15 results on '"Shu TAKIGAMI"'
Search Results
2. Fluctuation of CD9/SOX2-positive cell populations during the turnover of GH- and TSH-producing cells in the adult anterior pituitary gland
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Kotaro HORIGUCHI, Yuto TSUTSUI, Ken FUJIWARA, Takehiro TSUKADA, Takashi NAKAKURA, Saishu YOSHIDA, Rumi HASEGAWA, and Shu TAKIGAMI
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cluster of differentiation 9 (cd9) ,growth hormone (gh) ,pituitary ,sex-determining region y-box 2 (sox2) ,thyroid-stimulating hormone (tsh) ,Reproduction ,QH471-489 ,Internal medicine ,RC31-1245 - Abstract
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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- 2023
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3. Differentiation of stem progenitor CD9/SOX2-positive cells is promoted with increased prolactin-producing and endothelial cells in the pituitary
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Kotaro HORIGUCHI, Ken FUJIWARA, Takehiro TSUKADA, Takashi NAKAKURA, Saishu YOSHIDA, Rumi HASEGAWA, and Shu TAKIGAMI
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cd9 ,lactotrophs ,pituitary gland ,pregnancy ,stem cells ,Reproduction ,QH471-489 ,Internal medicine ,RC31-1245 - Abstract
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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- 2022
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4. Expression and functions of cluster of differentiation 9 and 81 in rat mammary epithelial cells
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Kotaro HORIGUCHI, Saishu YOSHIDA, Takehiro TSUKADA, Takashi NAKAKURA, Ken FUJIWARA, Rumi HASEGAWA, Shu TAKIGAMI, and Shunji OHSAKO
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cluster of differentiation (cd) 9 ,cd81 ,lactation ,mammary gland ,proliferation ,Reproduction ,QH471-489 ,Internal medicine ,RC31-1245 - Abstract
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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- 2020
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5. The multiciliated cells in Rathke’s cleft express CYP26A1 and respond to retinoic acid in the pituitary
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Kotaro Horiguchi, Ken Fujiwara, Takehiro Tsukada, Takashi Nakakura, Saishu Yoshida, Rumi Hasegawa, and Shu Takigami
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Male ,Histology ,Pituitary Gland, Anterior ,Pituitary Gland ,Animals ,Tretinoin ,Cell Biology ,Rats, Wistar ,Retinoic Acid 4-Hydroxylase ,Rats ,Pathology and Forensic Medicine - Abstract
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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- 2022
6. CD9-positive cells in the intermediate lobe of the pituitary gland are important supplier for prolactin-producing cells in the anterior lobe
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Ken Fujiwara, Rumi Hasegawa, Saishu Yoshida, Shu Takigami, Yoshito Takeda, Kotaro Horiguchi, Takehiro Tsukada, Takashi Nakakura, and Shunji Ohsako
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Male ,0301 basic medicine ,Pituitary gland ,Histology ,Cell ,Population ,Biology ,Tetraspanin 29 ,Pathology and Forensic Medicine ,Prolactin cell ,Mice ,03 medical and health sciences ,0302 clinical medicine ,SOX2 ,medicine ,Animals ,Rats, Wistar ,Progenitor cell ,education ,education.field_of_study ,Cell Biology ,Prolactin ,Rats ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Pituitary Gland ,embryonic structures ,Stem cell ,030217 neurology & neurosurgery - Abstract
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 S100β 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 S100β/SOX2-positive cells of primary and secondary niches (named CD9/CD81/S100β/SOX2-positive cell), and the cells located in the AL-side niches exhibit plasticity and multipotency. However, it is unclear whether CD9/CD81/S100β/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/S100β/SOX2-positive cells from the IL-side primary niche. They had a higher level of S100β and SOX2 mRNA and a greater pituisphere forming capacity than those of CD9/CD81/S100β/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/S100β/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/S100β/SOX2-positive cells in the MCL of the IL-side are potential suppliers of adult core stem cells in the AL.
- Published
- 2021
7. CX3CL1/CX3CR1-signalling in the CD9/S100β/SOX2-positive adult pituitary stem/progenitor cells modulates differentiation into endothelial cells
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Ken Fujiwara, Takehiro Tsukada, Yukio Kato, Shunji Ohsako, Rumi Hasegawa, Kotaro Horiguchi, Saishu Yoshida, Takako Kato, Takashi Yashiro, and Shu Takigami
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Male ,0301 basic medicine ,Chemokine ,Histology ,CX3C Chemokine Receptor 1 ,S100 Calcium Binding Protein beta Subunit ,Tetraspanin 29 ,Vascular remodelling in the embryo ,03 medical and health sciences ,SOX2 ,Precursor cell ,CX3CR1 ,Animals ,Rats, Wistar ,Progenitor cell ,Receptor ,CX3CL1 ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chemokine CX3CL1 ,SOXB1 Transcription Factors ,Stem Cells ,Endothelial Cells ,Cell Differentiation ,Cell Biology ,Rats, Inbred F344 ,Rats ,Cell biology ,Medical Laboratory Technology ,030104 developmental biology ,Pituitary Gland ,embryonic structures ,biology.protein ,Signal Transduction - Abstract
Approximately 8% of CD9-, S100β- and SOX2-triple positive (CD9/S100β/SOX2-positive) stem/progenitor cells in the anterior lobe of the rat pituitary gland have previously been shown to differentiate into endothelial cells in vitro, suggesting that they play a role in vascularisation as tissue-resident vascular precursor cells. In the present study, we focused on chemokine ligands to further characterise the CD9/S100β/SOX2-positive cells and found that they distinctively express CX3C chemokine ligand 1 (Cx3cl1). Immunohistochemical analysis of the anterior lobe showed that CX3CL1-positive cells comprised 7.8% in CD9-positive cells. By cultivation of the CD9-positive cells on laminin-coated plates, we observed that the expression levels of Cx3cl1 decreased, while those of Sox18, an endothelial cell-progenitor marker, and Cx3cr1, a CX3CL1 receptor, increased. Furthermore, in a rat model of prolactinoma, the most common pituitary tumour, which is accompanied by frequent neo-vasculogenesis in the anterior lobe, we have confirmed a decrease in Cx3cl1 expression and an increase in Cx3cr1 expression, as well as a prominent increase in Sox18 expression. These findings suggest that CX3CL1/CX3CR1 signalling in CD9/S100β/SOX2-positive cells plays an important role in resupplying endothelial cells for vascular remodelling in the anterior lobe.
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- 2020
8. Expression and functions of cluster of differentiation 9 and 81 in rat mammary epithelial cells
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Takashi Nakakura, Rumi Hasegawa, Shunji Ohsako, Takehiro Tsukada, Ken Fujiwara, Kotaro Horiguchi, Shu Takigami, and Saishu Yoshida
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Mammary gland ,Proliferation ,Biology ,Real-Time Polymerase Chain Reaction ,Tetraspanin 29 ,Tetraspanin 28 ,Mammary Glands, Animal ,CD81 ,Tetraspanin ,Pregnancy ,Lactation ,medicine ,Animals ,RNA, Small Interfering ,Rats, Wistar ,Diethylstilbestrol ,Cell Proliferation ,Cluster of differentiation ,Gene Expression Profiling ,Prolactin receptor ,Estrogen Receptor alpha ,Cell Differentiation ,Epithelial Cells ,Rats ,Cell biology ,Ki-67 Antigen ,medicine.anatomical_structure ,embryonic structures ,Pregnancy, Animal ,Immunohistochemistry ,Female ,Original Article ,Cluster of differentiation (CD) 9 ,Animal Science and Zoology ,Estrogen receptor alpha - Abstract
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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- 2020
9. CD9-positive cells in the intermediate lobe migrate into the anterior lobe to supply endocrine cells
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Rumi Hasegawa, Shunji Ohsako, Takashi Nakakura, Takehiro Tsukada, Saishu Yoshida, Kotaro Horiguchi, Ken Fujiwara, and Shu Takigami
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0301 basic medicine ,Male ,Histology ,Enteroendocrine cell ,Tetraspanin 29 ,03 medical and health sciences ,Tissue culture ,Downregulation and upregulation ,Cell Movement ,Pituitary Gland, Anterior ,Animals ,Progenitor cell ,Rats, Wistar ,Molecular Biology ,030102 biochemistry & molecular biology ,Cluster of differentiation ,Chemistry ,Cell migration ,Cell Biology ,Cell biology ,Rats ,Medical Laboratory Technology ,030104 developmental biology ,embryonic structures ,Endocrine Cells ,Developmental biology ,CD81 - Abstract
The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL), and secretes important hormones for growth, sexual development, metabolism, and reproduction. In the marginal cell layer (MCL) facing Rathke's cleft between the IL and AL, cluster of differentiation (CD) 9-, CD81-, S100β-, and SOX2-quadruple positive (CD9/CD81/S100β/SOX2-positive) cells in the adult IL are settled as tissue-resident stem/progenitor cells supplying hormone-producing cells to the AL. However, it is unclear how CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL migrate into the AL across Rathke's cleft. In the present study, we performed chimeric pituitary tissue culture using S100β/GFP-transgenic rats and Wistar rats, and traced the footprint of S100β/GFP-expressing cells. We detected IL-side S100β/GFP-expressing cells in the AL tissue, demonstrating that these cells migrate from the IL to the AL. However, the cells failed to migrate in the opposite direction. Consistently, scanning electron microscopic analysis revealed well-developed cytoplasmic protrusions in the IL-side MCL, but not in the AL-side MCL, suggesting that IL-side CD9/CD81/S100β/SOX2-positive cells had higher migratory activity. We also searched for a specific marker for IL-side CD9/CD81/S100β/SOX2-positive cells and identified tetraspanin 1 (TSPAN1) from microarray analysis. Downregulation of Tspan1 by specific siRNA impaired cell migration and significantly reduced expression of snail family transcriptional repressor 2 (Slug), a marker of epithelial-mesenchymal transition (EMT). Therefore, CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL can be stem/progenitor cells that provide stem/progenitor cells to the AL-side MCL via SLUG-mediated EMT and cell migration.
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- 2021
10. Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells
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Rumi Hasegawa, Takashi Nakakura, Takehiro Tsukada, Saishu Yoshida, Shu Takigami, Shunji Ohsako, Kotaro Horiguchi, and Ken Fujiwara
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0301 basic medicine ,Male ,Pituitary gland ,Histology ,Cell ,Biology ,Antibodies ,Tetraspanin 29 ,03 medical and health sciences ,Mice ,SOX2 ,medicine ,Animals ,Progenitor cell ,Molecular Biology ,Mice, Inbred ICR ,030102 biochemistry & molecular biology ,Cluster of differentiation ,Cell growth ,Stem Cells ,Cell Differentiation ,Cell Biology ,Immunohistochemistry ,Cell biology ,Medical Laboratory Technology ,030104 developmental biology ,medicine.anatomical_structure ,Pituitary Gland ,embryonic structures ,Stem cell ,Developmental biology - Abstract
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 S100β. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100β/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/S100β/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 S100β. 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/S100β/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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- 2020
11. Identification of THY1 as a novel thyrotrope marker and THY1 antibody-mediated thyrotrope isolation in the rat anterior pituitary gland
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Shu Takigami, Takako Kato, Rumi Hasegawa, Takehiro Tsukada, Shunji Ohsako, Takashi Nakakura, Naoko Kanno, Yukio Kato, Kotaro Horiguchi, and Saishu Yoshida
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Male ,0301 basic medicine ,medicine.medical_specialty ,Pituitary gland ,Biophysics ,Thyrotropin ,Cell Separation ,In situ hybridization ,urologic and male genital diseases ,Biochemistry ,03 medical and health sciences ,Anterior pituitary ,Antigen ,Pituitary Gland, Anterior ,Thyrotropic cell ,Internal medicine ,medicine ,Animals ,CD90 ,Rats, Wistar ,Molecular Biology ,Thymocytes ,biology ,Cell Biology ,Juxtacrine signalling ,Molecular biology ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,CD18 Antigens ,biology.protein ,Thy-1 Antigens ,Antibody ,Biomarkers - Abstract
Contact-dependent (juxtacrine) signaling is important for local cell-to-cell interaction and has received attention in recent years regarding its role in pituitary function, differentiation, and development. This study investigated one of the juxtacrine-related molecules, thymocyte differentiation antigen 1 (THY1), in the anterior lobe of the rat pituitary gland. Western blot analysis revealed expression of the THY1 protein in the adult rat anterior lobe. We also found that the THY1 ligand, integrin-β2 (ITGB2), is also expressed in the pituitary gland. In situ hybridization and immunohistochemical analyses showed that both THY1 mRNA and protein were present in almost, if not all, thyroid-stimulating hormone (TSH)-immunopositive cells (thyrotropes) and that ITGB2 was co-expressed in these cells. As THY1 appeared to represent a novel marker for thyrotropes, we then attempted to isolate these cells from various anterior lobe cells by the use of a THY1 antibody and a pluriBead-cascade cell isolation system. This technology allowed the isolation of thyrotropes with 83% purity at about 17-fold enrichment. Furthermore, the isolated THY1-immunopositive cells had higher Tsh mRNA levels compared with THY1-immunonegative cells and released TSH in response to thyrotropin-releasing hormone. These findings indicated that THY1 represents a potent thyrotrope marker and that the thyrotrope isolation method using the THY1 antibody may serve as a powerful tool to analyze their function including juxtacrine regulation through THY1/ITGB2 interaction.
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- 2016
12. Isolation and characterization of cluster of differentiation 9-positive ependymal cells as potential adult neural stem/progenitor cells in the third ventricle of adult rats
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Rumi Hasegawa, Takako Kato, Saishu Yoshida, Kotaro Horiguchi, Yukio Kato, Shu Takigami, and Shunji Ohsako
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0301 basic medicine ,Male ,Histology ,Ependymal Cell ,Biology ,Tetraspanin 29 ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Neural Stem Cells ,Neurosphere ,Ependyma ,medicine ,Animals ,Progenitor cell ,Rats, Wistar ,In Situ Hybridization ,Cell Proliferation ,Third Ventricle ,Third ventricle ,Cluster of differentiation ,Stem Cells ,Cell Differentiation ,Cell Biology ,Immunohistochemistry ,Oligodendrocyte ,Cell biology ,Rats ,030104 developmental biology ,medicine.anatomical_structure ,Neuron ,030217 neurology & neurosurgery ,Astrocyte - Abstract
Ependymal cells located above the ventricular zone of the lateral, third, and fourth ventricles and the spinal cord are thought to form part of the adult neurogenic niche. Many studies have focused on ependymal cells as potential adult neural stem/progenitor cells. To investigate the functions of ependymal cells, a simple method to isolate subtypes is needed. Accordingly, in this study, we evaluated the expression of cluster of differentiation (CD) 9 in ependymal cells by in situ hybridization and immunohistochemistry. Our results showed that CD9-positive ependymal cells were also immunopositive for SRY-box 2, a stem/progenitor cell marker. We then isolated CD9-positive ependymal cells from the third ventricle using the pluriBead-cascade cell isolation system based on antibody-mediated binding of cells to beads of different sizes and their isolation with sieves of different mesh sizes. As a result, we succeeded in isolating CD9-positive populations with 86% purity of ependymal cells from the third ventricle. We next assayed whether isolated CD9-positive ependymal cells had neurospherogenic potential. Neurospheres were generated from CD9-positive ependymal cells of adult rats and were immunopositve for neuron, astrocyte, and oligodendrocyte markers after cultivation. Thus, based on these findings, we suggest that the isolated CD9-positive ependymal cells from the third ventricle included tanycytes, which are special ependymal cells in the ventricular zone of the third ventricle that form part of the adult neurogenic and gliogenic niche. These current findings improve our understanding of tanycytes in the adult third ventricle in vitro.
- Published
- 2019
13. CXCL10/CXCR3 signaling mediates inhibitory action by interferon-gamma on CRF-stimulated adrenocorticotropic hormone (ACTH) release
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Takehiro Tsukada, Takashi Yashiro, Kozue Tateno, Shunji Ohsako, Kotaro Horiguchi, Masashi Higuchi, Shu Takigami, Ken Fujiwara, Rumi Hasegawa, Takako Kato, Saishu Yoshida, and Yukio Kato
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Male ,0301 basic medicine ,Agonist ,endocrine system ,medicine.medical_specialty ,Pro-Opiomelanocortin ,Receptors, CXCR3 ,Histology ,Corticotropin-Releasing Hormone ,medicine.drug_class ,medicine.medical_treatment ,S100 Calcium Binding Protein beta Subunit ,Adrenocorticotropic hormone ,CXCR3 ,Pathology and Forensic Medicine ,Interferon-gamma ,03 medical and health sciences ,Paracrine signalling ,Adrenocorticotropic Hormone ,Anterior pituitary ,Pituitary Gland, Anterior ,Internal medicine ,medicine ,Animals ,CXCL10 ,Rats, Wistar ,Cells, Cultured ,Inflammation ,Chemistry ,hemic and immune systems ,Cell Biology ,Rats ,Chemokine CXCL10 ,030104 developmental biology ,Endocrinology ,Cytokine ,medicine.anatomical_structure ,Corticotropic cell ,Rats, Transgenic ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
Secretion of hormones by the anterior pituitary gland can be stimulated or inhibited by paracrine factors that are produced during inflammatory reactions. The inflammation cytokine interferon-gamma (IFN-γ) is known to inhibit corticotropin-releasing factor (CRF)-stimulated adrenocorticotropin (ACTH) release but its signaling mechanism is not yet known. Using rat anterior pituitary, we previously demonstrated that the CXC chemokine ligand 10 (CXCL10), known as interferon-γ (IFN-γ) inducible protein 10 kDa, is expressed in dendritic cell-like S100β protein-positive (DC-like S100β-positive) cells and that its receptor CXCR3 is expressed in ACTH-producing cells. DC-like S100β-positive cells are a subpopulation of folliculo-stellate cells in the anterior pituitary. In the present study, we examine whether CXCL10/CXCR3 signaling between DC-like S100β-positive cells and ACTH-producing cells mediates inhibition of CRF-activated ACTH-release by IFN-γ, using a CXCR3 antagonist in the primary pituitary cell culture. We found that IFN-γ up-regulated Cxcl10 expression via JAK/STAT signaling and proopiomelanocortin (Pomc) expression, while we reconfirmed that IFN-γ inhibits CRF-stimulated ACTH-release. Next, we used a CXCR3 agonist in primary culture to analyze whether CXCL10 induces Pomc-expression and ACTH-release using a CXCR3 agonist in the primary culture. The CXCR3 agonist significantly stimulated Pomc-expression and inhibited CRF-induced ACTH-release, while ACTH-release in the absence of CRF did not change. Thus, the present study leads us to an assumption that CXCL10/CXCR3 signaling mediates inhibition of the CRF-stimulated ACTH-release by IFN-γ. Our findings bring us to an assumption that CXCL10 from DC-like S100β-positive cells acts as a local modulator of ACTH-release during inflammation.
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- 2015
14. Isolation and characterisation of CD9-positive pituitary adult stem/progenitor cells in rats
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Shu Takigami, Yukio Kato, Takehiro Tsukada, Rumi Hasegawa, Ken Fujiwara, Shunji Ohsako, Takashi Nakakura, Takako Kato, Saishu Yoshida, Kotaro Horiguchi, Takashi Yashiro, and Ken Arae
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0301 basic medicine ,Male ,Pituitary gland ,Endothelium ,Cellular differentiation ,lcsh:Medicine ,S100 Calcium Binding Protein beta Subunit ,Bone morphogenetic protein ,Tetraspanin 29 ,Article ,03 medical and health sciences ,Pituitary Gland, Anterior ,medicine ,Animals ,Prolactinoma ,Progenitor cell ,Rats, Wistar ,lcsh:Science ,Cells, Cultured ,Cell Proliferation ,Multidisciplinary ,Cluster of differentiation ,biology ,lcsh:R ,Cell Differentiation ,In vitro ,Cell biology ,Rats ,Adult Stem Cells ,030104 developmental biology ,medicine.anatomical_structure ,embryonic structures ,biology.protein ,lcsh:Q ,Endothelium, Vascular ,Antibody - Abstract
S100β protein and SOX2-double positive (S100β/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 S100β/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 S100β/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.
- Published
- 2017
15. Degradation of serum microRNAs during transient storage of serum samples at 4℃
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Toshiko Aiso, Hiroaki Ohnishi, Akiko Yamaki, and Shu Takigami
- Subjects
0301 basic medicine ,business.industry ,Clinical Biochemistry ,Pilot Projects ,General Medicine ,Serum samples ,Real-Time Polymerase Chain Reaction ,Specimen Handling ,Cold Temperature ,03 medical and health sciences ,Circulating MicroRNA ,MicroRNAs ,030104 developmental biology ,0302 clinical medicine ,Transient storage ,030220 oncology & carcinogenesis ,microRNA ,Immunology ,Medicine ,Humans ,business - Abstract
Background Numerous studies demonstrate the potential of circulating microRNAs as non-invasive biomarkers for several diseases. Circulating microRNAs are much more stable than mRNAs and remain largely intact even after prolonged incubation at room temperature. However, recent reports show that microRNAs in serum or plasma samples have diverse stabilities. The aim of this pilot study is to evaluate the stabilities of miR-92a, miR-122 and miR-145 in serum during transient storage at 4℃ before freezing. Methods Serum samples were stored for 24 h at 4℃, and then RNA was extracted from whole serum or extracellular vesicles in serum. Total Exosome Isolation Reagent (from serum) was used for the fractionation of extracellular vesicles. Reverse transcription and real-time PCR of microRNAs were performed using the TaqMan MicroRNA Assays for miR-92a, miR-122 and miR-145. Results MiR-122 and miR-145 were degraded rapidly in serum; the concentrations dropped to 35.9% ( P Conclusions MiR-122 and miR-145 in serum are extremely unstable and could be degraded during transient storage of serum at 4℃ prior to freezing.
- Published
- 2017
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