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Endocrinology Vol. 144, No. 4 1202-1210
Copyright © 2003 by The Endocrine Society

ARTICLE

Novel Neurotrophin-1/B Cell-Stimulating Factor-3 (Cardiotrophin-Like Cytokine) Stimulates Corticotroph Function via a Signal Transducer and Activator of Transcription-Dependent Mechanism Negatively Regulated by Suppressor of Cytokine Signaling-3

Christoph J. Auernhammer1, Nicola B. Isele1, Florian B. Kopp, Gerald Spoettl, Neziha Cengic, Matthias M. Weber, Giorgio Senaldi and Dieter Engelhardt

Department of Internal Medicine II (C.J.A., N.B.I., F.B.K., G.S., N.C., D.E.), Klinikum Grosshadern, Ludwig-Maximilians-Universität, Munich 81366, Germany; Department of Internal Medicine II (M.M.W.), Universität zu Köln, Cologne 51109, Germany; and Amgen Inc. (G.S.), Thousand Oaks, California 91320

Address all correspondence and requests for reprints to: Christoph Auernhammer, M.D., Department of Internal Medicine II, Klinikum Grosshadern, Ludwig-Maximilians-Universität, Marchioninistrasse 15, Munich 81377, Germany. E-mail: christoph.auernhammer{at}med2.med.uni-muenchen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3) is a recently cloned gp130 cytokine, acting through the tripartite ciliary neurotrophic factor receptor (CNTFR) {alpha}/leukemia inhibitory factor receptor (LIFR)/gp130 receptor complex. The aim of the current study was to investigate the role of NNT-1/BSF-3 in corticotroph cell function and further characterize NNT-1/BSF-3 signaling pathways. Using RT-PCR, expression of ciliary neurotrophic factor receptor {alpha}, leukemia inhibitory factor receptor, and gp130 could be demonstrated in mRNA derived from murine corticotroph AtT-20 cells and murine pituitary tissue. Incubation of AtT-20 cells with 10 ng/ml recombinant human NNT-1/BSF-3 rapidly induced tyrosine-phosphorylation of signal transducer and activator of transcription (STAT)3 and STAT1 at 5 and 10 min. Proopiomelanocortin promoter activity and suppressor of cytokine signaling (SOCS)-3 promoter activity were significantly stimulated by NNT-1/BSF-3 4.0 ± 0.3- and 5.9 ± 0.2-fold, respectively. In comparison with untreated control, NNT-1/BSF-3 significantly stimulated ACTH secretion at 24 and 48 h 1.7 ± 0.2-fold and 1.5 ± 0.1-fold above baseline. In comparison with mock-transfected cells, stable overexpression of SOCS-3 in AtT-20 cells abolished NNT-1/BSF-3-induced STAT1 and STAT3 phosphorylation and almost completely inhibited STAT-dependent proopiomelanocortin promoter and SOCS-3 promoter activities. In addition, NNT-1/BSF-3-induced ACTH secretion at 48 h was significantly attenuated by SOCS-3 overexpression. In summary, we have shown that NNT-1/BSF-3 is a modulator of corticotroph cell function, which is negatively regulated by SOCS-3. Our data indicate that the activation of the Jak-STAT cascade is essential for corticotroph NNT-1/BSF-3 signaling. Further studies will have to investigate the possible in vivo role of NNT-1/BSF-3 as a neuroimmunoendocrine modulator of hypothalamus-pituitary-adrenal axis stress response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SIX MEMBERS OF the gp130 cytokine family have been known until recently, namely IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor (CNTF). All gp130 cytokines signal through receptor complexes constituted by ligand-specific receptor subunits and the common receptor subunit gp130 (1, 2, 3). Binding of a gp130 cytokine to its specific receptor complex activates the Janus kinases (Jak)-signal transducer and activator of transcription (STAT) signaling cascade. Briefly, autophosphorylation of Jaks is followed by tyrosine phosphorylation of STAT factors. Phosphorylated STATs homo- or heterodimerize by binding each other with their Src homology 2 domains. Subsequently, STAT dimers act as transcription factors, translocating to the nucleus and binding to specific STAT-binding elements in the promoter region of various genes. Negative regulation of Jak-STAT signaling is mediated by different intracellular mechanisms including suppressor of cytokine signaling proteins (SOCS; Refs. 1, 2, 3).

A novel gp130 cytokine was recently cloned and descriptively named novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3; GenBank accession no. AF176912; Ref. 4). The same cytokine was also independently described and because of its homology to cardiotrophin-1 named cardiotrophin-like cytokine (GenBank accession no. AF172854; Ref. 5). NNT-1/BSF-3 could be demonstrated to be a second functional ligand to the tripartite CNTF receptor (CNTFR) complex [CNTFR{alpha}/LIF receptor (LIFR)/gp130 (Refs. 6, 7, 8)]. Despite harboring a signal peptide sequence, NNT-1/BSF-3 does not appear to be secreted from cells as a single peptide (4, 6, 7). Secretion of NNT-1/BSF-3 seems to require its heterodimerization with either cytokine-like factor-1 (CLF; Refs. 6 and 9) or soluble CNTFR{alpha} (7). Heterodimerization, however, does not seem to be required for NNT-1/BSF-3 binding to the CNTFR complex on cell surface and further activation of Jak-STAT signaling (4, 5, 7). In neuroblastoma cell lines harboring the CNTFR complex (CNTFR{alpha}/LIFR/gp130) NNT-1/BSF-3 alone or in complex with CLF stimulates tyrosine phosphorylation of Jak1 (8), Jak2 (8), Tyk2 (8), gp130 (4, 5, 6, 8), LIFR (4, 6, 8), STAT1 (5, 8), and STAT3 (4, 6, 7, 8) as well as activation of the MAPKs Erk1 and Erk2 (8) and phosphatidylinositol 3 kinase pathway (8) but does not stimulate tyrosine phosphorylation of STAT2, STAT4, STAT5, and STAT6 (8). NNT-1/BSF-3 is highly expressed in tissues of the immune system, e.g. spleen (4, 5), lymph node (4), peripheral blood leukocytes (5). NNT-1/BSF-3 alone (4) or forming complexes with either sCNTFR{alpha} (7) or CLF (6, 8) promotes in vitro motor neuron survival (4, 7) and induces proliferation of leukemic cells (4, 6, 8). In vivo NNT-1/BSF-3 induces secretion of acute phase proteins and corticosterone, weight loss, and enlargement of secondary lymphoid organs (4).

Several gp130 cytokines are immunoneuroendocrine modulators of corticotroph function (1, 2, 10, 11). The proopiomelanocortin (POMC) promoter harbors a functional STAT-binding element, and cytokine-induced activation of the JAK-STAT cascade results in POMC promoter activation and gene expression (12, 13). Corticotroph SOCS-3 is a STAT-inducible, short-lived protein with negative autoregulatory properties on cytokine-induced Jak-STAT signaling (14). Especially LIF action on corticotroph function has been widely investigated using murine corticotroph AtT-20 cells as a model (2, 14). Activation of corticotroph JAK-STAT signaling is essential for LIF-mediated POMC gene expression and ACTH secretion because it can be inhibited by SOCS-3 overexpression. In addition, LIF-mediated POMC gene expression and ACTH secretion can also be attenuated by dominant negative STAT3 mutants (2, 14).

In the current study, we investigated the putative role of the novel gp130 cytokine NNT-1/BSF-3 as a corticotroph stimulus. We could show expression of CNTFR{alpha} on corticotroph AtT-20 cells and demonstrate activation of corticotroph cell function by the CNTFR{alpha}/LIFR/gp130 complex with ligands NNT-1/BSF-3 and CNTF. NNT-1/BSF-3 stimulates STAT3 and STAT1 phosphorylation as well as STAT-dependent SOCS-3 gene and POMC gene expression in corticotrophs. NNT-1/BSF-3 was also shown to stimulate ACTH secretion from corticotrophs. Overexpression studies with SOCS-3 in AtT-20 cells demonstrate that NNT-1/BSF-3 induces STAT phosphorylation, which is completely suppressed by SOCS-3, and that the effects on corticotroph POMC gene expression are at least in part Jak-STAT dependent. Our findings show that the novel gp130 cytokine NNT-1/BSF-3 is a potent stimulator of corticotroph function by Jak-STAT-dependent mechanisms and that NNT-1/BSF-3 signaling is negatively regulated by SOCS-3. NNT-1/BSF-3 is a new player in the immunoneuroendocrine regulation of corticotroph function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
AtT-20/D16v-F2 cells were originally derived from ATCC (Manassas, VA). Recombinant human (rh) NNT-1/BSF-3 was expressed in Escherichia coli as described recently (4), and concentration was determined by UV (280 nm) spectrophotometry using a standard preparation as a reference. The rhCNTF and murine LIF were purchased from R \|[amp ]\| D Systems (Minneapolis, MN). DMEM, fetal calf serum, and other supplements were from PAA Laboratories (Linz, Austria). TRIZOL, Moloney murine leukemia virus reverse transcriptase, and TOPO-TA PCR2.1 vector were from Invitrogen (Karlsruhe, Germany). GeneAmp PCR system 2400 and AmpliTaq DNA polymerase were from Perkin-Elmer (Foster City, CA). Superfect, QIAEX II, and QIAquick nucleotide (nt) removal kit were from QIAGEN (Hilden, Germany). DECAprimeII random primed DNA labeling kit and mouse ß-actin DECAprobe template was from Ambion, Inc. (Austin, TX). QuikHyb Rapid solution was from Stratagene (La Jolla, CA). The Hybond-N+ membrane was from Amersham Biosciences (Freiburg, Germany). The ({alpha}-32P)CTP was from Perkin-Elmer Life Sciences (Zaventem, Belgium). Biomax MS and XOMAT-AR films were from Eastman Kodak Co. (Rochester, NY). Antibodies against phosphorylated STAT (pSTAT)3, pSTAT1, and ERK1/ERK2 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against pSTAT5 and pERK1/ERK2 were from Cell Signaling, Cummings Center (Beverly, MA). Antibodies against STAT3, STAT1, and STAT5 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase conjugated secondary antibodies to mouse or rabbit IgG and chemiluminescent substrate SuperSignal West Dura extended duration substrate were from Pierce Chemical Co. (Rockford, IL).

Cell culture
AtT-20/D16v-F2 cells were cultured as described recently (15, 16, 17). Briefly, cells were grown in complete medium (DMEM supplemented with 10% fetal calf serum) in a 5% CO2 atmosphere. Stable transfection and characterization of individual clones of AtT-20 cells mock transfected (AtT-20M) or overexpressing SOCS-3 (AtT-20S) has been described earlier (14). Individual cell clones were grown with G418 1 mg/ml as selection medium. Stock solution of rhNNT-1/BSF-3 contained 5 mM sodium citrate, pH 4. This buffer was also used to dissolve all other cytokines used and was added in equal volumes to untreated control cells.

Templates for probes and Northern blot analysis
SOCS-3 probe template was generated by repeatedly performed PCR from the previously described (15) 747-nt fragment of murine SOCS-3 (nt 15–762, GenBank accession no. U88328) inserted into pCR3.1 vector. POMC probe template was generated by repeatedly performed PCR from a 526-nt fragment of murine POMC fragment (nt 58–583, GenBank accession no. NM_008895) inserted into pCR2.1 vector. All fragments were verified by sequence analysis as described below. PCR products were electrophoresed on an agarose gel and specific bands gel purified by QIAEX II. The ß-actin DECAprobe template was the 1.076-kb fragment of the mouse ß-actin gene.

For Northern blot experiments, 1.5–1.8 x 106 cells were seeded in 100-mm dishes and grown for 48–72 h in 6.0 ml complete medium, followed by serum-free conditions (medium with 0.1% BSA) for 16 h. Subsequently medium was replaced by fresh serum-free medium, and incubation with the different cytokines tested was performed for the indicated time points. RNA extraction was performed using TRIZOL according to the manufacturer’s protocols. Northern blot analysis was performed as described recently (15, 16). Briefly, electrophoresis of 20 µg total RNA on a 1% agarose, 6.4% formaldehyde gel was followed by transfer to a Hybond-N nylon membrane and UV cross-linking. Probes were labeled with ({alpha}-32P)CTP by DECAprimeII random primed DNA labeling kit. Prehybridization and hybridization were performed with QuikHyb Rapid, following the manufacturer’s protocols. Autoradiographs were exposed to Biomax MS film (Kodak) at -70 C for 12–72 h.

RT-PCR and sequencing
Reverse transcription (RT) was performed with Moloney murine leukemia virus reverse transcriptase, according to the manufacturer’s instructions. PCR was performed on a GeneAmp PCR system 2400 using AmpliTaq DNA polymerase. After an initial denaturation step (94 C, 3 min), 40 PCR cycles followed (denaturation 94 C, 1 min; annealing at appropriate temperature, 1 min; extension 72 C, 1 min), and finally PCR was terminated by a single elongation step at 72 C for 10 min. Dimethylsulfoxide at a final concentration of 5% was added. A 567-nt fragment of the murine CNTFR{alpha} cDNA (nt 359–926, GenBank accession no. NM_016673) was generated with a specific primer pair, sense 5'-GCTCAACGGCTCTCAGCTGATACTG-3' and antisense 5'-CACATGCT GCCATTGGTCCAGGATG-3' at an annealing temperature of 58 C. A 315-nt fragment of the murine LIFR cDNA (nt 2618–2935, GenBank accession no. NM_013584) was generated with a specific primer pair, sense 5'-GAGCATCCTTTGCTATCGGAAGC-3' and antisense 5'-CGTTATTTCCTCCTCGATGATGG-3' at an annealing temperature of 56 C. A 689-nt fragment of the murine gp130 cDNA (nt 1863–2552, GenBank accession no. X62646) was generated with a specific primer pair, sense 5'-CGTACACAGATGAAGGTGGGAAAG-3' and antisense 5'-CTTCAGGCTGACTGCAGTTCTGC-3' at an annealing temperature of 58 C. Specific bands were gel purified using QIAEX II and cloned in pCR2.1 vector. Cycle sequencing was performed (Medigenomix, Munich, Germany) using big dye terminators and analyzing the reactions on an ABI Prism 3700 DNA Analyzer (Applied Biosystems, Foster City, CA).

Luciferase assay
For luciferase assay experiments 0.3–0.4 x 105 cells were seeded in 6-well plates and grown for 48 h in 2 ml complete medium. Afterward, transient transfection of cells with luciferase constructs (1.0 µg DNA per well) was performed with Superfect as described recently (15, 16, 17). Incubation with the transfection reagents for 6 h was followed by incubation with serum-free medium for another 16–24 h. Subsequently medium was replaced by fresh serum-free medium, and incubation with the different cytokines tested was performed for 6 h. Subsequently, supernatants were harvested and luciferase activities quantitated. A -706/+64 rat POMC promoter-luciferase construct in PGEM7Z vector was kindly provided by Dr. Malcolm Low (Vollum Institute, Portland, OR). A -2757/+929 murine SOCS-3 promoter-luciferase construct in pGL3Basic vector has been described by us recently (17) and constant transfection efficiency in AtT-20 cells has been demonstrated (17). In the current experiments, luciferase activities were measured in untreated controls and cells treated with NNT-1/BSF3, CNTF, or LIF at a concentration of 10 ng/ml each. The relative increase of untreated vs. cytokine-stimulated luciferase activity was calculated for each experiment. Because only relative increases of luciferase activity were calculated, an internal transfection control was not performed.

Protein extraction and Western blotting
For Western blot experiments, 1.5–1.8 x 106 cells were seeded in 100 mm-dishes and grown for 48–72 h in 6 ml complete medium, followed by incubation with serum-free medium (DMEM with 0.1% BSA) for 16 h. Subsequently medium was replaced by fresh serum-free medium, and incubation with the different cytokines tested was performed for the indicated time points. Then cells were lysed in 500 µl lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, and 2 mM sodium orthovanadate, pH 7.4) containing 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 2 µg/ml aprotinin, and 20 µM leupeptin. The lysates were centrifuged at 13,000 x g for 10 min at 4 C and supernatants were diluted 1:1 with sodium dodecyl sulfate (SDS) sample buffer (0.25 M Tris HCl, 40% glycerol, 2% SDS, 1% dithiothreitol, bromophenol blue, pH 8.8). Samples were boiled for 5 min and separated on a 10% SDS polyacrylamide gel. Proteins were electrotransferred in 60 min onto polyvinyl difluoride membranes (Immobilon, Millipore GmbH, Schwalbach, Germany) using a semidry Western blot technique. Membranes were blocked for 30 min in TBS-T buffer (0.02 M Tris-HCl, 0.15 M NaCl, 2% Tween 20. 0.01% sodium azide). The blocked membranes were incubated overnight in appropriate dilutions (PBS-T; 0.05 M sodium phosphate, 0.15 M NaCl, 2% Tween 20, pH 7) of antibodies against pSTAT1 (1:10,000), pSTAT3 (1:40,000), pSTAT5 (1:5,000), and pERK1/ERK2 (1:40,000). After washing with PBS, the membranes were incubated with a peroxidase conjugated secondary antibody (goat antimouse or donkey antirabbit, depending on the primary antibody; 1:25,000 each) for 2 h. The blots were washed and immersed in the chemiluminescent substrate for 30 min and exposed to XOMAT-AR film. Afterward the membranes were stripped and incubations with antibodies to STAT-1 (1:20,000), STAT3 (1:40,000), STAT5 (1:40,000), and ERK1/ERK2 (1:40,000) were performed as described above.

ACTH assay
After 24 and 48 h, supernatants from AtT-20 cells were collected and ACTH was measured with a commercial RIA (Diagnostic System Laboratories, Webster, TX).

Statistical analysis
Densitometry of specific bands in Western and Northern blots was performed using NIH Image 1.59 software. Statistical analysis was performed using JMP 5.0.1 software (SAS Institute Inc., Cary, NC). Statistical analysis between means of several treatment groups and the mean of a single control group was performed with Dunnett’s test for multiple comparisons. Statistical analysis between means of all pairs of treatment groups was performed with Tukey-Kramer honestly significant difference (HSD) test for multiple comparisons. Statistical analysis of pairwise comparisons between the mean of a single treatment group and the mean of a single control group was performed with t test. All values shown are mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CNTFR{alpha}, LIFR, and gp130 mRNA
Using RT-PCR, expression of CNTFR{alpha}, LIFR, and gp130 could be demonstrated in mRNA derived from murine corticotroph AtT-20 cells and pituitary tissue of C57BL/6 mice, respectively (Fig. 1Go). A 567-nt fragment of the murine CNTFR{alpha} cDNA (nt 359–926, GenBank accession no. NM_016673), a 315-nt fragment of the murine LIFR cDNA (nt 2618–2935, GenBank accession no. NM_013584), and a 689-nt fragment of the murine gp130 cDNA (nt 1863–2552, GenBank accession no. X62646) were generated. Appropriate-sized bands generated from corticotroph mRNA were cloned in pCR2.1 vector and full-length sequenced to verify their specificity.



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  		Figure 1. Expression of CNTFR{alpha}, LIFR, and gp130 mRNA mRNA in murine corticotrophs and pituitary tissue. RT-PCR was performed with RNA extracted from murine corticotroph AtT-20 cells and pituitary tissue. RT+, With RT; RT-, without RT. Each RT-PCR was repeated from three different samples. A 567-nt fragment of the murine CNTFR{alpha} cDNA, 315-nt fragment of the murine LIFR cDNA, and 689-nt fragment of the murine gp130 cDNA were generated. Specificity of RT-PCR-generated bands was verified by sequencing.

 
STAT protein phosphorylation
No pSTAT3 and pSTAT1 was detectable in untreated controls (Fig. 2Go). Using densitometry analysis, the ratio of pSTAT protein vs. total STAT protein (pSTAT/STAT ratio) was calculated for each treatment group. The pSTAT/STAT ratio of the LIF-treated group (positive control) was set as 1.0 each. Relative STAT phosphorylation levels of other treatment groups were calculated in comparison with this positive control. The significance of cytokine-stimulated tyrosine phosphorylation of STAT3 and STAT1 vs. untreated control was determined by Dunnett’s test. Using LIF (10 ng/ml) as a positive control, a significant stimulation of pSTAT3 (P < 0.001) and pSTAT1 (P < 0.05) levels vs. untreated controls was observed at 5 and 10 min, respectively (Fig. 2Go). Also, incubation with 10 ng/ml NNT-1/BSF-3 caused a significant stimulation of pSTAT3 (P < 0.001) and pSTAT1 (P < 0.001) levels at 5 and 10 min vs. untreated controls, respectively. The NNT-1/BSF-3-induced pSTAT3/STAT3 ratio appeared even higher than the LIF-induced ratio with 1.3 ± 0.2 and 1.1 ± 0.1 at 5 and 10 min, respectively. Similarly, the NNT-1/BSF-3 induced pSTAT1/STAT1 ratio appeared higher than the LIF-induced ratio with 2.1 ± 0.4 and 1.5 ± 0.4 at 5 and 10 min, respectively (Fig. 2Go). Incubation with 10 ng/ml CNTF caused a significant stimulation of pSTAT3 at 5 min (P < 0.05) and 10 min (P < 0.001) vs. untreated controls. However, the CNTF-induced pSTAT3/STAT3 ratio appeared lower than the LIF-induced ratio with 0.5 ± 0.1 and 0.6 ± 0.1 at 5 and 10 min, respectively. No significant stimulation of pSTAT1 was observed following incubation with CNTF (Fig. 2Go).



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  		Figure 2. NNT-1/BSF-3 and CNTF induce STAT protein activation in corticotrophs. Corticotroph AtT-20 cells were incubated with 10 ng/ml NNT-1/BSF-3, CNTF, or LIF (positive control) for 5 and 10 min, respectively. Tyrosine phosphorylated STAT3 and STAT1 were demonstrated by specific antibodies. Using antibodies against total STAT3 and STAT1, equal protein loading was verified. Using densitometry analysis, the ratio of pSTAT protein vs. total STAT protein (STAT) was calculated for each treatment group. The ratio pSTAT vs. STAT of the LIF-treated group (positive control) was set as 1.0. Relative STAT phosphorylation levels of other treatment groups are shown in comparison with the positive control. Shown are the mean values of five independently performed experiments (upper panels). Asterisks indicate significance of cytokine stimulated tyrosine phosphorylation of STAT3 and STAT1 vs. untreated control, determined by Dunnett’s test: *, P < 0.05; ***, P < 0.001. A representative Western blot of five independently performed experiments is shown (lower panels).

 
We could not detect tyrosine phosphorylation of STAT5 or ERK1 and ERK2 in AtT-20 cells following incubation with either of the above-mentioned cytokines (data not shown). Sensitivity of our different phosphotyrosine assays was proven by specific positive controls (data not shown).

SOCS-3 gene expression
SOCS-3 promoter activity of a -2757/+929 murine SOCS-3-luc construct (17) was significantly stimulated 5.9 ± 0.2-fold by NNT-1/BSF-3 (P < 0.01), 2.4 ± 0.2-fold by CNTF (P < 0.05), and 9.6 ± 1.7-fold by LIF (P < 0.001), respectively (Fig. 3AGo). Following stimulation with NNT-1/BSF-3, CNTF, and LIF for 60 min, SOCS-3 mRNA expression was induced 11.00-fold (P < 0.001), 7.25-fold (P > 0.05), and 13.25-fold (P < 0.01) above untreated control (Fig. 3BGo). Although NNT-1/BSF-3 and LIF showed a similar potency to induce SOCS-3 mRNA expression, CNTF appeared a weaker stimulus of SOCS-3 mRNA expression (Fig. 3BGo).



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  		Figure 3. NNT-1/BSF-3 and CNTF induce SOCS-3 promoter activity and mRNA expression in corticotrophs. A, AtT-20 cells were transiently transfected with a -2757/+929 murine SOCS-3 promoter-luciferase construct and incubated with 10 ng/ml NNT-1/BSF-3, CNTF, or LIF (positive control) for 6 h, respectively. Reporter gene activity of unstimulated controls was taken as 1.0 and activity of stimulated cells was calculated as fold increase. Mean values of four independently performed experiments are shown. Each experiment was performed with n = 3 per treatment group. Asterisks indicate significance of cytokine-stimulated SOCS-3 promoter activity vs. untreated control, determined by Dunnett’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, SOCS-3 mRNA expression was determined by Northern blot analysis following stimulation of AtT-20 cells with 10 ng/ml NNT-1/BSF-3, CNTF, or LIF (positive control) for 60 min. Using densitometry analysis, the ratio of SOCS-3 mRNA vs. ß-actin mRNA (SOCS-3/ß-actin ratio) was calculated for each treatment group. The SOCS-3/ß-actin ratio of the LIF-treated group (positive control) was set as 1.0. Relative SOCS-3 mRNA expression levels of other treatment groups were calculated in comparison with this positive control. Shown are the mean values of five independently performed experiments (upper panel). Asterisks indicate significance of cytokine-stimulated SOCS-3 mRNA expression vs. untreated control, determined by Dunnett’s test: **, P < 0.01; ***, P < 0.001. A representative experiment of five independently performed experiments is shown (lower panel).

 
POMC gene expression and ACTH secretion
POMC promoter activity of a -706/+64 rat POMC promoter-luciferase construct was significantly stimulated 4.0 ± 0.3-fold by NNT-1/BSF-3 (P < 0.001), 2.5 ± 0.1-fold by CNTF (P < 0.01), and 3.6 ± 0.2-fold by LIF (P < 0.001), respectively (Fig. 4AGo). Following stimulation with NNT-1/BSF-3, CNTF, and LIF for 24 h, POMC mRNA expression was induced 1.25-fold (n.s.), 1.90-fold (P < 0.05), and 2.40-fold (P < 0.01) above untreated control (Fig. 4BGo).



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  		Figure 4. NNT-1/BSF-3 and CNTF induce POMC promoter activity and POMC mRNA expression in corticotrophs. A, AtT-20 cells were transiently transfected with a -706/+64 rat POMC promoter-luciferase construct and incubated with 10 ng/ml NNT-1/BSF-3, CNTF, or LIF (positive control) for 6 h, respectively. Reporter gene activity of unstimulated controls was taken as 1.0, and activity of stimulated cells was calculated as fold increase. Mean values of four independently performed experiments are shown. Each experiment was performed with n = 3 per treatment group. Asterisks indicate significance of cytokine-stimulated POMC promoter activity vs. untreated control, determined by Dunnett’s test: **, P < 0.01; ***, P < 0.001. B, POMC mRNA expression was determined by Northern blot analysis following stimulation of AtT-20 cells with 10 ng/ml NNT-1/BSF-3, CNTF, or LIF (positive control) for 24 h. Using densitometry analysis, the ratio of POMC mRNA vs. ß-actin mRNA (POMC/ß-actin ratio) was calculated for each treatment group. The POMC/ß-actin ratio of the LIF-treated group (positive control) was set as 1.0. Relative POMC mRNA expression levels of other treatment groups were calculated in comparison with this positive control. Shown are the mean values of five independently performed experiments (upper panel). Asterisks indicate significance of cytokine-stimulated POMC mRNA expression vs. untreated control, determined by Dunnett’s test: *, P < 0.05; **, P < 001; n.s., not significant. A representative experiment of five independently performed experiments is shown (lower panel).

 
Mean basal ACTH secretion of AtT-20 cells incubated for 24 and 48 h was 180 ± 40 pg/ml and 550 ± 110 pg/ml, respectively. ACTH secretion at 24 and 48 h was significantly stimulated by 10 ng/ml NNT-1/BSF-3 to 1.75 ± 0.2- and 1.50 ± 0.1-fold above baseline (Fig. 5Go). Similarly, CNTF stimulated ACTH secretion at 24 h 1.70 ± 0.15- and at 48 h 1.40 ± 0.10-fold above baseline. The positive control LIF stimulated ACTH secretion at 24 h 2.05 ± 0.25- and at 48 h 1.75 ± 0.10-fold above baseline (Fig. 5Go).



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  		Figure 5. NNT-1/BSF-3 and CNTF induce ACTH secretion in corticotrophs. AtT-20 cells were stimulated with 10 ng/ml NNT-1/BSF-3, CNTF, or LIF (positive control) for 24 and 48 h, respectively. ACTH secretion was determined by RIA. Mean basal ACTH secretion of AtT-20 cells incubated for 24 and 48 h was 180 ± 40 pg/ml and 550 ± 110 pg/ml, respectively. ACTH secretion of unstimulated cells was taken as 1.0 and secretion of stimulated cells was calculated as fold increase. Mean values of three independently performed experiments are shown. Each experiment was performed with n = 5 per treatment group. Asterisks indicate significance of cytokine-stimulated ACTH secretion vs. untreated control, determined by Dunnett’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
SOCS-3 inhibits NNT-1/BSF-3-induced activation of corticotroph AtT-20 cells
AtT-20M cells showed a significant induction of STAT3 and STAT1 phosphorylation by incubation with NNT-1/BSF-3 for 10 min. In contrast, stable overexpression of AtT-20S cells resulted in complete inhibition of NNT-1/BSF-3 induced tyrosine phosphorylation of STAT3 and STAT1 (Fig. 6Go).



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  		Figure 6. Inhibition of NNT-1/BSF-3-induced STAT activation by SOCS-3. Different cell clones of mock-transfected AtT-20 cells (M) and stably SOCS-3-overexpressing AtT-20 cells (S) were incubated with 10 ng/ml NNT-1/BSF-3 for 10 min. Tyrosine phosphorylated STAT3 and STAT1 were demonstrated by specific antibodies (upper bands). Using antibodies against total STAT3 and STAT1, equal protein loading was verified (lower bands). A representative experiment of three independently performed experiments is shown.

 
NNT-1/BSF-3 significantly induced SOCS-3 promoter activity in different mock-transfected AtT-20M cells 7.0- to 12.0-fold above control, whereas in SOCS-3-overexpressing AtT-20S cells, we observed an almost complete inhibition to only 1.3- to 1.6-fold above control (Fig. 7AGo). SOCS-3 mRNA expression was strongly stimulated by NNT-1/BSF-3 in AtT-20M cells, whereas no stimulation was observed in AtT-20S cells (Fig. 7BGo).



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  		Figure 7. Inhibition of NNT-1/BSF-3-induced SOCS-3 promoter activity and endogenous SOCS-3 mRNA expression by SOCS-3 overexpression. A, Different cell clones of mock-transfected AtT-20 cells (M) and stably SOCS-3-overexpressing AtT-20 cells (S) were transiently transfected with a -2757/+929 murine SOCS-3 promoter-luciferase construct. Subsequently, cells were incubated with 10 ng/ml NNT-1/BSF-3 for 6 h. For each cell clone, reporter gene activity of unstimulated cells ({square}, white bars) was taken as 1.0 and activity of stimulated cells (, striped bars) was calculated as fold increase. Mean values of three independently performed experiments are shown. Each experiment was performed with n = 3 per treatment group and per clone. Asterisks indicate significance of NNT-1/BSF-3-stimulated SOCS-3 promoter activity vs. untreated control for each single cell clone, determined by Dunnett’s test: *, P < 0.05; ***, P < 0.001. The written P value demonstrates the significance between NNT-1/BSF-3-stimulated SOCS-3 promoter activity of all M clones vs. all S clones, determined by Tukey-Kramer HSD test. B, Different cell clones of mock-transfected AtT-20 cells (M) and stably SOCS-3-overexpressing AtT-20 cells (S) were incubated with 10 ng/ml NNT-1/BSF-3 for 45 min. SOCS-3 mRNA expression was determined by Northern blot analysis. A representative experiment of three independently performed experiments is shown.

 
Similarly, NNT-1/BSF-3 significantly induced POMC promoter activity in different mock-transfected AtT-20M cells 2.4 to 6.9 above control, whereas in SOCS-3-overexpressing AtT-20S cells, there was a complete inhibition to only 1.0- to 1.2-fold above control (Fig. 8AGo). In different mock-transfected AtT-20M cells, ACTH secretion was significantly stimulated by NNT-1/BSF-3 vs. respective untreated controls (M3: 335 ± 12 vs. 227 ± 17 pg/ml, P < 0.001; M6: 284 ± 9 vs. 226 ± 12 pg/ml, P < 0.05; M9: 331 ± 11 vs. 270 ± 10 pg/ml, P < 0.05; Fig. 8BGo). In contrast, in different SOCS-3-overexpressing AtT-20S cells, ACTH secretion was not significantly stimulated by NNT-1/BSF-3 vs. respective untreated controls (S8: 191 ± 14 vs. 163 ± 12 pg/ml; S11: 140 ± 12 vs. 140 ± 13 pg/ml; S17: 165 ± 15 vs. 155 ± 16 pg/ml; Fig. 8BGo).



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  		Figure 8. Inhibition of NNT-1/BSF-3-induced POMC promoter activity and ACTH secretion by SOCS-3 overexpression. A, Different cell clones of mock-transfected AtT-20 cells (M) and stably SOCS-3-overexpressing AtT-20 cells (S) were transiently transfected with a -706/+64 rat POMC promoter-luciferase construct. Subsequently, cells were incubated with 10 ng/ml NNT-1/BSF-3 for 6 h. For each cell clone, reporter gene activity of unstimulated cells ({square}, white bars) was taken as 1.0 and activity of stimulated cells (, striped bars) was calculated as fold increase. Mean values of three independently performed experiments are shown. Each experiment was performed with n = 3 per treatment group and per clone. Asterisks indicate significance of NNT-1/BSF-3-stimulated POMC promoter activity vs. untreated control for each single cell clone, determined by Dunnett’s test: ***, P < 0.001; n.s., not significant. The written P value demonstrates the significance between NNT-1/BSF-3-stimulated POMC promoter activity of all M clones vs. all S clones, determined by Tukey-Kramer HSD test. B, Different cell clones of mock-transfected AtT-20 cells (M) and stably SOCS-3-overexpressing AtT-20 cells (S) were incubated with 10 ng/ml NNT-1/BSF-3 for 48 h. ACTH secretion was determined by RIA. Mean values of four independent experiments performed with n = 6 per treatment group and per clone are shown. Pairwise comparison of NNT-1/BSF-3-stimulated ACTH level (, striped bars) vs. untreated control ({square}, white bars) was performed for each single cell clone. Asterisks indicate significance determined by t test: ***, P < 0.001; *, P < 0.05; n.s., not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that the novel gp130 cytokine NNT-1/BSF-3 stimulates corticotroph cell function. All the components of the tripartite CNTFR{alpha}/LIFR/gp130 receptor complex are expressed on corticotrophs. Stimulation by NNT-1/BSF-3 involves such complex and Jak-STAT signaling and is negatively regulated by SOCS-3.

Expression of all components of the tripartite CNTFR{alpha}/LIFR/gp130 receptor complex is essential for CNTF and NNT-1/BSF-3 signaling (8). In human pituitary tissues, expression of CNTFR{alpha} (18, 19), LIFR (18, 20), and gp130 (20) has been found by RT-PCR. Functional CNTFR is also expressed in normal rat pituitary cells and rat somatomammotropic GH3 cells as well as murine folliculostellate TtT/GF cells (19, 21). Our RT-PCR data now demonstrate expression of all components of the tripartite CNTFR{alpha}/LIFR/gp130 receptor complex in murine corticotroph AtT-20 cells as well as murine pituitary tissue (Fig. 1Go). These findings characterize corticotrophs as CNTFR{alpha}+, LIFR+, gp130+ cells, which might therefore be responsive to the two CNTFR complex ligands CNTF (22) and NNT-1/BSF-3 (6, 7, 8).

Effects of NNT-1/BSF-3 and CNTF on STAT phosphorylation and STAT-dependent gene expression were examined in murine corticotroph AtT-20 cells. LIF was used as a positive control (2, 14). NNT-1/BSF-3 significantly stimulated tyrosine phosphorylation of corticotroph STAT3 and STAT1, respectively (Fig. 2Go). In comparison, CNTF demonstrated only a significant effect on STAT3 phosphorylation but failed to induce STAT1 phosphorylation (Fig. 2Go). The predicted molecular masses are 20 kDa for LIF (3), 22 kDa for NNT-1/BSF-3 (4), and 22.9 kDa for CNTF (3). In a comparative dose-response study, we could demonstrate equimolar quantities of 5.0 nM (approximately 10 ng/ml) of LIF, NNT-1/BSF-3, and CNTF, respectively, to exhibit maximal stimulation of corticotroph STAT3 phosphorylation. Stimulation with even higher concentrations resulted in a plateau of STAT3 phosphorylation status (Auernhammer, C. J., F. B. Kopp, F. Dorn, N. B. Isele, G. Spoettl, N. Cengic, M. M. Weber, G. Senaldi, and D. Engelhardt, manuscript in preparation). Therefore, all experiments in this study were performed with the putative maximal stimulatory dose of 10 ng/ml for each cytokine tested. No constant effect of NNT-1/BSF-3 on tyrosine phosphorylation of STAT5 and the MAPKs ERK1 and ERK2 could be documented (data not shown), whereas sensitivity of the pSTAT5 and pERK blots was verified by positive controls (data not shown). Similar to our results, in different neuroblastoma cell lines, NNT-1/BSF-3 stimulates STAT3 and STAT1 phosphorylation (4, 5, 6, 7, 8) but has no effect on STAT5 phosphorylation (8). In contrast to our results, NNT-1/BSF-3 has been shown to stimulate ERK1/ERK2 phosphorylation in these neuroblastoma cell lines (8). A weak induction of ERK2 tyrosine phosphorylation by LIF has also been previously reported in AtT-20 cells (23). However MAPK activity is of no functional significance in LIF-induced activation of corticotrophs (23).

NNT-1/BSF-3 signaling in neuroepithelial cells has recently been demonstrated to be STAT3 dependent, using dominant negative STAT3 mutants or inactivating mutations in the STAT-binding element of the promoter region of a specific reporter gene (24). A functional STAT-binding element has been characterized in the untranslated 5'-region of the SOCS-3 (nt -72 to -64, GenBank accession no. AF117732) and the POMC gene (nt -399 to -379, GenBank accession no. X03171), respectively (12, 13, 14, 17). Promoter constructs of both genes were used in reporter gene assays to measure functional STAT activation by NNT-1/BSF-3 and CNTF in corticotrophs. LIF (14, 15) and CNTF (25) are known stimuli of SOCS-3 mRNA expression in vivo and in vitro. In our experiments NNT-1/BSF-3 stimulated STAT-dependent SOCS-3 promoter activity (Fig. 3AGo) and rapid expression of SOCS-3 mRNA (Fig. 3BGo) in corticotrophs with similar potency as LIF, whereas CNTF seemed less potent. Also, NNT-1/BSF-3 stimulated STAT-dependent POMC promoter activity with similar potency as LIF, whereas CNTF seemed less potent (Fig. 4AGo). In contrast, POMC gene expression at 24 h was potently stimulated by LIF and CNTF, whereas NNT-1/BSF-3 showed only modest nonsignificant effects (Fig. 4BGo). However, ACTH secretion was significantly stimulated by incubation with NNT-1/BSF-3 and CNTF to a similar extent, whereas LIF was even a little bit more potent (Fig. 5Go). The observed discrepancies among POMC promoter activity, POMC gene expression, and ACTH secretion might be explained in part by the following variables: 1) The -706/+64 rat POMC promoter-luciferase construct we used encompasses only a limited 5'-region of the POMC gene, whereas other positive and negative regulatory elements of POMC gene expression may be located outside this region; and 2) because of the previous observation that POMC mRNA expression following LIF stimulation of AtT-20 cells for 1–24 h demonstrates a peak at 24 h (23). Therefore, in the current study, only 24 h POMC mRNA expression was examined, whereas the maximum peak of NNT-1/BSF-3-induced POMC mRNA expression might be at a different time point. To summarize, our data demonstrate NNT-1/BSF-3 and CNTF to be able to stimulate corticotroph Jak-STAT signaling, STAT-dependent SOCS-3 gene expression, and STAT-dependent POMC gene expression and ACTH secretion. Because NNT-1/BSF-3 and CNTF signal by the tripartite CNTFR (6, 7, 8), our data suggest the CNTFR complex to exert a functional role in corticotroph stimulation by these cytokines.

In a next series of experiments, we investigated whether NNT-1/BSF-3 signaling can be regulated by SOCS-3. SOCS-3 is expressed in hypothalamic and pituitary tissues and inhibits Jak-STAT signaling mediated by a variety of different cytokines, thus acting as a putative regulator of neuroimmunoendocrine circuits (14, 26). Inhibitory effects of SOCS-3 have also been reported on the Jak-STAT signaling cascade of several gp130 cytokines including LIF and CNTF (14, 25, 26). In our current study, overexpression of SOCS-3 in AtT-20 cells abrogated NNT-1/BSF-3-induced STAT3 and STAT1 phosphorylation (Fig. 6Go). Also, STAT-dependent SOCS-3 promoter activity (Fig. 7AGo) and gene expression (Fig. 7BGo) was abolished by SOCS-3 overexpression. Similarly, STAT-dependent POMC promoter activity (Fig. 8AGo) was abolished by SOCS-3 overexpression, whereas NNT-1/BSF-3-induced ACTH secretion was significantly decreased (Fig. 8BGo). These findings demonstrate NNT-1/BSF-3-induced JAK-STAT signaling to be negatively regulated by SOCS-3. Because SOCS-3 inhibits STAT-dependent gene expression in corticotrophs (13), our data also indicate NNT-1/BSF-3 effects on the corticotroph to be JAK-STAT dependent. Further experiments using SOCS-3 promoter constructs and POMC promoter constructs harboring inactivating mutations in their respective STAT-binding region (12, 13, 17) might provide additional data on the role of NNT-1/BSF-3-induced STAT activation in corticotroph function.

In summary, we have shown that the novel gp130 cytokine NNT-1/BSF-3 is a potent modulator of corticotroph function. NNT-1/BSF-3 stimulates in murine corticotroph AtT-20 cells JAK-STAT signaling, STAT-dependent SOCS-3, and POMC gene expression and ACTH secretion. Similar effects could also be shown for CNTF. Thus, our data indicate the known ligands of the tripartite CNTFR{alpha}/LIFR/gp130 receptor complex, namely NNT-1/BSF-3 and CNTF, to be functional modulators of corticotroph function. Furthermore, we could demonstrate that NNT-1/BSF-3 signaling is negatively regulated by SOCS-3 and the effects of NNT-1/BSF-3 are dependent on activation of the Jak-STAT cascade. Although CNTF has no signal peptide and may not be actively secreted from cells but passively released following cell injury (8, 19), NNT-1/BSF-3 can be secreted from cells as a heteromeric complex together with CLF or CNTFR{alpha} (6, 7). We have found inducible expression of NNT-1/BSF-3 mRNA in the murine pituitary folliculostellate cell line TtT/GF (Vlotides, G., S. Hengge, D. Engelhardt, G. K. Stalla, and C. J. Auernhammer, manuscript in preparation) and NNT-1/BSF-3 might therefore act as a paracrine factor on corticotrophs. NNT-1/BSF-3 is a novel immunoneuroendocrine stimulus of corticotroph function demonstrating once more the complexity of the immunoneuroendocrine interface-modulating hypothalamus-pituitary-adrenal axis stress response.


   Acknowledgments
 
We gratefully acknowledge skillful technical assistance by S. Hengge.


   Footnotes
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (AU 139/2-1) and Friedrich-Baur Stiftung (0055/2001).

1 C.J.A. and N.B.I. contributed equally to this manuscript. Back

This work contains parts of the unpublished doctoral thesis of N.B.I. at the Ludwig-Maximilians-University of Munich, Germany.

Abbreviations: AtT-20M, AtT-20 cells mock transfected; AtT-20S, AtT-20 cells overexpressing suppressor of cytokine signaling-3; CLF, cytokine-like factor-1; CNTF, ciliary neurotrophic factor; CNTFR, CNTF receptor; HSD, honestly significant difference; Jak, Janus kinase; LIF, leukemia inhibitory factor; LIFR, LIF receptor; NNT-1/BSF-3, novel neurotrophin-1/B cell-stimulating factor-3; nt, nucleotide; POMC, proopiomelanocortin; pSTAT, phosphorylated STAT; pSTAT/STAT ratio, ratio of phosphorylated STATprotein vs. total STAT protein; rh, recombinant human; RT, reverse transcription; SDS, sodium dodecyl sulfate; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

Received September 5, 2002.

Accepted for publication December 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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