Positive regulation of Hedgehog signaling via phosphorylation of GLI2/GLI3 by DYRK2 kinase.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

DYRK2 maintains genome stability via neddylation of cullins in response to DNA damage.

Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8), an ubiquitin-like protein, is an essential regulator of the DNA damage response. Numerous studies have shown that neddylation (conjugation of NEDD8 to target proteins) dysfunction causes several human diseases, such as cancer. Hence clarifying the regulatory mechanism of neddylation could provide insight into the mechanism of genome stability underlying the DNA damage response (DDR) and carcinogenesis. Here, we demonstrate that dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a novel regulator of neddylation and maintains genome stability. Deletion of DYRK2 leads to persistent DNA double-strand breaks (DSBs) and subsequent genome instability. Mechanistically, DYRK2 promotes neddylation through forming a complex with NAE1, which is a component of NEDD8-activating enzyme E1, and maintaining its protein level by suppressing polyubiquitylation. The present study is the first to demonstrate that DYRK2 controls neddylation and is necessary for maintaining genome stability. This article has an associated First Person interview with the first author of the paper.

Inhibition of protein kinase C delta leads to cellular senescence to induce anti-tumor effects in colorectal cancer.

Protein kinase C delta (PKCδ) is a multifunctional serine-threonine kinase implicated in cell proliferation, differentiation, tumorigenesis, and therapeutic resistance. However, the molecular mechanism of PKCδ in colorectal cancer (CRC) remains unclear. In this study, we showed that PKCδ acts as a negative regulator of cellular senescence in p53 wild-type (wt-p53) CRC. Immunohistochemical analysis revealed that PKCδ levels in human CRC tissues were higher than those in the surrounding normal tissues. Deletion studies have shown that cell proliferation and tumorigenesis in wt-p53 CRC is sensitive to PKCδ expression. We found that PKCδ activates p21 via a p53-independent pathway and that PKCδ-kinase activity is essential for p21 activity. In addition, both repression of PKCδ expression and inhibition of PKCδ activity induced cellular senescence-like phenotypes, including increased senescence-associated ß-galactosidase (SA-ß-gal) staining, low LaminB1 expression, large nucleus size, and senescence-associated secretory phenotype (SASP) detection. Finally, a kinase inhibitor of PKCδ suppressed senescence-dependent tumorigenicity in a dose-dependent manner. These results offer a mechanistic insight into CRC survival and tumorigenesis. In addition, a novel therapeutic strategy for wt-p53 CRC is proposed.

Missing pieces of the pituitary puzzle: participation of extra-adenohypophyseal placode-lineage cells in the adult pituitary gland.

The pituitary gland is a major endocrine tissue composing of two distinct entities, the adenohypophysis (anterior pituitary, cranial placode origin) and the neurohypophysis (posterior pituitary, neural ectoderm origin), and plays important roles in maintaining vital homeostasis. This tissue is maintained by a slow, consistent cell-renewal system of adult stem/progenitor cells. Recent accumulating evidence shows that neural crest-, head mesenchyme-, and endoderm lineage cells invade during pituitary development and contribute to the maintenance of the adult pituitary gland. Based on these novel observations, this article discusses whether these lineage cells are involved in pituitary organogenesis, maintenance, regeneration, dysplasia, or tumors.

Fluctuation of CD9/SOX2-positive cell populations during the turnover of GH- and TSH-producing cells in the adult anterior pituitary gland.

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.

Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by the Unconventional Secretion of Cytosolic Proteins.

Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.

Enforced dual-specificity tyrosine-regulated kinase 2 expression by adenovirus-mediated gene transfer inhibits tumor growth and metastasis of colorectal cancer.

Colorectal cancer is one of the most common gastrointestinal tumors with good outcomes; however, with distant metastasis, the outcomes are poor. Novel treatment methods are urgently needed. Our in vitro studies indicate that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor in colorectal cancer by regulating cell survival, proliferation, and apoptosis induction. In addition, DYRK2 expression is decreased in tumor tissues compared to nontumor tissues in colorectal cancer, indicating a correlation with clinical prognosis. In this context, we devised a novel therapeutic strategy to overexpress DYRK2 in tumors by adenovirus-mediated gene transfer. The present study shows that overexpression of DYRK2 in colon cancer cell lines by adenovirus inhibits cell proliferation and induces apoptosis in vitro. Furthermore, in mouse subcutaneous xenograft and liver metastasis models, enforced expression of DYRK2 by direct or intravenous injection of adenovirus to the tumor significantly inhibits tumor growth. Taken together, these findings show that adenovirus-based overexpression of DYRK2 could be a novel gene therapy for liver metastasis of colorectal cancer.

Extracellular PKCδ signals to epidermal growth factor receptor for tumor proliferation in liver cancer cells.

Protein kinase C delta (PKCδ) is a multifunctional PKC family member and has been implicated in many types of cancers, including liver cancer. Recently, we have reported that PKCδ is secreted from liver cancer cells, and involved in cell proliferation and tumor growth. However, it remains unclear whether the extracellular PKCδ directly regulates cell surface growth factor receptors. Here, we identify epidermal growth factor receptor (EGFR) as a novel interacting protein of the cell surface PKCδ in liver cancer cells. Imaging studies showed that secreted PKCδ interacted with EGFR-expressing cells in both autocrine and paracrine manners. Biochemical analysis revealed that PKCδ bound to the extracellular domain of EGFR. We further found that a part of the amino acid sequence on the C-terminal region of PKCδ was similar to the putative EGFR binding site of EGF. In this regard, the point mutant of PKCδ in the binding site lacked the ability to bind to the extracellular domain of EGFR. Upon an extracellular PKCδ-EGFR association, ERK1/2 activation, downstream of EGFR signaling, was apparently induced in liver cancer cells. This study indicates that extracellular PKCδ behaves as a growth factor and provides a molecular basis for extracellular PKCδ-targeting therapy for liver cancer.

The multiciliated cells in Rathke's cleft express CYP26A1 and respond to retinoic acid in the pituitary.

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.

Differentiation of stem progenitor CD9/SOX2-positive cells is promoted with increased prolactin-producing and endothelial cells in the pituitary.

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.

Expression and localization of tight junction-related proteins in adult rat pituitary stem/progenitor cell niches.

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100ß, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.

Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells.

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.

New insights into the role and origin of pituitary S100ß-positive cells.

In the anterior pituitary, S100ß protein (S100ß) has been assumed to be a marker of folliculo-stellate cells, which are one of the non-hormone-producing cells existing in the parenchyma of the adult anterior lobe and are composed of subpopulations with various functions. However, recent accumulating studies on S100ß-positive cells, including non-folliculo-stellate cells lining the marginal cell layer (MCL), have shown the novel aspect that most S100ß-positive cells in the MCL and parenchyma of the adult anterior lobe are positive for sex determining region Y-box 2 (SOX2), a marker of pituitary stem/progenitor cells. From the viewpoint of SOX2-positive cells, the majority of these cells in the MCL and in the parenchyma are positive for S100ß, suggesting that S100ß plays a role in the large population of stem/progenitor cells in the anterior lobe of the adult pituitary. Reportedly, S100ß/SOX2-double positive cells are able to differentiate into hormone-producing cells and various types of non-hormone-producing cells. Intriguingly, it has been demonstrated that extra-pituitary lineage cells invade the pituitary gland during prenatal pituitary organogenesis. Among them, two S100ß-positive populations have been identified: one is SOX2-positive population which invades at the late embryonic period through the pituitary stalk and another is a SOX2-negative population that invades at the middle embryonic period through Atwell's recess. These two populations are likely the substantive origin of S100ß-positive cells in the postnatal anterior pituitary, while S100ß-positive cells emerging from oral ectoderm-derived cells remain unclear.

CD9-positive cells in the intermediate lobe of the pituitary gland are important supplier for prolactin-producing cells in the anterior lobe.

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.

CX3CL1/CX3CR1-signalling in the CD9/S100ß/SOX2-positive adult pituitary stem/progenitor cells modulates differentiation into endothelial cells.

Approximately 8% of CD9-, S100ß- and SOX2-triple positive (CD9/S100ß/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/S100ß/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/S100ß/SOX2-positive cells plays an important role in resupplying endothelial cells for vascular remodelling in the anterior lobe.

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.

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.

AMP-activated protein kinase activation reduces the transcriptional activity of the murine luteinizing hormone ß-subunit gene.

Malnutrition is one of the factors that induces reproductive disorders. However, the underlying biological processes are unclear. AMP-activated protein kinase (AMPK) is an enzyme that plays crucial role as a cellular energy sensor. In the present study, we examined the effects of AMPK activation on the transcription of the murine gonadotropin subunit genes Cga, Lhb, and Fshb, and the gonadotropin-releasing hormone receptor Gnrh-r. Real-time PCR and transcription assay using LßT2 cells demonstrated that 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, repressed the expression of Lhb. Next, we examined deletion mutants of the upstream region of Lhb and found that the upstream regulatory region of Lhb (-2527 to -2198 b) was responsible for the repression by AICAR. Furthermore, putative transcription factors (SP1, STAT5a, and TEF) that might mediate transcriptional control of the Lhb repression induced by AICAR were identified. In addition, it was confirmed that both AICAR and a competitive inhibitor of glucose metabolism, 2-deoxy-D-glucose, induced AMPK phosphorylation in LßT2 cells. Therefore, the upstream region of Lhb is one of the target sites for glucoprivation inducing AMPK activation. In addition, AMPK plays a role in repressing Lhb expression through the distal -2527 to -2198 b region.

Expression and functions of cluster of differentiation 9 and 81 in rat mammary epithelial cells.

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.

Involvement of GPR4 in increased growth hormone and prolactin expressions by extracellular acidification in MtT/S cells.

Hormone-secreting pituitary adenomas show unregulated hormonal hypersecretion and cause hyperpituitarism. However, the mechanism of the unregulated hormone production and secretion has not yet been fully elucidated. Solid tumors show reduced extracellular pH, partly due to lactate secretion from anaerobic glycolysis. It is known that extracellular acidification affects hormone secretion. However, whether and how the extracellular acidification influences the unregulated hormone production and secretion remain unknown. In the present study, we found that GPR4, a proton-sensing G protein-coupled receptor, was highly expressed in MtT/S cells, a growth hormone-producing and prolactin-producing pituitary tumor cell line. When we reduced the extracellular pH, growth hormone and prolactin mRNA expressions increased in the cells. Both increased expressions were partially suppressed by a GPR4 antagonist. We also found that extracellular acidification enhanced growth hormone-releasing factor-induced growth hormone secretion from MtT/S cells. These results suggest that GPR4 may play a role in hypersecretion of the hormone from hormone-producing pituitary tumors. A GPR4 antagonist will be a useful tool for preventing the hypersecretion.