain the trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated an active state of the promoter. A novel cis-regulatory region with active histone marks was identified ∼21 kbp upstream of the variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromosome conformation capture analysis confirmed looping of this distant regulatory region into the proximal promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close proximity to SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.

RESUMEN

O-GlcNAcylation is a dynamic post-translational modification where the sugar, O-linked ß-N-acetylglucosamine (O-GlcNAc) is added to or removed from various cytoplasmic, nuclear, and mitochondrial proteins. This modification is regulated by only two enzymes: O-GlcNAc transferase (OGT), which adds O-GlcNAc, and O-GlcNAcase (OGA), which removes the sugar from proteins. O-GlcNAcylation is integral to maintaining normal cellular function, especially in processes such as nutrient sensing, metabolism, transcription, and growth and development of the cell. Aberrant O-GlcNAcylation has been associated with a number of pathological conditions, including, neurodegenerative diseases, cancer, diabetes, and obesity. However, the role of O-GlcNAcylation in immune cell growth/proliferation, or other immune responses, is currently incompletely understood. In this review, we highlight the effects of O-GlcNAcylation on certain cells of the immune system, especially those involved in pro-inflammatory responses associated with diabetes and obesity.

Asunto(s)

Acetilglucosamina/inmunología , Sistema Inmunológico/inmunología , Animales , Diabetes Mellitus/genética , Diabetes Mellitus/inmunología , Humanos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/inmunología , Obesidad/genética , Obesidad/inmunología

RESUMEN

Interleukin 33 (IL33) signaling has been implicated in the establishment and maintenance of pregnancy and in pregnancy disorders. The goal of this project was to evaluate the role of IL33 signaling in rat pregnancy. The rat possesses hemochorial placentation with deep intrauterine trophoblast invasion; features also characteristic of human placentation. We generated and characterized a germline mutant rat model for IL33 using CRISPR/Cas9 genome editing. IL33 deficient rats exhibited deficits in lung responses to an inflammatory stimulus (Sephadex G-200) and to estrogen-induced uterine eosinophilia. Female rats deficient in IL33 were fertile and exhibited pregnancy outcomes (gestation length and litter size) similar to wild-type rats. Placental weight was adversely affected by the disruption of IL33 signaling. A difference in pregnancy-dependent adaptations to lipopolysaccharide (LPS) exposure was observed between wild-type and IL33 deficient pregnancies. Pregnancy in wild-type rats treated with LPS did not differ significantly from pregnancy in vehicle-treated wild-type rats. In contrast, LPS treatment decreased fetal survival rate, fetal and placental weights, and increased fetal growth restriction in IL33 deficient rats. In summary, a new rat model for investigating IL33 signaling has been established. IL33 signaling participates in the regulation of placental development and protection against LPS-induced fetal and placental growth restriction.

Asunto(s)

Retardo del Crecimiento Fetal/metabolismo , Interleucina-33/metabolismo , Enfermedades Placentarias/metabolismo , Complicaciones Infecciosas del Embarazo/metabolismo , Transducción de Señal , Animales , Femenino , Retardo del Crecimiento Fetal/etiología , Retardo del Crecimiento Fetal/patología , Interleucina-33/genética , Lipopolisacáridos/toxicidad , Mutación , Enfermedades Placentarias/etiología , Enfermedades Placentarias/patología , Embarazo , Complicaciones Infecciosas del Embarazo/etiología , Complicaciones Infecciosas del Embarazo/patología , Resultado del Embarazo , Ratas , Ratas Sprague-Dawley

RESUMEN

DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

RESUMEN

Investigating a protein of interest that runs at the same molecular weight as antibody heavy chain is a frequent deterrent to its evaluation by immunoprecipitation. Methods of minimizing the detection of the immunoprecipitating antibody are available. However, these still present a barrier to evaluating if intracellular proteins are modified by the O-GlcNAc post-translation protein modification due to interfering glycosylation on antibodies. IdeZ protease specifically cleaves antibody at the hinge region, allowing collapse of the antibody fragments to 25 kDa after denaturation. Thus, this proteolytic method uniquely allows evaluation of O-GlcNAcylation of proteins of interest formerly obscured by antibody heavy chain.

Asunto(s)

Acetilglucosamina/química , Cadenas Pesadas de Inmunoglobulina/química , Péptido Hidrolasas/química , Procesamiento Proteico-Postraduccional , Proteolisis , Acetilglucosamina/análisis , Glicosilación , Humanos , Cadenas Pesadas de Inmunoglobulina/análisis , Inmunoprecipitación

RESUMEN

Chronic, low-grade inflammation increases the risk for atherosclerosis, cancer, and autoimmunity in diseases such as obesity and diabetes. Levels of CD4+ T helper 17 (Th17) cells, which secrete interleukin 17A (IL-17A), are increased in obesity and contribute to the inflammatory milieu; however, the relationship between signaling events triggered by excess nutrient levels and IL-17A-mediated inflammation is unclear. Here, using cytokine, quantitative real-time PCR, immunoprecipitation, and ChIP assays, along with lipidomics and MS-based approaches, we show that increased levels of the nutrient-responsive, post-translational protein modification, O-GlcNAc, are present in naive CD4+ T cells from a diet-induced obesity murine model and that elevated O-GlcNAc levels increase IL-17A production. We also found that increased binding of the Th17 master transcription factor RAR-related orphan receptor γ t variant (RORγt) at the IL-17 gene promoter and enhancer, as well as significant alterations in the intracellular lipid microenvironment, elevates the production of ligands capable of increasing RORγt transcriptional activity. Importantly, the rate-limiting enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase 1 (ACC1), is O-GlcNAcylated and necessary for production of these RORγt-activating ligands. Our results suggest that increased O-GlcNAcylation of cellular proteins may be a potential link between excess nutrient levels and pathological inflammation.

Asunto(s)

Ácidos Grasos/biosíntesis , Interleucina-17/metabolismo , Células Th17/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Acilación/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Ácidos Grasos/análisis , Femenino , Humanos , Interleucina-17/genética , Lipidómica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Obesidad/metabolismo , Obesidad/patología , Regiones Promotoras Genéticas , Unión Proteica , Piranos/farmacología , Células Th17/citología , Tiazoles/farmacología , Activación Transcripcional/efectos de los fármacos

RESUMEN

Basal-like breast cancers are an aggressive breast cancer subtype, which often lack estrogen receptor, progesterone receptor, and Her2 expression, and are resistant to antihormonal and targeted therapy, resulting in few treatment options. Understanding the underlying mechanisms that regulate progression of basal-like breast cancers would lead to new therapeutic targets and improved treatment strategies. Breast cancer progression is characterized by inflammatory responses, regulated in part by chemokines. The CCL2/CCR2 chemokine pathway is best known for regulating breast cancer progression through macrophage-dependent mechanisms. Here, we demonstrated important biological roles for CCL2/CCR2 signaling in breast cancer cells. Using the MCF10CA1d xenograft model of basal-like breast cancer, primary tumor growth was significantly increased with cotransplantation of patient-derived fibroblasts expressing high levels of CCL2, and was inhibited with CRISP/R gene ablation of stromal CCL2. CRISP/R gene ablation of CCR2 in MCF10CA1d breast cancer cells inhibited breast tumor growth and M2 macrophage recruitment and validated through CCR2 shRNA knockdown in the 4T1 model. Reverse phase protein array analysis revealed that cell-cycle protein expression was associated with CCR2 expression in basal-like breast cancer cells. CCL2 treatment of basal-like breast cancer cell lines increased proliferation and cell-cycle progression associated with SRC and PKC activation. Through pharmacologic approaches, we demonstrated that SRC and PKC negatively regulated expression of the cell-cycle inhibitor protein p27KIP1, and are necessary for CCL2-induced breast cancer cell proliferation. IMPLICATIONS: This report sheds novel light on CCL2/CCR2 chemokine signaling as a mitogenic pathway and cell-cycle regulator in breast cancer cells.

Asunto(s)

Neoplasias de la Mama/metabolismo , Proteína Quinasa C/metabolismo , Receptores CCR2/metabolismo , Familia-src Quinasas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Quimiocina CCL2/metabolismo , Progresión de la Enfermedad , Activación Enzimática , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Receptores CCR2/genética , Transducción de Señal

RESUMEN

Identifying novel pathways that promote robust function and longevity of cytotoxic T cells has promising potential for immunotherapeutic strategies to combat cancer and chronic infections. We show that sprouty 1 and 2 (Spry1/2) molecules regulate the survival and function of memory CD8+ T cells. Spry1/2 double-knockout (DKO) ovalbumin (OVA)-specific CD8+ T cells (OT-I cells) mounted more vigorous autoimmune diabetes than WT OT-I cells when transferred to mice expressing OVA in their pancreatic ß-islets. To determine the consequence of Spry1/2 deletion on effector and memory CD8+ T cell development and function, we used systemic infection with lymphocytic choriomeningitis virus (LCMV) Armstrong. Spry1/2 DKO LCMV gp33-specific P14 CD8+ T cells survive contraction better than WT cells and generate significantly more polyfunctional memory T cells. The larger number of Spry1/2 DKO memory T cells displayed enhanced infiltration into infected tissue, demonstrating that absence of Spry1/2 can result in increased recall capacity. Upon adoptive transfer into naive hosts, Spry1/2 DKO memory T cells controlled Listeria monocytogenes infection better than WT cells. The enhanced formation of more functional Spry1/2 DKO memory T cells was associated with significantly reduced mTORC1 activity and glucose uptake. Reduced p-AKT, p-FoxO1/3a, and T-bet expression was also consistent with enhanced survival and memory accrual. Collectively, loss of Spry1/2 enhances the survival of effector CD8+ T cells and results in the formation of more protective memory cells. Deleting Spry1/2 in antigen-specific CD8+ T cells may have therapeutic potential for enhancing the survival and functionality of effector and memory CD8+ T cells in vivo.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/inmunología , Linfocitos T CD8-positivos/fisiología , Memoria Inmunológica/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Activación de Linfocitos/genética , Proteínas de la Membrana/inmunología , Fosfoproteínas/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Traslado Adoptivo/métodos , Animales , Linfocitos T CD8-positivos/trasplante , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/orina , Modelos Animales de Enfermedad , Femenino , Humanos , Memoria Inmunológica/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Listeria monocytogenes/inmunología , Listeria monocytogenes/aislamiento & purificación , Listeriosis/inmunología , Listeriosis/microbiología , Listeriosis/terapia , Activación de Linfocitos/inmunología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas , Quimera por Trasplante

RESUMEN

The rapidly expanding field of immunometabolism focuses on how metabolism controls the function of immune cells. CD4+ T cells are essential for the adaptive immune response leading to the eradication of specific pathogens. However, when T cells are inappropriately over-active, they can drive autoimmunity, allergic disease, and chronic inflammation. The mechanisms by which metabolic changes influence function in CD4+ T cells are not fully understood. The post-translational protein modification, O-GlcNAc (O-linked ß-N-acetylglucosamine), dynamically cycles on and off of intracellular proteins as cells respond to their environment and flux through metabolic pathways changes. As the rate of O-GlcNAc cycling fluctuates, protein function, stability, and/or localization can be affected. Thus, O-GlcNAc is critically poised at the nexus of cellular metabolism and function. This review highlights the intra- and extracellular metabolic factors that influence CD4+ T cell activation and differentiation and how O-GlcNAc regulates these processes. We also propose areas of future research that may illuminate O-GlcNAc's role in the plasticity and pathogenicity of CD4+ T cells and uncover new potential therapeutic targets.

Asunto(s)

Acetilglucosamina/inmunología , Procesamiento Proteico-Postraduccional/inmunología , Acetilglucosamina/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Humanos , Activación de Linfocitos/inmunología

RESUMEN

Ductal carcinoma in situ (DCIS) is the most common form of breast cancer, with 50,000 cases diagnosed every year in the United States. Overtreatment and undertreatment remain significant clinical challenges in patient care. Identifying key mechanisms associated with DCIS progression could uncover new biomarkers to better predict patient prognosis and improve guided treatment. Chemokines are small soluble molecules that regulate cellular homing through molecular gradients. CCL2-mediated recruitment of CCR2+ macrophages are a well-established mechanism for metastatic progression. Although the CCL2/CCR2 pathway is a therapeutic target of interest, little is known about the role of CCR2 expression in breast cancer. Here, using a mammary intraductal injection (MIND) model to mimic DCIS formation, the role of CCR2 was explored in minimally invasive SUM225 and highly invasive DCIS.com breast cancer cells. CCR2 overexpression increased SUM225 breast cancer survival and invasion associated with accumulation of CCL2 expressing fibroblasts. CCR2-deficient DCIS.com breast cancer cells formed fewer invasive lesions with fewer CCL2+ fibroblasts. Cografting CCL2-deficient fibroblasts with DCIS.com breast cancer cells in the subrenal capsule model inhibited tumor invasion and survival associated with decreased expression of aldehyde dehydrogenase (ALDH1), a proinvasive factor, and decreased expression of HTRA2, a proapoptotic serine protease. Through data mining analysis, high expression of CCR2 and ALDH1 and low HTRA2 expression were correlated with poor prognosis of breast cancer patients.Implications: This study demonstrates that CCR2 overexpression in breast cancer drives early-stage breast cancer progression through stromal-dependent expression of CCL2 with important insight into prognosis and treatment of DCIS. Mol Cancer Res; 16(2); 296-308. ©2017 AACR.

Asunto(s)

Neoplasias de la Mama/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Quimiocina CCL2/metabolismo , Fibroblastos/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Progresión de la Enfermedad , Femenino , Fibroblastos/citología , Fibroblastos/patología , Regulación Neoplásica de la Expresión Génica , Serina Peptidasa A2 que Requiere Temperaturas Altas/metabolismo , Humanos , Isoenzimas/metabolismo , Ratones , Invasividad Neoplásica , Estadificación de Neoplasias , Trasplante de Neoplasias , Pronóstico , Retinal-Deshidrogenasa/metabolismo , Transducción de Señal , Análisis de Supervivencia , Células Tumorales Cultivadas

RESUMEN

GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure.

Asunto(s)

Factor de Transcripción GATA2/fisiología , Factor de Transcripción GATA3/fisiología , Placenta/fisiología , Células Madre/citología , Trofoblastos/citología , Animales , Diferenciación Celular , Linaje de la Célula , Implantación del Embrión , Desarrollo Embrionario , Femenino , Eliminación de Gen , Humanos , Ratones , Ratones Noqueados , Embarazo , Preñez , Análisis de Secuencia de ARN

RESUMEN

Perfluorononanoic acid (PFNA) is a perfluoroalkyl substance (PFAS) that is structurally related to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Whereas PFOA and PFOS are known immunotoxicants, PFNA is less well characterized. Our previous study showed that PFNA has immunomodulatory effects on leukocyte populations and immune function. The present studies sought to determine whether, and to what degree, the immune system recovered 28 days after PFNA exposure. None of the parameters measured had fully recovered. A few parameters had partially recovered, including decreased spleen size and the decreased ratio of the CD4+/CD8+ double-positive population in thymus. The majority of effects of PFNA remained unchanged 28 days after exposure, including decreased proportion of intact thymocytes (as determined by FSC vs SSC), alterations in the ratios of immune cell populations in spleen and the CD4+, CD8+ and double-negative populations in thymus. Notably, PFNA markedly increased the TNFα response to LPS in vivo, and no recovery was evident 28 days after exposure. The effect of PFNA on CD4+ T cells, CD8+ T cells and CD19+ cells was more pronounced in females. The current study demonstrates that a single high dose exposure to PFNA (e.g. as might occur accidentally in an occupational setting) has long-lasting effects on the immune system.

Asunto(s)

Fluorocarburos/farmacología , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/inmunología , Tamaño de los Órganos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Ácidos Grasos , Femenino , Citometría de Flujo , Fluorocarburos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Bazo/efectos de los fármacos , Timocitos/efectos de los fármacos

RESUMEN

Polycystic kidney disease (PKD) is characterized by slow expansion of fluid-filled cysts derived from tubules within the kidney. Cystic expansion results in injury to surrounding parenchyma and leads to inflammation, scarring and ultimately loss of renal function. Macrophages are a key element in this process, promoting cyst epithelial cell proliferation, cyst expansion and disease progression. Previously, we have shown that the microenvironment established by cystic epithelial cells can 'program' macrophages, inducing M2-like macrophage polarization that is characterized by expression of markers that include Arg1 and Il10 Here, we functionally characterize these macrophages, demonstrating that their differentiation enhances their ability to promote cyst cell proliferation. This observation indicates a model of reciprocal pathological interactions between cysts and the innate immune system: cyst epithelial cells promote macrophage polarization to a phenotype that, in turn, is especially efficient in promoting cyst cell proliferation and cyst growth. To better understand the genesis of this macrophage phenotype, we examined the role of IL-10, a regulatory cytokine shown to be important for macrophage-stimulated tissue repair in other settings. Herein, we show that the acquisition of the pathological macrophage phenotype requires IL-10 secretion by the macrophages. Further, we demonstrate a requirement for IL-10-dependent autocrine activation of the STAT3 pathway. These data suggest that the IL-10 pathway in macrophages plays an essential role in the pathological relationship between cysts and the innate immune system in PKD, and thus could be a potential therapeutic target.

Asunto(s)

Comunicación Autocrina , Diferenciación Celular , Interleucina-10/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Enfermedades Renales Poliquísticas/patología , Factor de Transcripción STAT3/metabolismo , Comunicación Autocrina/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Líquido Quístico/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Fenotipo , Enfermedades Renales Poliquísticas/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

otrganic perfluorochemicals (PFCs) have become an environmental concern due to widespread detection in human blood and experimental evidence for immune, developmental, and liver toxicity. Whereas the blood concentrations of many PFCs are declining, blood levels of Perfluorononanoic Acid (PFNA) are rising in the United States. The purpose of the present studies was to determine the effects of PFNA on lymphoid organs and immune cells of C57BL/6 mice. The present study demonstrates that PFNA produces immunotoxic effects in both male and female C57BL/6 mice as evidenced by splenic atrophy, decreased splenocyte numbers, and a marked reduction in thymocyte viability. The current study also demonstrates that the effects of PFNA on different leukocyte populations are not uniform. The CD4+CD8+ double-positive thymocytes were particularly sensitive to PFNA in which the proportion of this population was >95% decreased relative to the entire CD4+ thymocyte population in PFNA-treated mice. Interestingly, PFNA also markedly increased serum levels of TNFα in response to LPS in mice. Collectively, the present studies demonstrate that PFNA decreases lymphocyte viability and alters the immune response to LPS in C57BL/6 mice.