ARTICLE |
Correspondence to: Sergio Vidal, Dept. of Anatomy, Laboratory of Histology, U. of Santiago de Compostela, Campus Univeritario de Lugo, 27002 Lugo, Spain. E-mail: svidal@lugo.usc.es
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Summary |
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Vascular endothelial growth factor (VEGF) acts primarily as an endothelial cell mitogen via the specific receptors Flk-1 and Flt-1. To help further define the possible role of VEGF in the control of pituitary cell function, we examined Flk-1 expression in normal rat pituitaries and in GH3 cells. Flk-1 expression was studied by immunohistochemistry, in situ hybridization, and double-labeling immunofluorescence combined with confocal laser microscopy. In normal rat pituitaries, Flk-1-immunoreactive cells appeared widely distributed only in the anterior lobe and were not detected in the intermediate or posterior lobe. Apart from the adenohypophysial cells, Flk-1 immunopositivity was also evident in endothelial cells of many capillaries distributed throughout the gland. Immunohistochemistry also showed that majority of GH3 cells expressed Flk-1 protein. In situ hybridization showed conclusive staining with the antisense probe and confirmed the immunohistochemical results. The double immunofluorescence method revealed Flk-1 expression in all types of hormone-producing adenohypophysial cells but not in folliculostellate cells. The percentage of immunopositive cells varied among the various cell types. The present study demonstrates that pituitary cells are not only sources of VEGF but also targets of this multifunctional substance, supporting the concept that VEGF functions as an autocrine/paracrine factor in the pituitary. (J Histochem Cytochem 50:533540, 2002)
Key Words: GH3 cell line, rat, immunocytochemistry, pituitary, in situ hybridization, confocal microscopy, VEGF, VEGF receptors, Flk-1 receptor
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Introduction |
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Vascular endothelial growth factor (VEGF) is a multifunctional polypeptide initially thought to be mainly a mitogen specific for endothelial cells (
Although the high-affinity tyrosine kinase receptors Flk-1 (fetal liver kinase-1), the equivalent of human KDR (kinase insert domain-containing receptor), and Flt-1 (fms-like tyrosine kinase) are specific targets of VEGF activity, it appears that both the in vivo and in vitro effects of VEGF are mediated almost exclusively via the Flk-1 receptor (
In the rat, previous immunohistochemical studies have reported pituitary overexpression of both VEGF and Flk-1 receptor after 7 days of estrogen exposure. This observation suggests a regulatory role of the VEGF/Flk-1 system in estrogen-induced murine pituitary tumors (
To obtain deeper insight into the role of the VEGF/Flk-1 system in anterior pituitary cell activity, we investigated expression of Flk-1 receptor mRNA and of its protein in normal rat pituitaries and in GH3 cells, a rat pituitary tumor cell line secreting both prolactin and growth hormone. Immunohistochemistry combined with confocal laser microscopy was also used to identify those pituitary cells representing targets of VEGF activity.
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Materials and Methods |
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Animals and Tissue Preparation
All rats were handled in accordance with the principles and procedures of the Guiding Principles for the Care and Use of Animal Research (86/609/EU). Six 3-month-old female SpragueDawley rats (220250 g) in the proestrous phase of their cycle were housed at room temperature (RT) with a controlled photoperiod and were fed a diet of rat pellets and tapwater ad libitum. The stage of their cycle was determined by daily vaginal smears.
The animals were deeply anesthetized with pentobarbital and then perfused via the ascending aorta with 4% paraformaldehyde in PBS. Their pituitaries were immediately removed, bisected sagittally, and fixed by immersion in 4% paraformaldehyde in PBS at 4C for 2 hr. After fixation, half of each pituitary was dehydrated and embedded in paraffin using an automatic processor. The other half of the gland was immersed in 15% sucrose in 0.1 M phosphate buffer for at least 24 hr, then quickly frozen and cut into 10-µm-thick sections in a cryostat for co-localization studies of Flk-1, the VEGF receptor.
Cell Culture
Obtained from the American Type Culture Collection (ATCC; Rockville, MD), the GH3 cells were grown in 175-ml flasks containing DMEM supplemented with 10% (v/v) horse serum, 5% (v/v) fetal calf serum, 48 mg penicillin/liter, and 100 mg streptomycin/liter at 37C in an atmosphere of 5% CO2 in air at 100% humidity. The GH3 cells were plated in culture wells at a density of 1020 x 104 cells per well. To facilitate cell attachment, they were maintained for 48 hr in supplemented DMEM. Before fixation, cells were washed twice with Ham-I solution (122 mM NaCl, 30 mM HEPES, 1 mM NaPO4H2·2H2O, 3 mM KCl, pH 7.4). These conditions increased the uniformity of the results. Thereafter, cell aliquots were fixed in 4% paraformaldehyde for 30 min at RT and attached to poly-L-lysine coated slides.
Immunohistochemistry and In Situ Hybridization
IHC and ISH procedures were performed to assess localization of Flk-1 protein and its mRNA in serial sections of paraffin-embedded rat pituitaries and in the GH3 cell line.
For the ICC demonstration of the Flk-1 receptor, a monoclonal antibody directed against a peptide sequence of the carboxyl terminal portion of the mouse Flk-1 receptor (Santa Cruz Biotechnology, SC-6251; Santa Cruz, CA) was used. Preliminary titration experiments determined the optimal working dilution (1:100). To ensure specificity, control tests included both replacement of Flk-1 antibody with TRIS-buffered saline (TBS) and preabsorption of the Flk-1 antibody with homologous antigen (Santa Cruz Biotechnology SC-315P). Immunolabeling was completely abolished in both controls.
ICC studies utilized the streptavidinbiotinperoxidase complex technique (
ISH was performed using an oligonucleotide probe complementary to regions 309338 of mFlk-1. The antisense stand sequence of the oligonucleotide is 5'-CTC GTT CCA CGA CGA CCG GCA GCG GGA CAC-3'. This was synthesized by the solid-phase cyanoethyl phosphoramidiete method on an automated DNA synthesizer (Gene Assembler; Pharmacia, Hoefer Pharmacia Biotech, San Francisco, CA) and then purified by gel electrophoresis on 20% polyacrylamide gels. Paraffin sections were dewaxed in xylene, rehydrated, and transferred to diethylpyrocarbonate water (DEPC water). Then the sections were pretreated with proteinase K (1 µg/ml) at 37C for 15 min before prehybridization for 1 hr at RT in hybridization mixture not containing the probe. Overnight hybridization was then performed with the digoxigenin-labeled oligonucleotide probe at 37C. Hybridized sections were then washed three times (5 min each) with 2 x SSC, followed by 1 x SSC and finally by 0.5 x SSC. After a rinse in Buffer A (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) the sections were bathed in blocking reagent (Buffer A with 1% normal sheep serum and 0.3% Triton X-100) for 30 min and incubated with anti-digoxigeninalkaline phosphatase complex (1:200; Boehringer Mannheim 1093274, Mannheim, Germany) for 3 hr. Three washes in Buffer A and one in Buffer C (50 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5) were followed by incubation in a substrate solution containing 4-nitroblue tetrazolium chloride, 5-bromo-4-chloro-3 indolyl phosphate, and levamisole, all in Buffer C. The reaction was stopped in Buffer C. The slides were then counterstained with nuclear fast red, coverslipped, and examined.
A digoxigenin-labeled oligonucleotide sense probe (5'-GAGCAAGGTGCTGCTGGCCGTCGCCCTGTG-3') served as a control to confirm the specificity of the ISH procedure.
Double-labeling Immunofluorescence and Confocal Laser Microscopy
Double-labeling immunofluorescence was applied to specifically identify the cell type(s) expressing Flk-1. Double immunostaining was performed on cryostat sections by combining monoclonal anti-Flk-1 antibody with a rabbit polyclonal antibody against pituitary hormones or S-100 protein, a marker of folliculostellate cells. Specifics of the various antisera employed were as follows: rabbit antisera directed against rat prolactin (PRL) (dilution 1:500; National Hormone and Pituitary Program, NIDDK-AFP-4251091, Baltimore, MD), rat growth hormone (GH) (dilution 1:500; UCB Bioproducts, i0538/002, Braine-L'Alleud, Belgium), human adrenocorticotropic hormone (ACTH) (dilution 1:400; Sigma A-0673), rat thyroid-stimulating hormone (ß-TSH) (dilution 1:400; National Hormone and Pituitary Program, NIDDK-AFP-1274789), human luteinizing hormone (ß-LH) (dilution 1:400; National Hormone and Pituitary Program, NIDDK-AFP-5595), human follicle-stimulating hormone (ß-FSH) (dilution 1:400; National Hormone and Pituitary Program, NIDDK-AFP-20102688), and bovine S-100 protein (1:400, Sigma S-2644). After rinsing in TBS, the double-stained sections were incubated at RT for 1 hr with FITC- conjugated donkey anti-mouse (dilution 1:250; Jackson Immunoresearch Laboratories 715-095-151, West Grove, PA) and rhodamine red-X-conjugated donkey anti-rabbit (dilution 1:250, Jackson Immunoresearch Laboratories 711-085-152). After rinsing in TBS, the sections were mounted in a mixture of PBS and glycerol (1:3) containing 0.1% p-phenilenediamine to prevent fading of the immunofluorescence reaction. Sections were examined on an MRC 1024 ES confocal laser scanning system (Bio-Rad Laboratories; Hercules, CA). The excitation wavelength was 488 nm for FITC and 514 nm for rhodamine red-X-induced fluorescence. Specificity studies carried out by omitting primary antisera or by preabsorbing the primary antisera with homologous antigen excess; all showed the absence of the fluorescent signal. As an additional control, we tested whether the secondary antisera used in the double labeling were specific and did or did not crossreact with each other. To test for this possibility, one of the primary antisera was incubated alone and revealed by both secondary antisera. The nonspecific staining was totally abolished without alteration of the specific staining.
Double immunofluorescence was used to determine the proportions of Flk-1-immunopositive cells, those immunoreactive for either adenohypophysial hormones or S-100 protein. In each section, 15 randomly selected fields were studied on a confocal laser scanning system at a magnification of x60. Approximately 100 cells were assessed in each field; only nucleated cells were counted.
Data were tested for statistical significance using the SPSS Statistical Computer Program (SPSS; Chicago, IL). As a multiple comparison method, all data were evaluated by one-way ANOVA and the Student's t-test. Only differences of p<0.05 were considered statistically significant.
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Results |
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Pituitary Glands
In the six rat pituitaries, Flk-1-immunoreactive cells appeared widely distributed only in the anterior lobe and were not detected in the intermediate or posterior lobes. Immunoreactivity was moderate to strong and involved the entire cytoplasm (Fig 1A and Fig 1B). Apart from the adenohypophysial cells, Flk-1 immunopositivity was also evident in endothelial cells of many capillaries distributed throughout the gland (Fig 1B).
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ISH showed conclusive staining with the antisense probe, whereas the sense probe preparation was negative. Hybridization labeling was in the same pattern as the IHC results. Thus, Flk-1 mRNA signal appeared widely expressed throughout the adenohypophysis. In positive cells, the signal was moderate to strong and cytoplasmic. The signal was also detected in endothelial cells of most capillaries throughout the gland (Fig 1C).
GH3 Cells
IHC showed that the majority of GH3 cells expressed Flk-1 protein. The pattern of immunopositivity was diffuse, moderate to strong cytoplasmic staining. ISH also demonstrated Flk-1 mRNA in the majority of GH3 cells. Strong cytoplasmic labeling was observed (Fig 1D).
Double-labeling Immunofluorescence and Confocal Laser Microscopy
Because the Flk-1 receptor was diffusely distributed in the normal adenohypophysis, double-labeling immunofluorescence combined with confocal laser microscopy was applied to identify which specific cells expressed the receptor (Fig 2 and Fig 3). The double-immunofluorescence method demonstrated that all types of hormone-producing adenohypophysial cells were immunopositive for Flk-1. In contrast, the protein was not detected in folliculostellate cells. The percentage of immunopositive cells varied considerably among the various cell types (Fig 4). Co-localization of Flk-1 and pituitary hormones was particularly abundant in somatotrophs and lactotrophs (6050%). Significantly fewer cells were immunopositive among corticotrophs (4030%), thyrotrophs (105%), and gonadotrophs (105%) (Fig 4).
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Discussion |
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In addition to adenohypophysial hormones, pituitary cells contain various other substances including many growth factors, cytokines, angiogenic substances, and neuropeptides. Frequent expression of the corresponding receptors on adenohypophysial cells enables these factors to influence cell growth and function by auto- or paracrine mechanisms (
Our study demonstrates that pituitary cells are not only sources of VEGF but also targets of this multifunctional substance. It also supports the concept that this cytokine functions as an autocrine/paracrine factor in the pituitary. Previous studies have shown that certain angiogenic factors, including epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), play a regulatory role in adenohypophysial hormone gene expression. Although on the basis of our data it is not possible to determine the biological significance of Flk-1 expression in pituitary cells, it appears that the Flk-1 receptor acts as a key modulator of VEGF activity not only in endothelial cells but in endocrine cells as well. Supporting this idea are previous studies showing that the VEGF/Flk-1 system plays a regulatory role in several physiological and pathological processes. In the fetal rat pancreas,
Our study demonstrates that in the normal rat adenohypophysis Flk-1 receptor is expressed in all but folliculostellate cells. The absence of Flk-1 receptor in pituitary folliculostellate cells is intriguing because this cell type is the main source of VEGF in the normal adenohypophysis (
Our findings of Flk-1 receptor expression in GH3 cells implicates VEGF in the regulation of neoplastic anterior pituitary cell function.
Clearly, further studies are required to clarify the functional role of the VEGF/Flk-1 system in the control secretory activity and growth in normal and neoplastic pituitary tissue. Such studies may bring about the use of angiogenic and anti-angiogenic factors in the control of medical and neoplastic diseases of the pituitary gland.
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Acknowledgments |
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Supported in part by a grant from the Ministerio de Ciencia y Tecnología Dirección General de Investigación (BFI2001-3336-C02-02) and by a generous donation from Mr and Mrs Jarislowski and the Lloyd CarrHarris Foundation. Dr Sergio Vidal was supported by a research grant from University of Santiago de Compostela Spain.
We thank Dr Albina Román for technical assistance and the staff of St. Michael's Hospital Health Sciences Library for their contribution in this study.
Received for publication July 9, 2001; accepted November 28, 2001.
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