Abstract
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.
Introduction
The anterior pituitary gland regulates homeostasis by meticulous adjustment of hormonal secretion. Folliculostellate (FS) cells are present in the anterior pituitary gland but do not secrete classical hormones. Although FS cells are agranular, evidence suggests that they are important in coordinating anterior pituitary function via the homotypic cellular network through gap junction communication in the gland (Fauquier et al. 2001, Shirasawa et al. 2004). Formation of clusters and elongated cytoplasmic processes are the structural hallmarks of FS cells (Soji & Herbert 1989). However, FS cells are known for another unique feature: apposition of their cytoplasmic processes and the extracellular matrix (ECM) of the basement membrane (Inoue et al. 1999, Shirasawa et al. 2004).
In a series of experiments that investigated the influence on FS cells of laminin – an ECM component of the basement membrane – we found that FS cells exhibited a motile phenotype and enhanced proliferation and that they established numerous interconnections, which closely resembled their cellular arrangement in vivo (Horiguchi et al. 2010, 2011a,b). The high motility of FS cells on laminin suggested that, to migrate on the ECM, they must express matrix-degrading enzymes. Among the enzymes capable of ECM degradation is the matrix metalloproteinase (MMP) family. MMP-9 has been extensively studied under numerous physiological and pathological conditions. In addition to its well-known proteolytic action, MMP-9 modulates cell motility and proliferation (Moon et al. 2003, Cauwe et al. 2009, Sans-Fons et al. 2010). Despite many studies of MMP-9 at the molecular and cellular levels in various organs, MMP-9 expression and function in FS cells of the anterior pituitary have not been investigated. Thus, the role of MMP-9 in FS cell motility and proliferation under the influence of laminin needs to be clarified.
We investigated MMP-9 expression and localization in FS cells. In addition, we studied the effect of laminin on MMP-9 expression in primary culture and attempted to confirm the role of MMP-9 in FS cell interconnection and proliferation.
Materials and Methods
Animals
Transgenic S100b-green fluorescent protein (GFP) rats (Itakura et al. 2007), which express GFP under the promoter control of the S100b protein gene (a marker of FS cells), were donated by Prof. K Inoue of Saitama University and bred in our laboratory. Eight- to ten-week-old male rats weighing 250–300 g were given ad libitum access to food and water and housed under a 12 h light:12 h darkness cycle. Rats were sacrificed by exsanguination from the right atrium under deep Nembutal anesthesia and then perfused with Ca2+- and Mg2+-free Hanks' solution for primary culture or with 4% paraformaldehyde (PFA) in 0.05 M phosphate buffer (pH 7.4) for immunohistochemistry. All animals were treated in accordance with the Jichi Medical University Guidelines for Animal Experimentation.
Cell culture
Anterior pituitary cells of S100b-GFP male rats were dispersed as described in a previous report (Horiguchi et al. 2008). The dispersed cells were then sorted by a MoFlo XDP (Beckman Coulter, Inc., Fullerton, CA, USA) into GFP-positive (GFP+) and GFP-negative (GFP−) cell fractions. GFP+ cells were plated onto eight-well glass chamber slides (1 cm2/well; Nalge Nunc International, Rochester, NY, USA), with or without a coating of 10 μg/cm2 laminin (Millipore, Bedford, MA, USA), at a density of 1×105 cells/cm2 in 400 μl of Medium 199 with Earle's salts (Invitrogen) supplemented with 10% fetal bovine serum (Sigma–Aldrich Corp.), 0.5 U/ml penicillin, and 0.5 μg/ml streptomycin (Invitrogen). Cells were then incubated at 37 °C in an atmosphere of 5% CO2 and 95% air. For MMP-9 inhibition, cells were treated with 100 μM synthetic MMP-9/MMP-13 Inhibitor I (444252; Merck), and control groups were treated with an equal volume of dimethyl sulfoxide 9 (DMSO); (Wako, Osaka, Japan) in the medium from 0 h. For MMP-9 knockdown, a siRNA sequence against rat MMP-9 (SI102004247; Qiagen), with a nontargeting siRNA (1027283; Qiagen) as control, was transfected into cells by using an INTERFERin transfection reagent (PolyPlus Transfections, Inc., New York, NY, USA) 24 h after seeding. Cells were time-lapse recorded using a digital camera (ORCA-ER; Hamamatsu Photonics, Shizuoka, Japan) and MetaMorph Software (Molecular Devices Corp., Downingtown, PA, USA) from 2 to 72 h after seeding in a CO2 gas culture chamber (Sankei Corp., Tokyo, Japan) with a thermostat (Kokensha Engineering Corp., Tokyo, Japan) on a fluorescence-inverted microscope (IX71; Olympus Corp., Tokyo, Japan). Each observation was performed in triplicate.
RT-PCR and real-time RT-PCR
For RT-PCR, total RNA fractions were prepared with TRIzol reagent (Invitrogen) from anterior pituitary gland, primary culture of anterior pituitary cells, and the GFP+ and GFP− cell fractions of S100b-GFP male rats. Expression of MMP-9 and cyclin D1 mRNA in primary cultured FS cells was measured by real-time RT-PCR. Total RNA fractions were prepared with the RNeasy Mini kit (Qiagen) from cultured FS cells, and all RNA samples were incubated with RNase-free DNase I (1 U/tube; Promega Corp.) and heated to inactivate DNase I. cDNA was synthesized using a Superscript III RT kit with oligo-(dT)20 primer (Invitrogen). The PCR mix consisted of the RT reaction product, PCR buffer containing dNTPs, KOD Dash DNA Polymerase (2.5 U/μl; Toyobo, Osaka, Japan), and each oligonucleotide primer. The primer pairs used and putative product lengths were as follows: MMP-9 (GenBank accession no.
Quantitative real-time PCR (ABI PRISM 7900HT; Applied Biosystems, Carlsbad, CA, USA) was performed by using gene-specific primers and SYBR Premix Ex Taq (Takara, Tokyo, Japan) containing SYBR Green I. To amplify cDNA fragments, the following primers were used: MMP-9, forward: 5′-CCT GAA AAC CTC CAA CCT CA-3′, reverse: 5′-GGA CTG CTT CTC TCC CAT CA-3′ (100 bp); cyclin D1 (
Immunoblot analysis
Anterior pituitary gland, GFP+ and GFP− cell fractions separated by cell sorter and primary cultured GFP+ cells were lysed in RIPA buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% w/v SDS, 1% v/v Triton X-100; pH 7.5), and total protein was estimated by Bradford assay (Sigma). Twenty micrograms of protein from each sample were applied to 10% SDS–PAGE. Proteins were then transferred electrophoretically onto Immobilon-P transfer membranes (Millipore). Membranes were blocked with 5% skim milk in TBST (50 mM Tris, 100 mM NaCl, 0.1% v/v Tween 20; pH 7.4), probed overnight at room temperature with rabbit monoclonal anti-MMP-9 antibody diluted 1:15 000 (ab76003; Abcam, Inc.) or mouse anti-β-actin antibody (0.1 μg/ml; BioVision, Mountain View, CA, USA), diluted in Can Get Signal solution (Toyobo) followed by TBST washes, and incubated for 1 h with HRP-labeled secondary antibodies (Envision+System–HRP, anti-rabbit; Dako, Glostrup, Denmark). Immunoreactive bands were visualized by ECL Plus Western Blotting Detection Reagents (GE Healthcare, Mississauga, ON, Canada). Each blot was performed in triplicate.
Gelatin zymography
Gelatin zymography to detect MMP-9 activity was performed according to the method described in a previous report (Zhang & Gottschall 1997). In brief, protein samples from anterior pituitary gland and GFP+ and GFP− cell fractions were prepared using nonreducing sample buffer containing 0.5 M Tris–HCl (pH 6.8), 10% SDS, and glycerol. GFP+ cells were cultured as described above for 48 h. Then, medium was replaced with serum-free medium supplemented with 0.1% BSA (Roche Diagnostics), and cell culture was continued for 24 h. Conditioned medium was collected from each well of three experimental replicates and centrifuged at 15 000 g for 3 min at 4 °C to remove cellular debris. Samples were electrophoretically run in copolymerized 10% SDS–acrylamide (Bio-Rad Laboratories, Inc.) and 0.1% gelatin (Difco Laboratories, Detroit, MI, USA) gel. Gel was incubated in regeneration buffer containing 2.5% Triton X-100 for 2 h at room temperature followed by incubation in reaction buffer containing 10 mM CaCl2 for 24 h at 37 °C. To visualize gelatinolytic bands, the gel was stained with Coomassie Brilliant Blue (ATTO Corp., Tokyo, Japan).
Immunohistochemistry
After perfusion with 4% PFA in 0.05 M PB (pH 7.4), the pituitary glands were carefully dissected and fixed overnight in the same fixative solution at 4 °C. Next, tissues were immersed in 0.05 M PB (pH 7.2) containing 30% sucrose for 2 days at 4 °C, embedded in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan), and snap-frozen. Frontal sections (8 μm) were incubated in PBS containing 2% normal goat serum for 20 min at 30 °C to block nonspecific antigen binding, then incubated with anti-rat MMP-9 rabbit monoclonal antibody diluted 1:25 in PBS for two nights at 4 °C, followed by incubation with Alexa Fluor 568-conjugated goat anti-rabbit IgG diluted 1:200 (Invitrogen) for 30 min at 30 °C. For immunostaining of cultured GFP+ cells, cells were fixed with 4% PFA in 0.025 M PB (pH 7.4) for 20 min at room temperature, immersed in PBS containing 2% normal goat serum for 20 min at 30 °C, incubated with anti-rat MMP-9 rabbit monoclonal antibody diluted 1:100 overnight at room temperature, then probed with Alexa Fluor 568-conjugated goat anti-rabbit IgG. Absence of an observable nonspecific reaction was confirmed using normal rabbit serum instead of primary antibody.
Proliferation assay
To observe FS cell proliferation, the nucleotide analog 5-bromo-2′-deoxyuridine (BrdU; Sigma) was incorporated into primary culture and detected in fixed cells according to previously described procedures (Horiguchi et al. 2010). Thirty random fields were imaged per well using a confocal laser microscope with a 60-fold objective lens. Observations were done in triplicate for each experimental group.
Statistical analysis
Results are presented as mean±s.e.m. Student's t-test was used to compare differences between groups, which were considered to be statistically significant at a P value of <0.05.
Results
MMP-9 expression in anterior pituitary gland
Localization of MMP-9 in anterior pituitary gland was investigated immunohistochemically. Figure 1A shows hematoxylin–eosin staining of S100b-GFP rat pituitary gland. Transgenic S100b-GFP rats expressed GFP specifically in FS cells of the anterior pituitary (Fig. 1B). Frozen frontal sections of S100b-GFP rat pituitary tissue were probed with anti-rat MMP-9 rabbit monoclonal antibody (Fig. 1C and D). MMP-9 immunoreactivity was observed throughout the anterior pituitary, specifically in clusters of GFP+ cells and in their long cytoplasmic processes (Fig. 1E). Most FS cells showed MMP-9 immunoreactivity on their cell bodies and cytoplasmic extensions. Next, expression of MMP-9 gene in FS cells was examined by RT-PCR. MMP-9 was detected in anterior pituitary cells both in vivo and in vitro (Fig. 1F). Analysis of GFP+ and GFP− cell fractions showed identical results, with the exception of the S100b, which was not detected in the GFP− cell fraction (Fig. 1F). Real-time RT-PCR was performed to quantify the relative expression of MMP-9 in FS cells cultured on uncoated and laminin-coated surfaces (Fig. 1G). Expression of MMP-9 was markedly higher in GFP+ cells cultured on laminin than in those cultured on an uncoated surface (P<0.05).
MMP-9 expression in FS cells. (A) Pituitary cryosection from S100b-GFP rat stained with hematoxylin–eosin showing the anterior lobe (AL), intermediate lobe (IL), and posterior lobe (PL). Scale bar, 100 μm. (B) Pituitary cryosection from S100b-GFP rat showing GFP+ cells in anterior lobe (AL), intermediate lobe (IL), and posterior lobe (PL). In anterior lobe, S100b expression (green) is limited to FS cells. Scale bar, 100 μm. (C) S100b-GFP rat anterior pituitary showing clusters of GFP+ FS cells. (D) MMP-9 immunoreactivity (red) in anterior pituitary section. (E) Overlay image of (C) and (D), MMP-9 immunoreactivity (red) is observed in most GFP+ cells (green). Scale bars for C-E, 10 μm. (F) Expression of MMP-9 gene in anterior pituitary. Total RNA fractions extracted from anterior pituitary tissue (tissue), GFP− and GFP+ cell fractions (by cell sorter), and primary culture of GFP+ cells on uncoated (GFP+ uncoated) and laminin-coated (GFP+ laminin) surfaces were analyzed by RT-PCR of MMP-9, S100b, and GAPDH. (G) Expression of MMP-9 mRNA as determined by real-time RT-PCR after 72 h incubation on uncoated (uncoated) and laminin-coated (laminin) surfaces, normalized to internal control (GAPDH; mean±s.e.m., n=3, *P<0.05). (H) Immunoblotting showing MMP-9 expression in anterior pituitary tissue (tissue), GFP− and GFP+ cell fractions, and primary cultured GFP+ cells on uncoated (GFP+ uncoated) and laminin-coated (GFP+ laminin) surfaces. (I) Gelatin zymography showing MMP-9 activity in anterior pituitary cell fractions (GFP−, GFP+) and conditioned medium of FS cells (GFP+) cultured on uncoated (GFP+ uncoated) and laminin-coated (GFP+ laminin) surfaces.
Citation: Journal of Endocrinology 212, 3; 10.1530/JOE-11-0433
Expression of MMP-9 was confirmed by immunoblot analysis of anterior pituitary cell lysate in vivo and in vitro. We detected an immunoreactive band at 82 kDa, which corresponds to the size of activated MMP-9 in anterior pituitary tissue (Fig. 1H). Blotting of the GFP+ cell fraction protein showed a strong immunoreactive band, corresponding to the 82 kDa protein, and a weaker band of 92 kDa that corresponds to the molecular weight of latent MMP-9 (Fig. 1H). No immunoreactive band appeared in the sample of the GFP− cell fraction (Fig. 1H). GFP+ cells of primary culture on uncoated surface showed very weak immunoreactive bands. However, two bands of MMP-9 appeared for GFP+ cells on laminin-coated primary culture (Fig. 1H). MMP-9, also known as 92 kDa gelatinase B, can digest gelatin, i.e. denatured collagen. Gelatin zymography was performed to detect this specific property of MMP-9. Protein samples were collected with nonreducing sample buffer from GFP− and GFP+ cell fractions. The only clear gelatinolytic band for the GFP+ cell fraction corresponded to 82 kDa (Fig. 1I). We then investigated whether FS cells secreted MMP-9 into the media. Serum-free media of FS cells cultured in the absence or presence of laminin were collected. The clear gelatinolytic band corresponding to 82 kDa was stronger for medium from FS cells conditioned in the presence of laminin (Fig. 1I).
MMP-9 localization on FS cells in primary culture
To observe MMP-9 localization on isolated FS cells in primary culture, a cell sorter was used to separate GFP+ FS cells from the anterior pituitary of S100b-GFP rats. These cells were then cultured on uncoated and laminin-coated chamber slides for 72 h. The shape of FS cells differed when grown on uncoated and laminin-coated surfaces (Fig. 2A–D; Horiguchi et al. 2010). On the uncoated surface, FS cells acquired a round shape and aggregated (Fig. 2A and B). Under the influence of laminin, FS cells appeared to flatten and form interconnections (Fig. 2C and D). Immunocytochemistry to detect MMP-9 distribution demonstrated that FS cells showed punctate MMP-9 immunoreactivity on the uncoated surface (Fig. 2B). However, under the influence of laminin, MMP-9 had a filament-like distribution on FS cells (Fig. 2D).
Immunocytochemistry of MMP-9 in primary cultured FS cells. (A and C) Phase contrast images. (B) Confocal image of FS cells cultured on uncoated surface. (D) Confocal image of FS cells cultured on laminin-coated surface. Note that MMP-9 immunoreactivity (red) is higher in FS cells (green) cultured on laminin-coated surface. Scale bar, 10 μm.
Citation: Journal of Endocrinology 212, 3; 10.1530/JOE-11-0433
Effect of MMP-9 inhibitor on FS cells in primary culture
To determine the role of MMP-9 on FS cells in the presence of laminin, we observed the behavior of living FS cells in primary culture with MMP-9 inhibitor (Fig. 3A and B). Cells were treated with 100 μM MMP-9 synthetic inhibitor or an equal volume of vehicle (DMSO; control group) from 0 to 72 h. In the control group, the behavior of FS cells was similar to that reported previously (Horiguchi et al. 2010, 2011a), i.e. by 72 h of culture, they attached to laminin by becoming flatter, showed remarkable locomotion, extended their cytoplasm toward other cells in their vicinity, and formed interconnections with other FS cells (Fig. 3A; Supplementary data Movie 1, see section on supplementary data given at the end of this article). In the group treated with MMP-9 inhibitor, FS cells did not fully flatten but did extend their cytoplasmic processes. In addition, although the motility of these cells was comparable to that of the control group, they only established sparse cellular interconnections by 72 h of culture (Fig. 3B; Supplementary data Movie 2, see section on supplementary data given at the end of this article). To investigate whether MMP-9 affects the proliferative capacity of FS cells, we performed BrdU incorporation in FS cells cultured without or with MMP-9 inhibitor. Figure 3C and D shows BrdU incorporation in FS cells cultured on the laminin-coated surface in the absence of MMP-9 inhibitor. In the presence of MMP-9 inhibitor (Fig. 3E and F), a marked decrease in BrdU-positive cells was observed. As compared with the control group, the ratio of BrdU-positive cells was lower on the laminin-coated surface with MMP-9 inhibitor (P<0.01; Fig. 3G).
Time-lapse images of FS cells isolated from S100b-GFP rat anterior pituitary cells in primary culture on laminin-coated surface, with or without MMP-9 inhibitor. Cells are time-lapse recorded at 15 min intervals from 2 to 72 h after plating. (A and B) GFP images superimposed on phase contrast images, using an inverted fluorescence microscope, on laminin-coated surface without (A) and with (B) MMP-9 inhibitor. 2, 24, 48, and 72 h: elapsed time from plating of cells. Higher magnification views are shown in the inset images. Scale bar, 100 μm. (C–F) BrdU incorporation into FS cells. (C and D) Control group treated with DMSO. BrdU immunoreactivity (red) is observed in FS cells. (E and F) Representative FS cells under the influence of MMP-9 inhibitor show very little proliferative activity. Scale bar, 10 μm. (G) Ratio of BrdU-positive FS cells cultured on laminin-coated surfaces in the absence (control) or presence of MMP-9 inhibitor (MMP-9 inhibitor; mean±s.e.m., n=3, **P<0.01).
Citation: Journal of Endocrinology 212, 3; 10.1530/JOE-11-0433
Knockdown of MMP-9 gene by siRNA
The use of siRNA to silence the expression of MMP-9 resulted in a marked change in FS cell shape. By 24 h of culture, plasma membrane projections were already established in most FS cells (Fig. 4A and B, 24 h). Transfection with MMP-9 siRNA from 24 to 72 h after plating led to retraction of these projections; i.e. FS cells adopted a round shape and detached from laminin (Fig. 4B, 48, 72 h; Supplementary data Movie 4, see section on supplementary data given at the end of this article). The number of cellular interconnections was also diminished, resulting in small, scattered clusters by 72 h of culture (Fig. 4B, 72 h). FS cells transfected for an identical period with nontargeting siRNA reconstructed interconnections (Fig. 4A, 72 h; Supplementary data Movie 3, see section on supplementary data given at the end of this article). Quantitative real-time RT-PCR analysis of MMP-9 expression showed that siRNA transfection decreased MMP-9 gene expression as compared with control (P<0.01; Fig. 4C). We also examined the effect of MMP-9 silencing on FS cell proliferation and found that MMP-9 downregulation decreased the ratio of BrdU-positive FS cells as compared with control (P<0.01; Fig. 4D). However, MMP-9 silencing did not significantly change the expression of cyclin D1 (Fig. 4E).
Time-lapse images of FS cells (in primary culture on laminin-coated surface) transfected with nontargeting siRNA or MMP-9 siRNA. (A and B) GFP images superimposed on phase contrast images on laminin-coated surface with nontargeting siRNA (A) and MMP-9 siRNA (B). 2, 24, 48, and 72 h: elapsed time from plating of cells. Higher magnification views are shown in the inset images. Scale bar, 100 μm. (C) Quantitative real-time RT-PCR shows that MMP-9 gene expression level is significantly lower in FS cells transfected with MMP-9 siRNA (MMP-9 siRNA) than in those transfected with nontargeting siRNA (control); results are normalized to internal control (GAPDH; mean±s.e.m., n=3, **P<0.01). (D) Ratio of BrdU-positive FS cells, cultured on laminin-coated surfaces, transfected with nontargeting siRNA (control) or MMP-9 siRNA (MMP-9 siRNA; *P<0.05). (E) Quantitative real-time RT-PCR shows that cyclin D1 expression is not affected by silencing MMP-9 expression; results are normalized to internal control (GAPDH; mean±s.e.m., n=3).
Citation: Journal of Endocrinology 212, 3; 10.1530/JOE-11-0433
Discussion
This study shows that MMP-9 is specifically expressed in FS cells of normal adult rat pituitary, that its expression is upregulated by laminin, and that it is involved in laminin-mediated morphological and proliferative changes in FS cells in vitro.
To clarify the mechanisms underlying the marked motility and proliferation of FS cells on laminin, we investigated MMP-9 expression in rat anterior pituitary and isolated FS cells, based on the techniques of our earlier study (Horiguchi et al. 2010). We found that FS cells express MMP-9 mRNA and protein (Fig. 1D and F). Other studies have used immunohistochemistry (Knappe et al. 2003) and zymography (Paez Pereda et al. 2000) to investigate MMP-9 expression in normal and tumorous human anterior pituitary glands. However, those earlier studies did not identify MMP-9-expressing cells in the gland.
We also observed that FS cells differentially expressed MMP-9 in the absence and presence of laminin and that this expression was increased by laminin (Fig. 1D–G). In several cell lines, laminin-derived peptide (Freitas et al. 2007) and whole laminin (Maity et al. 2011) induced MMP-9 secretion and activity upon interaction with integrin by utilizing the ERK pathway. The integrins comprise an α-subunit and β-subunit, which form a heterodimer. In mammals, 18 types of α-subunits and eight types of β-subunits are known; their various combinations give rise to 24 integrin heterodimers, which differ in ligand specificity (Hynes 2002). With respect to these ligand specificities, we recently reported that FS cells bind laminin through integrin-α3β1 and/or integrin-α6β1 (Horiguchi et al. 2010). Since laminin induces integrin β1 signaling in FS cells (Horiguchi et al. 2011b), we hypothesize that the increase in MMP-9 expression in the presence of laminin occurs via a similar pathway. Furthermore, the increases in MMP-9 mRNA expression and protein (in cell lysate and secreted into medium) in the presence of laminin parallel the phenotypic change in FS cell shape from round to stellate (Figs 1D–G and 2). Punctate MMP-9 immunoreactivity was observed in round FS cells on the uncoated surface. In contrast, filament-like signals were seen in clusters of stellate-shaped FS cells on laminin (Fig. 2). MMP-9 was spatially distributed along microfibers (Schumacher et al. 2005), and elevated MMP-9 expression was found to be associated with the invasive phenotype of cells (Peters et al. 1999). Sbai et al. (2010) showed that MMP-9 has a vesicular distribution and that the location of vesicles is associated with the cytoskeleton. Previously, we found that motile FS cells on laminin formed F-actin in their cytoplasmic processes during cellular interconnection (Horiguchi et al. 2010). It is thus possible that the differential spatial distribution of MMP-9 under the influence of laminin is related to variation in the pattern of cytoskeletal arrangement in FS cells.
To determine the role of MMP-9 in FS cells, we used MMP-9 inhibitor to observe the behavior of living FS cells in primary culture. Inhibition of MMP-9 abrogated FS cell interconnection on laminin (Fig. 3; Supplementary data Movie 2, see section on supplementary data given at the end of this article). Because MMP-13 is a secondary target for the inhibitor used in this study, we next ruled out the effect of MMP-13 inhibition on our observations by silencing MMP-9 expression. The results were similar to those observed with MMP-9 inhibitor, i.e. FS cells failed to reconstruct dense interconnections, due to loss of attachment to laminin (Fig. 4; Supplementary data Movie 4, see section on supplementary data given at the end of this article).
The interconnections between FS cells are believed to serve as a network for conveying cellular messages through gap junctions in the anterior pituitary gland (Soji & Herbert 1989, Fauquier et al. 2001, Shirasawa et al. 2004). Previously, we discovered that FS cells formed networks in the presence of laminin, a process mediated through integrin β1 signaling (Horiguchi et al. 2011a). Veeravalli et al. (2010) showed that MMP-9 silencing downregulates the α-subunit and β-subunit of integrin in vitro. Knocking down MMP-9 expression might inhibit integrin signaling in FS cells and therefore influence adhesion of FS cells to laminin. Our present results confirm that FS cells do at least partly depend on MMP-9 expression to interconnect and assemble their characteristic network on laminin.
MMP-9 promotes cell proliferation in several cell types (Dwivedi et al. 2009, Sans-Fons et al. 2010, Ingraham et al. 2011). The present study showed that MMP-9 inhibition suppressed proliferative activity of FS cells on laminin, which indicates that MMP-9 is necessary in FS cell division (Figs 3G and 4D). Proliferation of FS cells on laminin involves integrin β1, which is associated with caveolin-3, and uses MAPK signal transduction, which upregulates the cyclin D1 expression that drives the cell cycle (Horiguchi et al. 2011b). Despite the marked decrease in BrdU incorporation after MMP-9 silencing, cyclin D1 expression was unchanged in this study (Fig. 4). This unexpected finding suggests that MMP-9 does not affect the capacity of cells to progress from G1 to S phase via the caveolin-3-mediated signaling pathway, but rather that MMP-9 is necessary for FS cells to synthesize DNA. We are continuing to investigate the role of MMP-9 in FS cell proliferation.
In conclusion, the present study provides evidence that laminin promotes MMP-9 expression in FS cells of rat anterior pituitary gland and that MMP-9 is required for the interconnection and proliferation of FS cells. In the presence of laminin, increased MMP-9 expression appears to promote the characteristic features of FS cells in anterior pituitary gland. During the early postnatal period, a small number of FS cells appear sparsely in the anterior pituitary at day 10; during maturation, they gradually form a more interconnected cell network (Soji et al. 1997). It is tempting to speculate that MMP-9 contributes to the expansion of this FS cell network in vivo. Future study is required to identify the mechanisms involved in the network arrangement of FS cells.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-11-0433.
Declaration of interest
The authors hereby declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported in the manuscript entitled ‘Matrix metalloproteinase-9 expression in folliculostellate cells of rat anterior pituitary gland’.
Funding
This work was partially supported by a Grant-in-Aid for Scientific Research (C) (22590192), a Grant-in-Aid for Young Scientists (B) (22790190) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by promotional funds from the Keirin Race of the Japan Keirin Association.
Acknowledgements
We thank Prof. Kinji Inoue (Saitama University, Japan) for supplying the transgenic rats. We are grateful to Miss Megumi Yatabe for her excellent technical assistance and to Prof. Yutaka Hanazono and Mr Yutaka Furukawa for their support in fluorescence-activated cell sorting. We also thank David Kipler, ELS of Supernatant Communications for revising the language of the manuscript.
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