Folliculostellate (FS) cells of the anterior pituitary gland express a variety of regulatory molecules. Using transgenic rats that express green fluorescent protein specifically in FS cells, we recently demonstrated that FS cells in vitro showed marked changes in motility, proliferation, and that formation of cellular interconnections in the presence of laminin, a component of the extracellular matrix, closely resembled those observed in vivo. These findings suggested that FS cells express matrix metalloproteinase-9 (MMP-9), which assists their function on laminin. In the present study, we investigate MMP-9 expression in rat anterior pituitary gland and examine its role in motility and proliferation of FS cells on laminin. Immunohistochemistry, RT-PCR, immunoblotting, and gelatin zymography were performed to assess MMP-9 expression in the anterior pituitary gland and cultured FS cells. Real-time RT-PCR was used to quantify MMP-9 expression in cultured FS cells under different conditions and treatments. MMP-9 expression was inhibited by pharmacological inhibitor or downregulated by siRNA and time-lapse images were acquired. A 5-bromo-2′-deoxyuridine assay was performed to analyze the proliferation of FS cells. Our results showed that MMP-9 was expressed in FS cells, that this expression was upregulated by laminin, and that laminin induced MMP-9 secretion by FS cells. MMP-9 inhibition and downregulation did not impair FS motility; however, it did impair the capacity of FS cells to form interconnections and it significantly inhibited proliferation of FS cells on laminin. We conclude that MMP-9 is necessary in FS cell interconnection and proliferation in the presence of laminin.

Folliculo-stellate (FS) cells in the anterior pituitary gland appear to possess multifunctional properties. Recently, the development of transgenic rats (S100b–green fluorescent protein (GFP) rats) that express GFP specifically in FS cells in the anterior pituitary gland has allowed us to distinguish and observe living FS cells in other kinds of pituitary cells. We used S100b–GFP rats to investigate the topographic affinity of FS cells for other pituitary cells. We observed living FS cells in enzymatically dispersed anterior pituitary cells of S100b–GFP rats under a fluorescent microscope, and noted that FS cells markedly extended and contracted cytoplasmic processes and formed interconnections with neighboring FS cells. In addition, FS cells adhered to small clusters of GFP-negative cells, which were primarily hormone-producing cells, and these clusters further aggregated during the course of cytoplasmic contraction. In the presence of laminin, fibronectin, and varying types of collagen, FS cells showed marked changes in shape and specific proliferative activity; however, GFP-negative cells did not. On reverse transcription-PCR analysis and immunohistochemistry, FS cells were shown to express integrin subunits, which are the cell surface receptors for extracellular matrix (ECM). In the anterior pituitary gland, FS cells and the various types of hormone-producing cells generate a unique topography in the presence of basement membrane components and interstitial collagens. The novel characteristics of FS cells observed in the present study suggest that in the anterior pituitary gland, FS cells play important roles in determining and/or maintaining local cellular arrangement in the presence of ECM components.

Folliculo-stellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. FS cells connect to each other not only by mechanical means, but also by gap junctional cell-to-cell communication. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture markedly change their shape, and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. Morphological and functional changes in cells are believed to be partly modified by matricrine signaling, by which ECM components function as cellular signals. In the present study, we examined whether gap junction formation between FS cells is affected by matricrine cues. A cell sorter was used to isolate FS cells from male S100b-GFP rat anterior pituitary for primary culture. We observed that mRNA and protein levels of connexin 43 in gap junction channels were clearly higher in the presence of laminin. In addition, we confirmed the formation of gap junctions between FS cells in primary culture by electron microscopy. Interestingly, we also observed that FS cells in the presence of laminin displayed well-developed rough endoplasmic reticulum and Golgi apparatus. Our findings suggest that, in anterior pituitary gland, FS cells may facilitate functional roles such as gap junctional cell-to-cell communication by matricrine signaling.

Folliculostellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture exhibited marked proliferation in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In a process referred to as matricrine action, FS cells receive ECM as a signal through their receptors, which results in morphological and functional changes. In this study, we investigated matricrine signaling in FS cells and observed that the proliferation of FS cells is mediated by integrin β1, which is involved in various signaling pathways for cell migration and proliferation in response to ECM. Then, we analyzed downstream events of the integrin β1 signaling pathway in the proliferation of FS cells and identified caveolin 3 as a potential candidate molecule. Caveolin 3 is a membrane protein that binds cholesterol and a number of signaling molecules that interact with integrin β1. Using specific small interfering RNA of caveolin 3, the proliferation of FS cells was inhibited. Furthermore, caveolin 3 drove activation of the mitogen-activated protein kinase (MAPK) signaling cascades, which resulted in upregulation of cyclin D1 in FS cells. These findings suggest that matricrine signaling in the proliferation of FS cells was transduced by a caveolin 3-mediated integrin β1 signaling pathway and subsequent activation of the MAPK pathway.

The anterior pituitary gland comprises five types of endocrine cells plus non-endocrine cells including folliculostellate cells, endothelial cells, and capillary mural cells (pericytes). In addition to being controlled by the hypothalamic–pituitary–target organ axis, the functions of these cells are likely regulated by local cell and extracellular matrix (ECM) interactions. However, these complex interactions are not fully understood. We investigated folliculostellate cell-mediated cell-to-cell interaction. Using S100β-GFP transgenic rats, which express GFP in folliculostellate cells, we designed a three-dimensional cell culture to examine the effects of folliculostellate cells. Interestingly, removal of folliculostellate cells reduced collagen synthesis (Col1a1 and Col3a1). Because pericytes are important collagen-producing cells in the gland, we stained for desmin (a pericyte marker). Removal of folliculostellate cells resulted in fewer desmin-positive pericytes and less desmin mRNA. We then attempted to identify the factor mediating folliculostellate cell–pericyte interaction. RT-PCR and in situ hybridization revealed that the important profibrotic factor transforming growth factor beta-2 (TGFβ2) was specifically expressed in folliculostellate cells and that TGFβ receptor II was expressed in pericytes, endothelial cells, and parenchymal cells. Immunocytochemistry showed that TGFβ2 induced SMAD2 nuclear translocation in pericytes. TGFβ2 increased collagen synthesis in a dose-dependent manner. This action was completely blocked by TGFβ receptor I inhibitor (SB431542). Diminished collagen synthesis in folliculostellate cell-deficient cell aggregates was partially recovered by TGFβ2. TGFβ2-mediated folliculostellate cell–pericyte interaction appears to be essential for collagen synthesis in rat anterior pituitary. This finding sheds new light on local cell–ECM interactions in the gland.

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.