225 The extracellular matrix component laminin promotes formation in the rat anterior

Kotaro Horiguchi, Tom Kouki, Ken Fujiwara, Motoshi Kikuchi and Takashi Yashiro Division of Histology and Biology, Department of Anatomy, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan (Correspondence should be addressed to T Yashiro; Email: [email protected])

Abstract Folliculo-stellate (FS) cells in the gland are between FS cells is affected by matricrine cues. A cell believed to have multifunctional properties. FS cells connect sorter was used to isolate FS cells from male S100b-GFP rat to each other not only by mechanical means, but also by gap anterior pituitary for primary culture. We observed that junctional cell-to-cell communication. Using transgenic rats mRNA and protein levels of connexin 43 in gap junction that express green fluorescent protein (GFP) specifically in FS channels were clearly higher in the presence of laminin. In cells in the anterior pituitary gland (S100b-GFP rats), we addition, we confirmed the formation of gap junctions recently revealed that FS cells in primary culture markedly between FS cells in primary culture by electron microscopy. change their shape, and form numerous interconnections Interestingly, we also observed that FS cells in the presence with neighboring FS cells in the presence of laminin, an of laminin displayed well-developed rough endoplasmic extracellular matrix (ECM) component of the basement reticulum and Golgi apparatus. Our findings suggest that, in membrane. Morphological and functional changes in cells are anterior pituitary gland, FS cells may facilitate functional roles believed to be partly modified by matricrine signaling, by such as gap junctional cell-to-cell communication by which ECM components function as cellular signals. In the matricrine signaling. present study, we examined whether gap junction formation Journal of Endocrinology (2011) 208, 225–232

Introduction the basement membrane (Inoue et al. 1999, Shirasawa et al. 2004). Generally, FS cells can receive extracellular matrices The anterior pituitary gland comprises five types of (ECM) as signals that can exert functional changes, a process hormone-producing cells plus folliculo-stellate (FS) cells, called matricrine signaling (Koide & Ito 2000, Miyamoto which do not produce classical anterior pituitary hormones. et al. 2007). Based on the histological features of FS cells, we FS cells have a star-like appearance (Farquhar 1957, hypothesized that FS cells require matricrine signaling for Vila-Porcile 1972), and pseudolumina form in the center of their functional roles. FS cell clusters (Soji & Herbert 1989). It has been suggested Recently, Itakura et al. (2007) succeeded in producing an that FS cells are stem cells, , or cells that regulate S100b-GFP transgenic rat that expresses green fluorescent hormone release (Inoue et al. 1999, Allaerts & Vankelecom protein (GFP) specifically in FS cells in the anterior pituitary. 2005). In addition, gap junctions between FS-cells form Using S100b-GFP rat anterior pituitary cells, we found intercellular channels that facilitate transmission of ions and that gap junctional cell-to-cell communication was immu- small molecules between adjacent cells and allow direct nohistochemically reconstructed between FS cells in vitro cell–cell communication within the anterior pituitary gland (Horiguchi et al. 2008). Furthermore, using living-cell (Fauquier et al. 2001, Sato et al. 2005). It is also known that imaging, we revealed that FS cells under the influence of connexin 43, which is the protein subunit that forms gap ECM components may play important roles in determining junctions, is mainly expressed in the anterior pituitary (Meda and/or maintaining local cellular arrangement (Horiguchi et al. 1993). et al. 2010). The above-mentioned morphological and It has been shown that the lobular structures that are functional characteristics of FS cells lend weight to the surrounded by basement membrane are responsible for supposition that matricrine action influences cell-to-cell organizing the functional unit of the anterior pituitary communication within the anterior pituitary gland. In gland (Soji & Herbert 1989, Shirasawa et al. 2004). FS cells order to confirm this hypothesis, we attempted to determine are located in the core of these lobular structures, and, whether laminin, which is an ECM component of the interestingly, the cytoplasmic processes of FS cells attach to basement membrane, affects gap junction formation between

Journal of Endocrinology (2011) 208, 225–232 DOI: 10.1677/JOE-10-0297 0022–0795/11/0208–225 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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FS cells. Using the S100b-GFP transgenic rat, we were able to described above. We time-lapse recorded the cells by using observe the localization of connexin 43 in gap junction a digital camera (ORCA-ER; Hamamatsu Photonics, channels by immunohistochemistry, investigate the assembly Shizuoka, Japan) and MetaMorph software (Molecular of gap junctions by electron microscopy, and analyze Devices Corp., Downingtown, PA, USA). Each observation the expression of connexin 43 by real-time PCR and was performed in triplicate. western blotting. Immunocytochemistry Cultured cells fixed with 4% paraformaldehyde in 50 mM Materials and Methods cacodylate buffer for 20 min at room temperature were first immersed in PBS containing 2% normal goat serum for Animals 20 min at 30 8C, then incubated with anti-rat connexin Transgenic S100b-GFP rats express GFP under control of 43 mouse monoclonal antibody (0.5 mg/ml, Zymed Lab, the promoter of the S100b protein gene, a marker of FS South San Francisco, CA, USA) overnight at room cells. The rats were donated by Prof. K Inoue of Saitama temperature. After washing with PBS, cells were incubated University and were bred in our laboratory (Itakura et al. in PBS with Alexa Fluor 568-conjugated goat anti-mouse 2007). Eight- to ten-week-old male rats weighing 250 to IgG diluted to 1:200. Absence of an observable nonspecific 300 g were given ad libitum access to food and water and reaction was confirmed using normal mouse serum. housed under conditions of 12 h light:12 h darkness. The rats were killed by exsanguination from the right atrium under Electron microscopic observation deep Nembutal anesthesia, and were then perfused with C C Ca2 - and Mg2 -free Hanks’ solution for primary culture. For observation of gap junctions between FS cells, FS cells in All animals were treated in accordance with the Guidelines for primary culture on the uncoated and laminin-coated surfaces Animal Experimentation of Jichi Medical University, which were fixed with 2.5% glutaraldehyde in 0.1 M phosphate are based on the NIH Guidelines for the Care and Use of buffer, pH 7.4, for 1 h at 4 8C. Then, FS cells were postfixed Laboratory Animals. with 1% OsO4 in 0.1 M phosphate buffer for 1 h at 4 8C, dehydrated in a series of graded alcohols, and embedded in epoxy resin (Quetol 812; Nissin EM Co., Tokyo, Japan). FS Cell culture cells were sectioned into ultrathin slices with a Reichert- Anterior pituitary cells of male S100b-GFP rats were Nissei Ultracut S (Leica Microsystems, Wetzlar, Germany), dispersed as described previously (Horiguchi et al. 2008). stained with uranyl acetate and lead citrate, and then observed Dispersed cells were separated into GFP-positive and GFP- under a Hitachi H-7600 electron microscope (Hitachi). negative cells by a cell sorter (MoFlo XDP: Beckman Coulter, Inc., Fullerton, CA, USA). GFP-positive cells were plated Quantification of connexin 43 mRNA levels by real-time reverse onto 8-well glass chamber slides (1 cm2/well; Nalge Nunc transcription-PCR International, Rochester, NY, USA), with or without a coating of 10 mg/cm2 of laminin (Millipore, Bedford, MA, Total RNA fractions were prepared with Trizol reagent USA), at a density of 1!105 cells/cm2 in 400 ml of Medium (Invitrogen) from cultured cells and incubated with RNase- 199 with Earle’s salts (Invitrogen) supplemented with 10% free DNase I (1 U/tube; Promega Corp.). After inactivation fetal bovine serum (Sigma–Aldrich Corp.), 0.5U/ml of DNase I by heating for 10 min at 65 8C, cDNA was penicillin, and 0.5 mg/ml streptomycin (Invitrogen). In the synthesized using the Superscript III reverse transcription other experiment group, laminin-coated slides were treated in (RT) kit with oligo-(dT)20 primer (Invitrogen). Quantitative medium containing 5 mg/ml monoclonal anti-integrin b1 real-time PCR (ABI PRISM 7900HT; Applied Biosystems, antibody (clone Ha2/5, BD Biosciences, San Jose, CA, USA), Carlsbad, CA, USA) was performed by using gene-specific which was shown to block signaling mediated by integrin b1 primers and SYBR Premix Ex Taq (Takara, Tokyo, Japan) (Schultz & Armant 1995); hamster IgM served as control. containing SYBR Green I. The following primers were used Cells were then cultured for 72 h at 37 8C in a humidified to amplify cDNA fragments of connexin 43 (gap junction- atmosphere of 5% CO2 and 95% air. associated protein 1; GenBank accession no. NM_012567): forward 50-CTTCCTGCTCATCCAGTGGT-30 and reverse 50-GGACGTGAGAGGAAGCAGTC-30 (105 bp). Time-lapse observation of primary culture For normalization, we also quantified glyceraldehyde 0 The GFP-positive cells were cultured in a CO2 gas culture 3-phosphate dehydrogenase (M_17701): forward 5 -AAG- chamber (Sankei Corp., Tokyo, Japan) with a thermostat GGCTCATGACCACAGTC-30 and reverse 50-GGATGC- (Kokensha Engineering Corp., Tokyo, Japan) on a fluor- AGGGATGATGTTCT-30 (116 bp). Relative quantification escence-inverted microscope (IX71; Olympus Corp., Tokyo, was conducted using the standard curve method and was Japan). Culture conditions were the same as those performed in triplicate.

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Immunoblot analysis (Fig. 1BandSupplementary Movie 2, see section on supplementary data given at the end of this article). Later, After primary culture, cells were washed in PBS and lysed in the cytoplasmic processes of a large number of FS cells RIPA buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, interconnected (Fig. 1B). In contrast, only a few FS cells 0.1% w/v SDS, 1% v/v Triton X-100, pH 7.5), and total extended their cytoplasmic processes on the uncoated surface protein was estimated by the Bradford assay (Sigma). Twenty at 24 h (Fig. 1A and Supplementary Movie 1, see section on micrograms of protein from each sample was applied to 12% supplementary data given at the end of this article). At 72 h, SDS-PAGE. Proteins were then transferred electrophoreti- almost all FS cells on laminin were flattened, with cally to Immobilon-P transfer membrane (Millipore). interconnected cell bodies (Fig. 1B). However, FS cells on The membrane was blocked with 2% normal goat serum in the uncoated surface formed clusters of approximately ten TBST (50 mM Tris, 100 mM NaCl, 0.1% v/v Tween 20, pH cells (Fig. 1A). FS cells on the laminin-coated surface with 7.4) for 30 min. After washing with TBST, the membrane integrin b1 antibody aggregated and formed clusters of was incubated overnight with mouse monoclonal anti- approximately 12 cells at 2–24 h (Fig. 1D and Supplementary connexin 43 antibody (0.05 mg/ml; Zymed Lab) or mouse Movie 3, see section on supplementary data given at the end anti-b-actin antibody (0.1 mg/ml, BioVision, Mountain of this article). They remained clustered until 72 h of primary View, CA, USA), diluted in Can Get Signal solution culture (Fig. 1D). The motility of FS cells on laminin-coated (Toyobo, Osaka, Japan), followed by TBST washes, and surface with hamster IgM and those on laminin-coated incubated for 1 h with HRP-labeled secondary antibodies surface was similar (Fig. 1C). (EnvisionCSystem-HRP, anti-rabbit, Dako, Glostrup, Denmark). After washing with TBST, bands were visualized by 3,30-diaminobenzidine. The membrane was scanned, and Localization of connexin 43 on FS cells densitometric analysis was performed with ImageJ software We determined the localization of connexin 43 by (NIH, Bethesda, MD, USA). The results were normalized immunohistochemistry after 72 h in primary culture on with b-actin. Each analysis was performed in triplicate. both uncoated and laminin-coated surfaces. Immunoreactive connexin 43 was located mainly on cell bodies and partly in Statistical analysis the cytoplasmic processes at the interconnection of FS cells on both uncoated and laminin-coated surfaces (Fig. 2B, D, H, All results were presented as the meanGS.E.M. Statistical and J). Connexin 43 immunoreactivity of FS cells on the analysis was performed using ANOVA with Fisher’s protected laminin-coated surface was clearly greater than that observed least significant difference test (StatView 5.0 software, on the uncoated surface (Fig. 2B and D). In contrast, SAS Institute, Inc., Cary, NC, USA). Differences between treatment with anti-integrin b1 antibody completely blocked groups were considered to be statistically significant at a the increase in connexin 43 immunoreactivity between P value of !0.05. FS cells on laminin-coated surface with hamster IgM (Fig. 2H and J).

Results Quantification of connexin 43 mRNA and protein levels To determine whether laminin affects connexin 43 gene Time-lapse imaging of FS cells in primary culture expression in FS cells, we performed quantitative real-time A cell sorter was used to isolate FS cells from male S100b- RT-PCR. As compared with the uncoated surface, connexin GFP rat anterior pituitary for primary culture. At 2 and 72 h 43 expression was higher in cells on the laminin-coated of primary culture, the percentage of FS cells among all cells surface at 72 h (Fig. 2E). Using western blot analysis, was 96.0G0.4 and 96.4G0.2% (meanGS.E.M, nZ3) respect- connexin 43 immunoreactivity was also detected in FS cells ively. We observed the behavior of FS cells in primary culture as an w43-kDa band, which conforms to the expected size of on the uncoated and laminin-coated surfaces (Fig. 1A–D, rat connexin 43 (Fig. 2F). When normalized with b-actin, the Supplementary Movies 1–3, see section on supplementary expression of connexin 43 protein was greater in FS cells on data given at the end of this article). In culture for 2–24 h, we the laminin-coated surface (Fig. 2F). In contrast, at 72 h, the noted that FS cells on the uncoated surface were remarkably connexin 43 expression level on the laminin-coated surface motile as compared with cells on laminin-coated surface. with anti-integrin b1 antibody was not lower than that on the FS cells aggregated and formed clusters of approximately laminin-coated surface with hamster IgM (Fig. 2K and L). 12 cells on the uncoated surface at 24 h (Fig. 1A and Supplementary Movie 1, see section on supplementary data Electron microscopic observation of FS cells given at the end of this article). Although FS cells on the laminin-coated surface were solitary, paired, or formed small To verify the presence of gap junctions between FS cells, we clusters of only a few FS cells at 2 h (Fig. 1B), they flattened used an electron microscope to observe FS cells in primary and markedly extended their cytoplasmic processes at 24 h culture on the uncoated and laminin-coated surface. At gap www.endocrinology-journals.org Journal of Endocrinology (2011) 208, 225–232

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2 h 24 h 48 h 72 h A

B

C

D

Figure 1 Time-lapse images of FS cells isolated from male S100b-GFP rat anterior pituitary cells in primary culture on an uncoated surface, laminin-coated surface, laminin-coated surface with hamster IgM, and laminin- coated surface with integrin b1 antibody. Cells were time-lapse recorded at 5 min intervals from 2 to 72 h after cells were plated. (A–D) GFP images superimposed on phase contrast images, using a confocal laser microscope on an uncoated surface, laminin-coated surface, laminin-coated surface with hamster IgM, and laminin-coated surface with integrin b1 antibody respectively. (A–D) 2, 24, 48, and 72 h. The elapsed time from plating of cells was 2, 24, 48, and 72 h respectively. Scale bar, 100 mm.

junctions, the intercellular space is 2–4 nm, and the The S100b-GFP rat (Itakura et al. 2007) model is a membranes of cells are lined up in parallel. On the uncoated powerful tool for studying cell-to-cell communication surface, there were few gap junctions between FS cells between FS cells, and between FS cells and hormone- (Fig. 3). In contrast, we confirmed the presence of gap producing cells. In a recent study using this rat, we observed junctions between FS cells on the laminin-coated surface that FS cells in primary culture used their cytoplasmic (Fig. 4A and B). We also observed that FS cells displayed processes to interconnect and form cell clusters (i.e. FS cells developed rough endoplasmic reticulum and well-developed aggregated homophilically), and reported that these histo- Golgi apparatus in the presence of laminin, but not in its logical features in vitro provided a suitable environment absence (Figs 3 and 4). for cell-to-cell communication in the anterior pituitary (Horiguchi et al. 2008). In the present study, we observed how the histological features between FS cells were altered in Discussion primary culture without hormone-producing cells. We were able to obtain pure GFP-positive FS cells with a cell sorter, In the present study, we isolated pure FS cells from and to use them in primary culture. The histological S100b-GFP rat anterior pituitary to eliminate the physical features of FS cells were the same in the presence or absence and functional effects of the hormone-producing cells and of GFP-negative cells, which are primarily hormone- examined the direct involvement of laminin on gap producing cells. These findings suggest that primary culture junction formation between FS cells in primary culture. We of isolated FS cells was appropriate for analyzing FS cell-to-FS showed that laminin promoted gap junction formation cell communication in the absence of the effects of hormone- between FS cells. producing cells.

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AB GH

CD I J

E K 1·6 * 1·6

1·2 1·2

0·8 0·8 (fold change)

(fold change) 0·4 0·4

0 level 43 mRNA Connexin 0 Connexin 43 mRNA level 43 mRNA Connexin Uncoated LamininLaminin Laminin Ham IgM Itgb-1 AB FLLaminin Laminin Uncoated Laminin Ham IgM Itgb-1 AB Cx 43 Cx 43

β-Actin β-Actin 2·5 1·2 * 2 0·8 1·5 1 0·4 (fold change) 0·5 (fold change)

Connexin 43 protein level Connexin 0 43 protein level Connexin 0 Uncoated Laminin Laminin Laminin Ham IgM Itgb-1 AB Figure 2 Connexin 43 expression levels in primary culture of FS cells after 72 h of incubation. (A, B, C, D, G, H, I, and J) Immunocytochemistry for connexin 43 in primary culture of FS cells; (A and B) 72 h culture of FS cells on an uncoated surface; (C and D) 72 h culture of FS cells on the laminin-coated surface; (G and H) 72 h culture of FS cells with hamster IgM on the laminin-coated surface; (I and J) 72 h culture of FS cells with anti-integrin b1 antibody on the laminin-coated surface. (A, C, G, and I) Phase contrast images. (B, D, H, and J) Fluorescent images. Connexin 43 (red) immunoreactivity was observed between FS cells (green). Scale bar, 10 mm. (E and K) The expression of connexin 43 mRNA determined by real-time PCR was normalized with an internal control (GAPDH) after 72 h incubation. (E) Connexin 43 mRNA expression was higher on the laminin-coated surface (laminin) than on the uncoated surface (uncoated). (K) Connexin 43 mRNA expression was not lower on the laminin-coated surface with anti-integrin b1 antibody (laminin Itgb-1 AB) than on the laminin-coated surface with hamster IgM (laminin Ham IgM). Connexin 43 mRNA levels were calculated as ratios of uncoated (E) and laminin Ham IgM (K) values respectively. (F and L) The amount of connexin 43 protein was determined by western blotting. The upper panel shows connexin 43 and the lower panel shows b-actin, the loading control. Western blotting data from three experiments (meanGS.E.M., nZ3) were quantified by densitometry and normalized with b-actin. (F) Expression of connexin 43 in FS cells on the laminin-coated surface (laminin) was significantly higher than that in FS cells on the uncoated surface (uncoated) (*P!0.05). (L) Expression of connexin 43 in FS cells on the laminin-coated surface with anti-integrin b1 antibody (laminin Itgb-1 AB) was not significantly lower than that in FS cells on laminin-coated surface with hamster IgM (laminin Ham IgM). Connexin 43 protein levels were calculated as ratios of uncoated (F) and laminin Ham IgM (L) values respectively. www.endocrinology-journals.org Journal of Endocrinology (2011) 208, 225–232

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A under the influence of laminin is partly due to the up-regulation of connexin 43 mRNA and protein levels. Laminin transduces signals mediated by integrins, which are the principal ECM receptors, and regulates gene expression and protein synthesis (Belkin & Stepp 2000, Hynes 2002). Studies indicate that there are two possible cascades mediated by integrins for connexin 43 up-regulation. One possibility is that the connexin 43 gene is a downstream target for an outside-in integrin signaling cascade (Spragg & Kass 2005); the other is that laminin, via the integrin signaling cascade, promotes the assembly of cell-to-cell adhesion mediated by E-cadherin before gap junction assembly, thereby up-regulating connexin 43 (Jongen et al. 1991, B Frenzel & Johnson 1996). FS cells express integrin a3, a6, and b1, which are specific laminin receptors (Horiguchi et al. 2010), and E-cadherin (Kikuchi et al. 2006). As shown in Fig. 2, an antibody that inhibits the integrin b1 cascade did not block connexin 43 up-regulation. Connexin 43 up-regulation in the presence of laminin may be regulated by another integrin signaling cascade, such as integrin a6b4, and/or may depend on the mere increase of juxtaposed membranes that result from FS cell-to-FS cell adhesion

A

Figure 3 Electron micrograph of FS cells cultured on the uncoated surface for 72 h. (A) Lower magnification. (B) Higher magnification of the area indicated by the box in (A). Single arrow indicates the pseudolumen with microvilli. The Golgi apparatus is poorly developed (double arrows). Scale bar, 500 nm.

In a previous study using living-cell imaging of S100b-GFP rat anterior pituitary cells, we first examined whether FS cells respond to ECM components and observed that FS cells in primary culture formed numerous interconnections with neighboring FS cells (Horiguchi et al. 2010). In the present study, we observed the behavior of isolated FS cells in the B presence of laminin without hormone-producing cells. As shown in Fig. 1B, it is clear that laminin affects morpho- logical changes in FS cells and increases interconnections between them. Morand et al. (1996) showed that FS cells in vitro can transfer small molecules to neighboring FS cells through gap junction coupling in FS cell-to-FS cell communication. We observed that the immunoreactivity of connexin 43, which is the protein subunit forming the gap junction in the anterior pituitary gland (Meda et al. 1993) was greater on the laminin- coated surface than on the uncoated surface. Furthermore, fine structural formation of gap junctions between FS cells on the laminin-coated surface was confirmed by electron Figure 4 Electron micrograph of FS cells cultured on the laminin- microscopy. These cells had developed rough endoplasmic coated surface for 72 h. (A) Gap junctions (arrowhead) were reticulum and Golgi apparatus, as shown in Fig. 3. These frequently observed between FS cells. (B) Higher magnification of morphological findings were expected due to the hyperfunc- the area indicated by the box in A. In addition to gap junctions (arrowhead), well-developed Golgi apparatus, consisting of several tional status of FS cells in the presence of laminin, and they layers of Golgi lamellae (double arrows), and many vesicles and suggest that the increase in gap junction between FS cells vacuoles (arrow) in the Golgi area are present. Scale bar, 500 nm.

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Downloaded from Bioscientifica.com at 09/28/2021 04:28:14AM via free access ECM promote gap junction formation in FS cells . K HORIGUCHI and others 231 mediated by E-cadherin. In addition, it is interesting that References anti-integrin b1 antibody inhibited gap junction formation between juxtaposed membranes of FS cells (Fig. 2). Connexin Allaerts W & Vankelecom H 2005 History and perspectives of pituitary 43 delivery to the cell surface has been shown to be facilitated folliculo-stellate cell research. European Journal of Endocrinology 153 1–12. (doi:10.1530/eje.1.01949) by interactions with many connexin 43-binding proteins Belkin AM & Stepp MA 2000 Integrins as receptors for laminins. Microscopy (Giepmans 2004). Their genes may be a downstream target Research and Technique 51 280–301. (doi:10.1002/1097-0029(20001101) for the integrin b1 signaling cascade. It remains to be confir- 51:3!280::AID-JEMT7O3.0.CO;2-O) med whether connexin 43 up-regulation and gap junction Farquhar MG 1957 “Cotricotrophs” of the rat adenohypophysis as revealed formation in FS cells are caused by one or a combination of by electron microscopy. Anatomical Record 127 291. (doi:10.1002/ar. 1091270211) mechanisms. Nevertheless, to our knowledge, the present Fauquier T, Gue´rineau NC, McKinney RA, Bauer K & Mollard P 2001 study is the first to demonstrate directly that laminin–integrin Folliculostellate cell network: a route for long-distance communication in interaction triggers an increase in gap junction formation the anterior pituitary. PNAS 98 8891–8896. (doi:10.1073/pnas. in FS cells. 151339598) Data from recent morphological and electrophysiological Frenzel EM & Johnson RG 1996 Gap junction formation between cultured embryonic lens cells is inhibited by antibody to N-cadherin. Developmental studies have led to speculation regarding the function of FS Biology 179 1–16. (doi:10.1006/dbio.1996.0237) cells. One hypothesis maintains that FS cells interconnect via Giepmans BN 2004 Gap junctions and connexin-interacting proteins. gap junctions to form a network in anterior pituitary gland Cardiovascular Research 62 233–245. (doi:10.1016/j.cardiores.2003.12.009) that transmits signals regulating hormone release from Horiguchi K, Fujiwara K, Kouki T, Kikuchi M & Yashiro T 2008 Immunohistochemistry of connexin 43 throughout anterior pituitary gland hormone-producing cells, in addition to the established in a transgenic rat with green fluorescent protein-expressing folliculo- hypophyseal portal vein system (Shirasawa et al. 2004, 2007, stellate cells. Anatomical Science International 83 256–260. (doi:10.1111/ Sato et al. 2005). 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These findings suggest that FS cells in the Inoue K, Couch EF,Takano K & Ogawa S 1999 The structure and function of presence of laminin may assume functional roles such as FS folliculo-stellate cells in the anterior pituitary gland. Archives of Histology and Cytology 62 205–218. (doi:10.1679/aohc.62.205) cell-to-FS cell communication in the anterior pituitary gland. Itakura E, Odaira K, Yokoyama K, Osuna M, Hara T & Inoue K 2007 Generation of transgenic rats expressing green fluorescent protein in S-100b-producing pituitary folliculo-stellate cells and brain astrocytes. Supplementary data Endocrinology 148 1518–1523. (doi:10.1210/en.2006-1390) Jongen WM, Fitzgerald DJ, Asamoto M, Piccoli C, Slaga TJ, Gros D, This is linked to the online version of the paper at http://dx.doi.org/10.1677/ Takeichi M & Yamasaki H 1991 Regulation of connexin 43-mediated C JOE-10-0297. gap junctional intercellular communication by Ca2 in mouse epidermal cells is controlled by E-cadherin. Journal of Cell Biology 114 545–555. (doi:10.1083/jcb.114.3.545) Declaration of interest Kikuchi M, Yatabe M, Fujiwara K, Takigami S, Sakamoto A, Soji T & Yashiro T 2006 Distinctive localization of N- and E-cadherins in rat anterior pituitary gland. Anatomical Record. Part A, Discoveries in Molecular, The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Cellular, and Evolutionary Biology 288 1183–1189. (doi:10.1002/ar.a.20384) Koide T & Ito Y 2000 Juxtacrine and matricrine – the other ways of actions. Seikagaku 72 1259–1263. Meda P, Pepper MS, Traub O, Willecke K, Gros D, Beyer E, Nicholson B, Funding Paul D & Orci L 1993 Differential expression of gap junction connexins in endocrine and exocrine glands. Endocrinology 33 2371–2378. (doi:10.1210/ This work was partly supported by a Grant-in-Aid for Scientific Research (C) en.133.5.2371) (21570067), (22590192) and a Grants-in-Aid for Young Scientists (B) Miyamoto S, Nakamura M, Yano K, Ishii G, Hasebe T, Endoh Y, Sangai T, (22790190), from the Ministry of Education, Culture, Sports, Science, and Maeda H, Shi-Chuang Z, Chiba Tet al. 2007 Matrix metalloproteinase-7 Technology of Japan, by promotional funds for the Keirin Race of the Japan triggers the matricrine action of insulin-like growth factor-II via Keirin Association, and by Jichi Medical University Young Investigator Award proteinase activity on insulin-like growth factor binding protein 2 in the for Jichi Medical University, School of Medicine. extracellular matrix. Cancer Science 98 685–691. (doi:10.1111/j.1349-7006. 2007.00448.x) Morand I, Fonlupt P, Guerrier A, Trouillas J, Calle A, Remy C, Rousset B & Acknowledgements Munari-Silem Y 1996 Cell-to-cell communication in the anterior pituitary: evidence for gap junction-mediated exchanges between We thank Prof. K Inoue (Saitama University, Japan) for supplying the endocrine cells and folliculostellate cells. Endocrinology 137 3356–3367. transgenic rats. We are grateful to M Yatabe for her excellent technical (doi:10.1210/en.137.8.3356) assistance and to Y Furukawa for his technical support in fluorescence- Sato Y, Hashitani H, Shirasawa N, Sakuma E, Naito A, Suzuki H, Asai Y, activated cell sorting. We also thank David Kipler, ELS, of Supernatant Sato G, Wada I, Herbert DC et al. 2005 Intercellular communications Communications for revising the language of the manuscript. within the rat anterior pituitary XII: immunohistochemical and www.endocrinology-journals.org Journal of Endocrinology (2011) 208, 225–232

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physiological evidences for the gap junctional coupling of the study on the relationship between the agranular cells and GnRH neurons in folliculo-stellate cells in the rat anterior pituitary. Tissue and Cell 37 the dorsal pars tuberalis of the pituitary gland. Anatomical Record 290 281–291. (doi:10.1016/j.tice.2005.03.005) 1388–1398. (doi:10.1002/ar.20596) Schultz JF & Armant DR 1995 Beta 1- and beta 3-class integrins mediate Soji T & Herbert DC 1989 Intercellular communication between rat anterior fibronectin binding activity at the surface of developing mouse peri- pituitary cells. Anatomical Record 224 523–533. (doi:10.1002/ar.1092240410) implantation blastocysts. Regulation by ligand-induced mobilization of Spragg DD & Kass DA 2005 Controlling the gap: myocytes, matrix, and stored receptor. Journal of Biological Chemistry 270 11522–11531. (doi:10. mechanics. Circulation Research 96 485–487. (doi:10.1161/01.RES. 1074/jbc.270.19.11522) 0000160607.30286.54) Shirasawa N, Mabuchi Y, Sakuma E, Horiuchi O, Yashiro T, Kikuchi M, Vila-Porcile E 1972 The network of the folliculo-stellate cells and the follicles Hashimoto Y, Tsuruo Y, Herbert DC & Soji T 2004 Intercellular of the adenohypophysis in the rat (pars distalis). Zeitschrift fu¨r Zellforschung communication within the rat anterior pituitary gland: X. und Mikroskopische Anatomie 129 328–369. (doi:10.1007/BF00307293) Immunohistocytochemistry of S-100 and connexin 43 of folliculo-stellate cells in the rat anterior pituitary gland. Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology 278 462–473. (doi:10.1002/ar. Received in final form 26 November 2010 a.20040) Shirasawa N, Sakuma E, Wada I, Naito A, Horiuchi O, Mabuchi Y, Kanai M, Accepted 22 December 2010 Herbert DC & Soji T 2007 Intercellular communication within the rat Made available online as an Accepted Preprint anterior pituitary: XIV Electron microscopic and immunohistochemical 22 December 2010

Journal of Endocrinology (2011) 208, 225–232 www.endocrinology-journals.org

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