RESUMO
Maternal diabetes increases the risk of embryo resorptions and impairs embryo development. Decidualization is crucial for embryo development and regulated by mTOR signaling. However, little is known about how maternal diabetes affects the decidua at early postimplantation stages and whether dietary treatments enriched in polyunsaturated fatty acids (PUFAs) can prevent decidual alterations. Here, we determined resorption rates, decidual mTOR pathways and markers of decidual function and remodeling in diabetic rats fed or not with diets enriched in PUFAs exclusively during the early postimplantation period. Pregestational streptozotocin-induced diabetic Albino Wistar rats and controls were fed or not with diets enriched in 6% sunflower oil or 6% chia oil (enriched in n-6 or n-3 PUFAs, respectively) on days 7, 8 and 9 of pregnancy and evaluated on day 9 of pregnancy. Maternal diabetes induced an 11-fold increase in embryo resorptions, which was prevented by both PUFAs-enriched diets despite no changes in maternal glycemia. The activity of mTOR pathway was decreased in the decidua from diabetic rats, an alteration prevented by the PUFAs-enriched diets. PUFAs-enriched diets prevented increased expression of Foxo1 (a negative regulator of mTOR) and reduced expression of miR-21 (a negative regulator of Foxo1). These diets also prevented reduced markers of decidual function (leukemia inhibitory factor and IGFBP1 expression and MMPs activity) in diabetic rat decidua. We identified the early post implantation as a crucial stage for pregnancy success, in which dietary PUFAs can protect diabetic pregnancies from embryo resorptions, decidual mTOR signaling impairments, and altered markers of decidual function and remodeling.
Assuntos
Decídua/metabolismo , Gorduras na Dieta/administração & dosagem , Perda do Embrião/prevenção & controle , Ácidos Graxos Insaturados/farmacologia , Fenômenos Fisiológicos da Nutrição Pré-Natal , Serina-Treonina Quinases TOR/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Glicemia , Decídua/efeitos dos fármacos , Ácidos Graxos Insaturados/administração & dosagem , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Serina-Treonina Quinases TOR/genéticaRESUMO
To investigate the influence of Kuntai capsules on the expression level of leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-1)and epidermal growth factor (EGF) during the mouse's implantation window of superovulation period and controlled ovarian hyperstimulation period. 90 female mice were randomly divided into six groups in control, superovulation and controlled ovarian hyperstimulation (COH) conditions. The RNA expression of EGF, LIF and IGF-1 in the endometrium on the 4th day of pregnancy was detected, and the relative expression was compared. mRNA expression of these three factors in endometrium was significantly lower in superovulation and COH groups than control group (p<0.001). mRNA expression of these three factors in endometrium remained obviously lower in superovulation plus kuntai capsule group and COH plus kuntai capsule group than control group (p<0.01). mRNA expression of these three factors in endometrium was lower in control group than in the NS plus kuntai capsule group (p<0.05). Kuntai capsule cannot completely reverse the endometrial damages caused by superovulation and COH. Thus Kuntai capsule could partially improve a mouse's endometrial receptivity during the implantation window.
Para investigar la influencia de las cápsulas de Kuntai en el nivel de expresión del factor inhibidor de la leucemia (LIF), el factor de crecimiento similar a la insulina I (IGF-1) y el factor de crecimiento epidérmico (EGF) durante la ventana de implantación del ratón del período de superovulación y la hiperestimulación ovárica controlada período, se dividieron aleatoriamente 90 ratones hembra en seis grupos en condiciones de control, superovulación e hiperestimulación ovárica controlada (COH). Se detectó la expresión de ARN de EGF, LIF e IGF-1en el endometrio al cuarto día de embarazo, y se comparó la expresión relativa. La expresión de ARNm de estos tres factores en el endometrio fue significativamente menor en los grupos de superovulación y COH que en el grupo control (p<0,001). La expresión de ARNm de estos tres factores en el endometrio permaneció más baja en el grupo de cápsulas de superovulación más Kuntai y en el grupo de cápsulas de COH más Kuntai respecto del grupo control (p<0,01). La expresión de ARNm de estos tres factores en el endometrio fue menor en el grupo control que en el grupo de cápsula NS más Kuntai (p<0,05). La cápsula de Kuntai no pudo revertir completamente los daños endometriales causados por la superovulación y la COH. Por lo tanto, se sugiere que la cápsula de Kuntai podría mejorar parcialmente la receptividad endometrial de un ratón durante la ventana de implantación.
Assuntos
Animais , Feminino , Camundongos , Indução da Ovulação/métodos , Somatomedinas/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Fator de Crescimento Epidérmico/efeitos dos fármacos , Fator Inibidor de Leucemia/efeitos dos fármacos , Implantação do Embrião , Superovulação , Somatomedinas/genética , Somatomedinas/metabolismo , Cápsulas , Reação em Cadeia da Polimerase/métodos , Eletroforese , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismoRESUMO
Omega-3 (n-3) fatty acids modulate epigenetic changes critical to genesis and differentiation of neural cells. Conversely, maternal protein-malnutrition can negatively modify these changes. This study investigated whether a low n-6/n-3 ratio in a maternal diet could favor histone-3 (H3) modifications, gene transcription and differentiation in the offspring neural cells even under protein-deficiency. Female rats fed a control (Ct), or 3 types of multideficient diets differing in protein levels or linoleic/alpha-linolenic fatty acid ratios (RBD, RBD-C, RBD-SO) from 30 days prior to mating and during pregnancy. Cerebral cortex tissue and cortical cultures of progeny embryonic neurons and postnatal astrocytes were analyzed. H3K9 acetylation and H3K27 or H3K4 di-methylation levels were assessed by flow cytometry and/or immunocytochemistry. In astrocyte cultures and cortical tissue, the GFAP protein levels were assessed. Glial derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) gene expression were evaluated in the cortical tissue. GFAP levels were similar in astrocytes of Ct, RBD and RBD-C, but 65% lower in RBD-SO group. Higher levels of H3K9Ac were found in the neurons and H3K4Me2 in the astrocytes of the RBD group. No intergroup difference in the cortical GDNF mRNA expression or the H3K27Me2 levels in astrocytes was detected. LIF mRNA levels were higher in the RDB (P=.002) or RBD-C (P=.004) groups than in the control. The findings indicate the importance of dietary n-3 availability for the brain, even under a protein-deficient condition, inducing Histone modifications and increasing LIF gene transcription, involved in neural cell differentiation and reactivity.
Assuntos
Astrócitos/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Histonas/metabolismo , Fator Inibidor de Leucemia/genética , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Proteínas Alimentares/administração & dosagem , Epigênese Genética , Ácidos Graxos/análise , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteína Glial Fibrilar Ácida/metabolismo , Histonas/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Materna , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Gravidez , RatosRESUMO
Certain gene polymorphisms are associated with implantation failure and pregnancy loss. Studies of leukaemia inhibitory factor (LIF) gene polymorphisms are scarce. The LIF single nucleotide polymorphism (SNP) thymine (T)/guanine (G) (rs929271) was studied in women to determine whether an association existed with pregnancy outcomes after intracytoplasmic sperm injection (ICSI); 411 women who underwent ICSI were recruited. DNA was extracted from the peripheral blood, and the LIF gene SNP T/G (rs929271) was genotyped using real-time polymerase chain reaction. Participants were divided into three groups according to their LIF genotype: T/T (n = 168), T/G (n = 202) and G/G (n = 41). All IVF and ICSI procedures were carried out under the same clinical and laboratory conditions. The ICSI cumulative results (from fresh plus frozen cycles) of each genotype group were analysed. The G/G genotype in women was associated with a higher implantation rate (T/T: 15.9%, T/G: 16.2%, G/G: 27.0%; P < 0.05), ongoing pregnancy rate/patient (T/T: 31.5%, T/G: 36.1%, G/G: 53.7%; P < 0.05) and ongoing pregnancy rate/transfer (T/T: 18.5%, T/G: 20.2%, G/G: 36.7%; P < 0.05). LIF SNP T/G (rs929271) seems to be a susceptibility biomarker capable of predicting implantation efficiency and pregnancy outcomes.
Assuntos
Fator Inibidor de Leucemia/genética , Polimorfismo de Nucleotídeo Único , Resultado da Gravidez/genética , Técnicas de Reprodução Assistida , Adulto , Estudos de Casos e Controles , Implantação do Embrião/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Infertilidade/epidemiologia , Infertilidade/genética , Infertilidade/terapia , Pessoa de Meia-Idade , Gravidez , Resultado da Gravidez/epidemiologia , Taxa de Gravidez , Técnicas de Reprodução Assistida/estatística & dados numéricosRESUMO
OBJECTIVE: The aim of this study was to investigate the relationship between herpesvirus-associated ubiquitin-specific protease (HAUSP A/G, rs1529916), tumor protein p53 (TP53 Arg/Pro, rs1042522), leukemia inhibitory factor (LIF G/T, rs929271), glycoprotein 130 (gp130 A/T, rs1900173) and vascular endothelial growth factor (VEGF G/A, rs1570360) polymorphisms and recurrent implantation failure (RIF) in Brazilian women. SUBJECTS AND METHODS: A total of 120 women with RIF (i.e. those with ≥5 cleaved embryos transferred and a minimum of 2 failed in vitro fertilization/intracytoplasmic sperm injection attempts) were included. The control group involved 89 women who had experienced at least 1 live birth (without any infertility treatment). DNA was extracted from the peripheral blood of all participants, and the abovementioned single-nucleotide polymorphisms (SNPs) were genotyped by real-time polymerase chain reaction. The data were evaluated using Fisher's test. RESULTS: A significant difference between the RIF and control groups was found in the VEGF gene where the GG genotype showed a 2.1-fold increased chance of not being included in the RIF group, while the presence of an A allele increased this risk 1.6-fold. No significant differences were found for the other polymorphisms. CONCLUSION: This study showed an association between the VEGF -1154G/A polymorphism and RIF in Brazilian women.
Assuntos
Aborto Habitual/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Alelos , Brasil/epidemiologia , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genes p53/genética , Glicoproteínas/genética , Haplótipos , Humanos , Fator Inibidor de Leucemia/genética , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de UbiquitinaRESUMO
We used a simple and efficient method to construct a bicistronic eukaryotic expression vector pIRES2-LIF-NT-3. The leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3) genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The LIF cDNA fragment was inserted into the multiple cloning sites of a vector containing internal ribosome entry site and enhanced green fluorescent protein (EGFP) (pIRES2-EGFP) to generate the bicistronic eukaryotic expression plasmid pIRES2-LIF-EGFP. Next, the NT-3 cDNA fragment was cloned into pIRES2-LIF-EGFP in place of EGFP to create the plasmid pIRES2-LIF-NT-3. pIRES2-LIF-NT-3 was transfected into HEK293 cells and reverse transcription-polymerase chain reaction and Western blotting were used to test the co-expression of double genes. LIF and NT-3 genes were cloned and the DNA was sequenced. Sequencing analysis revealed that LIF and NT-3 were consistent with the sequence recorded in GenBank. Restriction analysis indicated that the LIF and NT-3 genes were inserted correctly into the expression vector pIRES2-EGFP. Following transfection of pIRES2-LIF-NT-3 into HEK293 cells, the double gene was expressed at the mRNA and protein levels. The LIF and NT-3 coexpression plasmid is a novel expression system that will enable further study of the functions of the LIF and NT-3 genes.
Assuntos
Clonagem Molecular/métodos , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Neurotrofina 3 , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Lipopolysaccharide (LPS) administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF), which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.
Assuntos
Anti-Inflamatórios/farmacologia , Perda do Embrião/induzido quimicamente , Perda do Embrião/prevenção & controle , Fator Inibidor de Leucemia/metabolismo , Lipopolissacarídeos/farmacologia , Progesterona/farmacologia , Animais , Anti-Inflamatórios/sangue , Suplementos Nutricionais , Perda do Embrião/sangue , Perda do Embrião/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Mifepristona/farmacologia , Óxido Nítrico/metabolismo , Gravidez , Progesterona/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Progesterona/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.
Assuntos
Clonagem Molecular/métodos , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Genoma Humano/genética , Fator Inibidor de Leucemia/genética , Mucosa Bucal/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Éxons/genética , Humanos , Fator Inibidor de Leucemia/química , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Murine embryonic stem cells (muESC) are maintained and expanded in vitro by culturing in the presence of leukemia inhibitory factor (LIF) or by coculturing on murine embryonic fibroblast (MEF). Previously we have shown that liver sinusoidal endothelial cells (LSEC) promote the survival, proliferation and differentiation of hematopoietic stem cells. In the present study we investigated whether LSEC might promote the survival and undifferentiated growth of muESC. For these purposes, muESC (CGR8 cell line) were cultured on LSEC monolayers (muESC/LSEC) or in the presence of conditioned medium from LSEC cultures (muESC/LSEC-CM), both in the absence of LIF. Microscopic observation showed the growth of undifferentiated ESC colonies in both muESC/LSEC or muESC/LSEC-CM cultures. A significant reduction in the growth of undifferentiated ESC colonies was observed when ESC were cultured in LSEC-CM previously incubated with anti-LIF. RT-PCR and Western blot analysis showed that LSEC constitutively express LIF at the mRNA and protein level. At different times of culture, muESC were harvested and analyzed for the expression of embryonic markers (SSEA-1 and Oct-4) and differentiation capacity. Flow cytometry analysis showed the presence of a higher percentage of muESC (>90%) expressing SSEA-1 in muESC/LSEC-CM, as compared with muESC/LSEC cocultures. muESC obtained from both types of cultures formed embryoid bodies in vitro, and form teratomas in testicles of mice. These results provide the first evidence that LSEC support the in vitro survival, self-renewal, undifferentiated growth and differentiation capacity of the muESC CGR8 cell line.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fator Inibidor de Leucemia/metabolismo , Fígado/citologia , Animais , Anticorpos Bloqueadores/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Meios de Cultivo Condicionados/farmacologia , Corpos Embrioides/citologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Inibidor de Leucemia/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Teratoma/metabolismo , Teratoma/patologia , Fatores de Transcrição/metabolismoRESUMO
Complementary DNA (cDNA) is valuable for investigating protein structure and function in the research of life science, but it is difficult to obtain by traditional reverse transcription. In this study, we employed a novel strategy to clone the human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA directly isolated from the mucous membrane of mouth. The hLIF sequence can be acquired within a few hours by means of amplification of each exon and splicing using overlap-PCR. Thus, the new approach developed in this study is simple, time- and cost-effective, and it is not limited to particular gene expression levels of each tissue.
Assuntos
Humanos , DNA Complementar/genética , Fator Inibidor de Leucemia/genética , Mucosa Bucal , Sequência de Bases , Clonagem Molecular , Splicing de RNA/genética , Éxons/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PROBLEM: Fetal implantation enhances the production of essential growth factors such as LIF (leukaemia inhibitory factor), hence we investigated the contribution of maternal CD4 cells, activated by paternal or trophoblast antigens and its modulation by VIP (vasoactive intestinal peptide) and progesterone. METHOD OF STUDY: We performed co cultures of trophoblast cells (Swan-71 cell line) or paternal antigens and PBMCs from patients with recurrent spontaneous abortions (RSA) and fertile women. RESULT: Fertile-CD4(+) LIF(+) cells were increased by VIP and progesterone in response to paternal and trophoblast antigens. Also MMP-9 activity was decreased and pSTAT3/STAT3 ratio was increased. RSA patients have decreased levels of LIF expression which could not be modulated by VIP and progesterone and displayed a reduced number of endometrial infiltrated CD4(+) LIF(+) cells compared with fertile women. CONCLUSION: The decrease of CD4(+) LIF(+) cells in RSA patients could be related with the exacerbated inflammatory response observed in the maternal-fetal dialogue model.
Assuntos
Aborto Habitual/imunologia , Aborto Habitual/fisiopatologia , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Fator Inibidor de Leucemia/metabolismo , Gravidez/imunologia , Trofoblastos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Implantação do Embrião/imunologia , Feminino , Humanos , Tolerância Imunológica , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/farmacologia , Leucócitos Mononucleares/imunologia , Masculino , Progesterona/metabolismo , Progesterona/farmacologia , Trofoblastos/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
The mammary epithelium undergoes cyclical periods of cellular proliferation, differentiation, and regression. During lactation, the signal transducer and activator of transcription factor (STAT)-5A and the glucocorticoid receptor (GR) synergize to induce milk protein expression and also act as survival factors. During involution, STAT3 activation mediates epithelial cell apoptosis and mammary gland remodeling. It has been shown that the administration of glucocorticoids at weaning prevents epithelial cell death, probably by extracellular matrix breakdown prevention. Our results show that the synthetic glucocorticoid dexamethasone (DEX) modulates STAT5A and STAT3 signaling and inhibits apoptosis induction in postlactating mouse mammary glands, only when administered within the first 48 h upon cessation of suckling. DEX administration right after weaning delayed STAT5A inactivation and degradation, preserving gene expression of target genes as ß-casein (bcas) and prolactin induced protein (pip). Weaning-triggered GR down-regulation is also delayed by the hormone treatment. Moreover, DEX administration delayed STAT3 activation and translocation into epithelial cells nuclei. In particular, DEX treatment impaired the increment in gene expression of signal transducer subunit gp130, normally up-regulated from lactation to involution and responsible for STAT3 activation. Therefore, the data shown herein indicate that glucocorticoids are able to modulate early involution by controlling the strong cross talk that GR, STAT5, and STAT3 pathways maintains in the mammary epithelium.
Assuntos
Dexametasona/farmacologia , Glândulas Mamárias Animais/fisiologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Fragmentação do DNA , Dexametasona/administração & dosagem , Feminino , Regulação da Expressão Gênica/fisiologia , Glucocorticoides/administração & dosagem , Glucocorticoides/farmacologia , Lactação/fisiologia , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genéticaRESUMO
BACKGROUND: Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. RESULTS: We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. CONCLUSION: Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glândulas Mamárias Animais/citologia , Estresse Mecânico , Animais , Linhagem Celular , Feminino , Expressão Gênica , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Gravidez , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , Fator de Transcrição STAT3/metabolismoRESUMO
BACKGROUND: Claudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy. METHODS: Endometrial tissue was collected from women undergoing IVF. Endometrial biopsies were obtained during the luteal phase preceding IVF, and were then used for tissue microarray (TMA) immunolabeling of CLDN4. Previously published LIF expression data were then combined with CLDN4 expression data, to determine CLDN4/LIF expression patterns. Associations between successful pregnancy after IVF and combined CLDN4/LIF expression patterns were evaluated. RESULTS: Four patterns of immunolabeling were observed in the endometrial samples: 16% showed weak CLDN4 and strong LIF (CLDN4-/LIF+); 20% showed strong CLDN4 and strong LIF (LIF+/CLDN4+); 28% showed strong CLDN4 and weak LIF (CLDN4+/LIF-); and 36% showed weak CLDN4 and weak LIF (CLDN4-/LIF-). Successful implantation after IVF was associated with CLDN4-/LIF+(p = 0.003). Patients showing this endometrial CLDN4-/LIF+ immunolabeling were also 6 times more likely to achieve pregnancy than patients with endometrial CLDN4+/LIF- immunolabeling (p = 0.007). CONCLUSION: The combined immunolabeling expression of CLDN4-/LIF+ in endometrial tissue is a potential biomarker for predicting successful pregnancy in IVF candidates.
Assuntos
Endométrio/metabolismo , Fertilização in vitro , Fator Inibidor de Leucemia/metabolismo , Proteínas de Membrana/metabolismo , Adulto , Biomarcadores/metabolismo , Coeficiente de Natalidade , Claudina-4 , Implantação do Embrião/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Infertilidade/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/fisiologia , Fase Luteal/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Gravidez , Resultado do TratamentoRESUMO
INTRODUCTION: It has been demonstrated that leukemia inhibitory factor (LIF) induces epithelium apoptosis through Stat3 activation during mouse mammary gland involution. In contrast, it has been shown that this transcription factor is commonly activated in breast cancer cells, although what causes this effect remains unknown. Here we have tested the hypothesis that locally produced LIF can be responsible for Stat3 activation in mouse mammary tumors. METHODS: The studies were performed in different tumorigenic and non-tumorigenic mammary cells. The expression of LIF and LIF receptor was tested by RT-PCR analysis. In tumors, LIF and Stat3 proteins were analyzed by immunohistochemistry, whereas Stat3 and extracellular signal-regulated kinase (ERK)1/2 expression and phosphorylation were studied by Western blot analysis. A LIF-specific blocking antibody was used to determine whether this cytokine was responsible for Stat3 phosphorylation induced by conditioned medium. Specific pharmacological inhibitors (PD98059 and Stat3ip) that affect ERK1/2 and Stat3 activation were used to study their involvement in LIF-induced effects. To analyze cell survival, assays with crystal violet were performed. RESULTS: High levels of LIF expression and activated Stat3 were found in mammary tumors growing in vivo and in their primary cultures. We found a single mouse mammary tumor cell line, LM3, that showed low levels of activated Stat3. Incidentally, these cells also showed very little expression of LIF receptor. This suggested that autocrine/paracrine LIF would be responsible for Stat3 activation in mouse mammary tumors. This hypothesis was confirmed by the ability of conditioned medium of mammary tumor primary cultures to induce Stat3 phosphorylation, activity that was prevented by pretreatment with LIF-blocking antibody. Besides, we found that LIF increased tumor cell viability. Interestingly, blocking Stat3 activation enhanced this effect in mammary tumor cells. CONCLUSION: LIF is overexpressed in mouse mammary tumors, where it acts as the main Stat3 activator. Interestingly, the positive LIF effect on tumor cell viability is not dependent on Stat3 activation, which inhibits tumor cell survival as it does in normal mammary epithelium.