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Endocrinology Vol. 141, No. 5 1746-1753
Copyright © 2000 by The Endocrine Society

ARTICLES

The gp130 Cytokines Interleukin-11 and Ciliary Neurotropic Factor Regulate through Specific Receptors the Function and Growth of Lactosomatotropic and Folliculostellate Pituitary Cell Lines1

Carolina Perez Castro, Alberto Carbia Nagashima, Marcelo Páez Pereda, Victoria Goldberg, Alberto Chervin, Penny Largen, Ulrich Renner, Günter K. Stalla and Eduardo Arzt2

Laboratorio de Fisiología y Biología Molecular, Department de Biología, FCEN, Universidad de Buenos Aires (C.P.C., A.C.N., E.A.), 1428 Buenos Aires, Argentina; Department of Endocrinology, Max Planck Institute of Psychiatry (M.P.P., P.L., U.R., G.K.S.), 80804 Munich, Germany; Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Buenos Aires (V.G), 1427 Buenos Aires, Argentina; Hospital Santa Lucía (A.C.), 1232 Buenos Aires, Argentina

Address all correspondence and requests for reprints to: Dr. E. Arzt, Laboratorio de Fisiología y Biología Molecular, Department de Biología, Facultad Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellon II, 1428 Buenos Aires, Argentina. E-mail: earzt{at}bg.fcen.uba.ar


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor and interleukin-6 (IL-6), which belong to the cytokine receptor family using the common gp130 signal transducer. We studied the actions of two other members of this family, IL-11 and ciliary neurotropic factor (CNTF), on folliculostellate (FS) cells (TtT/GF cell line) and lactosomatotropic cells (GH3 cell line). The messenger RNA (mRNA) for the {alpha}-chain specific for the IL-11 receptor (1.7 kb) and CNTF receptor (2 kb) are expressed on both cell types. In addition, we detected CNTF receptor mRNA in normal rat anterior pituitary cells. IL-11 (1.25–5 nM) dose dependently stimulated the proliferation of FS cells. CNTF, at doses from 0.4–2 nM, also significantly stimulated the growth of these cells. In addition, both cytokines significantly stimulated proliferation of lactosomatotropic GH3 cells, and CNTF stimulated hormone production (GH and PRL) at 24 h by these cells. At 16–72 h, IL-11 stimulates the secretion of the angiogenic factor vascular endothelial growth factor by FS cells. In addition, both GH3 and FS cells express CNTF mRNA. These data suggest that IL-11 and CNTF may act as growth and regulatory factors in anterior pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYTOKINES ARE NOW recognized to play an important role in modulating the neuroendocrine system, particularly with respect to hormone secretion from the pituitary gland, where they also act through autocrine/paracrine loops (1, 2). Two of the most potent cytokines acting in this way are interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), which belong to the cytokine receptor family that uses the common gp130 signal transducer. Other members of this family are IL-11, oncostatin M, ciliary neurotropic factor (CNTF), and cardiotropin-1 (3). All of these cytokines express specific receptors that use the gp130 protein as an initial cellular signal transducer without activating tyrosine kinases (4). Accordingly, these cytokines are grouped and referred to as the gp130 signal-mediated cytokines, gp130 cytokines, or IL-6 family of cytokines (5, 6).

IL-6 stimulates the release of PRL, GH, ACTH, FSH, and LH from normal rat pituitary cells (7, 8, 9). IL-6 also stimulates GH and PRL release from GH3 cells (10). One study shows that IL-6 receptors (IL-6-R) are expressed in rat anterior pituitary cells (11). The production of IL-6 protein and messenger RNA (mRNA) expression by normal rat anterior pituitary cells (12, 13, 14) and human pituitary adenomas (15, 16, 17, 18, 19) has been demonstrated by several groups. IL-6 production has been localized to folliculostellate (FS) cells of the pituitary (13, 20). The FS cell line obtained from a pituitary thyrotropic tumor, TtT/GF, releases IL-6 in response to vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) (21), and tumor necrosis factor-{alpha} (22). IL-1 is able to stimulate the release of IL-6 from cultures of pituitary adenomas and normal rat pituitaries (23, 24). IL-6 stimulates pituitary tumor cell growth and vascular endothelial growth factor (VEGF) production (10, 25). It has been shown that IL-6 expression may correlate with biological aggression in pituitary adenomas (18).

LIF-binding sites as well as LIF protein and mRNA have been demonstrated in developing human fetal pituitary and in normal and adenomatous adult human tissue (26). LIF receptor mRNA was also demonstrated in pituitary cells by RT-PCR and is induced, in vivo, by lipopolysaccharide (LPS) (27). Specific LIF-binding sites are also present in murine AtT-20 cells (26). In LIF gene knockout mice, a defect in activation of the hypothalamic-pituitary-adrenal axis was observed (28), whereas in mouse pituitary primary cell culture, LIF stimulates ACTH secretion (29). LIF protein has been shown to be secreted by bovine pituitary follicular cells in culture (30), and LIF mRNA was detected (31) in pituitary explant cultures. LIF mRNA was also induced in mice by ip injection of LPS, to a greater extent than LIF receptor mRNA (27).

Very recently, the expression and action of IL-11 on corticotropic cells have been demonstrated (32). IL-11 mRNA was detected by RT-PCR, and IL-11-R mRNA was detected by Northern blot in pituitary cells and AtT-20 cells. In these cells IL-11 stimulates ACTH secretion and POMC expression.

In the present work we describe the expression of specific receptor subunits of two different cytokines of the gp130 receptor-coupled family, IL-11 and CNTF, in somatomammotropic and FS pituitary cell lines and their actions in the regulation of hormone secretion, production of the angiogenic factor VEGF, and growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and stimulation
Materials and reagents, except where stated, were obtained from Seromed (Berlin, Germany), Life Technologies, Inc. (Karlsruhe, Ger-many), Flow (Meckenheim, Germany), Falcon (Heidelberg, Germany), and Nunc (Wiesbaden, Germany). Recombinant rat and human CNTF (R&D Systems, Inc., Minneapolis, MN) and recombinant human IL-11 (Roche Molecular Biochemicals, Mannheim, Germany) were used.

Two pituitary cell lines, GH3, a rat somatomammotropic pituitary tumor cell line, obtained from American Type Culture Collection (Manassas, VA), and TtT/GF cells, a FS cell line obtained from a mouse pituitary thyrotropic tumor (33), were used. They were cultured in DMEM (pH 7.3) supplemented with 10% FCS, 2.2 g/liter NaHCO3, 10 mM HEPES, 2 mM glutamine, 2.5 mg/liter Amphotericin, 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. For all experiments, using the two types of pituitary cultures, after washing the cells, medium was replaced by an experimental medium consisting, as indicated in each case, of the same supplemented DMEM with 1% or 2% FCS or without serum. For hormone determinations the experimental medium consisted of DMEM containing only 0.5 g/liter BSA, 2.2 g/liter NaHCO3, 30 µg/ml ascorbic acid, and 10 mM HEPES, pH 7.3. The monolayers were washed with PBS, and serum-free culture medium was added for 24 h to wash out the remaining serum. Cells were then washed again, and the stimulation was performed in serum-free culture medium. Except for the time-course studies, cells were stimulated for 24 h. 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.

Pituitary glands were obtained from adult male Sprague Dawley rats (180–250 g) after decapitation and cultured as previously described (10). Briefly, the tissue was washed with preparation buffer [137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 10 mM glucose, and 15 mM HEPES (pH 7.3)]. Sliced fragments were dispersed mechanically and enzymatically in preparation buffer containing 4 g/liter collagenase (Cooper Biochemicals, Malvern, PA), 10 mg/liter deoxyribonuclease II, 0.1 g/liter soybean trypsin inhibitor, and 1 g/liter hyaluronidase. Dispersed cells were centrifuged, resuspended, and cultured in DMEM supplemented with 2.2 g/liter NaHCO3, 10 mM HEPES, 2 mM essential vitamins, 5 mg/liter insulin, 20 mg/liter selenium, 5 mg/liter transferrin, and 30 pm T3 (Henning, Berlin, Germany), containing 10% FCS and 10,000 U/ml penicillin-streptomycin.

VEGF measurement
After 16- to 72-h simulation, the TtT/GF cell culture supernatants were harvested, and VEGF was measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Inc., Minneapolis, MN). The ELISA recognized all known secreted VEGF isoforms of both mouse and rat species. The detection limit of the assay was 3 pg/ml VEGF.

Determination of cell proliferation
Proliferation was measured with the wst-1 reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions. This compound is cleaved by the mitochondrial respiratory chain, and the product dye directly correlates with the number of viable cells in culture. The reaction product was measured in an ELISA plate reader at 450 nm.

Hormone determination
Hormones were measured by RIA as previously described (34). For rat GH and PRL, reagents were provided by Dr. A. F. Parlow from the National Hormone and Pituitary Program (Baltimore, MD).

Northern blot
Northern blot was performed as previously described (34, 35). Unless stated, reagents were from Sigma (St. Louis, MO), (Roche Molecular Biochemicals,) or Pharmacia Biotech (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, 5 x SSPE, 5 x Denhardt’s solution, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA), and then the probe was added for 12 h. Blots were washed at increasing salt and temperature stringency, with a final wash of 30 min at 60 C in 0.1 x SSC containing 0.1% SDS. Dried filters were exposed to Kodak XAR5 film (Eastman Kodak Co., Rochester, NY) at -70 C with intensifying screens for 2 days. A 1.6-kb murine IL-11-R {alpha}-chain complementary DNA (cDNA) fragment (36), a 1.56-kb human CNTF-R {alpha}-chain cDNA fragment (37), a 0.6-kb rat CNTF cDNA fragment (38), and a 1-kb PstI fragment of actin cDNA (39) were labeled with a random priming kit using [{alpha}-32P]deoxy-CTP (SA, 2–4 x 108 cpm/µg). The autoradiograms were scanned with a LKB Ultroscan II laser densitometer. The blots were reprobed after eluting the first probe with 5 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 0.1 x 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 control with the fragment of actin cDNA as probe was performed in each blot.

Statistics
Statistics were determined using ANOVA in combination with Scheffe’s test. Data are shown as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-11-R and CNTF-R in somatomammotropic and FS pituitary cell lines and in normal rat anterior pituitary cells
The mRNA for the {alpha}-chain specific for the IL-11-R is expressed in the FS TtT/GF cells as a single band of 1.7 kb (Fig. 1AGo). This {alpha}-chain is also expressed in GH3 cells (Fig. 1BGo). The specific {alpha}-chain for CNTF-R is also expressed in both TtT/GF (Fig. 2AGo) and GH3 (Fig. 2BGo) cells. It is expressed as a single 2-kb band (Fig. 2Go). It has recently been shown that normal pituitary cells express the IL-11-R (32). We examined whether the CNTF-R is also expressed in normal rat anterior pituitary cells and found that they express the specific 2-kb mRNA by Northern blot (Fig. 2CGo).



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  		Figure 1. Expression of IL-11-R {alpha} (1.7 kb) mRNA in pituitary cells lines. Northern blot analysis is shown using 30 µg RNA/lane as stated in Materials and Methods. A single band corresponding to the IL-11-R mRNA (1.7 kb) is shown in murine TTT/GF cells (A) and rat GH3 cells (B).

 


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  		Figure 2. Expression of CNTF-R {alpha} (2 kb) mRNA in pituitary cells lines and normal rat anterior pituitary cells. Northern blot analysis is shown using 30 µg RNA/lane as stated in Materials and Methods. A single band corresponding to the CNTF-R mRNA (2 kb) is shown in TTT/GF cells (A), GH3 cells (B), and normal rat anterior pituitary cells (C).

 
CNTF and IL-11 regulate FS and lactosomatotropic cell lines proliferation
IL-11, at doses ranging from 1.25–5 nM, dose dependently stimulated the proliferation of TtT/GF cells (Fig. 3AGo). CNTF, at doses of 0.4–2 nM, also significantly stimulated the growth of these cells (Fig. 3BGo), as did IL-6, which has been previously shown to stimulate the growth of these cells (40). Similar results were obtained with 2% FCS or in serum-free medium (data not shown). In addition, both CNTF and IL-11 significantly stimulated the proliferation of GH3 cells in serum-free medium, but at lower doses (CNTF at 0.04 nM and IL-11 at 0.25 nM; Fig. 4Go, A and B). For both cytokines, the effect was maximum at the lower dose used; saturation or sensitization of the receptor are putative reasons for this greater sensitivity (Fig. 4Go, A and B). As for the other actions described in this manuscript, effective IL-11 and CNTF concentrations are in the same range as those previously described for these cytokines (32, 41, 42, 43, 44).



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  		Figure 3. Effect of recombinant human IL-11 and recombinant human CNTF on the proliferation of TTT/GF cells. The cells were seeded at 2000 cells/well in multiwell plates, with 2% serum and treated with different doses of recombinant human IL-11 (A) or recombinant human CNTF (B), respectively, for 24 h. Proliferation was measured by the wst-1 method as detailed in Materials and Methods. Values represent the mean ± SEM of quadruplicate determinations from a single representative experiment (total of five for each cytokine). By ANOVA with Scheffe’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. corresponding basal). To compare serum’s effect, the same experiments were performed using serum-free medium. The results obtained were similar, but the effects observed with addition of 2% serum were more pronounced.

 


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  		Figure 4. Effect of recombinant human IL-11 and recombinant rat CNTF on the proliferation of GH3 cells. The cells were seeded at 9000 cells/well in 24-well plates with 10% serum. After attachment, cells were washed twice with PBS and incubated in serum-free medium for 24 h. Cells were treated with different doses of recombinant rat CNTF (A) or human IL-11 (B), respectively, for 24 h in serum-free medium. Proliferation was measured by the wst-1 method as detailed in Materials and Methods. Values represent the mean ± SEM of quadruplicate determinations of a single representative experiments (total of five for each cytokine). By ANOVA with Scheffe’s test: *, P < 0.05; **, P < 0.01 (vs. corresponding basal). Similar results were obtained at 72 h (data not shown).

 
IL-11 stimulates VEGF production by FS cells
FS cells are known to secrete the angiogenic factor VEGF (25). We found that IL-11 dose dependently stimulates this secretion at 16–72 h (Fig. 5Go, A and B). The effect was independent of the proliferative action, because it was exerted at 16 h, a time at which there was no evidence of cell growth (data not shown).



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  		Figure 5. Effect of recombinant human IL-11 on VEGF release from TTT/GF cells. Cells were seeded at 2000 cells/well in multiwell plates with 1% serum. After attachment, cells were washed twice with PBS and incubated in serum-free medium for 24 h. Cells were treated with different doses of recombinant human IL-11 for 24 h (A) or recombinant human IL-11 (5 mM) at different times (as indicated; B) in medium with 1% serum. The VEGF (picograms per ml) content was measured in the supernatants by ELISA. Values represent the mean ± SEM of quadruplicate determinations of a single representative experiment (total of four each time). By ANOVA with Scheffe’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. corresponding basal).

 
CNTF and IL-11 regulate GH and PRL secretion by GH3 cells
CNTF significantly stimulated PRL secretion by GH3 cells (Fig. 6AGo). The stimulatory effect was only observed at the highest doses tested. CNTF also induced a slight, but consistent, stimulation of GH production (Fig. 6BGo). This action was independent of the stimulation of proliferation, because doses that exert similar effects on proliferation have different actions on hormone secretion (0.04–0.4 nM is not active).



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  		Figure 6. Effect of recombinant rat CNTF on PRL (A) and GH (B) secretion in GH3 cells. Cells were treated with different doses of recombinant rat CNTF (as indicated). GH3 cells were seeded at 2 x 105 in 24-well plates. After serum depletion for 24 h, treatments were added with fresh serum-depleted medium. After 24 h, the supernatants were collected, and PRL and GH were measured by RIA. Values represent the mean ± SEM of one of four independently performed experiments, with four wells per treatment group. By ANOVA with Scheffe’s test: *, P < 0.05; ***, P < 0.001 (vs. corresponding basal).

 
CNTF mRNA expression in somatomammotropic and FS pituitary cells
It has recently been shown that pituitary cells express IL-11 mRNA (32). We examined whether they also express the mRNA for CNTF. Using Northern blot as the detection method, we observed that both GH3 and TtT/GF cells express a specific 1.2-kb CNTF mRNA (Fig. 7Go).



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  		Figure 7. Expression of CNTF mRNA in pituitary cells lines. Northern blot analysis is shown using 30 µg RNA/lane as stated in Materials and Methods. A single band corresponding to CNTF mRNA (1.2 kb) is shown in murine TTT/GF cells (A) and rat GH3 cells (B).

 
As for the CNTF-R, the CNTF mRNA signal is more intensive in GH3 than in TtT/GF cells (Figs. 2Go and 7Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Receptors for IL-11 and CNTF are expressed on both lactosomatotropic and FS cell lines. Both receptors are also expressed in normal pituitary cells and the AtT-20 corticotropic cell line (32). Although the gp130 signal transduction protein is ubiquitously expressed, expression of the specific cytokine-binding receptor subunits is restricted to specific cell types (3, 4, 5, 6). Thus, for example, CNTF-specific receptor subunit is expressed mostly on neuronal and muscle cells (37). In the absence of membrane-bound IL-11-R, cells can be stimulated with IL-11 and soluble IL-11R, using the cell gp130 (45, 46). Our findings, showing the expression of the {alpha}-subunit of the IL-11 and CNTF receptors in these pituitary cells, provide the basis for a specific direct action of these two gp130 ligands on these cells. The expression of the {alpha}-specific chain also provides the substrate for regulation of the response to these cytokines, as these cytokines may exert different biological responses according to the expression of the {alpha}-subunit. Moreover, the detection of CNTF mRNA, in conjunction with the recently described expression of IL-11 mRNA in anterior pituitary cells (32), strongly suggests that the actions of these cytokines may conform autocrine/paracrine loops. The fact that a FS cell line is involved in their actions strongly supports this idea, because FS cells have been postulated to be an important component of the integration of information in the anterior pituitary auto/paracrine loops (47). Functional studies must prove this in the normal anterior pituitary gland.

Pituitary FS cells were the first normal cell type in which VEGF was detected and are the only cell type within the normal pituitary that produces VEGF (48, 49). We previously reported that TtT/GF cells also produce VEGF and can be used as a model to study the regulation of VEGF secretion (25). As previously shown for IL-6 (25), we now provide evidence that IL-11 is a potent stimulator of VEGF secretion in TtT/GF cells. Angiogenesis is critically involved in tumor development, as vascularization is an essential step for tumor growth and expansion (50). Therefore, it is not surprising that, especially in well vascularized tumors, an elevated expression of VEGF is found (51, 52). The elevation of VEGF could play a role in pituitary adenoma angiogenesis. It has been reported that TtT/GF cells are essential for pituitary tumor formation in nude mice, probably by supporting angiogenesis (33). Although VEGF-producing FS cells are normally absent or rare within the adenomas (53, 54), it has been reported that between the adenoma and the normal pituitary tissue a transition zone exists that is extremely rich in FS cells (53, 55). The gp130 signal-transducer coupled cytokine family could enhance in a paracrine manner the release of VEGF from the FS cells in the transition zone. VEGF could subsequently induce endothelial cell proliferation and the sprouting of vessels into the pituitary adenoma.

We show for the first time that CNTF, like other cytokines (1, 2), stimulates both GH and PRL production by a lactosomatotropic cell line. The action is of low potency compared with the action on cell proliferation. In the case of IL-6 it was previously reported that no direct correlation between the effect on cell growth and hormone secretion was apparent; the action on cell proliferation was greater (10). Further studies on the coupling of the receptors for IL-6 and CNTF with the signals leading to cell division or hormone secretion are needed to understand this difference. The results for hormone secretion further support that the signaling of the activated immune system, through multiple cytokines, activates the secretion of hormones by the anterior pituitary. Although few studies have demonstrated the interaction of the different cytokines in these actions (56, 57) and particularly the gp130-mediated cytokine interaction on corticosterone production in vivo (58), the fine tuning regulation resulting from the putative multiple interactions remains to be established.

These two cytokines, IL-11 and CNTF, are able not only to regulate hormone and VEGF secretion, but also to influence another critical parameter of pituitary function with pathological consequences: the proliferation of these cells. IL-6 has been shown to regulate, in addition to hormone secretion and VEGF production (7, 8, 9, 10, 25), anterior pituitary cell growth. In the GH3 cell line it significantly stimulates [3H]thymidine incorporation and cell number (10, 59), it stimulates the growth of TtT/GF cells (40) and the MtT/E rat tumor pituitary cell line (60), it stimulates or inhibits c-fos expression in different pituitary adenomas (61), and it has also been shown to stimulate proliferation in some pituitary adenomas, but not in others (62). The pituitary FS cell line TtT/GF stimulation of somatotropic pituitary tumor cell (MtT/S) growth in nude mice (33) may involve the actions of these cytokines. IL-11 in conjunction with CNTF and IL-6 may have an important role, acting through the gp130 transducer, on the onset signaling of proliferation of pituitary cells. It is tempting to speculate that some of the genetic events underlying pituitary pathogenesis progression may involve genes regulating cellular growth in response to gp130 pathways.

In summary, our results show that IL-11 and CNTF receptors are expressed, and the two cytokines act on pituitary cell lines in at least three different ways: by stimulating hormone secretion, release of the angiogenic factor VEGF, and growth of these cells. These results together with those previously published (32), suggest that IL-11 and CNTF may act as auto/paracrine growth and regulatory factors in anterior pituitary cells.


   Acknowledgments
 
We thank Amgen, Inc., for providing us the rat CNTF cDNA for our experiments.


   Footnotes
 
1 This work was supported by grants from the Volkswagen Foundation (I/74 149), the Deutsche Forschungsgemeinschaft (Sta 285/7–3), the University of Buenos Aires, the Argentine National Research Council, and Agencia Nacional de Promoción Científica y Tecnológica-Argentina. Back

2 Member of the Argentine National Research Council. Back

Received October 21, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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