RESUMEN
The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.
RESUMEN
The functional maturation of the pituitary gland requires adequate cell differentiation and vascular network formation. Although spatiotemporal signaling and transcription factors are known to govern pituitary development, the involvement of primary cilia, nonmoving hair-like organelles, remains unclear. In this study, we uncovered the contribution of primary cilia to cell-type determination and vascular network formation during pituitary development. Homozygous knockout mice lacking a ciliary kinase, Dyrk2-/-, exhibit abnormalities in ciliary structure and pituitary hypoplasia, accompanied by varying degrees of failure in differentiation among all types of hormone-producing cells in the anterior lobe. Aberrations in cell differentiation in Dyrk2-/- mice arise from a decrease in the expression of crucial transcription factors, Lhx4, Lhx3, and Prop1, resulting from the inactivity of Hedgehog (Hh) signaling during the early stages of development. Furthermore, the loss of Dyrk2 results in vascular system abnormalities during the middle to late stages of development. Mechanistically, transcriptome analyses revealed the downregulation of vitronectin-integrin αvß3-VEGFR2 signaling, essential for orchestrating vascular development. Collectively, our findings demonstrate that primary cilia play a pivotal role as critical regulators of cell survival, cell determination, and angiogenesis during pituitary gland development through the activation of Hh signaling. These findings expand our understanding of the potential link between pituitary dysfunction in human disorders and ciliopathies.
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Diferenciación Celular , Cilios , Neovascularización Fisiológica , Hipófisis , Animales , Ratones , Angiogénesis , Cilios/metabolismo , Cilios/fisiología , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones Noqueados , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Hipófisis/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Quinasas DyrK/genéticaRESUMEN
INTRODUCTION: Axons of magnocellular neurosecretory cells project from the hypothalamus to the posterior lobe (PL) of the pituitary. In the PL, a wide perivascular space exists between the outer basement membrane (BM), where nerve axons terminate, and the inner BM lining the fenestrated capillaries. Hypothalamic axon terminals and outer BMs in the PL form neurovascular junctions. We previously had found that collagen XIII is strongly localized in the outer BMs. In this study, we investigated the role of collagen XIII in the PL of rat pituitaries. METHODS: We first studied the expression of Col13a1, the gene encoding the α1 chains of collagen XIII, in rat pituitaries via quantitative real-time polymerase chain reaction and in situ hybridization. We observed the distribution of COL13A1 in the rat pituitary using immunohistochemistry and immunoelectron microscopy. We examined the expression of Col13a1 and the distribution of COL13A1 during the development of the pituitary. In addition, we examined the effects of water deprivation and arginine vasopressin (AVP) signaling on the expression of Col13a1 in the PL. RESULTS: Col13a1 was expressed in NG2-positive pericytes, and COL13A1 signals were localized in the outer BM of the PL. The expression of Col13a1 was increased by water deprivation and was regulated via the AVP/AVPR1A/Gαq/11 cascade in pericytes of the PL. CONCLUSION: These results suggest that pericytes surrounding fenestrated capillaries in the PL secrete COL13A1 and are involved in the construction of neurovascular junctions. COL13A1 is localized in the outer BM surrounding capillaries in the PL and may be involved in the connection between capillaries and axon terminals.
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Colágeno Tipo XIII , Animales , Ratas , Masculino , Colágeno Tipo XIII/metabolismo , Sistemas Neurosecretores/metabolismo , Arginina Vasopresina/metabolismo , Ratas Wistar , Neurohipófisis/metabolismo , Hipófisis/metabolismo , Pericitos/metabolismo , Membrana Basal/metabolismoRESUMEN
The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke's cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.
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Adenohipófisis , Animales , Femenino , Ratas , Hormona del Crecimiento , Hipófisis/metabolismo , Adenohipófisis/metabolismo , Prolactina , Tirotropina , Tetraspanina 29/metabolismo , Factores de Transcripción SOXB1/metabolismoRESUMEN
Endothelial fenestrae are transcellular pores separated by diaphragms formed by plasmalemma vesicle-associated proteins (PLVAP) and function as channels for peptide hormones and other substances. Caveola, a key regulator of clathrin-independent endocytosis, may be involved in the invagination and fusion of plasma membranes, which are essential for fenestra formation. In this study, we first found that caveolin-1 and -2, the major components of caveolae, was localized in fenestrated endothelial cells in the anterior lobe of the rat pituitary by immunohistochemistry. As we also observed caveolae in the endothelial cells of the anterior lobe of the rat pituitary by transmission electron microscopy, we studied the relationship between the caveolae-mediated endocytosis pathway and fenestrae structure in cultured endothelial cells isolated from the anterior lobe of the rat pituitary (CECAL) by immunofluorescence staining and scanning electron microscopy. The inhibition of caveolae-mediated endocytosis by genistein enlarged the PLVAP-positive oval-shaped structure that represented the sieve plate and induced the formation of a doughnut-shaped bulge around the fenestra in CECAL. In contrast, the acceleration of caveolae-mediated endocytosis by okadaic acid induced the diffusion of PLVAP-positive signals in the cytoplasm and reduced the number of fenestrae in CECAL. These results indicate that the caveolae-mediated endocytosis pathway is involved in the fenestra homeostasis in the fenestrated endothelial cells of the rat pituitary.
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Caveolas , Células Endoteliales , Ratas , Animales , Caveolas/metabolismo , Células Endoteliales/metabolismo , Caveolina 1/metabolismo , Endotelio/metabolismo , Proteínas Portadoras/metabolismo , Endocitosis/fisiología , HomeostasisRESUMEN
Endothelial fenestrae are transcellular pores divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). They function as a channel for peptide hormones and other substances. Invagination of the plasma membrane is necessary for the fenestra formation. The actin cytoskeleton is essential for scission of endocytic vesicles from the invaginated plasma membrane. Therefore, we examined the involvement of the actin cytoskeleton in fenestra formation in cultured endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, using immunofluorescence and scanning electron microscopy. Inhibition of polymerization and depolymerization of the actin cytoskeleton by latrunculin A and jasplakinolide, respectively, remarkably increased the PLVAP-positive sieve plate area and number of fenestrae. Jasplakinolide significantly affected the arrangement of the fenestra on the cell surface, resulting in parallel serpentine furrows of the fenestra. These results suggest that the actin cytoskeleton not only induces fenestra formation but also regulates cell arrangement. Dynamin is a scission protein of the invaginated plasma membrane and interacts with the actin cytoskeleton. We found that dynamin2 is mainly expressed in the endothelial cells of the rat AL. We then investigated the function of dynamin2 by the treatment with dyngo-4a, a potent inhibitor of dynamin1 and dynamin2, on the fenestra formation. As a result, the PLVAP-positive area is significantly increased by the treatment. These results show that the actin-dynamin2 interaction is essential for the control of the fenestra formation in endothelial cells of rat AL. In conclusion, the actin cytoskeleton and dynamin2 function as regulators of endothelial fenestra formation.
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Actinas , Dinamina II , Células Endoteliales , Animales , Ratas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , Dinamina II/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.
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Adenohipófisis , Prolactina , Animales , Diferenciación Celular/fisiología , Células Endoteliales , Femenino , Antígeno Ki-67 , Hipófisis , Embarazo , Ratas , Ratas Wistar , Factores de Transcripción SOXB1/inmunología , Células Madre , Tetraspanina 29/inmunologíaRESUMEN
The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary.
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Adenohipófisis , Tretinoina , Animales , Masculino , Hipófisis/metabolismo , Adenohipófisis/metabolismo , Ratas , Ratas Wistar , Ácido Retinoico 4-Hidroxilasa/metabolismo , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Obesity-associated type 2 diabetes mellitus is associated with the development of insulin resistance. Among several metabolites, resolvins that are metabolites of eicosapentaenoic acid have been shown to exert insulin-sensitizing effects; however, the role of resolvin E3 (RvE3) in glucose metabolism has not been studied. In this study, the effect of RvE3 on glucose metabolism in mice with high-fat diet-induced obesity and 3T3L1 adipocytes was studied. C57BL/6 mice fed a high-fat diet were administered RvE3, for which insulin tolerance, oral glucose tolerance tests, and the homeostasis model assessment of insulin resistance, were performed. RvE3 treatment significantly improved insulin sensitivity and glucose tolerance and regulated protein kinase B (Akt) phosphorylation in the adipose tissue. Moreover, RvE3 treatment enhanced the insulin-stimulated glucose transporter 4 (Glut4) translocation, glucose uptake, phosphatidylinositol-3-kinase (PI3K) activity, and Akt phosphorylation in 3T3L1 adipocytes, whereas a PI3K inhibitor inhibited the enhanced insulin-stimulated glucose uptake induced by RvE3. These findings indicate that RvE3 likely improves insulin sensitivity, resulting in the upregulation of glucose uptake in adipocytes by activating the PI3K/Akt signaling pathways. Collectively, the findings of this study show that RvE3 may play a role in glucose homeostasis and could be used as a potential therapeutic target for developing treatments for obesity-associated diabetes.
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Adipocitos/efectos de los fármacos , Ácidos Grasos Insaturados/farmacología , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Transportador de Glucosa de Tipo 4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
A supply of hormone-producing cells from stem/progenitor cells is critical to sustain the endocrine activity of the pituitary gland. In the adenohypophysis composing the anterior and intermediate lobe (AL and IL, respectively), stem/progenitor cells expressing sex-determining region Y-box 2 (SOX2) and S100ß are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). Our previous studies using mice and rats indicated that the tetraspanin superfamily CD9 and CD81 are expressed in S100ß/SOX2-positive cells of primary and secondary niches (named CD9/CD81/S100ß/SOX2-positive cell), and the cells located in the AL-side niches exhibit plasticity and multipotency. However, it is unclear whether CD9/CD81/S100ß/SOX2-positive cells in the IL-side primary niche are stem/progenitor cells for the AL or IL. Here, we successfully isolated pure CD9/CD81/S100ß/SOX2-positive cells from the IL-side primary niche. They had a higher level of S100ß and SOX2 mRNA and a greater pituisphere forming capacity than those of CD9/CD81/S100ß/SOX2-positive cells isolated from the AL. They also had capacity to differentiate into all types of adenohypophyseal hormone-producing cells, concomitantly with the loss of CD9 expression. Loss of CD9 and CD81 function in CD9/CD81/S100ß/SOX2-positive cells by siRNA treatment impaired prolactin cell differentiation. Consistently, in the pituitary gland of CD9/CD81 double knockout mice, dysgenesis of the MCL and a lower population of prolactin cells were observed. These results suggest that the CD9/CD81/S100ß/SOX2-positive cells in the MCL of the IL-side are potential suppliers of adult core stem cells in the AL.
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Hipófisis/anatomía & histología , Prolactina/metabolismo , Tetraspanina 29/metabolismo , Animales , Masculino , Ratones , Ratas , Ratas WistarRESUMEN
Endothelial fenestrae are the transcellular pores existing on the capillary walls which are organized in clusters referred to as sieve plates. They are also divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). In this study, we examined the involvement of fibronectin signaling in the formation of fenestra and diaphragm in endothelial cells. Results showed that Itga5 and Itgb1 were expressed in PECAM1-positive endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, and integrin α5 was localized at the fenestrated capillaries of the rat pituitary and cultured PECAM1-positive endothelial cells isolated from AL (CECAL). Inhibition of both integrin α5ß1 and FAK, a key molecule for integrin-microtubule signaling, respectively, by ATN-161 and FAK inhibitor 14, caused the delocalization of PLVAP at the sieve plates and depolymerization of microtubules in CECAL. Paclitaxel prevented the delocalization of PLVAP by the inhibition of integrin α5ß1. Microtubule depolymerization induced by colcemid also caused the delocalization of PLVAP. Treatment of CECAL with ATN-161 and colcemid caused PLVAP localization at the Golgi apparatus. The localization of PLVAP at the sieve plates was inhibited by BFA treatment in a time-dependent manner and spread diffusely to the cytoplasm. These results indicate that a constant supply of PLVAP proteins by the endomembrane system via the Golgi apparatus is essential for the localization of PLVAP at sieve plates. In conclusion, the endomembrane transport pathway from the Golgi apparatus to sieve plates requires microtubule cytoskeletons, which are regulated by fibronectin-integrin α5ß1 signaling.
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Células Endoteliales/metabolismo , Fibronectinas/metabolismo , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Transducción de SeñalRESUMEN
Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle-associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin-511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary.
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Capilares/metabolismo , Células Endoteliales/metabolismo , Fibronectinas/metabolismo , Animales , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Endoteliales/ultraestructura , Masculino , Proteínas de la Membrana/metabolismo , Hipófisis/citología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Ratas WistarRESUMEN
SOX2-positive cells are stem/progenitor cells that supply hormone-producing cells; they are found in the anterior lobe of the rodent pituitary gland. However, they are likely composed of several subpopulations. In rats, a SOX2-positive cell populations can be distinguished by the presence of S100ß. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100ß/SOX2-positive cells. Recently, CD9/CD81 double-knockout mice were generated. Although they grew normally until 3 weeks after birth, they exhibited atrophy of the pituitary gland. These findings suggested that CD9/CD81/S100ß/SOX2-positive cells in the mouse pituitary are adult stem/progenitor cells. To substantiate this hypothesis, we examined CD9 and CD81 expression in the adult and developing anterior lobe. Immunohistochemistry showed that CD9/CD81-positive cells began appearing from postnatal day 0 and settled in the stem cell niches (marginal cell layer and parenchyma) of the adult anterior lobe while expressing S100ß. We next isolated CD9 -positive cells from the adult anterior lobe, using the anti-CD9 antibody for cell characterisation. The cells in culture formed free-floating three-dimensional clusters (pituispheres); moreover, induction into all types of hormone-producing cells was successful. Furthermore, reduction of CD9 and CD81 mRNAs by siRNAs inhibited cell proliferation. These findings indicate that CD9/CD81/S100ß/SOX2-positive cells may play a role as adult stem/progenitor cells in SOX2-positive subpopulations, thus supplying hormone-producing cells in the postnatal anterior lobe. Furthermore, CD9 and CD81 are implicated in cell proliferation. The current findings provide novel insights into adult pituitary stem/progenitor cells.
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Hipófisis/citología , Células Madre/citología , Tetraspanina 29/inmunología , Animales , Anticuerpos/inmunología , Diferenciación Celular , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Hipófisis/inmunología , Células Madre/inmunologíaRESUMEN
Being delivered as a low birthweight (LBW) infant is a risk factor for elevated blood pressure and future problems with cardiovascular and cerebellar diseases. Although premature babies are reported to have low numbers of nephrons, some unclear questions remain about the mechanisms underlying elevated blood pressure in full-term LBW infants. We previously reported that glucocorticoids increased miR-449a expression, and increased miR-449a expression suppressed Crhr1 expression and caused negative glucocorticoid feedback. Therefore, we conducted this study to clarify the involvement of pituitary miR-449a in the increase in blood pressure caused by higher glucocorticoids in LBW rats. We generated a fetal low-carbohydrate and calorie-restricted model rat (60% of standard chow), and some individuals showed postnatal growth failure caused by growth hormone receptor expression. Using this model, we examined how a high-fat diet (lard-based 45kcal% fat)-induced mismatch between prenatal and postnatal environments could elevate blood pressure after growth. Although LBW rats fed standard chow had slightly higher blood pressure than control rats, their blood pressure was significantly higher than controls when exposed to a high-fat diet. Observation of glomeruli subjected to periodic acid methenamine silver (PAM) staining showed no difference in number or size. Aortic and cardiac angiotensin II receptor expression was altered with compensatory responses. Blood aldosterone levels were not different between control and LBW rats, but blood corticosterone levels were significantly higher in the latter with high-fat diet exposure. Administration of metyrapone, a steroid synthesis inhibitor, reduced blood pressure to levels comparable to controls. We showed that high-fat diet exposure causes impairment of the pituitary glucocorticoid negative feedback via miR-449a. These results clarify that LBW rats have increased blood pressure due to high glucocorticoid levels when they are exposed to a high-fat diet. These findings suggest a new therapeutic target for hypertension of LBW individuals.
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Presión Sanguínea/fisiología , Retroalimentación Fisiológica/fisiología , Glucocorticoides/sangre , Enfermedades de la Hipófisis/sangre , Enfermedades de la Hipófisis/fisiopatología , Hipófisis/fisiología , Animales , Peso al Nacer/efectos de los fármacos , Peso al Nacer/fisiología , Presión Sanguínea/efectos de los fármacos , Corticosterona/sangre , Dieta Alta en Grasa/efectos adversos , Femenino , Humanos , Hipertensión/sangre , Hipertensión/fisiopatología , Recién Nacido de Bajo Peso/sangre , Recién Nacido de Bajo Peso/fisiología , Recién Nacido , Masculino , Metirapona/uso terapéutico , Enfermedades de la Hipófisis/tratamiento farmacológico , Hipófisis/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/sangre , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Ratas , Ratas WistarRESUMEN
Cluster of differentiation (CD) 9 and CD81 are closely-related members of the tetraspanin family that consist of four-transmembrane domain proteins. Cd9 and Cd81 are highly expressed in breast cancer cells; however, their expression in healthy mammary glands is unclear. In this study, we performed quantitative real-time PCR to analyze the expression levels of Cd9 and Cd81. Histological techniques were employed to identify Cd9- and Cd81-expressing cells in rat mammary glands during pregnancy and lactation. It was observed that Cd9 and Cd81 were expressed in the mammary glands, and their expression levels correlated with mammary gland development. To identify cells expressing Cd9 and Cd81 in the mammary glands, we performed double immunohistochemical staining for CD9 and CD81, prolactin receptor long form, estrogen receptor alpha, or Ki67. The results showed that CD9 and CD81 were co-expressed in proliferating mammary epithelial cells. Next, we attempted to isolate CD9-positive epithelial cells from the mammary gland using pluriBead cell-separation technology based on antibody-mediated binding of cells to beads of different sizes, followed by isolation using sieves with different mesh sizes. We successfully isolated CD9-positive epithelial cells with 96.8% purity. In addition, we observed that small-interfering RNAs against Cd9 and Cd81 inhibited estrogen-induced proliferation of CD9-positive mammary epithelial cells. Our current findings may provide novel insights into the proliferation of mammary epithelial cells during pregnancy and lactation as well as in pathological processes associated with breast cancer.
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Células Epiteliales/citología , Perfilación de la Expresión Génica , Glándulas Mamarias Animales/metabolismo , Tetraspanina 28/biosíntesis , Tetraspanina 29/biosíntesis , Animales , Diferenciación Celular , Proliferación Celular , Dietilestilbestrol , Receptor alfa de Estrógeno/biosíntesis , Femenino , Antígeno Ki-67/biosíntesis , Lactancia , Embarazo , Preñez , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Mammalian Hedgehog (Hh) signaling plays key roles in embryogenesis and uniquely requires primary cilia. Functional analyses of several ciliogenesis-related genes led to the discovery of the developmental diseases known as ciliopathies. Hence, identification of mammalian factors that regulate ciliogenesis can provide insight into the molecular mechanisms of embryogenesis and ciliopathy. Here, we demonstrate that DYRK2 acts as a novel mammalian ciliogenesis-related protein kinase. Loss of Dyrk2 in mice causes suppression of Hh signaling and results in skeletal abnormalities during in vivo embryogenesis. Deletion of Dyrk2 induces abnormal ciliary morphology and trafficking of Hh pathway components. Mechanistically, transcriptome analyses demonstrate down-regulation of Aurka and other disassembly genes following Dyrk2 deletion. Taken together, the present study demonstrates for the first time that DYRK2 controls ciliogenesis and is necessary for Hh signaling during mammalian development.
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Cilios/metabolismo , Proteínas Hedgehog/fisiología , Organogénesis/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Animales , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
We investigated the effects of resolvin E (RvE) 1, RvE2, and RvE3 on IL-4- and IL-33-stimulated bone marrow-derived dendritic cells (BMDCs) from house dust mite (HDM)-sensitized mice. We also investigated the role of RvE3 in a murine model of HDM-induced airway inflammation. In vitro, BMDCs from HDM-sensitized mice were stimulated with IL-4 and IL-33 and then treated with RvE1, RvE2, RvE3, or vehicle. RvE1, RvE2, and RvE3 suppressed IL-23 release from BMDCs. In vivo, RvE3 administrated to HDM-sensitized and challenged mice in the resolution phase promoted a decline in total numbers of inflammatory cells and eosinophils, reduced levels of IL-23 and IL-17 in lavage fluid, and suppressed IL-23 and IL-17A mRNA expression in lung and peribronchial lymph nodes. RvE3 also reduced resistance in the lungs of HDM-sensitized mice. A NanoBiT ß-arrestin recruitment assay using human embryonic kidney 293 cells revealed that pretreatment with RvE3 suppressed the leukotriene B4 (LTB4)-induced ß-arrestin 2 binding to LTB4 receptor 1 (BLT1R), indicating that RvE3 antagonistically interacts with BLT1R. Collectively, these findings indicate that RvE3 facilitates the resolution of allergic airway inflammation, partly by regulating BLT1R activity and selective cytokine release by dendritic cells. Our results accordingly identify RvE3 as a potential therapeutic target for the management of asthma.-Sato, M., Aoki-Saito, H., Fukuda, H., Ikeda, H., Koga, Y., Yatomi, M., Tsurumaki, H., Maeno, T., Saito, T., Nakakura, T., Mori, T., Yanagawa, M., Abe, M., Sako, Y., Dobashi, K., Ishizuka, T., Yamada, M., Shuto, S., Hisada, T. Resolvin E3 attenuates allergic airway inflammation via the interleukin-23-interleukin-17A pathway.
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Asma/inmunología , Ácidos Grasos Insaturados/inmunología , Interleucina-17/inmunología , Subunidad p19 de la Interleucina-23/inmunología , Transducción de Señal/inmunología , Animales , Asma/patología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Femenino , Regulación de la Expresión Génica/inmunología , Leucotrieno B4/inmunología , Ratones , Ratones Endogámicos BALB C , Pyroglyphidae/inmunología , Receptores de Leucotrieno B4/inmunología , Arrestina beta 2/inmunologíaRESUMEN
Brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs) have been proposed as a new blood-brain barrier model, but their transport function has not been fully clarified. Therefore, in this study, we investigated the gene expression and function of transporters in hiPS-BMECs by means of quantitative reverse transcription-PCR, in vitro transcellular transport studies, and uptake experiments. mRNAs encoding ABC and SLC transporters, such as BCRP, MCT1, CAT1, and GLAST, were highly expressed in hiPS-BMECs. Transcellular transport studies showed that prazosin, [14C]l-lactate, [3H]l-arginine, and [3H]l-glutamate (substrates of BCRP, MCT1, CAT1, and GLAST, respectively) were transported asymmetrically across the hiPS-BMEC monolayer. Substrates of LAT1, OCTN2, CAT1, GLAST, MCT1, and proton-coupled organic cation (H+/OC) antiporter were taken up by hiPS-BMECs in a time-, temperature-, and concentration-dependent manner, and the uptakes were markedly decreased by inhibitors of the corresponding transporter. These results indicate that hiPS-BMECs express multiple nutrient and drug transporters.
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Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Arginina/farmacocinética , Diferenciación Celular , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ácido Glutámico/farmacocinética , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Ácido Láctico/farmacocinética , Proteínas de Transporte de Membrana/genética , Microvasos/citología , Prazosina/farmacocinética , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Acetylation of α-tubulin is a well-studied posttranscriptional modification, which is mostly catalyzed by α-tubulin N-acetyltransferase (ATAT1). ATAT1 possibly affects various cellular functions related with microtubules, such as intracellular transport, cell motility, cilia formation, and neuronal signaling. Here, we analyzed the subcellular localization of immunolabeled ATAT1 in human fibroblast KD cells through the cell cycle using confocal laser scanning microscopy. ATAT1 dramatically changed its localization through the cell cycle, depending on the mitotic phase. In interphase, immunolabeled ATAT1 was observed in centrioles, nuclei, and basal bodies if the cells projected primary cilia. ATAT1 was intensely detected as clusters in the nuclei in the G1-G2 phase. In telophase, ATAT1 colocalized with chromatids and spindle poles, and ultimately migrated to the daughter nucleus, newly synthesized centrioles, and midbody. The nucleolus is a core region of ribosomal RNA transcription, and the midbody is associated with severing and depolymerizing of microtubules in the stembody. The specific distributions of ATAT1 through the cell cycle suggest multiple functions of ATAT1, which could include acetylation of microtubules, RNA transcription activity, severing microtubules, and completion of cytokinesis.
Asunto(s)
Acetiltransferasas/metabolismo , Ciclo Celular , Fibroblastos/metabolismo , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Transcripción Genética , Acetilación , Línea Celular , Fibroblastos/fisiología , Humanos , Transporte de ProteínasRESUMEN
S100ß protein and SOX2-double positive (S100ß/SOX2-positive) cells have been suggested to be adult pituitary stem/progenitor cells exhibiting plasticity and multipotency. The aim of the present study was to isolate S100ß/SOX2-positive cells from the adult anterior lobes of rats using a specific antibody against a novel membrane marker and to study their characteristics in vitro. We found that cluster of differentiation (CD) 9 is expressed in the majority of adult rat S100ß/SOX2-positive cells, and we succeeded in isolating CD9-positive cells using an anti-CD9 antibody with a pluriBead-cascade cell isolation system. Cultivation of these cells showed their capacity to differentiate into endothelial cells via bone morphogenetic protein signalling. By using the anterior lobes of prolactinoma model rats, the localisation of CD9-positive cells was confirmed in the tumour-induced neovascularisation region. Thus, the present study provides novel insights into adult pituitary stem/progenitor cells involved in the vascularisation of the anterior lobe.