Reduced Expression of the Cytokine Transducer gp130 Inhibits Hormone Secretion, Cell Growth and Tumor Development of Pituitary Lactosomatotrophic GH3
Abstractr:%t), 百拇医药
Two of the most potent cytokines that regulate anterior pituitary cell function are leukemia inhibitory factor and IL-6. These and others like IL-11 and ciliary neurotrophic factor are referred to as the gp130 cytokines because they share the gp130 glycoprotein as a common receptor initial signal transducer. We and others have shown that gp130 cytokines and their receptors are expressed and functional in normal and tumoral anterior pituitary cells. To study the role of gp130 cytokines in tumorigenic process, we generated gp130 cDNA gp130 sense and gp130 antisense (gp130-AS) transfected stable clones derived from lactosomatotroph GH3 cells. We examined hormone secretion and cell proliferation of these clones as well as their tumorigenic properties in athymic nude mice. Although gp130-AS clones, which have low gp130 levels and impaired signal transducer and activator of transcription 3 activity and suppressor of cytokine signaling-3 expression, showed reduced proliferation and hormone secretion (GH and prolactin) in response to gp130 cytokines, they had a normal response to gp130-independent stimuli. Moreover, gp130-AS clones showed a severely impaired in vivo tumor development. In contrast, the overexpressing gp130 clones (gp130 sense) showed no differences, compared with cells transfected with control vector. Thus, the present study provides new evidence supporting a link between gp130 and pituitary abnormal growth.
Introductionm6#3i, 百拇医药
FOR IL-6 AND OTHER cytokines that belong to the IL-6 cytokine family, their receptor cytoplasmic domains are not required for cytokine-mediated signal transduction, which is mediated by another membrane glycoprotein-denominated gp130 (1, 2). The gp130 glycoprotein alone does not bind IL-6 or other cytokine of its family, and the binding of these cytokines (i.e. IL-6) triggers the association of their {alpha} -subunits with gp130 (i.e. IL-6R{alpha} chain with gp130). All those cytokines that make use of the gp130 chain belong to the gp130 cytokine family, which is composed by IL-6, leukemia inhibitory factor (LIF), IL-11, oncostatin M, ciliary neurotrophic factor (CNTF), cardiotropin-1, cardiotrophin-like related cytokine, and stimulating neurotrophin-1/B cell-stimulating factor-3 cell (3). Gp130 mainly activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and also induces a family of cytokine-inducible signaling inhibitors including among others suppressor of cytokine signaling-3 (SOCS-3; Ref. 4).
Considering that gp130 is ubiquitously expressed, the responsiveness of a given cell type to gp130 cytokines is therefore mainly determined by expression of the respective specific {alpha} -subunits and the cytokines themselves. This takes place in the pituitary gland in which specific receptors for IL-6 (5), LIF (6), IL-11 (7, 8), and CNTF (8) have been described, providing the basis for the regulatory role of these cytokines in both hormone secretion and cell growth (9). In addition, the JAK/STAT/SOCS-3 pathway has also been described in pituitary cells (10, 11).hm:#, 百拇医药
It has been demonstrated that these cytokines regulate hormone secretion in pituitary cell lines, normal anterior pituitary cells, and human tumor pituitary cells. For example, IL-6 stimulates the release of prolactin (PRL), GH, and LH (12, 13). IL-6 and LIF are also powerful stimuli for ACTH secretion by corticotrophs in vitro and in vivo in different species (13, 14, 15, 16). Induced expression of proopiomelanocortin mRNA by IL-6 (17) as well as the enhanced ACTH secretion and proopiomelanocortin expression by IL-11 was reported (7). Similarly, IL-11 and CNTF regulate the secretion of GH and PRL in lactosomatotrophic cells (GH3 cell line) (8), human pituitary adenomas (18), and vascular endothelial growth factor (VEGF) release in folliculostellate (FS) cells (TtT/GF cell line) (8). The role of gp130 has not, however, been studied so far.
Besides the effects of gp130 cytokines on the secretion of pituitary hormones, gp130 cytokines also have influence on pituitary cell growth. Thus, we previously demonstrated that IL-6 regulates pituitary cell growth (19). Intriguingly, IL-6 stimulates DNA synthesis and cell number of tumor GH3 cells, yet at the same cytokine concentrations, it inhibits the growth of normal rat pituitary cells (19). IL-6 has been also reported to stimulate the growth of TtT/GF cells (20) as well as the proliferation of the MtT/E rat tumor pituitary cell line (21). Accordingly, TtT/GF cells, which also express IL-6, has been shown to stimulate the somatotrophic tumor cells (MtT/S) pituitary tumor growth in nude mice (22). In different tumors (ACTH-, PRL-, GH-secreting and nonfunctioning adenomas), IL-6 has been shown to have either inhibitory or stimulatory effects. However, the type of response does not seem to be associated with tumor type or size. By using blocking antibodies in a functional assay to study intrapituitary action, it has been demonstrated that endogenous IL-6 regulates the expression of c-fos in a similar manner (23). CNTF and IL-11 stimulate the proliferation of FS and GH3 cells (8). The fact that, in addition, these cytokines (IL-6, LIF, IL-11, and CNTF) are expressed in normal and tumoral pituitary cells (9, 13, 14, 15, 16) suggests that they have important paracrine/autocrine actions in the pituitary gland, which could be mediated by gp130.
Based on this evidence, we generated stable GH3 clones expressing different gp130 levels to study the role of the cytokine common transducer gp130 glycoprotein in the regulation of lactosomatotrophic function and growth.68jvp, 百拇医药
Materials and Methods68jvp, 百拇医药
Cell culture and stimulation68jvp, 百拇医药
Materials and reagents, except where stated, were obtained from Seromed (Berlin, Germany), Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Frederick, MD), Sigma, (St. Louis, MO), and Nunc (Wiesbaden, Germany). Recombinant rat CNTF (R&D Systems Inc., Minneapolis, MN) and TRH (Bachem, Bubendorf, Switzerland) were used.68jvp, 百拇医药
GH3, a rat lactosomatotrophic pituitary tumor cell line obtained from the American Type Culture Collection (Manassas, VA) (19), and GH3 gp130 clones, generated as indicated below, were used. They were cultured in DMEM (pH 7.3) supplemented with 10% fetal calf serum (FCS), 2.2 g/liter NaHCO3, 10 mM HEPES, 2 mM glutamine, 2.5 mg/liter Amphotericin B, 105 U/liter penicillin-streptomycin, 5 mg/liter insulin, 5 mg/liter transferrin, 20 mg/liter sodium selenite, and 30 pM T3 until they were confluent. The stable clones were maintained in selection medium with the antibiotic G418 (600 mg/ml). For proliferation and hormone secretion experiments, the cells were washed twice with PBS, and then medium was replaced by an experimental medium consisting of the same supplemented DMEM without FCS. Before and after the stimulation period, cell viability was routinely controlled to ensure that this parameter did not change during the experiment. Cell viability was determined microscopically after ethidium bromide/acridine orange staining.
Constructs1oh4^, 百拇医药
The murine gp130 expression vector (pBMG gp130) was generously provided by Dr. Matsumoto Hibi (24). The cDNA full-length (3-kb XhoI/XhoI fragment) was subcloned into pcDNA3 (Invitrogen) (pcDNA3/gp130 sense). The antisense expression vector was generated by subcloning of 0.9-kb XhoI/BamHI fragment into pcDNA3 expression vector in reverse orientation (pcDNA3/gp130 antisense). cDNA insertions were verified by sequencing.1oh4^, 百拇医药
To measure STAT3 activity, the p4xm 67 TATA-tk-LUC was used, provided generously by Dr. J. E. Darnell, Jr. (25). (DEI) 4-alb-LUC construct, used to evaluate NF-IL-6 activity was kindly provided by Dr. P. Johnson and consists of CCAAT/enhancer-binding protein (C/EBP) responsive DEI sites and an albumin minimal promoter coupled to the luciferase (LUC) gene (26). The LUC reporter gene under the control of 2.5 kb of the PRL promoter (PRL-LUC) construct was provided by Dr. A. Gutierrez-Hartmann (27).1oh4^, 百拇医药
GH3 gp130 stable clones
To obtain stable clones with over- or downexpressing gp130 protein, pcDNA3/gp130 sense, pcDNA3/gp130 antisense, or pcDNA3-control vector were used to transfect GH3 cells by Lipofectamine (Invitrogen). After 48 h of incubation, cells were subcultured and maintained in selection medium containing G418. The pcDNA3/gp130 sense, pcDNA3/gp130 antisense, and pcDNA3 stably transfected clones were denominated gp130-sense (gp130-S), gp130-antisense (gp130-AS), and GH3 control, respectively. The levels of gp130 in these clones were tested by Western blot. Southern blot analysis were performed and confirmed the presence of integrated transgenes in all selected clones, which are independent (i.e. the different clones carry gp130 cDNA in different regions of their genome). Results from the same representative clone for each construct are shown in all experiments. Similar results were observed with the different individual clones for each construct as indicated for each experiment.{$v$ft, 百拇医药
Western blot analysis
Cells were washed once with PBS (pH 7.0), and then cell lysates were prepared in standard cracking buffer and boiled for 3 min. Equal levels of protein (30 µg) were analyzed by 8% SDS-PAGE. Proteins were blotted onto nitrocellulose Western blotting membranes (Sigma) using standard procedures. A specific anti-gp130 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) previously used by others (28) was used. The antibodies were detected with alkaline phosphatase-conjugated antirabbit IgG (Santa Cruz Biotechnology, Inc.) followed by 5-bromo-4-chloro-3-indoly phosphate p-Toluidine and nitroblue tetrazolium chloride detection (Invitrogen)..)evn4, 百拇医药
Northern blot analysis.)evn4, 百拇医药
Total RNA extraction and Northern blot analysis were performed as described previously (29). Reagents were from Sigma, Roche Molecular Biochemicals (Mannheim, Germany), or Pharmacia (Uppsala, Sweden). Briefly, total RNA, isolated by the guanidine isothiocyanate phenol-chloroform extraction method, was denatured with glyoxal, electrophoresed on a 1.2% agarose gel, and transferred overnight to a nylon membrane. Filters were baked for 2 h at 80 C and stained with methylene blue. They were prehybridized for 1 h at 60 C (50% formamide, 5x sodium chloride/sodium phosphate/EDTA, 5x Denhardt’s solution, 0.1% sodium dodecyl sulfate, 100 µg/ml denatured salmon sperm DNA), and then the probe was added and hybridized for 12 h. Blots were washed at increasing salt and temperature stringency with a final wash of 30 min at 60 C in 0.1x saline sodium citrate buffer containing 0.1% sodium dodecyl sulfate. After washing, blots were exposed to XAR5 film (Kodak, Rochester, NY) at -70 C with intensifying screens for 6 d. A 0.5-kb SOCS-3 cDNA fragment (30) and a 1-kb PstI fragment of ß-actin cDNA (31) were labeled with a random-priming kit using {alpha} -32P-dCTP (specific activity, 2–4 x 108 cpm/µg). The blots were reprobed after stripping the first probe with 5 mM Tris/HCl (pH 8.0), 2 mM EDTA, and 0.1x Denhardt’s solution at 65 C for 2 h. After the previous signal was removed and confirmed by reexposure of the filter, the blots were prehybridized and hybridized following the methods described above. The loading control with the fragment of ß-actin cDNA as probe was performed in each blot. The mRNA levels were quantified by using phosphor imager analysis (Fuji Photo Film Co. Ltd., Aichi, Japan).
Cell proliferation[4kw, 百拇医药
Cell proliferation was measured with the WST-1 reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions, and the reaction product was measured in an ELISA plate reader at 450 nm (8, 32). Results were validated by total cell count. Acridine orange-ethidium bromide staining was used to rule out toxic effects.[4kw, 百拇医药
Hormone determination[4kw, 百拇医药
Hormones were measured by RIA as previously described (8, 19). For recombinant GH and PRL, reagents were kindly provided by Dr. A. F. Parlow from the National Hormone and Pituitary Program (Torrance, CA).[4kw, 百拇医药
Transfection of GH3 cells and reporter assays[4kw, 百拇医药
Cell transfection was performed with Lipofectamine using standard procedures, as previously described (32). After plating the cells in 6-well plates, the cells were transfected in DMEM without FCS for 6 h using 5 µl lipofectamine (Invitrogen) per well with 1.5 µg for each plasmid, in conjunction with a control Rous sarcoma virus (RSV)-ßgal construct (0.5 µg). Cells were then washed and left 20 h in DMEM supplemented with 10% FCS. Then cells were washed and stimulated for 6 h with CNTF (2 nM) in DMEM supplemented with 2% FCS. After treatment, LUC activity in cell lysates was measured as previously described (32) using the Luciferase measure kit (Promega Corp., Madison, WI) with a Junior luminometer (Berlthod, Bad Wildbad, Germany). The ß-gal activity was measured as previously described (32) as control for transfection efficiency. Results were standardized for ß-gal activity.
In vivo experiments in nude mice^d#@9|8, 百拇医药
GH3 cells were collected, washed twice with PBS, resuspended in DMEM, and injected (1 x 106 cells) into the flanks of adult (20 g) female N:NIH nude mice. The mice were divided into three groups: injected with GH3 control cells, injected with gp130-S cells, and injected with gp130-AS cells. Animals were examined for tumor formation every 2 d, and tumor growth was determined as described (32). The experiments were finished at 26 d after cell injection because of GH3 control mice morbidity.^d#@9|8, 百拇医药
All experimental protocols were approved by the Ethical Committee on Animal Care and Use, University of Buenos Aires, Argentina.^d#@9|8, 百拇医药
Immunohistochemistry for CD31 and determination of vascular density^d#@9|8, 百拇医药
For immunohistochemistry, 8-µm sections of shock-frozen GH3 tumor tissue were thaw mounted onto SuperFrost Plus slides (Menzel-Glaser, Hamburg, Germany), fixed in 4% paraformaldehyde in PBS, and stored in 96% ethanol at 4 C until use.
An antimouse CD31 (PECAM-1) antibody (Santa Cruz Biotechnology, Inc.) was used to detect intratumoral microvessels. Slides were first incubated in horse serum diluted 1:10 in Tris-based buffer (pH 7.6). Subsequently the slides were incubated overnight at 4 C with CD31 primary antibody diluted 1:500. After three washes in Tris-based buffer, the biotinylated horse antigoat secondary antibody (dilution: 1:300) (Vector Laboratories Inc., Burlingame, CA) was added for 30 min at room temperature. The slides were then washed again and were incubated for 30 min with the avidin-biotin-peroxidase complex (Vector Laboratories Inc.). Color development was performed using 1 mg/ml diaminobenzidine (Sigma) with 0.01% hydrogen peroxide applied for 45 sec. After washing in deionized water, the sections were counterstained with toluidine blue, fixed in Roti-Histol (Carl Roth, Karlsruhe, Germany) and coverslipped using Roti-Histokitt (Carl Roth). Negative controls were performed by omitting the primary antibody. No staining for CD31 has been detected in negative control cells.
For determination of vascular density, an investigator who was not informed about the type of tumor counted vessels inside an area delimited by an eyepiece graticule 12.5 x 12.5 mm divided in 10 x 10 squares (Zeiss, Munich, Germany) at a magnification x200 (x20 objective and x10 ocular). Vessel density was determined in six different tumors of each group (gp130-AS; gp130-S; GH3 control-derived tumors), and in each tumor both single cells and cell clusters positive for CD31 were counted in three different areas. Results of vessel counting are expressed as mean ± SEM of 18 areas counted.%v, 百拇医药
Statistics%v, 百拇医药
Statistics were performed by ANOVA in combination with the Scheffé’s test. Data are shown as mean ± SEM.%v, 百拇医药
Results%v, 百拇医药
Generation and characterization of gp130 stable cell lines%v, 百拇医药
To generate lactosomatotrophic GH3 stable clones that over- or downexpress the gp130 glycoprotein, GH3 cells were transfected with vectors containing gp130 cDNA in sense or antisense orientations. As shown in Fig. 1A, gp130 glycoproteins were expressed at higher levels in gp130-S clones, whereas they were downexpressed in gp130-AS clones, compared with GH3 cells stably transfected with control vector.
fig.ommitteedh^r#5]y, 百拇医药
Figure 1. Characterization of gp130-S and gp130-AS stable clones. A, Western blot analysis of GH3 lactosomatotrophic stable clones showing overexpressed or downexpressed gp130 protein levels. The bands corresponding to gp130 protein were detected by an anti-gp130 polyclonal antibody and are indicated by an arrow. GH3 control, GH3 cells transfected with pcDNA3/control vector; gp130-S, GH3 cells transfected with pcDNA3/gp130 sense vector; gp130-AS, GH3 cells transfected with pcDNA3/gp130 antisense vector. Molecular weight marker is indicated on the right. Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown. B, STAT-3-mediated transcriptional activation in response to CNTF in gp130-S and gp130-AS stable clones. GH3 stable lines were transiently transfected with plasmid DNA mixture containing 1.5 µg luciferase reporter construct [(4x) IRF-1] with four copies of the STAT-3-binding sequence from the interferon regulatory factor 1 (IRF-1) and 0.5 µg pRSV-Lac Z. At 20 h after transfection, cells were treated with CNTF (2 nM) (bars) for 6 h. The values corresponding to luciferase were normalized with respect to ß-galactosidase activity. Results are expressed as mean ± SEM (n = 4) of one representative of three independent experiments with similar results. By ANOVA with Scheffé’s test: *, P < 0.05 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
To test the signaling pathway downstream of gp130 in the stably transfected GH3 clones, they were transiently transfected with the STAT3-LUC construct. As shown in Fig. 1B, CNTF (2 nM) induced STAT3-transcriptional activity with respect to the basal condition in the GH3 control cells, which was slightly enhanced in the gp130-S clones. However, in the case of gp130-AS clones, LUC activity was efficiently blocked (Fig. 1B). Similar results were obtained with a C/EBP-LUC reporter construct (data not shown).*;%hr, 百拇医药
In addition, SOCS-3 mRNA was also stimulated in GH3 control and gp130-S clones in response to CNTF, whereas it was blunted in gp130-AS clones (Fig. 2).*;%hr, 百拇医药
fig.ommitteed*;%hr, 百拇医药
Figure 2. Effect of CNTF on SOCS-3 mRNA expression in GH3 control, gp130-S, and gp130-AS clones. Northern blot analysis is shown using 30 µg RNA per lane as stated in Materials and Methods. A, Bands corresponding to SOCS-3 mRNA (3.2 kb) or ß-actin from one representative experiment are shown. The cell lines were seeded at 6 x 105 cells/well and treated with CNTF (2 nM). Densitometric units of the bands relative to constitutive ß-actin expression are shown. B, Mean densitometric analysis of SOCS-3 mRNA content from three independent experiments including the one shown in A. Data are expressed as a ratio ± SEM of autoradiographic signals of SOCS-3 mRNA relative to ß-actin mRNA used as loading control. Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
Cell proliferation and hormone secretion inhibition in gp130-AS clones[5f/, http://www.100md.com
No differences in basal proliferation were observed among different clones (Fig. 3). CNTF significantly stimulated the growth of GH3 control cells, at the same doses previously reported (8), and a similar response was found in the gp130-S clones. In contrast, as shown in Fig. 3, the capacity of the gp130-AS clones to respond to the gp130 specific stimuli was abolished, whereas they were able to respond to a gp130-independent stimulus, TRH. Similar results were obtained in the hormone secretion experiments: Both CNTF and TRH significantly stimulated GH secretion from GH3 control and gp130-S cells, whereas the gp130-AS clones had a normal response only to TRH (Fig. 4).[5f/, http://www.100md.com
fig.ommitteed[5f/, http://www.100md.com
Figure 3. Proliferation of GH3 control, gp130-S, gp130-AS clones in response to CNTF and TRH. GH3 gp130 cell lines were seeded at 9000 cells/well in multiwell plates with 10% FCS. After attachment, cells were washed twice with PBS and incubated in FCS-free medium for 24 h. Cells were treated either with CNTF (2 nM) or TRH (100 nM) for 72 h. Proliferation was measured by the WST-1 colorimetric method as detailed in Materials and Methods. Values represent the mean ± SEM of quadruplicate determinations of a single representative experiment (total of five with similar results). By ANOVA with Scheffé’s test: *, P < 0.05; **, P < 0.01 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
fig.ommitteedqtk, 百拇医药
Figure 4. Effect of recombinant rat CNTF and TRH on GH secretion in GH3 control, gp130-S, and gp130-AS clones. Clones were treated with CNTF (2 nM) or TRH (100 nM) as indicated. GH3 cell lines were seeded at 2 x 105 cells in 24-well plates. After FCS depletion for 24 h, treatments were added with fresh FCS-depleted medium. After 24 h, the supernatants were collected, and GH was measured by RIA. Values represent the mean ± SEM of quadruplicate determinations of a single representative experiment (total of three with similar results). By ANOVA with Scheffé’s test: *, P < 0.05; **, P < 0.01 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.qtk, 百拇医药
However, gp130-AS clones showed a very low level of basal PRL secretion. Interestingly, the basal activity of the PRL promoter was blunted in the gp130-AS clones, showing they have a constitutive lower basal PRL-transcriptional activity (Fig. 5). No difference between GH3 control and gp130-S cells was observed in terms of hormone secretion (GH and PRL) and cell proliferation stimulated by either gp130 cytokine or TRH (Figs. 3–5).
fig.ommitteed$jl)in$, 百拇医药
Figure 5. PRL promoter activity and PRL secretion in GH3 control, gp130-S, and gp130-AS clones. A, GH3 stable lines were transiently transfected with plasmid DNA mixture containing 1.5 µg luciferase reporter construct of PRL-LUC and 0.5 µg pRSV-Lac Z. The values corresponding to luciferase were normalized with respect to ß-galactosidase activity, and results are expressed as mean ± SEM (n = 3) of one representative experiment of three independent experiments with similar results. B, Clones were treated with CNTF (2 nM) or TRH (100 nM) as indicated. GH3 cell lines were seeded at 2 x 105 cells in 24-well plates. After FCS depletion for 24 h, treatments were added with fresh FCS-depleted medium. After 24 h, the supernatants were collected and PRL was measured by RIA. Results are expressed as mean ± SEM (n = 4) of one representative of three independent experiments. By ANOVA with Scheffé’s test: *, P < 0.05; **, P < 0.01 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
In vivo tumor formation capacity is inhibited in gp130-AS clones00w^, 百拇医药
Based on the findings that GH3 gp130-AS cells had an impaired response to gp130 cytokine stimulation, we hypothesized that the capacity of gp130-AS cells to develop tumors would be inhibited. Therefore, we evaluated the tumor formation by injecting GH3 stables clones into nude mice. Within 14 d after injection, visible tumors were developed in mice injected either with GH3 control or GH3 gp130-S cells. These tumors grew very rapidly and reached considerable sizes at 26 d after injection (Fig. 6). On the contrary, in mice injected with gp130-AS cells, tumor formation was delayed until 23 d after injection, and they developed tumors of very small sizes. Thus, the growth rate and the tumor size were greatly reduced in comparison with both GH3 control and GH3 gp130-S groups (Fig. 6).00w^, 百拇医药
fig.ommitteed00w^, 百拇医药
Figure 6. GH3 cell tumor development is abolished in gp130-AS clones. A, Cells (1 x 106) were injected sc into athymic nude mice (n = 4, each group with the same clone) as described in Materials and Methods. Photographs were taken at d 26 after injection. B, The development of tumors in GH3 control and gp130-S-injected animals started to be apparent at d 14 after injection, whereas group gp130-AS showed delayed tumor formation (visible at d 23). In some cases, symbols corresponding to individual animals overlap. There were no significant differences between d 26 and d 36 in the animals injected with gp130-AS cells (data not shown). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown. The clones preserved the expression of gp130-S or gp130-AS after in vivo injection.
The counting of cells and cell clusters positive for the endothelial cell marker CD31 showed that the vessel density was significantly reduced in gp130-AS-derived tumors. Whereas the vessel density was similar in GH3 control-derived tumors (37.1 ± 5.4 vessels per area) and gp130-S derived-tumors (43.1 ± 6.4 vessels per area), only 12.9 ± 4.5 vessels per area could be detected in gp130-AS derived-tumors (Fig. 7).r'g)gt, 百拇医药
fig.ommitteedr'g)gt, 百拇医药
Figure 7. Density of microvessels in gp130-AS-derived tumors (A) in gp130-S-derived-tumors (B) and GH3 control-derived-tumors (C). Immunostaining for the endothelial cell marker CD31 (brown) is shown. The vessel density was significantly reduced (P < 0.001) by ANOVA with Scheffé’s test in gp130-AS-derived-tumors (with respect to the gp130-S-derived-tumors and GH3 control-derived-tumors). Corresponding negative controls are inserted at the right corner of each picture.r'g)gt, 百拇医药
Discussionr'g)gt, 百拇医药
Pathogenesis of pituitary tumors has been extensively studied to identify those activating oncogene mutations and/or inactivating tumor suppressor genes involved in tumorigenesis (33, 34). For example, G protein (Gs{alpha} ) mutations have been shown to occur only in a subset of sporadic GH-secreting pituitary adenomas, which represent a small percentage of all pituitary tumors (33, 34), suggesting that there may be other events responsible for pituitary tumorigenesis. A novel pituitary tumor-transforming gene involved in the generation of pituitary adenomas has been cloned from rat GH4 pituitary tumors (35). However, despite these discoveries, it is still an open question how pituitary tumors are formed during pathogenesis.
Several studies have postulated the possibility that gp130 cytokines, through the gp130 glycoprotein-mediated signal transduction cascades, could play an important role in the tumorigenic process in different tissues (36, 37, 38, 39). In agreement with this, IL-6 and IL-11 have been reported to stimulate growth of pituitary cells (8, 19, 20, 21) and release of angiogenic factors, such as VEGF by FS cells and TtT/GF cells (8, 40). Thus, the importance of the gp130 cytokines in function and growth of pituitary cells could indicate that some genetic events that occur during pituitary pathogenesis would be mediated by signaling response originated from gp130 cytokines. Therefore, the gp130 cytokines should be considered as feasible candidates to generate a favorable ambient for cell transformation events in the pituitary gland. In fact, in the present study, we demonstrated that reducing gp130 level inhibits tumor development of GH3 cells in vivo.\v*, 百拇医药
Koyama et al. (22) have shown that the MtT/S cell line needs the presence of TtT/GF to induce formation of tumor in nude mice. Although it is still uncertain how TtT/GF cells regulate MtT/S’s cell behavior, it is likely that the microenvironment determined by cellular interactions and extracellular matrix modifications would contribute to support their tumor development. In contrast, GH3 cells have innate capacity to develop tumors in nude mice (41, 42, 43). They respond to angiogenic and growth factors and do not need the presence of other pituitary cells (i.e. TtT/GF cells). Hence, GH3 cells represent a good model to study the influence of gp130 protein expression levels during tumor formation. Our results show that GH3 gp130-AS cells have lower gp130 signal cascade activation, showing reduced STAT-3 and C/EBP-activity and also SOCS-3 mRNA expression levels. It is important to notice that GH3 gp130-AS cells are still able to respond to gp130-independent stimuli, such as TRH, which indicates that the lower amount of gp130 protein is indeed specific and critical for gp130 cytokine actions. These clones have reduced proliferative activity. In accordance with our results, altered forms of gp130 that fail to induce activation of JAKs fail to transduce an IL-6 proliferative signal (44); in mouse B cells and an erythroleukemia-derived cell line expressing human gp130 glycoprotein, the presence of gp130 monoclonal antibodies completely abolished the biological response such as the cell growth induced by gp130 cytokines (45).
On the contrary, the overexpression of gp130 did not significantly modify the cellular behavior, which might indicate that pituitary gp130 endogenous levels fulfill for a normal functional cellular response. Similarly, in transgenic mice that overexpress gp130, the activation-signaling pathway downstream of gp130 in the heart is not enhanced (46), suggesting that gp130 overexpression does not always induce changes in the cell biology, which is in accordance with our results.j!(-}pj, 百拇医药
In the GH3 gp130-S-derived-tumors, vessel density was the highest, observing 2- to 3-fold more vessels in GH3 gp130-S-derived tumors, compared with GH3 gp130-AS- derived tumors. It is therefore clear that in GH3 gp130-AS-derived-tumors, neovascularization is impaired. However, we need to address further studies to determine whether this is a direct effect (suppression of gp130-mediated VEGF and/or basic fibroblast growth factor production because of gp130 blockade) or an indirect effect because of lower tumor cell growth associated with lack of tumor expansion and hypoxia-induced neoangiogenesis.
We show for the first time that the reduced level of gp130 protein in GH3 cells (gp130-AS cells) blocked the cell growth and hormone secretion stimulated by CNTF and have impaired tumor formation capacity in nude mice. These data contribute to support gp130 cytokines as candidates that would favor the development and/or growth of pituitary tumors.$1a[, 百拇医药
Acknowledgments$1a[, 百拇医药
We thank Dr. Matsumoto Hibi, Dr. J. E. Darnell, Jr., Dr. P. Johnson, Dr. A. Gutierrez-Hartmann, and Dr. Tracy Willson for generously providing the constructs used in this study.$1a[, 百拇医药
A.C.N. and E.A. are members of the Argentine National Research Council (Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina).$1a[, 百拇医药
Received August 26, 2002.$1a[, 百拇医药
Accepted for publication October 21, 2002.$1a[, 百拇医药
References$1a[, 百拇医药
Hibi M, Nakajima K, Hirano T 1996 IL-6 cytokine family and signal transduction: a model of the cytokine system. J Mol Med 74:1–12
Kishimoto T, Taga T, Akira S 1994 Cytokine signal transduction. Cell 76:253–262tyhp, 百拇医药
Kishimoto T, Akira S, Narazaki M, Taga T 1995 Interleukin-6 family of cytokines and gp130. Blood 86:1243–1254tyhp, 百拇医药
Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L 1998 Interleukin-6-type cytokine signaling through the gp130/Jak/STAT pathway. Biochem J 334:297–314tyhp, 百拇医药
Ohmichi M, Hirota K, Koike K, Kurachi H, Ohtsuka S, Matsuzaki N, Yamaguchi M, Miyake A, Tanizawa O 1992 Binding sites for interleukin-6 in the anterior pituitary gland. Neuroendocrinology 55:199–203tyhp, 百拇医药
Akita S, Webster J, Ren SG, Takino H, Said J, Zand O, Melmed S 1995 Human and murine pituitary expression of leukemia inhibitory factor. Novel intrapituitary regulation of adrenocorticotropin hormone synthesis and secretion. J Clin Invest 95:1288–1298tyhp, 百拇医药
Auernhammer CJ, Melmed S 1999 Interleukin-11 stimulates proopiomelanocortin gene expression and adrenocorticotropin secretion in corticotroph cells: evidence for a redundant cytokine network in the hypothalamo-pituitary-adrenal axis. Endocrinology 140:1559–1566
Perez Castro C, Carbia Nagashima A, Páez-Pereda M, Goldberg V, Chervin A, Largen P, Renner U, Stalla GK, Arzt E 2000 The gp130 cytokines interleukin-11 and ciliary neurotropic factor regulate through specific receptors the function and growth of lactosomatotropic and folliculostellate pituitary cell lines. Endocrinology 141:1746–1753n^t, 百拇医药
Arzt E 2001 Gp130 cytokine signaling in the pituitary gland: a paradigm for cytokine-neuro-endocrine pathways. J Clin Invest 108:1729–1733n^t, 百拇医药
Busquet C, Zatelli MC, Melmed S 2000 Direct regulation of pituitary proopiomelanocortin by STAT3 provides a novel mechanism for immuno-neuroendocrine interfacing. J Clin Invest 106:1417–1425n^t, 百拇医药
Auernhammer CJ, Melmed S 2001 The central role of SOCS-3 in integrating the neuro-immunoendocrine interface. J Clin Invest 108:1735–1740n^t, 百拇医药
Spangelo BL, Judd AM, Isakson PC, MacLeod RM 1989 Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 127:575–577n^t, 百拇医药
Spangelo BL, Gorospe WC 1995 Role of the cytokines in the neuroendocrine-immune system axis. Front Neuroendocrinol 16:1–12
Ray D, Melmed S 1997 Pituitary cytokine and growth factor expression and action. Endocr Rev 18:206–228p0[@o|@, 百拇医药
Arzt E, Páez-Pereda M, Perez Castro C, Pagotto U, Renner U, Stalla GK 1999 Pathophysiological role of the cytokine network in the anterior pituitary gland. Front Neuroendocrinol 20:71–95p0[@o|@, 百拇医药
Auernhammer CJ, Melmed S 2000 Leukemia-inhibitory factor-neuroimmune modulator of endocrine function. Endocr Rev 21:313–345p0[@o|@, 百拇医药
Páez-Pereda M, Lohrer P, Kovalovsky D, Perez Castro C, Goldberg V, Losa M, Chervín A, Berner S, Molina H, Stalla GK, Arzt E 2000 Interleukin-6 is inhibited by glucocorticoids and stimulates ACTH secretion and POMC expression in human corticotroph pituitary adenomas. Exp Clin Endocr Diabetes 108:202–207p0[@o|@, 百拇医药
Perez Castro C, Nagashima AC, Páez-Pereda M, Goldberg V, Chervín A, Carrizo G, Molina H, Renner U, Stalla Gk, Arzt E 2001 Effects of the gp130 cytokines CNTF and IL-11 on pituitary cells: CNTF receptors on human pituitary adenomas and stimulation of PRL and GH secretion in normal rat anterior pituitary aggregate cultures. J Endocrinol 169:539–547
Arzt E, Buric R, Stelzer G, Stalla J, Sauer J, Renner U, Stalla GK 1993 Interleukin involvement in anterior pituitary cell growth regulation: effects of interleukin-2 (IL-2) and IL-6. Endocrinology 132:459–467yyjj\0f, 百拇医药
Renner U, Gloddek J, Arzt E, Inoue K, Stalla GK 1997 Interleukin-6 is an autocrine growth factor for TtT/GF mouse pituitary cells. Exp Clin Endocrinol Diabetes 105:345–352yyjj\0f, 百拇医药
Sawada T, Koike K, Kanda Y, Ikegami H, Jikihara T, Maeda T, Osako Y, Hirota K, Miyake A 1995 Interleukin-6 stimulates cell proliferation of rat anterior pituitary clonal cell lines in vitro. J Endocrinol Invest 18:83–90yyjj\0f, 百拇医药
Koyama C, Matsumoto H, Sakai T, Wakabayashi K, Ito A, Couch EF, Inoue K 1995 Pituitary folliculo-stellate-like cells stimulate somatotropic pituitary tumor growth in nude mice. Endocr Pathol 6:67–75yyjj\0f, 百拇医药
Páez-Pereda M, Goldberg V, Chervín A, Carrizo G, Molina A, Andrada J, Sauer J, Renner U, Stalla GK, Arzt E 1996 Interleukin-2 (IL-2) and IL-6 regulate c-fos protooncogene expression in human pituitary adenoma explants. Mol Cell Endocrinol 124:33–42
Saito M, Yoshida K, Hibi M, Taga T, Kishimoto T 1992 Molecular cloning of a murine IL-6 receptor-associated signal transducer, gp130, and its regulated expression in vivo. J Immunol 148:4066–4071ez|zwp, 百拇医药
Wen Z, Zhong Z, Darnell JJ 1995 Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241–250ez|zwp, 百拇医药
Williams C, Baer M, Dillner A, Johnson P 1995 CRP2 (c/EBPß) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J 14:3170–3183ez|zwp, 百拇医药
Farrow KN, Manning N, Schaufele F, Gutierrez-Hartmann A 1996 The c-Jun {gamma} -domain inhibits neuroendocrine promoter activity in a DNA sequence and pituitary-specific manner. J Biol Chem 271:17139–17146ez|zwp, 百拇医药
Tanuma N, Nakamura K, Shima H, Kikuchi K 2000 Protein-tyrosine phosphatase PTP{epsilon} C inhibits Jak-STAT signaling and differentiation induced by interleukin-6 and leukemia inhibitory factor in M1 leukemia cells. J Biol Chem 275:28216–28221
Arzt E, Stelzer G, Renner U, Lange M, Müller OA, Stalla GK 1992 Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures. J Clin Invest 90:1944–1951\2, http://www.100md.com
Starr R, Willson TA, Viney EM, Murray LJL, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ 1997 A family of cytokine-inducible inhibitors of signaling. Nature 387:917–921\2, http://www.100md.com
Arzt E, Sauer J, Pollmacher T, Labeur M, Holsboer F, Reul JMHM, Stalla GK 1994 Glucocorticoids suppress interleukin-1 receptor antagonist synthesis following induction by endotoxin. Endocrinology 134:672–677\2, http://www.100md.com
Páez-Pereda M, Kovalovsky D, Hopfner U, Theodoropoulou M, Pagotto U, Uhl E, Losa M, Stalla J, Grubler Y, Missale C, Arzt E, Stalla G 2001 Retinoic acid prevents experimental Cushing syndrome. J Clin Invest 108:1123–1131\2, http://www.100md.com
Asa SL, Ezzat S 1998 The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 19:798–827\2, http://www.100md.com
Melmed S 2001 Acromegaly and cancer: not a problem? J Clin Endocrinol Metab 86:2929–2934
Pei L, Melmed S 1997 Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Mol Endocrinol 11:433–441#$3, http://www.100md.com
Horiguchi A, Oya M, Marumo K, Murai M 2002 STAT3, but not ERKs, mediates the IL-6-induced proliferation of renal cancer cells, ACHN and 769P. Kidney Int 61:926–938#$3, http://www.100md.com
Liu XH, Kirschenbaum A, Lu M, Yao S, Klausner A, Preston C, Holland JF, Levine AC 2002 Prostaglandin E(2) stimulates prostatic intraepithelial neoplasia cell growth through activation of the interleukin-6/GP130/STAT-3 signaling pathway. Biochem Biophys Res Commun 290:249–255#$3, http://www.100md.com
Campbell CL, Jiang Z, Savarese DM, Savarese TM 2001 Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma. Am J Pathol 158:25–32#$3, http://www.100md.com
Burger R, Guenther A, Bakker F, Schmalzing M, Bernand S, Baum W, Duerr B, Hocke GM, Steininger H, Gebhart E, Gramatzki M 2001 Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma. Hematol J 2:42–53
Gloddek J, Pagotto U, Páez-Pereda M, Arzt E, Stalla GK, Renner U 1999 Pituitary adenylate cyclase-activating polypeptide, interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate cells. J Endocrinol 160:483–490m'e:[l, 百拇医药
Leung CKH, Shiu PC 1981 Required presence of both estrogen and pituitary factors for the growth of human breast cancer cells in athymic nude mice. Cancer Res 41:546–551m'e:[l, 百拇医药
Yano T, Uozumi T, Kawamoto K, Mukada K, Onda J, Ito A, Fujimoto N 1991 Photodynamic therapy for rat pituitary tumor in vitro and in vivo using pheophorbide-A and white light. Lasers Surg Med 11:174–182m'e:[l, 百拇医药
Davalli AM, Galbiati F, Bertuzzi F, Polastri L, Pontiroli AE, Perego L, Freschi M, Pozza G, Folli F, Meoni C 2000 Insulin-secreting pituitary GH3 cells: a potential ß-cell surrogate for diabetes cell therapy. Cell Transplant 9:841–851m'e:[l, 百拇医药
Nakashima K, Taga T 1998 Gp130 and IL-6 family of cytokines: signaling mechanisms and thrombopoietic activities. Semin Hematol 35:210–221m'e:[l, 百拇医药
Taga T, Narazaki M, Yasukawa K, Saito T, Daisuke M, Hamaguchi M, Davis S, Shoyab M, Yancopoulos GD, Kishimoto T 1992 Functional inhibition of hematopoietic and neurotrophic cytokines by blocking the interleukin 6 signal transducer gp130. Proc Natl Acad Sci USA 89:10998–11001m'e:[l, 百拇医药
Tone E, Kunisawa K, Kumanogoh A, Negoro S, Funamoto M, Osugi T, Kishimoto T, Yamauchi-Takihara K 2000 Gp130-dependent signaling pathway is not enhanced in gp130 transgenic heart after LIF stimulation. Cytokine 12:1512–1518(Carolina Perez Castro Damiana Giacomini Alberto Carbia Nagashima Chiara Onofri Mariana Graciarena Ke)
Two of the most potent cytokines that regulate anterior pituitary cell function are leukemia inhibitory factor and IL-6. These and others like IL-11 and ciliary neurotrophic factor are referred to as the gp130 cytokines because they share the gp130 glycoprotein as a common receptor initial signal transducer. We and others have shown that gp130 cytokines and their receptors are expressed and functional in normal and tumoral anterior pituitary cells. To study the role of gp130 cytokines in tumorigenic process, we generated gp130 cDNA gp130 sense and gp130 antisense (gp130-AS) transfected stable clones derived from lactosomatotroph GH3 cells. We examined hormone secretion and cell proliferation of these clones as well as their tumorigenic properties in athymic nude mice. Although gp130-AS clones, which have low gp130 levels and impaired signal transducer and activator of transcription 3 activity and suppressor of cytokine signaling-3 expression, showed reduced proliferation and hormone secretion (GH and prolactin) in response to gp130 cytokines, they had a normal response to gp130-independent stimuli. Moreover, gp130-AS clones showed a severely impaired in vivo tumor development. In contrast, the overexpressing gp130 clones (gp130 sense) showed no differences, compared with cells transfected with control vector. Thus, the present study provides new evidence supporting a link between gp130 and pituitary abnormal growth.
Introductionm6#3i, 百拇医药
FOR IL-6 AND OTHER cytokines that belong to the IL-6 cytokine family, their receptor cytoplasmic domains are not required for cytokine-mediated signal transduction, which is mediated by another membrane glycoprotein-denominated gp130 (1, 2). The gp130 glycoprotein alone does not bind IL-6 or other cytokine of its family, and the binding of these cytokines (i.e. IL-6) triggers the association of their {alpha} -subunits with gp130 (i.e. IL-6R{alpha} chain with gp130). All those cytokines that make use of the gp130 chain belong to the gp130 cytokine family, which is composed by IL-6, leukemia inhibitory factor (LIF), IL-11, oncostatin M, ciliary neurotrophic factor (CNTF), cardiotropin-1, cardiotrophin-like related cytokine, and stimulating neurotrophin-1/B cell-stimulating factor-3 cell (3). Gp130 mainly activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and also induces a family of cytokine-inducible signaling inhibitors including among others suppressor of cytokine signaling-3 (SOCS-3; Ref. 4).
Considering that gp130 is ubiquitously expressed, the responsiveness of a given cell type to gp130 cytokines is therefore mainly determined by expression of the respective specific {alpha} -subunits and the cytokines themselves. This takes place in the pituitary gland in which specific receptors for IL-6 (5), LIF (6), IL-11 (7, 8), and CNTF (8) have been described, providing the basis for the regulatory role of these cytokines in both hormone secretion and cell growth (9). In addition, the JAK/STAT/SOCS-3 pathway has also been described in pituitary cells (10, 11).hm:#, 百拇医药
It has been demonstrated that these cytokines regulate hormone secretion in pituitary cell lines, normal anterior pituitary cells, and human tumor pituitary cells. For example, IL-6 stimulates the release of prolactin (PRL), GH, and LH (12, 13). IL-6 and LIF are also powerful stimuli for ACTH secretion by corticotrophs in vitro and in vivo in different species (13, 14, 15, 16). Induced expression of proopiomelanocortin mRNA by IL-6 (17) as well as the enhanced ACTH secretion and proopiomelanocortin expression by IL-11 was reported (7). Similarly, IL-11 and CNTF regulate the secretion of GH and PRL in lactosomatotrophic cells (GH3 cell line) (8), human pituitary adenomas (18), and vascular endothelial growth factor (VEGF) release in folliculostellate (FS) cells (TtT/GF cell line) (8). The role of gp130 has not, however, been studied so far.
Besides the effects of gp130 cytokines on the secretion of pituitary hormones, gp130 cytokines also have influence on pituitary cell growth. Thus, we previously demonstrated that IL-6 regulates pituitary cell growth (19). Intriguingly, IL-6 stimulates DNA synthesis and cell number of tumor GH3 cells, yet at the same cytokine concentrations, it inhibits the growth of normal rat pituitary cells (19). IL-6 has been also reported to stimulate the growth of TtT/GF cells (20) as well as the proliferation of the MtT/E rat tumor pituitary cell line (21). Accordingly, TtT/GF cells, which also express IL-6, has been shown to stimulate the somatotrophic tumor cells (MtT/S) pituitary tumor growth in nude mice (22). In different tumors (ACTH-, PRL-, GH-secreting and nonfunctioning adenomas), IL-6 has been shown to have either inhibitory or stimulatory effects. However, the type of response does not seem to be associated with tumor type or size. By using blocking antibodies in a functional assay to study intrapituitary action, it has been demonstrated that endogenous IL-6 regulates the expression of c-fos in a similar manner (23). CNTF and IL-11 stimulate the proliferation of FS and GH3 cells (8). The fact that, in addition, these cytokines (IL-6, LIF, IL-11, and CNTF) are expressed in normal and tumoral pituitary cells (9, 13, 14, 15, 16) suggests that they have important paracrine/autocrine actions in the pituitary gland, which could be mediated by gp130.
Based on this evidence, we generated stable GH3 clones expressing different gp130 levels to study the role of the cytokine common transducer gp130 glycoprotein in the regulation of lactosomatotrophic function and growth.68jvp, 百拇医药
Materials and Methods68jvp, 百拇医药
Cell culture and stimulation68jvp, 百拇医药
Materials and reagents, except where stated, were obtained from Seromed (Berlin, Germany), Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Frederick, MD), Sigma, (St. Louis, MO), and Nunc (Wiesbaden, Germany). Recombinant rat CNTF (R&D Systems Inc., Minneapolis, MN) and TRH (Bachem, Bubendorf, Switzerland) were used.68jvp, 百拇医药
GH3, a rat lactosomatotrophic pituitary tumor cell line obtained from the American Type Culture Collection (Manassas, VA) (19), and GH3 gp130 clones, generated as indicated below, were used. They were cultured in DMEM (pH 7.3) supplemented with 10% fetal calf serum (FCS), 2.2 g/liter NaHCO3, 10 mM HEPES, 2 mM glutamine, 2.5 mg/liter Amphotericin B, 105 U/liter penicillin-streptomycin, 5 mg/liter insulin, 5 mg/liter transferrin, 20 mg/liter sodium selenite, and 30 pM T3 until they were confluent. The stable clones were maintained in selection medium with the antibiotic G418 (600 mg/ml). For proliferation and hormone secretion experiments, the cells were washed twice with PBS, and then medium was replaced by an experimental medium consisting of the same supplemented DMEM without FCS. Before and after the stimulation period, cell viability was routinely controlled to ensure that this parameter did not change during the experiment. Cell viability was determined microscopically after ethidium bromide/acridine orange staining.
Constructs1oh4^, 百拇医药
The murine gp130 expression vector (pBMG gp130) was generously provided by Dr. Matsumoto Hibi (24). The cDNA full-length (3-kb XhoI/XhoI fragment) was subcloned into pcDNA3 (Invitrogen) (pcDNA3/gp130 sense). The antisense expression vector was generated by subcloning of 0.9-kb XhoI/BamHI fragment into pcDNA3 expression vector in reverse orientation (pcDNA3/gp130 antisense). cDNA insertions were verified by sequencing.1oh4^, 百拇医药
To measure STAT3 activity, the p4xm 67 TATA-tk-LUC was used, provided generously by Dr. J. E. Darnell, Jr. (25). (DEI) 4-alb-LUC construct, used to evaluate NF-IL-6 activity was kindly provided by Dr. P. Johnson and consists of CCAAT/enhancer-binding protein (C/EBP) responsive DEI sites and an albumin minimal promoter coupled to the luciferase (LUC) gene (26). The LUC reporter gene under the control of 2.5 kb of the PRL promoter (PRL-LUC) construct was provided by Dr. A. Gutierrez-Hartmann (27).1oh4^, 百拇医药
GH3 gp130 stable clones
To obtain stable clones with over- or downexpressing gp130 protein, pcDNA3/gp130 sense, pcDNA3/gp130 antisense, or pcDNA3-control vector were used to transfect GH3 cells by Lipofectamine (Invitrogen). After 48 h of incubation, cells were subcultured and maintained in selection medium containing G418. The pcDNA3/gp130 sense, pcDNA3/gp130 antisense, and pcDNA3 stably transfected clones were denominated gp130-sense (gp130-S), gp130-antisense (gp130-AS), and GH3 control, respectively. The levels of gp130 in these clones were tested by Western blot. Southern blot analysis were performed and confirmed the presence of integrated transgenes in all selected clones, which are independent (i.e. the different clones carry gp130 cDNA in different regions of their genome). Results from the same representative clone for each construct are shown in all experiments. Similar results were observed with the different individual clones for each construct as indicated for each experiment.{$v$ft, 百拇医药
Western blot analysis
Cells were washed once with PBS (pH 7.0), and then cell lysates were prepared in standard cracking buffer and boiled for 3 min. Equal levels of protein (30 µg) were analyzed by 8% SDS-PAGE. Proteins were blotted onto nitrocellulose Western blotting membranes (Sigma) using standard procedures. A specific anti-gp130 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) previously used by others (28) was used. The antibodies were detected with alkaline phosphatase-conjugated antirabbit IgG (Santa Cruz Biotechnology, Inc.) followed by 5-bromo-4-chloro-3-indoly phosphate p-Toluidine and nitroblue tetrazolium chloride detection (Invitrogen)..)evn4, 百拇医药
Northern blot analysis.)evn4, 百拇医药
Total RNA extraction and Northern blot analysis were performed as described previously (29). Reagents were from Sigma, Roche Molecular Biochemicals (Mannheim, Germany), or Pharmacia (Uppsala, Sweden). Briefly, total RNA, isolated by the guanidine isothiocyanate phenol-chloroform extraction method, was denatured with glyoxal, electrophoresed on a 1.2% agarose gel, and transferred overnight to a nylon membrane. Filters were baked for 2 h at 80 C and stained with methylene blue. They were prehybridized for 1 h at 60 C (50% formamide, 5x sodium chloride/sodium phosphate/EDTA, 5x Denhardt’s solution, 0.1% sodium dodecyl sulfate, 100 µg/ml denatured salmon sperm DNA), and then the probe was added and hybridized for 12 h. Blots were washed at increasing salt and temperature stringency with a final wash of 30 min at 60 C in 0.1x saline sodium citrate buffer containing 0.1% sodium dodecyl sulfate. After washing, blots were exposed to XAR5 film (Kodak, Rochester, NY) at -70 C with intensifying screens for 6 d. A 0.5-kb SOCS-3 cDNA fragment (30) and a 1-kb PstI fragment of ß-actin cDNA (31) were labeled with a random-priming kit using {alpha} -32P-dCTP (specific activity, 2–4 x 108 cpm/µg). The blots were reprobed after stripping the first probe with 5 mM Tris/HCl (pH 8.0), 2 mM EDTA, and 0.1x Denhardt’s solution at 65 C for 2 h. After the previous signal was removed and confirmed by reexposure of the filter, the blots were prehybridized and hybridized following the methods described above. The loading control with the fragment of ß-actin cDNA as probe was performed in each blot. The mRNA levels were quantified by using phosphor imager analysis (Fuji Photo Film Co. Ltd., Aichi, Japan).
Cell proliferation[4kw, 百拇医药
Cell proliferation was measured with the WST-1 reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions, and the reaction product was measured in an ELISA plate reader at 450 nm (8, 32). Results were validated by total cell count. Acridine orange-ethidium bromide staining was used to rule out toxic effects.[4kw, 百拇医药
Hormone determination[4kw, 百拇医药
Hormones were measured by RIA as previously described (8, 19). For recombinant GH and PRL, reagents were kindly provided by Dr. A. F. Parlow from the National Hormone and Pituitary Program (Torrance, CA).[4kw, 百拇医药
Transfection of GH3 cells and reporter assays[4kw, 百拇医药
Cell transfection was performed with Lipofectamine using standard procedures, as previously described (32). After plating the cells in 6-well plates, the cells were transfected in DMEM without FCS for 6 h using 5 µl lipofectamine (Invitrogen) per well with 1.5 µg for each plasmid, in conjunction with a control Rous sarcoma virus (RSV)-ßgal construct (0.5 µg). Cells were then washed and left 20 h in DMEM supplemented with 10% FCS. Then cells were washed and stimulated for 6 h with CNTF (2 nM) in DMEM supplemented with 2% FCS. After treatment, LUC activity in cell lysates was measured as previously described (32) using the Luciferase measure kit (Promega Corp., Madison, WI) with a Junior luminometer (Berlthod, Bad Wildbad, Germany). The ß-gal activity was measured as previously described (32) as control for transfection efficiency. Results were standardized for ß-gal activity.
In vivo experiments in nude mice^d#@9|8, 百拇医药
GH3 cells were collected, washed twice with PBS, resuspended in DMEM, and injected (1 x 106 cells) into the flanks of adult (20 g) female N:NIH nude mice. The mice were divided into three groups: injected with GH3 control cells, injected with gp130-S cells, and injected with gp130-AS cells. Animals were examined for tumor formation every 2 d, and tumor growth was determined as described (32). The experiments were finished at 26 d after cell injection because of GH3 control mice morbidity.^d#@9|8, 百拇医药
All experimental protocols were approved by the Ethical Committee on Animal Care and Use, University of Buenos Aires, Argentina.^d#@9|8, 百拇医药
Immunohistochemistry for CD31 and determination of vascular density^d#@9|8, 百拇医药
For immunohistochemistry, 8-µm sections of shock-frozen GH3 tumor tissue were thaw mounted onto SuperFrost Plus slides (Menzel-Glaser, Hamburg, Germany), fixed in 4% paraformaldehyde in PBS, and stored in 96% ethanol at 4 C until use.
An antimouse CD31 (PECAM-1) antibody (Santa Cruz Biotechnology, Inc.) was used to detect intratumoral microvessels. Slides were first incubated in horse serum diluted 1:10 in Tris-based buffer (pH 7.6). Subsequently the slides were incubated overnight at 4 C with CD31 primary antibody diluted 1:500. After three washes in Tris-based buffer, the biotinylated horse antigoat secondary antibody (dilution: 1:300) (Vector Laboratories Inc., Burlingame, CA) was added for 30 min at room temperature. The slides were then washed again and were incubated for 30 min with the avidin-biotin-peroxidase complex (Vector Laboratories Inc.). Color development was performed using 1 mg/ml diaminobenzidine (Sigma) with 0.01% hydrogen peroxide applied for 45 sec. After washing in deionized water, the sections were counterstained with toluidine blue, fixed in Roti-Histol (Carl Roth, Karlsruhe, Germany) and coverslipped using Roti-Histokitt (Carl Roth). Negative controls were performed by omitting the primary antibody. No staining for CD31 has been detected in negative control cells.
For determination of vascular density, an investigator who was not informed about the type of tumor counted vessels inside an area delimited by an eyepiece graticule 12.5 x 12.5 mm divided in 10 x 10 squares (Zeiss, Munich, Germany) at a magnification x200 (x20 objective and x10 ocular). Vessel density was determined in six different tumors of each group (gp130-AS; gp130-S; GH3 control-derived tumors), and in each tumor both single cells and cell clusters positive for CD31 were counted in three different areas. Results of vessel counting are expressed as mean ± SEM of 18 areas counted.%v, 百拇医药
Statistics%v, 百拇医药
Statistics were performed by ANOVA in combination with the Scheffé’s test. Data are shown as mean ± SEM.%v, 百拇医药
Results%v, 百拇医药
Generation and characterization of gp130 stable cell lines%v, 百拇医药
To generate lactosomatotrophic GH3 stable clones that over- or downexpress the gp130 glycoprotein, GH3 cells were transfected with vectors containing gp130 cDNA in sense or antisense orientations. As shown in Fig. 1A, gp130 glycoproteins were expressed at higher levels in gp130-S clones, whereas they were downexpressed in gp130-AS clones, compared with GH3 cells stably transfected with control vector.
fig.ommitteedh^r#5]y, 百拇医药
Figure 1. Characterization of gp130-S and gp130-AS stable clones. A, Western blot analysis of GH3 lactosomatotrophic stable clones showing overexpressed or downexpressed gp130 protein levels. The bands corresponding to gp130 protein were detected by an anti-gp130 polyclonal antibody and are indicated by an arrow. GH3 control, GH3 cells transfected with pcDNA3/control vector; gp130-S, GH3 cells transfected with pcDNA3/gp130 sense vector; gp130-AS, GH3 cells transfected with pcDNA3/gp130 antisense vector. Molecular weight marker is indicated on the right. Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown. B, STAT-3-mediated transcriptional activation in response to CNTF in gp130-S and gp130-AS stable clones. GH3 stable lines were transiently transfected with plasmid DNA mixture containing 1.5 µg luciferase reporter construct [(4x) IRF-1] with four copies of the STAT-3-binding sequence from the interferon regulatory factor 1 (IRF-1) and 0.5 µg pRSV-Lac Z. At 20 h after transfection, cells were treated with CNTF (2 nM) (bars) for 6 h. The values corresponding to luciferase were normalized with respect to ß-galactosidase activity. Results are expressed as mean ± SEM (n = 4) of one representative of three independent experiments with similar results. By ANOVA with Scheffé’s test: *, P < 0.05 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
To test the signaling pathway downstream of gp130 in the stably transfected GH3 clones, they were transiently transfected with the STAT3-LUC construct. As shown in Fig. 1B, CNTF (2 nM) induced STAT3-transcriptional activity with respect to the basal condition in the GH3 control cells, which was slightly enhanced in the gp130-S clones. However, in the case of gp130-AS clones, LUC activity was efficiently blocked (Fig. 1B). Similar results were obtained with a C/EBP-LUC reporter construct (data not shown).*;%hr, 百拇医药
In addition, SOCS-3 mRNA was also stimulated in GH3 control and gp130-S clones in response to CNTF, whereas it was blunted in gp130-AS clones (Fig. 2).*;%hr, 百拇医药
fig.ommitteed*;%hr, 百拇医药
Figure 2. Effect of CNTF on SOCS-3 mRNA expression in GH3 control, gp130-S, and gp130-AS clones. Northern blot analysis is shown using 30 µg RNA per lane as stated in Materials and Methods. A, Bands corresponding to SOCS-3 mRNA (3.2 kb) or ß-actin from one representative experiment are shown. The cell lines were seeded at 6 x 105 cells/well and treated with CNTF (2 nM). Densitometric units of the bands relative to constitutive ß-actin expression are shown. B, Mean densitometric analysis of SOCS-3 mRNA content from three independent experiments including the one shown in A. Data are expressed as a ratio ± SEM of autoradiographic signals of SOCS-3 mRNA relative to ß-actin mRNA used as loading control. Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
Cell proliferation and hormone secretion inhibition in gp130-AS clones[5f/, http://www.100md.com
No differences in basal proliferation were observed among different clones (Fig. 3). CNTF significantly stimulated the growth of GH3 control cells, at the same doses previously reported (8), and a similar response was found in the gp130-S clones. In contrast, as shown in Fig. 3, the capacity of the gp130-AS clones to respond to the gp130 specific stimuli was abolished, whereas they were able to respond to a gp130-independent stimulus, TRH. Similar results were obtained in the hormone secretion experiments: Both CNTF and TRH significantly stimulated GH secretion from GH3 control and gp130-S cells, whereas the gp130-AS clones had a normal response only to TRH (Fig. 4).[5f/, http://www.100md.com
fig.ommitteed[5f/, http://www.100md.com
Figure 3. Proliferation of GH3 control, gp130-S, gp130-AS clones in response to CNTF and TRH. GH3 gp130 cell lines were seeded at 9000 cells/well in multiwell plates with 10% FCS. After attachment, cells were washed twice with PBS and incubated in FCS-free medium for 24 h. Cells were treated either with CNTF (2 nM) or TRH (100 nM) for 72 h. Proliferation was measured by the WST-1 colorimetric method as detailed in Materials and Methods. Values represent the mean ± SEM of quadruplicate determinations of a single representative experiment (total of five with similar results). By ANOVA with Scheffé’s test: *, P < 0.05; **, P < 0.01 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
fig.ommitteedqtk, 百拇医药
Figure 4. Effect of recombinant rat CNTF and TRH on GH secretion in GH3 control, gp130-S, and gp130-AS clones. Clones were treated with CNTF (2 nM) or TRH (100 nM) as indicated. GH3 cell lines were seeded at 2 x 105 cells in 24-well plates. After FCS depletion for 24 h, treatments were added with fresh FCS-depleted medium. After 24 h, the supernatants were collected, and GH was measured by RIA. Values represent the mean ± SEM of quadruplicate determinations of a single representative experiment (total of three with similar results). By ANOVA with Scheffé’s test: *, P < 0.05; **, P < 0.01 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.qtk, 百拇医药
However, gp130-AS clones showed a very low level of basal PRL secretion. Interestingly, the basal activity of the PRL promoter was blunted in the gp130-AS clones, showing they have a constitutive lower basal PRL-transcriptional activity (Fig. 5). No difference between GH3 control and gp130-S cells was observed in terms of hormone secretion (GH and PRL) and cell proliferation stimulated by either gp130 cytokine or TRH (Figs. 3–5).
fig.ommitteed$jl)in$, 百拇医药
Figure 5. PRL promoter activity and PRL secretion in GH3 control, gp130-S, and gp130-AS clones. A, GH3 stable lines were transiently transfected with plasmid DNA mixture containing 1.5 µg luciferase reporter construct of PRL-LUC and 0.5 µg pRSV-Lac Z. The values corresponding to luciferase were normalized with respect to ß-galactosidase activity, and results are expressed as mean ± SEM (n = 3) of one representative experiment of three independent experiments with similar results. B, Clones were treated with CNTF (2 nM) or TRH (100 nM) as indicated. GH3 cell lines were seeded at 2 x 105 cells in 24-well plates. After FCS depletion for 24 h, treatments were added with fresh FCS-depleted medium. After 24 h, the supernatants were collected and PRL was measured by RIA. Results are expressed as mean ± SEM (n = 4) of one representative of three independent experiments. By ANOVA with Scheffé’s test: *, P < 0.05; **, P < 0.01 (vs. corresponding basal). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown.
In vivo tumor formation capacity is inhibited in gp130-AS clones00w^, 百拇医药
Based on the findings that GH3 gp130-AS cells had an impaired response to gp130 cytokine stimulation, we hypothesized that the capacity of gp130-AS cells to develop tumors would be inhibited. Therefore, we evaluated the tumor formation by injecting GH3 stables clones into nude mice. Within 14 d after injection, visible tumors were developed in mice injected either with GH3 control or GH3 gp130-S cells. These tumors grew very rapidly and reached considerable sizes at 26 d after injection (Fig. 6). On the contrary, in mice injected with gp130-AS cells, tumor formation was delayed until 23 d after injection, and they developed tumors of very small sizes. Thus, the growth rate and the tumor size were greatly reduced in comparison with both GH3 control and GH3 gp130-S groups (Fig. 6).00w^, 百拇医药
fig.ommitteed00w^, 百拇医药
Figure 6. GH3 cell tumor development is abolished in gp130-AS clones. A, Cells (1 x 106) were injected sc into athymic nude mice (n = 4, each group with the same clone) as described in Materials and Methods. Photographs were taken at d 26 after injection. B, The development of tumors in GH3 control and gp130-S-injected animals started to be apparent at d 14 after injection, whereas group gp130-AS showed delayed tumor formation (visible at d 23). In some cases, symbols corresponding to individual animals overlap. There were no significant differences between d 26 and d 36 in the animals injected with gp130-AS cells (data not shown). Results from one representative clone (same in all figures) from several gp130-S (n = 2) and gp130-AS (n = 3) stable clones with similar results are shown. The clones preserved the expression of gp130-S or gp130-AS after in vivo injection.
The counting of cells and cell clusters positive for the endothelial cell marker CD31 showed that the vessel density was significantly reduced in gp130-AS-derived tumors. Whereas the vessel density was similar in GH3 control-derived tumors (37.1 ± 5.4 vessels per area) and gp130-S derived-tumors (43.1 ± 6.4 vessels per area), only 12.9 ± 4.5 vessels per area could be detected in gp130-AS derived-tumors (Fig. 7).r'g)gt, 百拇医药
fig.ommitteedr'g)gt, 百拇医药
Figure 7. Density of microvessels in gp130-AS-derived tumors (A) in gp130-S-derived-tumors (B) and GH3 control-derived-tumors (C). Immunostaining for the endothelial cell marker CD31 (brown) is shown. The vessel density was significantly reduced (P < 0.001) by ANOVA with Scheffé’s test in gp130-AS-derived-tumors (with respect to the gp130-S-derived-tumors and GH3 control-derived-tumors). Corresponding negative controls are inserted at the right corner of each picture.r'g)gt, 百拇医药
Discussionr'g)gt, 百拇医药
Pathogenesis of pituitary tumors has been extensively studied to identify those activating oncogene mutations and/or inactivating tumor suppressor genes involved in tumorigenesis (33, 34). For example, G protein (Gs{alpha} ) mutations have been shown to occur only in a subset of sporadic GH-secreting pituitary adenomas, which represent a small percentage of all pituitary tumors (33, 34), suggesting that there may be other events responsible for pituitary tumorigenesis. A novel pituitary tumor-transforming gene involved in the generation of pituitary adenomas has been cloned from rat GH4 pituitary tumors (35). However, despite these discoveries, it is still an open question how pituitary tumors are formed during pathogenesis.
Several studies have postulated the possibility that gp130 cytokines, through the gp130 glycoprotein-mediated signal transduction cascades, could play an important role in the tumorigenic process in different tissues (36, 37, 38, 39). In agreement with this, IL-6 and IL-11 have been reported to stimulate growth of pituitary cells (8, 19, 20, 21) and release of angiogenic factors, such as VEGF by FS cells and TtT/GF cells (8, 40). Thus, the importance of the gp130 cytokines in function and growth of pituitary cells could indicate that some genetic events that occur during pituitary pathogenesis would be mediated by signaling response originated from gp130 cytokines. Therefore, the gp130 cytokines should be considered as feasible candidates to generate a favorable ambient for cell transformation events in the pituitary gland. In fact, in the present study, we demonstrated that reducing gp130 level inhibits tumor development of GH3 cells in vivo.\v*, 百拇医药
Koyama et al. (22) have shown that the MtT/S cell line needs the presence of TtT/GF to induce formation of tumor in nude mice. Although it is still uncertain how TtT/GF cells regulate MtT/S’s cell behavior, it is likely that the microenvironment determined by cellular interactions and extracellular matrix modifications would contribute to support their tumor development. In contrast, GH3 cells have innate capacity to develop tumors in nude mice (41, 42, 43). They respond to angiogenic and growth factors and do not need the presence of other pituitary cells (i.e. TtT/GF cells). Hence, GH3 cells represent a good model to study the influence of gp130 protein expression levels during tumor formation. Our results show that GH3 gp130-AS cells have lower gp130 signal cascade activation, showing reduced STAT-3 and C/EBP-activity and also SOCS-3 mRNA expression levels. It is important to notice that GH3 gp130-AS cells are still able to respond to gp130-independent stimuli, such as TRH, which indicates that the lower amount of gp130 protein is indeed specific and critical for gp130 cytokine actions. These clones have reduced proliferative activity. In accordance with our results, altered forms of gp130 that fail to induce activation of JAKs fail to transduce an IL-6 proliferative signal (44); in mouse B cells and an erythroleukemia-derived cell line expressing human gp130 glycoprotein, the presence of gp130 monoclonal antibodies completely abolished the biological response such as the cell growth induced by gp130 cytokines (45).
On the contrary, the overexpression of gp130 did not significantly modify the cellular behavior, which might indicate that pituitary gp130 endogenous levels fulfill for a normal functional cellular response. Similarly, in transgenic mice that overexpress gp130, the activation-signaling pathway downstream of gp130 in the heart is not enhanced (46), suggesting that gp130 overexpression does not always induce changes in the cell biology, which is in accordance with our results.j!(-}pj, 百拇医药
In the GH3 gp130-S-derived-tumors, vessel density was the highest, observing 2- to 3-fold more vessels in GH3 gp130-S-derived tumors, compared with GH3 gp130-AS- derived tumors. It is therefore clear that in GH3 gp130-AS-derived-tumors, neovascularization is impaired. However, we need to address further studies to determine whether this is a direct effect (suppression of gp130-mediated VEGF and/or basic fibroblast growth factor production because of gp130 blockade) or an indirect effect because of lower tumor cell growth associated with lack of tumor expansion and hypoxia-induced neoangiogenesis.
We show for the first time that the reduced level of gp130 protein in GH3 cells (gp130-AS cells) blocked the cell growth and hormone secretion stimulated by CNTF and have impaired tumor formation capacity in nude mice. These data contribute to support gp130 cytokines as candidates that would favor the development and/or growth of pituitary tumors.$1a[, 百拇医药
Acknowledgments$1a[, 百拇医药
We thank Dr. Matsumoto Hibi, Dr. J. E. Darnell, Jr., Dr. P. Johnson, Dr. A. Gutierrez-Hartmann, and Dr. Tracy Willson for generously providing the constructs used in this study.$1a[, 百拇医药
A.C.N. and E.A. are members of the Argentine National Research Council (Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina).$1a[, 百拇医药
Received August 26, 2002.$1a[, 百拇医药
Accepted for publication October 21, 2002.$1a[, 百拇医药
References$1a[, 百拇医药
Hibi M, Nakajima K, Hirano T 1996 IL-6 cytokine family and signal transduction: a model of the cytokine system. J Mol Med 74:1–12
Kishimoto T, Taga T, Akira S 1994 Cytokine signal transduction. Cell 76:253–262tyhp, 百拇医药
Kishimoto T, Akira S, Narazaki M, Taga T 1995 Interleukin-6 family of cytokines and gp130. Blood 86:1243–1254tyhp, 百拇医药
Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L 1998 Interleukin-6-type cytokine signaling through the gp130/Jak/STAT pathway. Biochem J 334:297–314tyhp, 百拇医药
Ohmichi M, Hirota K, Koike K, Kurachi H, Ohtsuka S, Matsuzaki N, Yamaguchi M, Miyake A, Tanizawa O 1992 Binding sites for interleukin-6 in the anterior pituitary gland. Neuroendocrinology 55:199–203tyhp, 百拇医药
Akita S, Webster J, Ren SG, Takino H, Said J, Zand O, Melmed S 1995 Human and murine pituitary expression of leukemia inhibitory factor. Novel intrapituitary regulation of adrenocorticotropin hormone synthesis and secretion. J Clin Invest 95:1288–1298tyhp, 百拇医药
Auernhammer CJ, Melmed S 1999 Interleukin-11 stimulates proopiomelanocortin gene expression and adrenocorticotropin secretion in corticotroph cells: evidence for a redundant cytokine network in the hypothalamo-pituitary-adrenal axis. Endocrinology 140:1559–1566
Perez Castro C, Carbia Nagashima A, Páez-Pereda M, Goldberg V, Chervin A, Largen P, Renner U, Stalla GK, Arzt E 2000 The gp130 cytokines interleukin-11 and ciliary neurotropic factor regulate through specific receptors the function and growth of lactosomatotropic and folliculostellate pituitary cell lines. Endocrinology 141:1746–1753n^t, 百拇医药
Arzt E 2001 Gp130 cytokine signaling in the pituitary gland: a paradigm for cytokine-neuro-endocrine pathways. J Clin Invest 108:1729–1733n^t, 百拇医药
Busquet C, Zatelli MC, Melmed S 2000 Direct regulation of pituitary proopiomelanocortin by STAT3 provides a novel mechanism for immuno-neuroendocrine interfacing. J Clin Invest 106:1417–1425n^t, 百拇医药
Auernhammer CJ, Melmed S 2001 The central role of SOCS-3 in integrating the neuro-immunoendocrine interface. J Clin Invest 108:1735–1740n^t, 百拇医药
Spangelo BL, Judd AM, Isakson PC, MacLeod RM 1989 Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 127:575–577n^t, 百拇医药
Spangelo BL, Gorospe WC 1995 Role of the cytokines in the neuroendocrine-immune system axis. Front Neuroendocrinol 16:1–12
Ray D, Melmed S 1997 Pituitary cytokine and growth factor expression and action. Endocr Rev 18:206–228p0[@o|@, 百拇医药
Arzt E, Páez-Pereda M, Perez Castro C, Pagotto U, Renner U, Stalla GK 1999 Pathophysiological role of the cytokine network in the anterior pituitary gland. Front Neuroendocrinol 20:71–95p0[@o|@, 百拇医药
Auernhammer CJ, Melmed S 2000 Leukemia-inhibitory factor-neuroimmune modulator of endocrine function. Endocr Rev 21:313–345p0[@o|@, 百拇医药
Páez-Pereda M, Lohrer P, Kovalovsky D, Perez Castro C, Goldberg V, Losa M, Chervín A, Berner S, Molina H, Stalla GK, Arzt E 2000 Interleukin-6 is inhibited by glucocorticoids and stimulates ACTH secretion and POMC expression in human corticotroph pituitary adenomas. Exp Clin Endocr Diabetes 108:202–207p0[@o|@, 百拇医药
Perez Castro C, Nagashima AC, Páez-Pereda M, Goldberg V, Chervín A, Carrizo G, Molina H, Renner U, Stalla Gk, Arzt E 2001 Effects of the gp130 cytokines CNTF and IL-11 on pituitary cells: CNTF receptors on human pituitary adenomas and stimulation of PRL and GH secretion in normal rat anterior pituitary aggregate cultures. J Endocrinol 169:539–547
Arzt E, Buric R, Stelzer G, Stalla J, Sauer J, Renner U, Stalla GK 1993 Interleukin involvement in anterior pituitary cell growth regulation: effects of interleukin-2 (IL-2) and IL-6. Endocrinology 132:459–467yyjj\0f, 百拇医药
Renner U, Gloddek J, Arzt E, Inoue K, Stalla GK 1997 Interleukin-6 is an autocrine growth factor for TtT/GF mouse pituitary cells. Exp Clin Endocrinol Diabetes 105:345–352yyjj\0f, 百拇医药
Sawada T, Koike K, Kanda Y, Ikegami H, Jikihara T, Maeda T, Osako Y, Hirota K, Miyake A 1995 Interleukin-6 stimulates cell proliferation of rat anterior pituitary clonal cell lines in vitro. J Endocrinol Invest 18:83–90yyjj\0f, 百拇医药
Koyama C, Matsumoto H, Sakai T, Wakabayashi K, Ito A, Couch EF, Inoue K 1995 Pituitary folliculo-stellate-like cells stimulate somatotropic pituitary tumor growth in nude mice. Endocr Pathol 6:67–75yyjj\0f, 百拇医药
Páez-Pereda M, Goldberg V, Chervín A, Carrizo G, Molina A, Andrada J, Sauer J, Renner U, Stalla GK, Arzt E 1996 Interleukin-2 (IL-2) and IL-6 regulate c-fos protooncogene expression in human pituitary adenoma explants. Mol Cell Endocrinol 124:33–42
Saito M, Yoshida K, Hibi M, Taga T, Kishimoto T 1992 Molecular cloning of a murine IL-6 receptor-associated signal transducer, gp130, and its regulated expression in vivo. J Immunol 148:4066–4071ez|zwp, 百拇医药
Wen Z, Zhong Z, Darnell JJ 1995 Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241–250ez|zwp, 百拇医药
Williams C, Baer M, Dillner A, Johnson P 1995 CRP2 (c/EBPß) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J 14:3170–3183ez|zwp, 百拇医药
Farrow KN, Manning N, Schaufele F, Gutierrez-Hartmann A 1996 The c-Jun {gamma} -domain inhibits neuroendocrine promoter activity in a DNA sequence and pituitary-specific manner. J Biol Chem 271:17139–17146ez|zwp, 百拇医药
Tanuma N, Nakamura K, Shima H, Kikuchi K 2000 Protein-tyrosine phosphatase PTP{epsilon} C inhibits Jak-STAT signaling and differentiation induced by interleukin-6 and leukemia inhibitory factor in M1 leukemia cells. J Biol Chem 275:28216–28221
Arzt E, Stelzer G, Renner U, Lange M, Müller OA, Stalla GK 1992 Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures. J Clin Invest 90:1944–1951\2, http://www.100md.com
Starr R, Willson TA, Viney EM, Murray LJL, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ 1997 A family of cytokine-inducible inhibitors of signaling. Nature 387:917–921\2, http://www.100md.com
Arzt E, Sauer J, Pollmacher T, Labeur M, Holsboer F, Reul JMHM, Stalla GK 1994 Glucocorticoids suppress interleukin-1 receptor antagonist synthesis following induction by endotoxin. Endocrinology 134:672–677\2, http://www.100md.com
Páez-Pereda M, Kovalovsky D, Hopfner U, Theodoropoulou M, Pagotto U, Uhl E, Losa M, Stalla J, Grubler Y, Missale C, Arzt E, Stalla G 2001 Retinoic acid prevents experimental Cushing syndrome. J Clin Invest 108:1123–1131\2, http://www.100md.com
Asa SL, Ezzat S 1998 The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 19:798–827\2, http://www.100md.com
Melmed S 2001 Acromegaly and cancer: not a problem? J Clin Endocrinol Metab 86:2929–2934
Pei L, Melmed S 1997 Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Mol Endocrinol 11:433–441#$3, http://www.100md.com
Horiguchi A, Oya M, Marumo K, Murai M 2002 STAT3, but not ERKs, mediates the IL-6-induced proliferation of renal cancer cells, ACHN and 769P. Kidney Int 61:926–938#$3, http://www.100md.com
Liu XH, Kirschenbaum A, Lu M, Yao S, Klausner A, Preston C, Holland JF, Levine AC 2002 Prostaglandin E(2) stimulates prostatic intraepithelial neoplasia cell growth through activation of the interleukin-6/GP130/STAT-3 signaling pathway. Biochem Biophys Res Commun 290:249–255#$3, http://www.100md.com
Campbell CL, Jiang Z, Savarese DM, Savarese TM 2001 Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma. Am J Pathol 158:25–32#$3, http://www.100md.com
Burger R, Guenther A, Bakker F, Schmalzing M, Bernand S, Baum W, Duerr B, Hocke GM, Steininger H, Gebhart E, Gramatzki M 2001 Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma. Hematol J 2:42–53
Gloddek J, Pagotto U, Páez-Pereda M, Arzt E, Stalla GK, Renner U 1999 Pituitary adenylate cyclase-activating polypeptide, interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate cells. J Endocrinol 160:483–490m'e:[l, 百拇医药
Leung CKH, Shiu PC 1981 Required presence of both estrogen and pituitary factors for the growth of human breast cancer cells in athymic nude mice. Cancer Res 41:546–551m'e:[l, 百拇医药
Yano T, Uozumi T, Kawamoto K, Mukada K, Onda J, Ito A, Fujimoto N 1991 Photodynamic therapy for rat pituitary tumor in vitro and in vivo using pheophorbide-A and white light. Lasers Surg Med 11:174–182m'e:[l, 百拇医药
Davalli AM, Galbiati F, Bertuzzi F, Polastri L, Pontiroli AE, Perego L, Freschi M, Pozza G, Folli F, Meoni C 2000 Insulin-secreting pituitary GH3 cells: a potential ß-cell surrogate for diabetes cell therapy. Cell Transplant 9:841–851m'e:[l, 百拇医药
Nakashima K, Taga T 1998 Gp130 and IL-6 family of cytokines: signaling mechanisms and thrombopoietic activities. Semin Hematol 35:210–221m'e:[l, 百拇医药
Taga T, Narazaki M, Yasukawa K, Saito T, Daisuke M, Hamaguchi M, Davis S, Shoyab M, Yancopoulos GD, Kishimoto T 1992 Functional inhibition of hematopoietic and neurotrophic cytokines by blocking the interleukin 6 signal transducer gp130. Proc Natl Acad Sci USA 89:10998–11001m'e:[l, 百拇医药
Tone E, Kunisawa K, Kumanogoh A, Negoro S, Funamoto M, Osugi T, Kishimoto T, Yamauchi-Takihara K 2000 Gp130-dependent signaling pathway is not enhanced in gp130 transgenic heart after LIF stimulation. Cytokine 12:1512–1518(Carolina Perez Castro Damiana Giacomini Alberto Carbia Nagashima Chiara Onofri Mariana Graciarena Ke)