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1 Department of Internal Medicine2 Graduate School3 Brain Korea 21 Project for Medical Science4 , Biochemistry and Molecular Biology5 Institute of Endocrine Research, Yonsei University College of Medicine, Seoul, South Korea6 Endocrinology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
(Correspondence should be addressed to E J Lee at Endocrinology, Internal Medicine, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea; Email: ejlee423{at}yuhs.ac)
* Y Lee and J M Kim contributed equally to this work and should be considered co-first authors 
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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After CXCL12 binding sites were discovered in the adult rat pituitary gland by autoradiographic assay using I131 labeled CXCL12 (Banisadr et al. 2000), a group demonstrated that CXCL12 stimulated cell proliferation and growth hormone (GH) secretion in the rat pituitary adenoma-derived cell line GH4C1 (Florio et al. 2006, Massa et al. 2006), suggesting that interaction between CXCL12 and CXCR4 regulates the function of the anterior pituitary gland. Furthermore, it has recently been reported that CXCL12 and CXCR4 are expressed in the posterior pituitary neurons and their interaction is important for regulating arginine vasopressin (AVP) secretion (Callewaere et al. 2006). In this study, we demonstrated the colocalization of CXCR4 with rat pituitary somatotroph and investigated the role of CXCL12/CXCR4 interaction on the secretion and production of GH and cell proliferation in both normal rat pituitary and rat pituitary somatotroph tumor GH3 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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Expression of Cxcl12 mRNA and Cxcr4 mRNA was analyzed by RT-PCR using total RNA isolated from rat pituitary tissues and GH3 cells. RNA was extracted using TRIZOL reagent (Invitrogen Corp.) as described by the manufacturer's instructions. 20 µg RNA were treated with RNase-free DNase-I (QIAGEN Korea Ltd) for 10 min at 25 °C. Random hexamers were used to synthesize single-stranded cDNA using 2 µg DNase-I treated RNA in a 20 µl reaction volume containing 50 mM of Tris–HCl, pH 8.3, 8 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 1 mM dNTPs, 22 U RNase inhibitor, and 10 U moloney murine leukemia virus (MMLV) RT (Promega) for 60 min at 37 °C. A portion (1/40) of cDNA solution was treated with 2.5 U Taq DNA polymerase (Promega Corp.) for amplification of CXCL12, CXCR4, and GAPDH as a control. Cycle conditions were: 2 min hot start at 96 °C, followed by 35 cycles of 1 min at 94 °C, 45 s at 55 °C, followed by 1 min at 72 °C, and extension at 72 °C for 15 min. An aliquot (20%) of each PCR product was resolved by electrophoresis on 1.5% agarose gels and DNA products were visualized with ethidium bromide. Oligonucleotides used for PCR amplification were: CXCL12, forward 5'-CAT GGA CGC CAA GGT CGT-3' and reverse 5'-TCC AGG TAC TCT TGG ATC CA-3'; CXCR4, forward 5'-CAA GCA AGG ATG TGA GTT CG-3' and reverse 5'-GCT GGA GTG AAA ACT TGA GG-3'; GAPDH (390 bp), forward 5'-CCA AAG TTG TCA TGG ATG AC-3' and reverse 5'-CCC TTC ATT GAC CTC AAC TA-3'.
Immunohistochemistry and immunofluorescence
Immunohistochemical study for the expression of CXCL12 and CXCR4 was performed using paraffin-embedded pituitary glands of Sprague–Dawley rats. 5 µm sections of the pituitary were prepared. After deparaffinization and hydration of the slides, peroxidase quenching was performed with 3% hydrogen peroxide in PBS for 10 min. After preincubation with serum blocking solution (Zymed Laboratories Inc., San Francisco, CA, USA) containing 5% horse serum, specimens were incubated with mouse monoclonal anti CXCR4 (5 µg/ml, sc-12764, Santa Cruz Biotechnology Inc., Burlingame, CA, USA) or anti-CXCL12 antibody (10 µg/ml, R&D System Inc., Minneapolis, MN, USA), which are compatible to human and rat species for 3 h at room temperature. After washing the slides with tris-buffered saline/0.025% Tween, biotinylated horse anti-mouse immunoglobulin (5 µg/ml, Vector Laboratories, Inc., Burlingame, CA, USA) and streptavidin-peroxidase conjugate (Vector Laboratories) were added sequently. DAB (diaminobenzidene; DAKO Corp., Carpinteria, CA, USA) was used as a chromogen. We also tested other anti-CXCR4 antibodies originated from goat (N-terminus specific, sc-6279, Santa Cruz) that can detect CXCR4 of the human, rat, and mouse. Immunoglobulin G originated from mouse and goat was used as negative controls against anti-CXCR4 antibody. We used two different tissue controls including the posterior lobe for positive control and adrenocorticotrophin (ACTH)-secreting human pituitary tumor for negative control. CXCR4 was detected in the posterior lobe (Fig. 1C), whereas this was not detected in ACTH secreting pituitary tumor tissues (data not shown).
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To detect the expression of CXCR4 or GHRHR in GH3-GHRHR cells, cells were plated on fibronectin-coated cover slips and washed with PBS twice, then fixed with 4% paraformaldehyde in PBS (pH 7.4) for 5 min. The slides were incubated with rabbit anti-human GHRH (hGHRH) receptor (1:1000; provided by Dr Bruce Gaylinn, University of Virginia, Charlottesville, VA, USA) or mouse monoclonal anti-CXCR4 at room temperature for 1 h. After washing, staining was performed using biotinylated secondary antibodies (Vector Laboratories), and streptavidin-FITC (1:100; Vector Laboratories). Images were obtained using a Zeiss microscope (Axioskop, Carl Zeiss Inc., Oberkochen, Germany).
Establishment of stable GH3 cells expressing GHRHR
GH3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco's Modified Eagle Medium (DMEM)/Ham's F-12 containing 10% FBS. All media were supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and maintained at 37 °C with 5% CO2. Stable GH3-GHRHR were selected. Briefly, GH3 cells at 70% confluency were transfected with pCDNA3 carrying hGHRHR cDNA (provided by Kelly E Mayo, Northwestern University, Chicago, IL, USA) using low-serum Opti-MEM (Invitrogen Corp.) and lipofectamine 2000 (Invitrogen Corp.) for 4–5 h. The transfected cells were washed and incubated in DMEM/Ham's F-12 medium (GIBCO BRL) containing 300 µg/ml of Geneticin (G418, Life Technologies). GH3-GHRHR cells that grew into monoclones were selected from the media and transferred to other plates. Each selected monoclone was harvested and the expression of GHRHR was confirmed by RT-PCR, western blot analysis, and immunofluorescent staining.
Western blot analysis
GH3-GHRHR cells in 10 cm plates were lysed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 5% phosphatase inhibitors, 0.5% protease inhibitors) and protein content was measured using Coomassie (Bradford, UK) Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of protein (10 µg) were heat denatured in 2X sample buffer (2% SDS, 62.5 mM of Tris, pH 6.8, 0.01% bromphenol blue, 1.43 mM mercaptoethanol, and 0.1% glycerol), separated on 10% SDS-polyacrylamide gel, transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad), and blotted with the appropriate antibodies: anti-hGHRH receptor antibody, p44/42 MAPK (ERK1/2) antibody, and phosphor-p44/42 MAPK antibody (Cell Signaling Technology Inc). Detection of immunocomplexes was performed by enhanced chemiluminescence system (Cell Signaling Technology Inc., Danvers, MA, USA).
Luciferase assay
To determine whether CXCL12 is involved in GH-gene promoter activity, GH3-GHRHR cells were seeded in 12-well plates at a density of 5x105 cells/well, then plasmids containing the human GH promoter (–610,+58 from transcription start site; Lee & Jameson 2002) luciferase constructs were transfected using lipofectamine 2000 according to the manufacturer's instructions. The transfected cells were treated with SDF1β (1.8x10–8 M, Human/Feline CXCL12/SDF-1β, 96% identical with rat, R&D Systems, Inc.), hGHRH (1–29; 10–9 M, Sigma–Aldrich Inc.), and somatostatin (10–7 M, Sigma–Aldrich) in serum-free medium for 16 h, then lysed for assay of luciferase activities (Luciferase Assay System, Promega). Relative light units were determined using MicroLumat LB96PEG&G luminometer (Berthold Technologies, Bad Wildbad, Germany). Luciferase activity was normalized as described previously (Ishikawa et al. 2004).
Real-time PCR
To assess the level of gene expression, real-time RT-PCR was carried out on the experimental samples using ABI 7300 Sequence Detection System (Perkin–Elmer Applied Biosystems, Foster City, CA, USA). The synthesized cDNAs, obtained by the method described above, were further amplified in triplicate by PCR using SYBR Green I as fluorescent dye and 1xPCR Master Mix (Takara Bio Inc., Tokyo, Japan) containing 300 nmol of forward and reverse primers in a final volume of 25 µl. GAPDH served as a reference gene used for normalization of GH mRNA level. The gene-specific primers used for GH amplification were: forward 5'- GCT GCG TTC TGC TTC TCA G-3' and reverse 5'- CCG AGG TAC CAA ACA TCA G-3'. PCR cycling conditions were: initial denaturation and enzyme activation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 1 min. Calculation of relative expression levels of the target mRNAs was conducted based on the cycle threshold (CT) method (Higuchi et al. 1993). The CT for each sample was calculated using ABI 7300 Real-time PCR detection system software with an automatic fluorescence threshold setting.
Primary rat pituitary cell cultures and measurement of GH
Anterior pituitary glands from male Sprague–Dawley rats (250–300 g, 8 weeks old, Orient Bio Inc., Kyoungki, Korea) were obtained after careful removal of the posterior pituitary described previously (Colin & Jameson 1998) and dispersed by enzymatic digestion for 15 min in 0.125% trypsin (Worthington Biochemical Corp., Lakewood, NJ, USA)/PBS solution containing no calcium and magnesium ions with shaking. Ten U/ml of DNAse-I (Sigma–Aldrich) were added to the solution and incubated for 2 min. Cells were treated with 0.125% collagenase type IV (Sigma–Aldrich) for 5 min and filtered and washed with DMEM (Hyclone, UT, USA), then plated in 12-well culture plates in a mixture of DMEM (Hyclone) supplemented with 10% FBS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin for 48 h.
Rat anterior pituitary cells were maintained in serum-free DMEM for 1 h before the experiment. Then, serum-free DMEM containing SDF1β (18 nM), GHRH (1 nM), or both CXCL12 and GHRH was replaced, aliquots were collected at 30, 60, and 120 min, and stored at –70 °C. The levels of GH in culture medium were measured using a rat GH RIA kit (LINCO Research, Inc., St Charles, MO, USA).
Animal experiment and protocol were approved by Institutional Animal Care and Use Committees of Yonsei University, College of Medicine.
BrdU incorporation analysis of GH3 cells
GH3-GHRHR cells were plated at 5x104/well in 96-well plates, serum starved for 48 h before being treated with SDF1β for 16 h, and in the last 2 h, cells were incorporated with BrdU. Cell proliferation was measured by BrdU incorporation assay according to the manufacturer's protocol (Chemicon International, Temecula, CA, USA).
Statistical analyses
Data are presented as the mean±S.D. The statistical significance of differences was analyzed by Student's t-test for comparison between control and experiment groups. P<0.05 was considered statistically significant. Statistical analyses were performed using SPSS for Windows software (version 12.0; SPSS, Chicago, IL, USA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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To evaluate the putative role of the CXCL12/CXCR4 system in the function of pituitary, expression of chemokine receptor CXCR4 and its natural ligand CXCL12 was studied in the pituitary gland of Sprague–Dawley rats and GH3 cells. RT-PCR analysis showed that Cxcr4 mRNA was detected in both rat pituitary and GH3 cells whereas Cxcl12 mRNA was expressed only in rat pituitary tissues that contained neurons of the posterior pituitary. Immunofluorescent staining showed that CXCR4 was localized along the membrane of GH3 cells (Fig. 1B), while CXCL12 was not detected (data not shown). Immunohistochemical staining of rat pituitary glands revealed that a number of cells were stained with anti-CXCL12 and anti-CXCR4 antibodies in the posterior pituitary whereas CXCL12 positive cells were not observed in the anterior and intermediate lobe. A large number of cells in the anterior and posterior lobe were stained with anti-CXCR4 antibody, whereas the intermediate lobe was not stained at all. The intensity of positivity in the anterior pituitary is stronger compared with that in the posterior pituitary (Fig. 1C). Using different antibody and control IgGs originating from mouse and goat, the same results were obtained (data not shown).
Colocalization of CXCR4 with GH in rat pituitary
Because a large number of anterior pituitary cells were positive for CXCR4 by immunohistochemical staining, we investigated whether they were somatotroph or lactotroph. Double immunofluorescent staining revealed that CXCR4 expression was strictly colocalized with GH whereas PRL was not colocalized at all (Fig. 2). This finding suggests that CXCR4 is specifically expressed in the somatotroph of the anterior pituitary.
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To investigate the role of CXCR4/CXCL12 interaction on GH gene activation in primary rat pituitary cell cultures and GH3 in comparison with GHRH and somatostatin, which are major regulators of production and secretion of the GH in the somatotroph, we examined the activity of the GH promoter by luciferase assay and the expression of GH mRNA by real-time PCR. Because GH3 cells do not possess receptors for GHRH (Zeytin et al. 1984), stably transfected GH3 cells with hGHRHR (GH3-GHRHR) cells were established. RT-PCR and western blot analysis were performed to assess hGHRHR expression in 4 clones obtained. An hGHRH receptor-specific band of 
52 kDa was detected in GH3-GHRHR cells and an additional band of higher molecular weight was also observed, presumably corresponding to a different form of glycosylated hGHRHR (Fig. 3A; Lee et al. 2001). Immunofluorescent staining showed that expression of hGHRH receptor was readily detected in membranes of GH3-GHRHR cells (clone 4; Fig. 3B). Experiments were then performed using clone 4.
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Effect of CXCL12 on GH secretion in primary rat anterior pituitary cells
To investigate whether CXCL12 stimulates GH secretion in primary rat anterior pituitary cells, measurement of GH in culture media was performed after treatment of SDF1β or GHRH (Fig. 5). The posterior pituitary was carefully removed before culturing to minimize the effect of endogenous CXCL12 secreted from the posterior pituitary. SDF1β significantly stimulated the secretion of GH at 30 and 60 min up to 2.5–3.5-fold compared with the control. GH was also increased after GHRH alone (2-fold) or co-treatment (3.0-fold) of SDF1β and GHRH.
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Proliferation of GH3-GHRHR cells was examined by BrdU incorporation assay after treatment of SDF1β, GHRH, or somatostatin for 16 h (Fig. 6A). GH3-GHRHR cells were maintained in serum-free medium to avoid a possible proliferative effect of various growth factors. As previously reported GHRH has a mitogenic effect on GH3 cells expressing GHRH receptors (Lee et al. 2001), SDF1β and GHRH significantly stimulated the proliferation of GH3-GHRHR cells by 30% compared with the control whereas somatostatin reduced cell proliferation slightly.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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However, few studies to date have investigated the possible role of CXCL12/CXCR4 signal in the pituitary. Since low densities of CXCL12 binding sites were detected in rat pituitary gland (Banisadr et al. 2000), Florio et al. only reported that SDF1
induces cell proliferation and GH secretion in the rat pituitary adenoma-derived cell line GH4C1 (Florio et al. 2006, Massa et al. 2006). The GH4C1 cell line, established from GH3 cells that produce PRL and large amounts of GH, expresses low basal levels of GH but higher PRL levels (Tashjian et al. 1968, 1970). According to the evidence that GH4C1 cell responses to IGF1 were opposite to those of GH3 cells or primary rat pituitary cells (Castillo & Aranda 1997), GH3 cells may be more similar to normal somatotrophs in characteristics compared with GH4C1 cells. Our experiments were conducted using primary rat pituitary cells and GH3 cells, which are known as valuable sources for studies of hormonal secretory physiology and signal transduction pathways (Lee et al. 2001).
Expression of CXCL12 and CXCR4 was determined in the pituitary of Sprague–Dawley rats and GH3 pituitary tumor cell lines in our study. In contrast to the finding that rat pituitary cells produced Cxcl12 and Cxcr4 mRNA, only 7-transmembrane receptor CXCR4 was localized along membranes of GH3 cells. Immunostaining showed that CXCL12- and CXCR4-positive cells were observed in the posterior pituitary glands, which contained neurons projected from hypothalamic nuclei, and was consistent with a previous report (Callewaere et al. 2006). However, in our results, the anterior pituitary was strongly stained with anti-CXCR4 antibodies. The anterior pituitary consists of various types of endocrine cells. Among them, somatotrophs and lactotrophs are 2 major cell components. However, it is notable that only somatotrophs possess receptors for CXCL12, confirmed by double immunofluorescent staining. This result was demonstrated for the first time in the rat pituitary gland. This result suggests that CXCR4 could play a role in normal physiological function of the somatotroph.
In our study, SDF1β increased transcription of GH mRNA by activating GH promoter in GH3 cells expressing GHRHR. There are two splice forms of CXCL12, designated as SDF1 and SDF1β, which are identical in the first 89 amino acid (AA) sequences, but SDF1β has 4 additional AA at the C-terminus (Tashiro et al. 1993, Shirozu et al. 1995). The first 17AA, which are important to the binding ability of CXCL12 to CXCR4 (Loetscher et al. 1998, Doranz et al. 1999), are identical in both isoforms while SDF1 is more sensitive to proteolysis by serum CD26/dipeptidyl peptidase than SDF1β (De La Luz Sierra et al. 2004), implying the differences in functions or regulatory roles in vivo. Furthermore, SDF1β is extensively expressed throughout the rat brain, especially in cerebral endothelial cells, while SDF1 is localized in specific neuronal structures such as cingulate (Stumm et al. 2002). In the present experiments, we chose SDF1β, which showed about a 2-fold potency, compared with SDF1 (Crump et al. 1997), and are generally distributed over entire brain area.
We observed that the activation of GH promoter or GH mRNA expression by SDF1β in GH3-GHRHR cells was higher than by GHRH. This difference might be due to the relatively low concentration of GHRH (1 nM) compared with the relatively higher concentration of SDF1β (18 nM) although we used 1 nM GHRH before (Lee et al. 2001). Barbieri et al. (2007) showed that similar levels of GH increases were achieved using 12.5 nM of SDF1 or 300 nM of GHRH in GH4C1 cells. It is intriguing to predict the actual concentrations of chemokine CXCL12 and hormone GHRH in the microenvironment of the pituitary in vivo.
We examined the effects of SDF1β on synthesis and secretion of GH in primary rat anterior pituitary cells for the first time. Although anterior pituitary glands consist of heterogeneous groups of endocrine cells, only somatotrophs in the anterior pituitary expressed the receptor CXCR4, according to double immunofluorescent studies (Fig. 2). After the administration of SDF1β, transcription of GH mRNA and release of GH were significantly increased compared with those induced by GHRH. Higher GH secretion was maintained up to 2 h by time-dependent manner after treatment (data at 120 min not shown). Interestingly, no synergistic or additive effects on GH secretion or GH mRNA transcription were observed after administration of both SDF1β and GHRH, suggesting that CXCL12/CXCR4 signaling and GHRH-mediated pathway may possess the same intracellular mediators or interfere with each other. After GHRH binds to Gs protein-coupled GHRHR in the membrane of somatotrophs, activated adenylyl cyclase produces intracellular cAMP, followed by secretion of GH. Although SDF1β induces G inhibitory protein-linked reduction in concentrations of cAMP in cytoplasm (Zheng et al. 1999, Peng et al. 2004, Florio et al. 2006), increased intracellular calcium levels stimulated by SDF1β may be related to GH release. Further study is needed to clarify this mechanism.
We demonstrated the mitogenic effect of SDF1β in GH3 cells using BrdU incorporation assay. GH3 cell proliferation was significantly increased after treatment with SDF1β, which induced phosphorylation of ERK1/2. Ca++-independent activation of ERK1/2 and Ca++-dependent stimulation of cytosolic tyrosine kinase, Pyk2, were reported to be important SDF1-related intracellular signaling pathways that mediate growth of pituitary GH4C1 cells (Florio et al. 2006, Massa et al. 2006). Furthermore, CXCL12 increases the percentage of cells in the S phase and inhibits tumor cell apoptosis by activation of NF-
B (Helbig et al. 2003, Kryczek et al. 2007). Based on evidence that various kinds of tumor cell proliferations are associated with CXCL12 (Scotton et al. 2002, Barbero et al. 2003, Hall & Korach 2003), development of the pituitary tumor may be related to CXCL12-mediated intracellular pathways. Pituitary tumors are known to derive from the transformed single pituicyte in which initiating mutations of candidate genes such as Ras or Gsp have caused a gain of proliferative functions, followed by clonal expansion stimulated by promoting factors such as locally produced growth factors and cytokines (Faglia & Spada 2001, Asa & Ezzat 2002).
It has been demonstrated that neurons from hypothalamic nuclei and posterior pituitary produce CXCL12, which could modulate AVP secretion induced by autocrine mechanism (Callewaere et al. 2006, 2007). From our immunohistochemical analyses showing that anterior pituitary glands did not express CXCL12 but CXCR4, the function of anterior pituitary cells might be controlled by external CXCL12 originating from hypothalamic neurons or systemic circulation via blood vessels (Barbieri et al. 2007). Disruption of these putative pathways related to CXCL12/CXCR4 interactions might play a role in the development of pituitary adenoma. Further studies are required to prove this hypothesis.
In conclusion, we investigated the expression of CXCR4 and CXCL12 and their physiological roles in normal rat pituitary cells and GH-producing GH3 tumor cells. CXCL12/CXCR4 interaction plays an important role in the synthesis and release of GH and cell proliferation. These results may suggest that CXCL12/CXCR4 signaling could be a promising target for the treatment of GH-producing pituitary tumors, and should be considered in future studies.
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Declaration of interest |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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Funding |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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Received in final form 1 August 2008
Accepted 22 August 2008
Made available online as an Accepted Preprint 27 August 2008
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