RESUMO
In brief: Fibroblast growth factor-2 (FGF2) is essential for early placenta development in sheep. This study shows that the mechanistic target of rapamycin is the key modulator of trophoblast adaptive response under FGF2 modulation. Abstract: During the early stage of placentation in sheep, normal conceptus development is affected by trophoblast cell functionality, whose dysregulation results in early pregnancy loss. Trophoblast metabolism is supported mainly by histotrophic factors, including fibroblast growth factor-2 (FGF2), which are involved in cell differentiation and function through the modulation of specific cellular mechanisms. The mechanistic target of rapamycin (mTOR) is known as a cellular 'nutrient sensor', but its downstream regulation remains poorly understood. The hypothesis was that during trophoblast development, the FGF2 effect is mediated by mTOR signalling pathway modulation. Primary trophoblast cells from 21-day-old sheep placenta were characterised and subjected to FGF2 and rapamycin treatment to study the effects on cell functionality and gene and protein expression profiles. The model showed mainly mononuclear cells with epithelial cell-like growth and placental morphological properties, expressing typical trophoblast markers. FGF2 promoted cell proliferation and migration under normal culture conditions, whereas mTOR inhibition reversed this effect. When the mTOR signalling pathway was activated, FGF2 failed to influence invasion activity. mTOR inhibition significantly reduced cell motility, but FGF2 supplementation restored motility even when mTOR was inhibited. Interestingly, mTOR inhibition influenced endocrine trophoblast marker regulation. Although FGF2 supplementation did not affect ovine placenta lactogen expression, as observed in the control, interferon-tau was drastically reduced. This study provides new insights into the mechanism underlying mTOR inhibitory effects on trophoblast cell functionality. In addition, as mTOR is involved in the expression of hormonal trophoblast markers, it may play a crucial role in early placenta growth and fetal-maternal crosstalk.
Assuntos
Placentação , Trofoblastos , Gravidez , Animais , Ovinos , Feminino , Trofoblastos/metabolismo , Placenta/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Sirolimo/farmacologia , Sirolimo/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
In the adult female, within the estrous cycle, the mammary gland undergoes multiple rounds of growth, with increased cellular proliferation, and involution, with increased apoptosis. The increase in proliferation is elicited by endocrine (Estrogen, Progesterone), as well as locally produced (epidermal growth factor, insulin-like growth factor, etc) growth factors. Among the genes that are modulated during cellular proliferation, immediate early genes play a fundamental role, being rapidly upregulated and then downregulated within the G0/G1 phase of the cell cycle, allowing the progression to the subsequent phases. Egrs (1-4) are immediate early genes that encode for transcription factors that promote, within different cell types and depending on the strength and duration of the stimuli, several different responses like mitogenesis, differentiation, apoptosis or even anti-apoptosis. In this work we have studied the mechanisms of modulation of the Egr family, in mammary epithelial cells of different origin (bovine, canine, feline, murine). Following stimulation with growth medium, Egr mRNA expression showed a strong upregulation reaching a peak at 45-60min, that rapidly declined. Among several cytokines, particularly important for mammary morphogenesis, that we have tested (EGF, IGF-I, insulin, estrogen, progesterone), only EGF upregulated Egrs to levels close to those elicited by growth medium. In order to understand how the Egr transcription factors were regulated, we have inhibited Erk 1/2 and PI3K, molecules that drive two major intracellular signaling pathways. Inhibition of the Erk 1/2 pathway totally abolished Egr upregulation mediated by growth medium or EGF. On the other hand, the PI3K-Akt pathway played a minor role on Egr levels, with a strong inhibitory effect on cat GH2 cells only, that could be ascribed to reduced Erk phosphorylation following PI3K inhibition. Finally we showed that addition of growth medium also upregulated that the mammary luminal marker cytokeratin 18, but only in the murine NMuMG cell line. This is the first manuscript describing how the Egr transcription factors are expressed in mammary epithelial cells of domestic animals and which growth factors and signaling pathways modulate their expression.
Assuntos
Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Glândulas Mamárias Animais/citologia , Família Multigênica , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Queratina-18/genética , Queratina-18/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Silymarin, a naturally acknowledged hepatoprotector used in humans to treat liver diseases has been tested in murine (HC11) and bovine (BME-UV) mammary epithelial cell lines to evaluate a possible direct effect on cell growth and differentiation in mammary gland. Silymarin enhanced cell proliferation (p < 0.05) from 10 to 1000 ng/ml in association with growth factors, (up to 20%) or alone (up to 15%) versus controls. Furthermore, silymarin (100 ng/ml) was able to increase (p < 0.05) beta-casein gene expression alone or in association with prolactin (5 microg/ml). These effects may be related with protein kinase B (AKT) activation induced by silymarin treatment (p < 0.05) and/or by a dose-related inhibitory effect (p < 0.05) on caspase-3 activity related to a protective role in cell apoptosis. These data suggest that silymarin should be considered a candidate to support mammary gland activity during a lactogenetic state.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Glândulas Mamárias Animais/citologia , Silimarina/farmacologia , Animais , Antioxidantes/farmacologia , Caseínas/genética , Caseínas/metabolismo , Caspase 3/metabolismo , Bovinos , Crescimento Celular , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Prolactina/farmacologia , Transdução de SinaisRESUMO
Cellular adaptation to a hypoxic microenvironment is essential for tumour progression and is largely mediated by HIF-1α and hypoxia-regulated factors, including CXCR4, VEGF-A and GLUT-1. In human osteosarcoma, hypoxia is associated with resistance to chemotherapy as well as with metastasis and poor survival, whereas little is known about its role in canine osteosarcoma (cOSA). This study aimed primarily to evaluate the prognostic value of several known hypoxic markers in cOSA. Immunohistochemical analysis for HIF-1α, CXCR4, VEGF-A and GLUT-1 was performed on 56 appendicular OSA samples; correlations with clinicopathological features and outcome was investigated. The second aim was to investigate the in vitro regulation of markers under chemically induced hypoxia (CoCl2). Two primary canine osteosarcoma cell lines were selected, and Western blotting, immunofluorescence and qRT-PCR were used to study protein and gene expression. Dogs with high-grade OSA (35.7%) were more susceptible to the development of metastases (P = 0.047) and showed high HIF-1α protein expression (P = 0.007). Moreover, HIF-1α overexpression (56%) was correlated with a shorter disease-free interval (DFI; P = 0.01), indicating that it is a reliable negative prognostic marker. The in vitro experiments identified an accumulation of HIF-1α in cOSA cells after chemically induced hypoxia, leading to a significant increase in GLUT-1 transcript (P = 0.02). HIF-1α might be a promising prognostic marker, highlighting opportunities for the use of therapeutic strategies targeting the hypoxic microenvironment in cOSA. These results reinforce the role of the dog as a comparative animal model since similar hypoxic mechanisms are reported in human osteosarcoma.
Assuntos
Neoplasias Ósseas/veterinária , Hipóxia Celular/fisiologia , Doenças do Cão/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Osteossarcoma/veterinária , Animais , Biomarcadores Tumorais/análise , Neoplasias Ósseas/química , Neoplasias Ósseas/fisiopatologia , Linhagem Celular Tumoral , Doenças do Cão/patologia , Cães , Feminino , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Imuno-Histoquímica/veterinária , Masculino , Metástase Neoplásica/fisiopatologia , Osteossarcoma/química , Osteossarcoma/fisiopatologia , Prognóstico , Receptores CXCR4/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.
Assuntos
Bovinos/fisiologia , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Glândulas Mamárias Animais/citologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Bovinos/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The aim of this work is to investigate on plasma profiles of leptin and estradiol 17beta during the interpartum period and leptin concentrations in the milk and in the colostrum during the period from parturition to the successive delivery in mare. Leptin plasma concentration varied from 5.1+/-2.3 ng/ml after the first parturition (week 0) to 3.0+/-0.7 at week 21 (p<0.05), then it increased to maximal level at week 49 (6.9+/-1.0 ng/ml, p<0.05). Leptin concentration in the colostrum and in the milk has been significantly (p<0.05) higher than that in plasma samples at week 1 (milk 8.8+/-2.3 versus plasma 5.2+/-0.6 ng/ml) and between week 12 and 17. This difference may be explained with a local leptin production at mammary level and supports a role of leptin in the mammary gland and/or in foal intestine. Estradiol 17beta increased from week 15 (17.9+/-2.3 pg/ml) up to 487.9+/-67.7 pg/ml at week 43. Plasma estradiol 17beta rise anticipated by 4 weeks plasma leptin increase and it does not seem to be positively correlated to leptin secretion.
Assuntos
Estradiol/sangue , Cavalos/metabolismo , Leptina/análise , Leite/química , Período Pós-Parto/sangue , Prenhez/sangue , Animais , Colostro/química , Colostro/metabolismo , Feminino , Leptina/sangue , Leite/metabolismo , Gravidez , Fatores de TempoRESUMO
The met proto-oncogene product Met is a member of the family of tyrosine kinase growth factor receptors, and hepatocyte growth factor/scatter factor (HGF/SF) has been identified as its only ligand. Bovine Met and HGF/SF have been recently cloned and their expression has been characterized in the mammary gland, but no data regarding the biological effects of this ligand/receptor couple in bovine mammary cells are yet available. We examined the role of HGF/SF and its receptor in a bovine mammary epithelial cell line (BME-UV). Expression of Met at the mRNA level in BME-UV mammary epithelial cells evaluated by real-time PCR was similar to the expression in MDCK cells, a widely used model for Met biology. Met expression in BME-UV at the protein level was confirmed by western blot. The analysis of some signal transductional pathways downstream from the Met receptor revealed that HGF/SF addition to BME-UV cells induced activation of the extracellular signal-regulated kinase 1/2 proliferative pathway and the Akt antiapoptotic pathway. The BME-UV cells treated with HGF responded with increased proliferation, cell scatter, and motility. Met activation by HGF induced degradation of the extracellular matrix and migration through matrigel coated transwells. Moreover, BME-UV cells included in a 3-dimensional matrix of collagen and treated with HGF developed tubular structures, reminiscent of the mammary gland ducts. These data indicate that HGF and Met might be important regulators of mammary gland growth, morphogenesis, and development in the bovine.
Assuntos
Bovinos , Células Epiteliais/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Glândulas Mamárias Animais/citologia , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Cães , Fator de Crescimento Epidérmico/farmacologia , Matriz Extracelular/efeitos dos fármacos , Feminino , Expressão Gênica , Fator de Crescimento de Hepatócito/fisiologia , Rim , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/fisiologia , RNA Mensageiro/análiseRESUMO
Satellite cells are adult stem cells located between the basal lamina and sarcolemma of muscle fibers. Under physiological conditions, satellite cells are quiescent, but they maintain a strong proliferative potential and propensity to differentiate, which underlies their critical role in muscle preservation and growth. MicroRNAs (miRNAs) play essential roles during animal development as well as in stem cell self-renewal and differentiation regulation. MiRNA-1, miRNA-133a and miRNA-206 are closely related muscle-specific miRNAs, and are thus defined myomiRNAs. MyomiRNAs are integrated into myogenic regulatory networks. Their expression is under the transcriptional and post-transcriptional control of myogenic factors and, in turn, they exhibit widespread control of muscle gene expression. Very little information is available about the regulation and behavior of satellite cells in large farm animals, in particular during satellite cell differentiation. Here, we study bovine satellite cells (BoSCs) undergoing a differentiation process and report the expression pattern of selected genes and miRNAs involved. Muscle samples of longissimus thoracis from Holstein adult male animals were selected for the collection of satellite cells. All satellite cell preparations demonstrated myotube differentiation. To characterize the dynamics of several transcription factors expressed in BoSCs, we performed real-time PCR on complementary DNA generated from the total RNA extracted from BoSCs cultivated in growth medium (GM) or in differentiation medium (DM) for 4 days. In the GM condition, BoSCs expressed the satellite cell lineage markers as well as transcripts for the myogenic regulatory factors. At the time of isolation from muscle, PAX7 was expressed in nearly 100% of BoSCs; however, its messenger RNA (mRNA) levels dramatically decreased between 3 and 6 days post isolation (P<0.01). MyoD mRNA levels increased during the 1st day of cultivation in DM (day 7; P<0.02), showing a gradual activation of the myogenic gene program. During the subsequent 4 days of culture in DM, several tested genes, including MRF4, MYOG, MEF2C, TMEM8C, DES and MYH1, showed increased expression (P<0.05), and these levels remained high throughout the culture period investigated. Meanwhile, the expression of genes involved in the differentiation process also miRNA-1, miRNA-133a and miRNA-206 were strongly up-regulated on the 1st day in DM (day 7; P<0.05). Analysis revealed highly significant correlations between myomiRNAs expression and MEF2C, MRF4, TMEM8C, DES and MYH1 gene expression (P<0.001). Knowledge about the transcriptional changes correlating with the growth and differentiation of skeletal muscle fibers could be helpful for developing strategies to improve production performance in livestock.
Assuntos
Bovinos/fisiologia , Diferenciação Celular/fisiologia , Fatores de Diferenciação de Crescimento/metabolismo , MicroRNAs/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Fatores de Diferenciação de Crescimento/genética , Masculino , MicroRNAs/genética , Fibras Musculares Esqueléticas/metabolismo , Fatores de Regulação Miogênica/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Células Satélites de Músculo Esquelético/citologia , TranscriptomaRESUMO
We have investigated the mechanism by which two oncogenic mutations (M1268T and D1246H/N; Amino-acids are numbered according to Schmidt et al., 1999) affecting conserved residues in the catalytic domain of the Met receptor, activate its transforming potential. Both mutations were previously found in tumorigenic forms of the Ret and Kit receptors, respectively. The mutated residues are located either in the P+1 loop (M) or within the activation loop (A-loop) (D), which in a number of receptor tyrosine kinases harbors a pair of tandem tyrosines (Y1252-1253 in Met). Ligand-induced dimerization promotes their phosphorylation, and locks the A-loop into an open conformation. When unphosphorylated, the tandem tyrosines inhibit enzymatic activity by blocking the active site. Upon Y-->F mutation of Y1252-1253, neither ligand binding nor Tpr-mediated dimerization can release this block. Here we show that the M1268T mutation partially rescues the kinase activity (and the transforming ability) of the Y1252-1253F Tpr-Met mutant, but is completely dependent on dimerization for its effect. In contrast, the two D1246H/N mutants strictly depend on Y1252-1253 for activity. Surprisingly, however, they constitutively activate the isolated cytoplasmic TK domain of Met (Cyto-Met). These data indicate that the two mutations operate via distinct mechanisms.
Assuntos
Transformação Celular Neoplásica/genética , Sequência Conservada , Proteínas de Drosophila , Mutação , Proteínas Proto-Oncogênicas c-met/genética , Sequência de Aminoácidos , Dimerização , Ligantes , Complexo de Proteínas Formadoras de Poros Nucleares , Fosforilação , Conformação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Nuclear matrix attachment regions (MAR) have been implicated in the regulation of gene expression. We have identified a region within the proximal 3'-flanking sequences of the ovine beta-lactoglobulin (betalg) gene that interacts with the nuclear matrix in vitro. No equivalent region was detected in the 5' flanking region. We have investigated the role of this element in regulating betalg expression in vitro and in vivo. Removal of the MAR did not affect the frequency of betalg transgene expression at the mRNA level, but betalg transgenes that lacked the MAR were expressed at a lower level than wild-type betalg transgenes. In neither in-vitro HC11 transfection experiments nor transgenic mice was hormonal induction of betalg expression significantly affected by MAR removal. Nuclear run-on analysis demonstrated that the impaired basal expression of betalg transgene loci lacking the MAR was due to a reduced transcription rate. Thus, the single MAR enhances the basal transcriptional potential of the betalg gene.
Assuntos
Lactoglobulinas/genética , Matriz Nuclear/metabolismo , Transgenes/genética , Animais , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , DNA Recombinante/genética , Dexametasona/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Camundongos , Camundongos Transgênicos , Prolactina/farmacologia , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Sequências Reguladoras de Ácido Nucleico/fisiologia , Análise de Sequência de DNA , Ovinos , Transcrição GênicaRESUMO
HIV-gp120 sensitizes Th1 clones from seronegative donors to apoptosis, which occurs through two distinct events: expression of CD95L followed by its interaction with CD95 to trigger cell death. gp120-apoptosis of the Th1 clone 103 was inhibited by Cyclosporin A, the PTK inhibitors Genistein and PNU152518, as well as the anti-oxidants Ascorbic Acid and Glutathione. Cyclosporin A interfered with CD95L expression, Ascorbic Acid and Glutathione inhibited cell death triggered by CD95/CD95L interaction; Genistein and PNU152518 acted on both steps. The occurrence of oxidative stress during CD95-dependent apoptosis was supported by the direct evidence of ROI production.
Assuntos
Apoptose , Proteína gp120 do Envelope de HIV/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células Th1/virologia , Receptor fas/metabolismo , Ácido Ascórbico/farmacologia , Células Clonais , Ciclosporina/farmacologia , Proteína Ligante Fas , Expressão Gênica , Genisteína , Glutationa/farmacologia , Proteína gp120 do Envelope de HIV/genética , Humanos , Isoflavonas/farmacologia , Células Jurkat , Glicoproteínas de Membrana/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Células Th1/metabolismo , Células Th1/fisiologiaRESUMO
HIV protein gp120 in combination with T cell antigen receptor (TCR) triggering induces apoptosis (gp120-apoptosis) in Th1 cells. Gp120-apoptosis occurs by induction of Fas-L and subsequent triggering of the Fas apoptotic pathway. Here, through the use of several compounds inhibiting induction of Fas-L, we show that, in a Th1 clone, a protein kinase C (PKC) independent pathway activated by TCR stimulation is distinguishible from a PKC dependent pathway activated by either phorbol 12-myristate 13-acetate (PMA)/ionomycin or asynchronous stimulation of TCR and CD4 as occurs in gp120-apoptosis.
Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteína gp120 do Envelope de HIV/farmacologia , Ionomicina/farmacologia , Glicoproteínas de Membrana/genética , Proteína Quinase C/metabolismo , Células Th1/fisiologia , Antígenos de Superfície/genética , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Clonais , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Ligante Fas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Ativação Linfocitária , Glicoproteínas de Membrana/biossíntese , Naftalenos/farmacologia , Receptores de Antígenos de Linfócitos T/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Células Th1/citologia , Células Th1/imunologiaRESUMO
Leptin and its receptors have been shown to be expressed in several tissues, suggesting that this protein might be effective not only at the CNS level but also peripherally. We have previously reported that leptin and its long form receptor are expressed in the mouse mammary epithelial cell line HC11. In this study, we report a specific relationship among leptin, prolactin (PRL), interleukin-6 (IL-6), and tumor necrosis-alpha (TNF-alpha) in the modulation of the suppressor of cytokine signaling 1 (SOCS-1). Furthermore, we show that leptin and PRL are able to effectively enhance SOCS-1 gene expression in the HC11 cell line. Finally, high concentrations of leptin (100 nM) and/or PRL significantly (p<0.05) reduce the inhibitory effect of IL-6 (10 and 100 ng/ml) and TNF-alpha (10 and 100 ng/ml) on beta-casein gene expression in HC11 cells transfected with pbetacCAT, a chimeric rat-beta casein gene promoter-cloramphenicol acetyl transferase (CAT) gene construct. These results provide evidence that leptin may be an important mediator in regulating mammary gland growth and development and that this role may be related to the immune factors that are involved in inflammation.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Leptina/farmacologia , Glândulas Mamárias Animais/efeitos dos fármacos , Prolactina/farmacologia , Proteínas Repressoras/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteínas de Transporte/genética , Caseínas/genética , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Leptina/genética , Leptina/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Prolactina/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , Fatores de TempoRESUMO
A small column containing 2 mM CH-Sepharose 4B-immobilized trypsin was connected to a flow injection device equipped for potentiometric measurements (0.01-2 mM protons) and for post-column analysis by spectrophotometry and capillary electrophoresis (CE). The device was engaged with N alpha-benzoyl-L-arginine pNO2-anilide (BAPNA), beta-lactoglobulin (beta-Lac) and peptides of V8-protease predigested beta-Lac. At a given flow rate, the reaction with BAPNA or beta-Lac (below 2 mM) produced about 1 proton per substrate molecule in each sample (linear relation to substrate amount); with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalents per substrate molecule (hyperbolic dependence). Final experiments demonstrated that the reactor gave a correct estimate of available lysine in peptides of beta-Lac modified with 5-nitrosalicylaldehyde. The data could be predicted by a kinetic model describing the reactor performance in 'single turnover' conditions. The interplay between resident time and the non-catalytic amount of trypsin prevented each enzyme molecule from recycling as well as each substrate molecule (containing one or more cleavage sites) from encountering the enzyme more than once. In conclusion, both from the experimental and the theoretical point of view, this work permitted the analysis of trypsin behaviour in some extreme working conditions and indicates how to modulate the performance of an endoprotease-based reactor. A brief discussion on potential applications in protein mapping and tagging and in the quantitative analysis of protein bioavailability by means of a biosensorial strategy is also described.
Assuntos
Análise de Injeção de Fluxo , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Benzoilarginina Nitroanilida/metabolismo , Técnicas Biossensoriais , Bovinos , Eletroforese Capilar , Enzimas Imobilizadas , Análise de Injeção de Fluxo/instrumentação , Hidrólise , Cinética , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Dados de Sequência Molecular , Pâncreas/enzimologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Potenciometria , Serina Endopeptidases/metabolismo , EspectrofotometriaRESUMO
Excessive meat tenderization in cured raw Parma ham has recently been correlated with abnormal levels of cathepsin B in freshly slaughtered thigh meat. We have developed a visual assay employing the substrate Z-Arg-Arg-NNapOMe for the quantitative detection of active cathepsin B levels in pork thigh muscle homogenates. The work was based on a kinetic characterization, in steady state condition, of pig muscle cathepsin B with several peptidyl chromophoric substrate analogs. This assay can easily and safely be performed by non-specialized personnel directly in the slaughterhouse or in the factory, for an early quality evaluation of thighs selected for Parma ham production. Our characterization has further indicated that the catalytic properties of porcine muscle cathepsin B and those of isoforms from other animal and plant species are practically identical. This is particularly evident in the commercially available bovine spleen isoform, which was employed as a model enzyme in most of the experiments.
RESUMO
Mammary stem cells provide opportunities for the cyclic remodelling of the bovine mammary gland. Therefore, understanding the character and regulation of mammary stem cells is important for increasing animal health and productivity. The exciting possibility that stem cell expansion can influence milk production is currently being investigated by several researchers. In fact, appropriate regulation of mammary stem cells could hopefully benefit milk yield, persistency of lactation, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and regulate the function of bovine mammary stem cells. However, research on mammary stem cells requires tissue biopsies, which represents a limitation for the management of animal welfare. Interestingly, different studies recently reported the identification of putative mammary stem cells in human breast milk. The possible identification of primitive cell types within cow's milk may provide a non-invasive source of relevant mammary cells for a wide range of applications. In this review, we have summarized the main achievements in this field for dairy cow science and described the interesting perspectives open to manipulate milk persistency during lactation and to cope with oxidative stress during the transition period by regulating mammary stem cells.
Assuntos
Células-Tronco Adultas/citologia , Bovinos , Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Células-Tronco Adultas/metabolismo , Animais , Bovinos/fisiologia , Proliferação de Células , Indústria de Laticínios , Células Epiteliais/metabolismo , Feminino , Lactação , Glândulas Mamárias Animais/efeitos dos fármacos , Leite/metabolismoRESUMO
Basal-like breast cancer is an aggressive subtype of mammary carcinoma. Despite expressing basal markers, typical of mammary stem cells, this tumor has been proposed to originate from luminal progenitors, which are downstream of stem cells along the mammary epithelial hierarchy. This suggests that committed luminal progenitors may reacquire basal, stem-like characteristics, but the mechanisms that regulate this transition remain unclear. Using mouse models, we found that luminal progenitors express high levels of the Met receptor for hepatocyte growth factor (HGF), as compared with the other mammary epithelial sub-populations. Constitutive activation of Met led luminal progenitors to attain stem cell properties, including enhanced clonogenic activity in vitro and de novo ability to reconstitute mammary glands in repopulation assays in vivo. Moreover, in response to Met signaling, luminal progenitors gave rise to hyperplastic ductal morphogenesis and preferentially underwent basal lineage commitment at the expense of luminal cell-fate specification. Opposite and symmetric results were produced by systemic pharmacological inhibition of Met. Hence, Met signaling targets luminal progenitors for expansion, impairs their differentiation toward the mature luminal phenotype and enables their commitment toward the basal lineage. These results emphasize a critical role for Met in promoting deregulated proliferation and basal plasticity of normal luminal progenitors in the mammary gland, a complex of events that may be required for sustaining the functional and phenotypic properties of basal-like breast tumors.
Assuntos
Neoplasias da Mama/patologia , Diferenciação Celular/genética , Proliferação de Células , Células Epiteliais/fisiologia , Glândulas Mamárias Animais/fisiologia , Neoplasia de Células Basais/patologia , Proteínas Proto-Oncogênicas c-met/fisiologia , Animais , Neoplasias da Mama/genética , Linhagem da Célula/genética , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Neoplasia de Células Basais/genética , Fenótipo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/genética , Células-Tronco/metabolismo , Células-Tronco/fisiologiaAssuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glândulas Mamárias Animais/citologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Bovinos , Linhagem Celular , Proliferação de Células , Células Epiteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
MicroRNAs (miRNAs) are approximately 22 nucleotide endogenous RNA molecules which exert their functions by base pairing with messenger RNAs (mRNAs), thereby regulating protein-coding gene expression. In eukaryotic cells, miRNAs play important roles in regulating biological processes such as proliferation, differentiation, apoptosis, and stem cell self-renewal. miRNAs are encoded by the genome, and more than 1,000 human miRNAs have been identified so far. miRNAs are predicted to target -60% of human mRNAs and are expressed in all animal cells. Unique expression domains, targets, and gain- and loss-of-function phenotypes of particular miRNAs have important implications for directed to control differentiation of stem cell populations. Many cancers show variations in miRNA levels, and more specifically an overall downregulation, when compared to their normal counterparts. Therefore, miRNAs may be used as potential therapeutic agents to correct aberrant transcript levels found in the signaling pathways of cancer. This review examines the most recent acquisition on the role of miRNAs in regulating the cell cycle, with particular emphasis on their effects on cell proliferation and differentiation. The second part explores specifically the role of these factors in the physiological regulation of embryonic stem cells, of cellular reprogramming and their involvement in the activation of stem cells in adult tissues. In the third part, the article discusses some issues that relate to the role of miRNAs in the development of neoplastic diseases, focusing on aspects of the genetic and transcriptional alterations that determine the beginning and the development of tumor process, with emphasis on, looking to emphasize their involvement in the activation of adult cancer stem cells.