菊地 元史

Psychoneuroimmunological studies have clearly demonstrated that both cellular and humoral immunity are related to major depression. Soluble ST2 is regarded as a key molecule regulating immune system as well as cell proliferation. Indeed, soluble ST2 is reported to reduce IL-33-induced IL-6 and TNF-α production in macrophages and IL-33-induced IL-5 and IL-13 production in type 2 innate lymphoid cells. Elevated serum concentrations of soluble ST2 have been reported in patients with neuropsychiatric disorders, suggesting pathophysiological roles of soluble ST2 in behavioral phenotypes. Nevertheless, the relation between soluble ST2 and depressive behavior remain to be uncovered. To complement this point, we performed broad behavioral phenotyping, utilizing transgenic mice with a high concentration of serum ST2 in the present study. Soluble ST2 overexpression mice (ST2 Tg mice) were generated on a C3H/HeJ background. ST2 Tg mice crossed onto the BALB/c genetic background were used. Before starting tests, each mouse was observed in a clean cage for a general health check and neurological screening tests. In Experiment I, comprehensive behavioral phenotyping was performed to reveal the role of soluble ST2 on sensorimotor functions, anxiety-like behaviors, depression-like behaviors, social behaviors, and learning and memory functions. In Experiment II, to confirm the role of soluble ST2 on depression-like behaviors, a depression test battery (two bottle choice test, forced swimming test, and tail suspension test) was applied. The general health check indicated good general health and normal gross appearance for ST2 Tg mice. Further, the neurological reflexes of all the mice were normal. We found that soluble ST2 overexpression resulted in decreased social interaction. Moreover, depression-like behaviors of ST2 Tg mice were observed in two well-established behavioral paradigms, the forced swimming test and the tail suspension test. Nevertheless, hedonic reaction to sucrose was observed in ST2 Tg mice similar to WT mice. These results suggest the depression in the ST2 Tg mice. In conclusion, through a series of experiments, we established the animal model for assessing role of soluble ST2 in neuropsychiatric disorders, and revealed the possible involvement of soluble ST2 in depressive behavior.

Laminin, a major basement membrane protein, comprises three subunit chains: α, β, and γ chains. Among these chains, only the laminin α chain is capable of signaling via laminin receptors. Although laminin isoforms containing the α5 chain were reported to be the first laminin produced during rat anterior pituitary gland development, the functions of these isoforms are unknown. We used immunohistochemical techniques to localize the laminin α5 chain and its specific receptor, basal cell adhesion molecule (BCAM), in fetal and adult pituitary gland. Laminin α5 chain immunoreactivity was observed in the basement membrane of the primordial adenohypophysis at embryonic days 12.5 to 19.5. Double immunostaining showed that BCAM was present and co-localized with the laminin α5 chain in the tissue. Quantitative analysis showed that the laminin α5 chain and BCAM were expressed in the anterior pituitary gland during postnatal development and in adulthood (postnatal day 60). In the adult gland, co-localization of the laminin α5 chain and BCAM was observed, and BCAM was detected in both the folliculo-stellate cells and endothelial cells. These results suggest that laminin α5 chain signaling via BCAM occurs in both the fetal adenohypophysis and adult anterior pituitary gland.

Retinoic acid (RA) is an important signaling molecule in embryonic development and adult tissue. The actions of RA are mediated by the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR), which regulate gene expression. RAR and RXR are widely expressed in the anterior pituitary gland. RA was reported to stimulate growth hormone (GH) gene expression in the anterior pituitary cells. However, current evidence is unclear on the role of RA in gene expression of growth hormone-releasing hormone receptor (Ghrh-r), growth hormone secretagogue receptor (Ghs-r) and somatostatin receptors (Sst-rs). Using isolated anterior pituitary cells of rats, we examined the effects of RA on gene expression of these receptors and GH release. Quantitative real-time PCR revealed that treatment with all-trans retinoic acid (ATRA; 10(-6) M) for 24 h increased gene expression levels of Ghrh-r and Ghs-r; however, expressions of Sst-r2 and Sst-r5 were unchanged. Combination treatment with the RAR-agonist Am80 and RXR-agonist PA024 mimicked the effects of ATRA on Ghrh-r and Ghs-r gene expressions. Exposure of isolated pituitary cells to ATRA had no effect on basal GH release. In contrast, ATRA increased growth hormone-releasing hormone (GHRH)- and ghrelin-stimulated GH release from cultured anterior pituitary cells. Our results suggest that expressions of Ghrh-r and Ghs-r are regulated by RA through the RAR-RXR receptor complex and that RA enhances the effects of GHRH and ghrelin on GH release from the anterior pituitary gland.

The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components.

Midkine (MK) belongs to a family of secreted heparin-binding growth factors and is highly expressed in various tissues during development. MK has multiple functions, such as regulation of cell proliferation, migration, survival and differentiation. We recently reported that MK mRNA is strongly expressed in the developing rat pituitary gland. In the adult pituitary, however, expression of MK and its receptor and the characteristics of the cells that produce them, have not been determined. Therefore, in this study, we investigate whether MK and its receptor, protein tyrosine phosphatase receptor-type Z (Ptprz1), are present in the adult rat pituitary. In situ hybridization, real-time reverse transcription-PCR and immunoblotting were performed to assess MK and Ptprz1 expression. We also characterize MK- and Ptprz1-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for each pituitary hormone or S100 protein [a marker of folliculostellate (FS) cells]. MK-expressing cells were located in the anterior and posterior lobes but not in the intermediate lobe. Double-staining and immunoblotting revealed that MK mRNA and protein were only expressed in FS cells in the anterior pituitary. Regarding Ptprz1 expression, Ptprz1 mRNA was detected in adrenocorticotropic hormone (ACTH) cells and growth hormone (GH) cells but not in prolactin cells, thyroid-stimulating hormone cells, luteinizing hormone cells, or FS cells. These findings suggest that MK produced in FS cells acts locally on ACTH cells and GH cells via Ptprz1 in the adult rat anterior pituitary.

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke's pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.

Cell-matrix interaction is required for tissue development. Laminin, a major constituent of the basement membrane, is important for structural support and as a ligand in tissue development. Laminin has 19 isoforms, which are determined by combinational assembly of five α, three β, and three γ chains (eg, laminin 121 is α1, β2, and γ1). However, no report has identified the laminin isoforms expressed during pituitary development. We used in situ hybridization to investigate all laminin chains expressed during rat anterior pituitary development. The α5 chain was expressed during early pituitary development (embryonic day 12.5-15.5). Expression of α1 and α4 chains was noted in vasculature cells at embryonic day 19.5, but later diminished. The α1 chain was re-expressed in parenchymal cells of anterior lobe from postnatal day 10 (P10), while the α4 chain was present in vasculature cells from P30. The α2 and α3 chains were transiently expressed in vasculature cells and anterior lobe, respectively, only at P30. Widespread distribution of β and γ chains was also observed during development. These findings suggest that numerous laminin isoforms are involved in anterior pituitary gland development and that alteration of the expression pattern is required for proper development of the gland.

The anterior pituitary gland is organized tissue comprising hormone-producing cells and folliculostellate (FS) cells. FS cells interconnect to form a meshwork, and their cytoplasmic processes are anchored by a basement membrane containing laminin. Recently, we developed a three-dimensional (3D) cell culture that reproduces this FS cell architecture. In this study of the novel function of FS cells, we used transgenic rats that express green fluorescent protein in FS cells for the 3D culture. Anterior pituitary cells were cultured with different proportions of FS cells (0%, 5%, 10%, and 20%). Anterior pituitary cells containing 5-20% FS cells formed round/oval cell aggregates, whereas amorphous cell aggregates were formed in the absence of FS cells. Interestingly, immunohistochemistry showed laminin-immunopositive cells instead of extracellular laminin deposition in FS cell-deficient cell aggregates. Double-immunostaining revealed that these laminin-immunopositive cells were gonadotrophs. Laminin mRNA expression did not differ in relation to the presence or absence of FS cells. When anterior pituitary cells with no FS cells were cultured with FS cell-conditioned medium, the proportion of laminin-immunopositive cells was lower than in control. These results suggest that a humoral factor from FS cells is required for laminin release from gonadotrophs.

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

In the anterior pituitary gland, folliculo-stellate cells and five types of hormone-producing cells are surrounded by an extracellular matrix (ECM) essential for these cells to perform their respective roles. Syndecans-type I transmembrane cell-surface heparan sulfate proteoglycans act as major ECM coreceptors via their respective heparan sulfate chains and efficiently transduce intracellular signals through the convergent action of their transmembrane and cytoplasmic domains. The syndecans comprise four family members in vertebrates: syndecan-1, -2, -3 and -4. However, whether syndecans are produced in the pituitary gland or whether they have a role as a coreceptor is not known. We therefore used (1) reverse transcription plus the polymerase chain reaction to analyze the expression of syndecan genes and (2) immunohistochemical techniques to identify the cells that produce the syndecans in the anterior pituitary gland of adult rat. Syndecan-2 mRNA expression was clearly detected in the corticotropes of the anterior pituitary gland. Moreover, the expression of syndecan-2 in the developing pituitary gland had a distinct temporospatial pattern. To identify the cells expressing syndecan-2 in the developing pituitary gland, we used double-immunohistochemistry for syndecan-2 and the cell markers E-cadherin (immature cells) and Ki-67 (proliferating cells). Some E-cadherin- and Ki-67-immunopositive cells expressed syndecan-2. Therefore, syndecan-2 expression occurs in developmentally regulated patterns and syndecan-2 probably has different roles in adult and developing anterior pituitary glands.

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components-collagens and laminin-were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization.

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

Laminin is a key component of the basement membrane and is involved in the structural scaffold and in cell proliferation and differentiation. Research has identified 19 laminin isoforms, which are assemblies of α, β, and γ chains (eg, the α1, β1, and γ1 chains form the laminin 111 isoform). Although laminin is known to be present in the anterior pituitary, the specific laminin isoforms have not been identified. This study used molecular biological and histochemical techniques-namely, RT-PCR, immunohistochemistry, and in situ hybridization-to identify the laminin isoforms and laminin-producing cells in rat anterior pituitary. RT-PCR showed that laminin α1, α3, and α4 genes were expressed in anterior pituitary. Immunohistochemistry revealed laminin α1 in gonadotrophs and laminin α4 in almost all vascular endothelial cells. Laminin α3 was seen in a subset of vascular endothelial cells. We then performed in situ hybridization to localize β and γ chains in these cells and found that laminin β1, β2, and γ1 were expressed in gonadotrophs and that laminin β1 and γ1 were expressed in endothelial cells. In conclusion, we identified gonadotroph-type (laminin 111 and 121) and vascular-type (laminin 411 and 311) laminin isoforms in rat anterior pituitary.

Folliculostellate (FS) cells in the anterior pituitary gland appear to have multifunctional properties. FS cells connect to each other at gap junctions and thereby form a histological and functional network. We have performed a series of studies on network formation in FS cells and recently reported that FS cells markedly prolong their cytoplasmic processes and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In this study, we investigated the mechanism of this extension of FS cell cytoplasmic processes under the influence of laminin and found that laminin promoted stress fiber formation within FS cells. Next, we noted that formation of stress fibers in FS cells was mediated by syndecan-4, a transmembrane proteoglycan that binds ECM and soluble factors via their extracellular glycosaminoglycan chain. We then observed that expressions of syndecan-4 and α-actinin (a microfilament bundling protein that cross-links actin stress fibers in FS cells) were upregulated by laminin. Using specific siRNA of syndecan-4, actin polymerization of FS cells was inhibited. Our findings suggest that FS cells received a signal from laminin-syndecan-4 interaction, which resulted in morphological changes, and that the formation of a morphological and functional network in FS cells was transduced by a syndecan-4-dependent mechanism in the presence of ECM.

The anterior pituitary gland is composed of five types of hormone-producing cells plus folliculostellate (FS) cells, which do not produce classical anterior pituitary hormones. FS cells are interconnected by cytoplasmic processes and encircle hormone-producing cells or aggregate homophilically. Using living-cell imaging of primary culture, we recently reported that some FS cells precisely extend their cytoplasmic processes toward other FS cells and form interconnections with them. These phenomena suggest the presence of a chemoattractant factor that facilitates the interconnection. In this study, we attempted to discover the factor that induces interconnection of FS cells and succeeded in identifying chemokine (CXC)-L12 and its receptor CXCR4 as potential candidate molecules. CXCL12 is a chemokine of the CXC subfamily. It exerts its effects via CXCR4, a G protein-coupled receptor. The CXCL12/CXCR4 axis is a potent chemoattractant for many types of neural cells. First, we revealed that CXCL12 and CXCR4 are expressed by FS cells in rat anterior pituitary gland. Next, to clarify the function of the CXCL12/CXCR4 axis in FS cells, we observed living anterior pituitary cells in primary culture with specific CXCL12 inhibitor or CXCR4 antagonist and noted that extension of cytoplasmic processes and interconnection of FS cells were inhibited. Finally, we examined FS cell migration and invasion by using Matrigel matrix assays. CXCL12 treatment resulted in markedly increased FS cell migration and invasion. These data suggest that FS cells express chemokine CXCL12 and its receptor CXCR4 and that the CXCL12/CXCR4 axis evokes interconnection of FS cells.