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Endocrinology, doi:10.1210/en.2003-0813
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Endocrinology Vol. 145, No. 2 716-727
Copyright © 2004 by The Endocrine Society

Expression of Novel Neurotrophin-1/B-Cell Stimulating Factor-3 (NNT-1/BSF-3) in Murine Pituitary Folliculostellate TtT/GF Cells: Pituitary Adenylate Cyclase-Activating Polypeptide and Vasoactive Intestinal Peptide-Induced Stimulation of NNT-1/BSF-3 Is Mediated by Protein Kinase A, Protein Kinase C, and Extracellular-Signal-Regulated Kinase1/2 Pathways

George Vlotides, Kathrin Zitzmann, Sabine Hengge, Dieter Engelhardt, Gunter K. Stalla and Christoph J. Auernhammer

Department of Internal Medicine II (G.V., K.Z., S.H., D.E., C.J.A.), Klinikum der Ludwig-Maximilians-Universität München, Standort Grosshadern, Munich 81377, Germany; and Department of Neuroendocrinology (G.K.S.), Max-Planck Institute of Psychiatry, Munich 80804, Germany

Address all correspondence and requests for reprints to: Christoph J. Auernhammer, M.D., Department of Internal Medicine II, Klinikum der Ludwig-Maximilians-Universität München, Standort Grosshadern, Marchioninistrasse 15, Munich 81377, Germany. E-mail: christoph.auernhammer{at}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 gp130 cytokine potently stimulating corticotroph proopiomelanocortin gene expression and ACTH secretion by a Janus kinase-signal transducer and activator of transcription (JAK-STAT)-dependent mechanism. In the current study, we examined the regulation of NNT-1/BSF-3 mRNA expression in murine pituitary folliculostellate TtT/GF cells using Northern blot technique. A 5- to 9-fold and a 4- to 7-fold induction in NNT-1/BSF-3 mRNA expression was observed between 2 and 6 h stimulation with the protein kinase C (PKC) stimulus phorbol-12-myristate-13-acetate (100 nM) and the protein kinase A (PKA) stimulus Bu2cAMP (5 mM), respectively. Pituitary adenylate cyclase-activating polypeptide (PACAP-38, 50 nM) and vasoactive intestinal peptide (VIP, 50 nM) also stimulated NNT-1/BSF-3 mRNA expression 5- to 9-fold between 2 and 6 h. Preincubation with PKC and PKA inhibitors such as H-7 (20 µM), GF109203X (50 µM), and H-89 (50 µM) decreased the stimulatory effects of PACAP and VIP. Both PACAP-38 and VIP also rapidly induced ERK1/2 phosphorylation and their stimulatory effect on NNT-1/BSF-3 mRNA expression was reduced by the MAPK kinase/ERK kinase (MEK) inhibitor U0126 (10 µM). Dexamethasone (10-7 M) was a potent inhibitor of phorbol-12-myristate-13-acetate-induced NNT-1/BSF-3 expression. RT-PCR analysis demonstrated TtT/GF cells to express the short and the hop variant but not the hip variant of the PACAP-1 receptor (PAC1-R). In addition, TtT/GF cells express the VIP/PACAP-2 receptor (VPAC2-R). In summary, NNT-1/BSF-3 is expressed in pituitary folliculostellate TtT/GF cells and induced by PKC-, PKA-, and ERK1/2-dependent mechanisms. The novel gp130 cytokine NNT-1/BSF-3 derived from folliculostellate cells might act as a paracrine neuroimmunoendocrine modulator of pituitary corticotroph function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IL-6 FAMILY OF CYTOKINES encompassed until recently six members, namely oncostatin M (OSM), IL-6, IL-11, ciliary neurotrophic factor (CNTF), cardiotrophin-1, and leukemia inhibitory factor. Because all these cytokines share gp130 as a critical transducer in their respective receptor complexes, they are also referred to as the gp130 family of cytokines (1, 2). Receptor-specific ligand binding induces autophosphorylation of Janus kinases (JAK), followed by tyrosine phosphorylation of signal transducer and activator of transcription (STAT) factors. Homo- or heterodimerized STAT factors translocate to the nucleus and bind to specific STAT binding elements in the promoter region of target genes (1).

Recently, a novel gp130 cytokine was described by Senaldi et al. (3) and due to its B cell stimulating properties was named novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3). Shi et al. (4) independently cloned the same cytokine and named it cardiotrophin-like cytokine due to its similarity to cardiotrophin-1. Strong expression of NNT-1/BSF-3 has been observed in lymph nodes and spleen, but NNT-1/BSF-3 was also detected in numerous other organ systems (3, 4). NNT-1/BSF-3 contains a putative signal peptide and would be expected to enter into the classical secretory pathway. However, complex formation of NNT-1/BSF-3 with either cytokine-like factor-1 (CLF-1) or soluble CNTF receptor-{alpha} (sCNTFR{alpha}) seems to be required for cellular secretion (5, 6, 7, 8).

Because many gp130 cytokines are involved in the immunoneuroendocrine interface (9, 10, 11), we recently examined the putative role of NNT-1/BSF-3 on pituitary corticotroph function. NNT-1/BSF-3 was demonstrated to be a potent direct modulator of corticotroph proopiomelanocortin gene expression and ACTH secretion by Janus kinase/STAT-dependent mechanisms (12).

In the current study, we investigated the expression of NNT-1/BSF-3 and CLF-1 in murine pituitary folliculostellate TtT/GF cells. We show a several-fold up-regulation of NNT-1/BSF-3 expression by protein kinase C (PKC) or protein kinase A (PKA) stimuli such as phorbol 12-myristate-13-acetate (PMA) or N6,2'-O-dibutyryladenosine-3',5'-cyclic-monophosphate (Bu2cAMP). Pituitary-adenylate-cyclase-activating polypeptide (PACAP)-38 and vasoactive-intestinal-peptide (VIP), two peptides of the secretin/VIP/glucagon family also induce NNT-1/BSF-3 mRNA expression, in a time- and dose-dependent manner. We further demonstrate that PACAP and VIP-induced NNT-1/BSF-3 expression is mediated by PKC, PKA, and ERK1/2 pathways. In contrast to NNT-1/BSF-3, CLF-1 mRNA is constantly expressed in TtT/GF cells at a high level. Our data suggest a possible paracrine mode of action of NNT-1/BSF-3 in the pituitary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM and fetal calf serum were from PAA Laboratories (Linz, Austria). Penicillin/streptomycin were from Life Technologies, Inc. (Karlsruhe, Germany), and amphotericin B was purchased from Biochrom (Berlin, Germany). Plastic ware for cell culture was from Becton Dickinson (Heidelberg, Germany). Lipopolysaccharide (LPS), PMA, Bu2cAMP, TGFß1, interferon-{gamma}, D-600 and dexamethasone were from Sigma (St. Louis, MO). PACAP-38, VIP, PACAP 6–38, angiotensin II, and vasopressin were from Bachem (Heidelberg, Germany). H-89, H-7, and GF109203X were purchased from Biomol (Hamburg, Germany). Human CNTF and murine IL-6, IL-1ß, and OSM were from R&D Systems (Minneapolis, MN). U0126 was purchased from Promega (Mannheim, Germany).

Antibody against phosphorylated ERK (pERK)1 and pERK2 was from Cell Signaling, Cummings Center (Beverly, MA). Antibody against ERK1/ERK2 was from Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish peroxidase-conjugated secondary antibody to rabbit IgG and chemiluminescent substrate SuperSignal West Dura extended duration substrate were from Pierce Chemical Co. (Rockford, IL).

Cell culture
TtT/GF cells were cultured in DMEM medium supplemented with 10% fetal calf serum, 2 mM glutamine, 1% penicillin/streptomycin, and 0.4% amphotericin B in a 5% CO2 atmosphere. For the experiments, the cells were plated in 100-mm dishes at a density of approximately 1.6 x 106 cells. They were grown for 48 h in complete medium, followed by serum depletion (medium with 0.2% BSA) for a further 20 h. Then stimulatory agents were added with fresh serum-depleted medium and samples were collected at appropriate time points.

RT-PCR and sequencing
RNA extraction was performed using TRIZOL reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Reverse transcription (RT) of 1.0 µg total RNA derived from folliculostellate TtT/GF cells or murine pituitary tissue was performed with Muloney murine leukemia virus-reverse transcriptase (Invitrogen, Karlsruhe, Germany) following the manufacturer’s recommendations. PCR of 2 µl of cDNA sample was performed on a GeneAmp PCR System 2400 (PerkinElmer, Cologne, Germany) in a total reaction volume of 50 µl with 10% GeneAmp 10 x PCR buffer (PerkinElmer) containing 15 mM MgCl2, 5% dimethylsulfoxide, 0.2 mM of each deoxynucleotide triphosphate, 1.25 U AmpIITaq DNA polymerase (PerkinElmer), and 100 pmol of sense and antisense primers (Thermo Hybaid Biotechnologie, Ulm, Germany). 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 the reaction was terminated by a single elongation step at 72 C for 7 min.

A 479-nt fragment of the murine NNT-1/BSF-3 cDNA (nt 259–738, GenBank accession no. AF176913.1) was generated with a specific primer pair (sense 5'-CAGCTTAGCTGGGACCTACCTG-3' and antisense 5'-GAAGCTGCTGGAGGCTGCATC-3') at an annealing temperature of 58 C.

A 352-nt fragment of the murine CLF-1 cDNA (nt 208–560, GenBank accession no. XM_147360) was created using a specific primer pair (sense 5'-AGCAGTCAGGAGACAATCTG-3' and antisense 5'-GAGGACATCAGATCTTGCTG-3') at an annealing temperature of 58 C.

A 455-nt fragment of the murine c-fos cDNA (nt 716-1171, GenBank accession no. NM_010234) was created using a specific primer pair (sense 5'-CTGGAGTTTATTTTGGCAGCC-3' and antisense 5'-GGTGAAGACAAAGGAAGACG-3') at an annealing temperature of 60 C.

To identify PACAP-1 receptor (PAC1-R) mRNA, a specific primer pair was synthesized (sense 5'-CTTGTACAGAAGCTGCAGTCCCCAGACATG-3' and antisense 5'-CCGGTGCTTGAAGTCCATAGTGAAGTAACGGTTCACCTT-3') with sequences present in all splice variants of rat PAC1-R cDNA. As described by Zhou et al. (13), this primer pair generates a 303-nt fragment corresponding to the short variant PAC1-Rs, a 384-nt fragment corresponding to PAC1-R-hop2, a 387-nt fragment corresponding to PAC1-R-hop1 or PAC1-R-hip and a 471-nt fragment corresponding to PAC1-R-hip-hop1 (13) at an annealing temperature of 64 C. Because PAC1-R-hop1 and PAC1-R-hip have the same size cassette (84 nt), specific sense primers were synthesized based on the sequences of the hop (5'-TGCAGAAATGCTACTGCAAGCCACAGCC-3') and hip (5'-TAAGACTGAGAGTCCCCAAGAAAACCCG-3') cassette (13). These sense primers paired with the antisense PAC1-R primer generates a 324-nt fragment corresponding to the PAC1-R-hop1 (or hop2) and a 324-nt fragment corresponding to the PAC1-R-hip (or 408-nt fragment corresponding to the PAC1-R-hip-hop1), respectively (13).

A 298-nt fragment of the murine VIP/PACAP-1-receptor (VPAC1-R) (sense 5'-GCCCCCATCCTCCTCTCCATC-3', antisense 5'-TCCGCCTGCACCTCACCATTG-3') and a 296-nt fragment of the murine VIP/PACAP-2 receptor (VPAC2-R) (sense 5'-GTCAACTTTGCCCTCTCCATCA-3', antisense 5'-GCCTCTCCACCTTCTTTTCAGT-3') (14) were generated at an annealing temperature of 62 and 60 C, respectively.

Electrophoresis of PCR products was performed on a 1.2% agarose gel and specific bands were gel purified by Quiaex (Qiagen, Hilden, Germany). Cycle sequencing was performed by Medigenomix (Munich, Germany).

Templates for probes and Northern blot analysis
Probe templates were generated by repeatedly performed PCR from the 479-, 352-, and 455-nt fragments of murine NNT-1/BSF-3, CLF-1, and c-fos (described above) inserted into pCR 2.1 vectors (Invitrogen, Carlsbad, CA). The ß-actin DECA probe template was a 1.076-kb fragment of the mouse ß-actin gene (Ambion, Cambridgeshire, UK).

RNA extraction was performed using TRIZOL reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Northern blot analysis was performed as described recently (12). In brief, 20 µg total RNA were electrophoresed on a 1% agarose, 6.4% formaldehyde gel, transferred to a Hybond-N+ membrane (Amersham-Pharmacia, Freiburg, Germany), and UV-cross-linked. Probes were labeled with ({alpha}-32P)CTP using the DECAprimeII DNA labeling kit (Ambion). QIAquick nucleotide removal kit (Qiagen) was used to clean the probe. Prehybridization and hybridization of the membranes were performed using QuickHyb solution (Stratagene Europe, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Membranes were exposed to Biomax MS films (Kodak, Rochester, NY) for 1–4 d at -70 C.

Protein extraction and Western blotting
For Western blot experiments, approximately 1.6 x 106 cells were seeded in 100-mm dishes and grown for 48 h in 5 ml complete medium, followed by incubation with serum-free medium (DMEM with 0.2% BSA) for 20 h. Then, stimulatory agents were added with fresh serum-depleted medium, and the samples were collected at appropriate 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 2% nonfat dry milk in PBS-T buffer (0.05 M sodium phosphate, 0.15 M NaCl, 0.1% Tween 20, 0.01% sodium azide). The blocked membranes were incubated overnight with antibody against pERK1/ERK2 (1:40,000; in 2% nonfat dry milk in PBS-T buffer). After washing with PBS, the membranes were incubated with a peroxidase-conjugated secondary antibody (goat antirabbit, 1:25,000 each) for 2 h (2% nonfat dry milk in PBS-T buffer, without sodium azide). 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 antibody to ERK1/ERK2 (1:40,000) were performed as described above.

Statistical analysis
National Institutes of Health Image 1.59 software was used for densitometric analysis of specific bands in Northern and Western blots, and statistical analysis was performed with JMP 5.0.1 software. The data are expressed as the mean ± SE of independently performed experiments. The mean of a single treatment group was compared with the mean of a single control group with t test and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of NNT-1/BSF-3 and CLF-1 mRNA in TtT/GF cells and mouse pituitary tissue
Using RT-PCR, expression of both NNT-1/BSF-3 and CLF-1 was detected in murine folliculostellate TtT/GF cells and pituitary tissue of C57BI6 mice (Fig. 1Go). A 479-nt fragment of the murine NNT-1/BSF-3 cDNA (nt 259–738, GenBank accession no. AF176913.1) and a 352-nt fragment of the murine CLF-1 cDNA (nt 208–560, GenBank accession no. XM_147360) were generated. Expected bands were gel purified, cloned into pCR2.1 vectors, and full length sequenced.



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  		FIG. 1. Expression of NNT-1/BSF-3 and CLF-1 mRNA in murine folliculostellate TtT/GF cells and murine pituitary tissue. RT-PCR was repeatedly performed from three different RNA samples. RT+, with RT; RT-, without RT. A 479-nt fragment of the murine NNT-1/BSF-3 and a 352-nt fragment of the murine CLF-1 cDNA were generated and verified by sequencing.

 
Fetal calf serum induces NNT-1/BSF-3 mRNA expression in TtT/GF cells
Incubation of serum-starved TtT/GF cells with fetal calf serum (0, 1, 2, 4, and 8%, respectively) showed a concentration-dependent induction of NNT-1/BSF-3 mRNA peaking at 2 h (Fig. 2Go).



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  		FIG. 2. Time- and dose-dependent effects of fetal calf serum on NNT-1/BSF-3 and CLF-1 mRNA expression in TtT/GF cells. TtT/GF cells were serum starved for 20 h and subsequently treated with 1, 2, 4, and 8% fetal calf serum. Total RNA was extracted at the time points indicated. NNT-1/BSF-3 mRNA expression was determined by Northern blot technique. Subsequently, the membranes were stripped and reblotted with a specific probe for CLF-1. Equal loading of each band was verified by subsequent reblotting for ß-actin. A representative experiment of two independently performed experiments is shown (lower panel). Using densitometric analysis, the ratio of NNT-1:BSF-3 mRNA vs. ß-actin mRNA was calculated for each treatment group. The NNT-1:BSF-3 / ß-actin ratio of the 1-h control group was set as 1.0. Relative NNT-1:BSF-3 mRNA expression levels of all other groups were calculated in comparison with this basal control group. Shown are mean values ± SE of two independently performed experiments (upper panel).

 
Bu2cAMP and PMA induce NNT-1/BSF-3 mRNA expression in TtT/GF cells
The PKA stimulus Bu2cAMP (5 mM) induced NNT-1/BSF-3 mRNA expression 2.5 ± 0.4-fold at 1 h (n.s.), 4.3 ± 1.5-fold (P < 0.05) at 2 h, 4.3 ± 3.3-fold (P < 0.05) at 4 h, and 6.9 ± 4.9-fold (P < 0.001) at 6 h, respectively (Fig. 3AGo). In a dose-response curve (0.01 to 10 mM), only concentrations of 5 and 10 mM Bu2CAMP significantly stimulated NNT-1/BSF-3 expression around 7-fold above baseline (Fig. 3BGo).



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  		FIG. 3. Time- and dose-dependent effects of Bu2cAMP on NNT-1/BSF-3 and CLF-1 mRNA expression in TtT/GF cells. TtT/GF cells were serum starved for 20 h and subsequently treated with Bu2cAMP in various concentrations. Total RNA was extracted at the time points indicated. NNT-1/BSF-3 mRNA expression was determined by Northern blot technique. Subsequently, the membranes were stripped and reblotted with a specific probe for CLF-1. Equal loading of each band was verified by subsequent reblotting for ß-actin. The ratio of NNT-1/BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1/BSF-3/ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A, Time-dependent effects of Bu2cAMP (5 mM) from 0.5 to 24 h. A representative experiment of three independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of three independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05; ***, P < 0.001. B, Dose-dependent effects of 10–10000 µM Bu2cAMP. TtT/GF cells were treated with the indicated Bu2cAMP concentrations for 5 h. A representative experiment of two independently performed experiments is shown.

 
The PKC stimulus PMA (100 nM) induced NNT-1/BSF-3 mRNA expression 5.1 ± 3-fold (P < 0.05) at 2 h, 8.7 ± 7-fold (P < 0.001) at 4 h, and 5.1 ± 2.5-fold (P < 0.05) at 6 h, respectively (Fig. 4AGo). In a dose-response curve (0.1 to 1000 nM) PMA at concentrations as low as 10 nM up to 1000 nM dose-dependently stimulated NNT-1/BSF-3 expression. NNT-1/BSF-3 expression peaked 30-fold above baseline at a concentration of 1000 nM PMA (Fig. 4BGo).



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  		FIG. 4. Time- and dose-dependent effects of PMA on NNT-1/BSF-3 and CLF-1 mRNA expression in TtT/GF cells. Inhibition of PMA-induced NNT-1/BSF-3 mRNA expression by dexamethasone. TtT/GF cells were serum starved for 20 h and subsequently treated with PMA in various concentrations. Total RNA was extracted at the time points indicated. NNT-1/BSF-3 mRNA expression was determined by Northern blot technique. Subsequently, the membranes were stripped and reblotted with a specific probe for CLF-1. Equal loading of each band was verified by subsequent reblotting for ß-actin. The ratio of NNT-1/BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1/BSF-3:ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A, Time-dependent effects of PMA (100 nM) from 0.5 to 24 h. A representative experiment of three independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of three independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05; ***, P < 0.001. B, Dose-dependent effects of 0.1–1000 nM PMA. TtTGF cells were treated with the indicated PMA concentrations for 4 h. A representative experiment of two independently performed experiments is shown. C, TtT/GF cells were treated with 10-7 M dexamethasone for 1 h before stimulation with 100 nM PMA for 4 h. A representative experiment of three independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of three independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05.

 
Dexamethasone inhibits PMA-induced NNT-1/BSF-3 expression in TtT/GF cells
Pretreatment of TtT/GF cells with the synthetic glucocorticoid dexamethasone (10-7 M) for 1 h completely abolished PMA-induced NNT-1/BSF-3 expression. PMA alone caused a 3.2 ± 2-fold induction of NNT-1/BSF-3 (P < 0.05). Pretreatment with dexamethasone significantly reduced PMA-induced NNT-1/BSF-3 to 0.9 ± 0.5-fold (not significantly different from baseline) (Fig. 4CGo).

PACAP-38 induces NNT-1/BSF-3 mRNA expression in TtT/GF cells
PACAP-38 (50 nM) increased NNT-1/BSF-3 mRNA expression 5.0 ± 3-fold at 2 h (n.s.), 8.6 ± 7.1-fold (P < 0.01) at 4 h, 7.1 ± 6.3-fold (P < 0.05) at 6 h, and 6.6 ± 6.5-fold (P < 0.05) at 24 h, respectively (Fig. 5AGo). In a dose-response curve 1–100(1–100,000 pM) concentrations of 10 and 100 nM PACAP potently stimulated NNT-1/BSF-3 expression 5.6-fold and 7.3-fold above baseline (Fig. 5BGo).



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  		FIG. 5. Time- and dose-dependent effects of PACAP-38 on NNT-1/BSF-3 and CLF-1 mRNA expression in TtT/GF cells. TtT/GF cells were serum starved for 20 h and subsequently treated with PACAP-38 in various concentrations. Total RNA was extracted at the time points indicated. NNT-1/BSF-3 mRNA expression was determined by Northern blot technique. Subsequently, the membranes were stripped and reblotted with a specific probe for CLF-1. Equal loading of each band was verified by subsequent reblotting for ß-actin. The ratio of NNT-1/BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1/BSF-3:ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A, Time-dependent effects of PACAP-38 (50 nM) from 0.5 to 24 h. A representative experiment of three independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of three independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05; ***, P < 0.001. B, Dose-dependent effects of 1–100,000 pM PACAP-38. TtT/GF cells were treated with the indicated PACAP-38 concentrations for 4 h. A representative experiment of two independently performed experiments is shown.

 
Inhibitors H-89, GF109203X, and H-7 decrease PKA- and PKC-induced NNT-1/BSF-3 expression in TtT/GF cells
At first we tested whether pretreatment (2 h before stimulation) of the TtT/GF cells with the PKA inhibitor H-89 (50 µM) and the PKC inhibitor GF109203X (50 µM) would inhibit the effect of the respective inducers Bu2cAMP and PMA. Although the Bu2cAMP-induced NNT-1/BSF-3 mRNA increase was reduced to 64% by H-89 pretreatment, the PMA-induced NNT-1/BSF-3 mRNA increase was reduced to 20% by GF109203X pretreatment (Fig. 6AGo).



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  		FIG. 6. Effects of the PKA inhibitor H-89, the PKC inhibitor GF109203X, and the PKA and PKC inhibitor H-7 on NNT-1/BSF-3 mRNA expression. TtT/GF cells were treated with H-89 (50 µM) or GF109203X (50 µM) for 2 h or H-7 (20 µM) for 1 h before stimulation with Bu2cAMP (5 mM), PMA (100 nM), or PACAP-38 (50 nM) for 4 h. Total RNA was extracted and NNT-1/BSF-3 mRNA was detected with Northern blot technique. The ratio of NNT-1/BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1/BSF-3:ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A, Inhibition of Bu2cAMP- and PMA-induced NNT-1/BSF-3 mRNA expression by H-89 and GF109203X, respectively. A representative experiment of two independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of two independently performed experiments is shown (upper panel). B, Inhibition of PACAP-38-induced NNT-1/BSF-3 mRNA expression by H-89 and GF109203X, respectively. A representative experiment of five independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of five independently performed experiments is shown (upper panel). C, Inhibition of PACAP-38-induced NNT-1/BSF-3 mRNA expression by H-7. A representative experiment of four independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of four independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05.

 
Next we examined the effect of both inhibitors on the PACAP-38-induced elevation of NNT-1/BSF-3 mRNA levels. As demonstrated in Fig. 6BGo, preincubation with H-89 (µM) caused only a modest reduction of the PACAP-induced NNT-1/BSF-3 mRNA increase to 73%, whereas GF109203X (50 µM) potently suppressed the PACAP-38-induced NNT-1/BSF-3 mRNA increase to 30%. Pretreatment with both inhibitors caused PACAP-38-stimulated NNT-1/BSF-3 expression to be no longer significantly different from baseline. Preincubation (1 h before stimulation) with the PKA and PKC inhibitor H-7 (20 µM) more potently down-regulated PACAP-38-induced NNT-1/BSF-3 levels (3.7 ± 2.2-fold) to 1.3 ± 0.8-fold (no longer significantly different from baseline) (Fig. 6CGo).

VIP induces NNT-1/BSF-3 mRNA expression in TtT/GF cells
VIP (50 nM) increased NNT-1/BSF-3 mRNA expression 4.9 ± 3-fold at 2 h (n.s.), 6.3 ± 4.5-fold (P < 0.05) at 4 h, 8.2 ± 5.8-fold (P < 0.01) at 6 h, and 6.1 ± 3.9-fold (P < 0.05) at 24 h, respectively (Fig. 7AGo). In a dose-response curve 1–100(1–100,000 pM), concentrations of 10 nM and 100 nM VIP potently stimulated NNT-1/BSF-3 expression 11.9-fold and 16.5-fold above baseline (Fig. 7BGo).



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  		FIG. 7. Time- and dose-dependent effects of VIP on NNT-1/BSF-3 mRNA expression in TtT/GF cells. Inhibition of VIP-induced NNT-1/BSF-3 mRNA by the receptor antagonist PACAP 6–38. TtT/GF cells were serum starved for 20 h and subsequently treated with VIP in various concentrations. Total RNA was extracted at the time points indicated. Treatment with PACAP 6–38 (1 µM) was performed for 15 min before stimulation with VIP. NNT-1/BSF-3 mRNA expression was determined by Northern blot technique. Equal loading of each band was verified by subsequent reblotting for ß-actin. The ratio of NNT-1/BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1/BSF-3:ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A, Time-dependent effects of VIP (50 nM) from 0.5 to 24 h. A representative experiment of three independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of three independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05; **, P < 0.01. B, Dose-dependent effects of 1–100,000 pM VIP. TtT/GF cells were treated with the indicated VIP concentrations for 4 h. A representative experiment of two independently performed experiments is shown. C, Inhibition of VIP-induced NNT-1/BSF-3 mRNA by PACAP 6–38. TtT/GF cells were treated with 1 µM PACAP 6–38 for 15 min before stimulation with 10 nM VIP for 4 h. A representative experiment of four independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of four independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05; **, P < 0.01.

 
PACAP 6–38 inhibits VIP-induced NNT-1/BSF-3 expression in TtT/GF cells
Pretreatment of TtT/GF cells with the receptor antagonist PACAP 6–38 (1 µM) for 15 min potently down-regulated VIP-induced NNT-1/BSF-3 expression. VIP alone (10 nM; 4 h) caused a 4.4 ± 2.9-fold induction of NNT-1/BSF-3 (P < 0.01). Pretreatment with PACAP 6–38 significantly reduced VIP-induced NNT-1/BSF-3 to 1.7 ± 0.8-fold (not significantly different from baseline) (Fig. 7CGo).

Inhibition of VIP-induced NNT-1/BSF-3 expression in TtT/GF cells by H-89, GF109203X, and H-7
As demonstrated in Fig. 8Go, stimulation with VIP alone (50 nM) for 4 h induced NNT-1/BSF-3 expression 7.3 ± 1.2-fold (P < 0.001). Pretreatment with the PKA inhibitor H-89 (50 µM) and the PKC inhibitor GF109203X (50 µM) for 2 h significantly reduced VIP-induced NNT-1/BSF-3 mRNA increase to 64 and 16%, respectively. Preincubation with the PKA and PKC inhibitor H-7 (20 µM) for 1 h completely abolished VIP-induced NNT-1/BSF-3 expression.



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  		FIG. 8. Effects of the PKA inhibitor H-89, the PKC inhibitor GF109203X, and the PKA and PKC inhibitor H-7 on VIP-induced NNT-1/BSF-3 mRNA expression. TtT/GF cells were treated with H-89 (50 µM) or GF109203X (50 µM) for 2 h or H-7 (20 µM) for 1 h before stimulation with VIP (50 nM) for 4 h. Total RNA was extracted and NNT-1/BSF-3 mRNA was detected with Northern blot technique. The ratio of NNT-1/BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1/BSF-3:ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A representative experiment of three independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of three independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. ***, P < 0.001.

 
Involvement of the MAPK ERK1/2 in PACAP-38 and VIP-induced NNT-1/BSF-3 mRNA expression in TtT/GF cells
PACAP-38 (50 nM) induced ERK1/2 phosphorylation 4.7 ± 4.9-fold at 2 min (P < 0.05), 5.1 ± 1.8-fold (P < 0.05) at 5 min, 4.9 ± 3.1-fold (P < 0.05) at 10 min, and 2.2 ± 1.8-fold (n.s.) at 15 min, respectively (Fig. 9AGo). PACAP-38 alone caused a 4.5 ± 2.2-fold induction of NNT-1/BSF-3 (P < 0.05). Pretreatment with the MAPK kinase/ERK kinase (MEK) inhibitor U0126 reduced PACAP-induced NNT-1/BSF-3 to 2.4 ± 2.2-fold (not significantly different from baseline) (Fig. 9BGo).



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  		FIG. 9. Involvement of the MAPK pathway in the PACAP-38-induced NNT-1/BSF-3 mRNA expression. A, Time course of PACAP-38 effects on ERK1/2 phosphorylation. TtT/GF cells were incubated in the presence or absence of 50 nM PACAP-38, and protein extraction was performed at indicated time points. pERK1/2 was demonstrated by specific antibodies. Equal loading of each band was verified by the use of antibodies against total ERK1/2. Using densitometric analysis, the ratio of pERK1/2 protein vs. total ERK1/2 protein was calculated for each treatment group. The ratio of pERK1/2 vs. ERK1/2 of the 2-min control group was set as 1. Relative pERK1/2:ERK1/2 levels of all other groups were calculated in comparison with this control group. A representative experiment of four independently performed experiments is shown (lower panel). Relative ERK1/2 phosphorylation (mean values ± SE) of four independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05. B, TtT/GF cells were treated with 10 µM U0126 for 30 min before stimulation with 50 nM PACAP-38 for 4 h. Total RNA was extracted and NNT-1/BSF-3 mRNA was detected with Northern blot technique. The ratio of NNT-1:BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1/BSF-3:ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A representative experiment of four independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of four independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05.

 
Similar to PACAP-38, VIP (50 nM) also induced ERK1/2 phosphorylation 1.4 ± 0.7-fold at 2 min (n.s.), 3.9 ± 2.9-fold (P < 0.05) at 5 min, 3.9 ± 2.7-fold (n.s.) at 10 min, and 3 ± 1.8-fold (n.s.) at 15 min, respectively (Fig. 10AGo). Stimulation with VIP alone caused a 6.6 ± 4-fold induction of NNT-1/BSF-3 (P < 0.05). Pretreatment with U0126 reduced VIP-induced NNT-1/BSF-3 to 4.4 ± 2.7-fold (not significantly different from baseline) (Fig. 10BGo).



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  		FIG. 10. Involvement of the MAPK pathway in the VIP-induced NNT-1/BSF-3 mRNA expression. A, Time course of VIP effects on ERK1/2 phosphorylation. TtT/GF cells were incubated in the presence or absence of 50 nM VIP, and protein extraction was performed at indicated time points. pERK1/2 was demonstrated by specific antibodies. Equal loading of each band was verified by the use of antibodies against total ERK1/2. Using densitometric analysis, the ratio of pERK1/2 protein vs. total ERK1/2 protein was calculated for each treatment group. The ratio of pERK1/2 vs. ERK1/2 of the 2-min control group was set as 1. Relative pERK1/2:ERK1/2 levels of all other groups were calculated in comparison with this control group. A representative experiment of three independently performed experiments is shown (lower panel). Relative ERK1/2 phosphorylation (mean values ± SE) of three independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. *, P < 0.05. B, TtT/GF cells were treated with 10 µM U0126 for 30 min before stimulation with 50 nM VIP for 4 h. Total RNA was extracted and NNT-1/BSF-3 mRNA was detected with Northern blot technique. The ratio of NNT-1/BSF-3 mRNA vs. ß-actin mRNA was calculated using densitometric analysis for each treatment group. The NNT-1:BSF-3:ß-actin ratio of the control group was set as 1.0. Relative NNT-1/BSF-3 mRNA expression levels of all other groups were calculated in comparison with this control group. A representative experiment of four independently performed experiments is shown (lower panel). Relative NNT-1/BSF-3 mRNA expression (mean values ± SE) of four independently performed experiments is shown (upper panel). Significant differences vs. the control group are indicated with asterisks. **, P < 0.01.

 
CLF-1 mRNA expression
TtT/GF cells constitutively express abundant amounts of CLF-1 mRNA under basal conditions (Figs. 2–5GoGoGoGo). Incubation with fetal calf serum, Bu2CAMP, PMA, PACAP-38, or dexamethasone revealed no significant effect on the level of CLF-1 expression (Figs. 2–5GoGoGoGo).

Expression of PACAP and VIP receptors in TtT/GF cells and mouse pituitary tissue
Using RT-PCR, we examined the expression of PAC1-R splice variants in folliculostellate TtT/GF cells and mouse pituitary tissue (Fig. 11Go). Using the primer pair common to all splice variants of PAC1-R, we detected in both pituitary tissue and TtT/GF cells the 303-nt fragment corresponding to the short variant PAC1-Rs and a second larger fragment representing either the 384-nt fragment corresponding to PAC1-R-hop2, or the 387-nt fragment corresponding either to PAC1-R-hop1 or PAC1-R-hip (Fig. 11Go). To discriminate between PAC1-R-hop1 and PAC1-R-hip, we performed PCR with specific sense primers based on the sequence of the hop and hip cassette. Both pituitary tissue and TtT/GF cells express the PAC1-R-hop splice variant, whereas PAC1-R-hip was present only in murine pituitary tissue but not in TtT/GF cells.



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  		FIG. 11. Expression of PAC1-R splice variants, VPAC1-R, and VPAC2-R in murine pituitary tissue and folliculostellate TtT/GF cells. RT-PCR was repeatedly performed from three different RNA samples. RT+, with RT; RT-, without RT. From the bands generated, the 303-nt band corresponding to the PAC1-Rs, the 324-nt band corresponding to the PAC1-R-hop splice variant, the 324-nt band corresponding to the PAC1-R-hip splice variant, the 298-nt band corresponding to the VPAC1-R, and the 296-nt band corresponding to the VPAC2-R were verified by sequencing.

 
A 296-nt fragment of the murine VPAC2-R was detected in both pituitary tissue and TtT/GF cells, whereas the 298-nt fragment of the murine VPAC1-R was consistently generated only in murine pituitary tissue. Expected bands were gel purified and full length sequenced.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that murine pituitary folliculostellate TtT/GF cells exhibit regulated expression of the novel gp130 cytokine NNT-1/BSF-3 by PACAP and VIP, two peptides belonging to the secretin/VIP/glucagon family. Furthermore, we show that NNT-1/BSF-3 expression is induced by PKC-, PKA-, and ERK1/2-dependent pathways and inhibited by dexamethasone.

The novel cytokine NNT-1/BSF-3 belonging to the gp130 family of cytokines has only recently been identified (3, 4). NNT-1/BSF-3 mRNA expression has been reported in lymph nodes, spleen, peripheral lymphocytes and leukocytes, bone marrow, liver, lung, ovary, uterus, and placenta (3, 4). However, no data on the regulation of NNT-1/BSF-3 expression have been published so far. Experimental work has mainly been focused on NNT-1/BSF-3 signal transduction and its pluripotent properties in several target tissues. NNT-1/BSF-3 supports the survival of chicken embryo motor and sympathetic neurons (3), induces astrocyte differentiation of fetal neuroepithelial cells (15), and has potent effects on B cell function (3, 16). NNT-1/BSF-3 increases serum corticosterone levels in mice in vivo (3), and we recently demonstrated NNT-1/BSF-3 to be a potent STAT-dependent activator of pituitary corticotroph function in vitro (12).

In search of possible physiological sources of NNT-1/BSF-3 in the pituitary, we examined the expression of NNT-1/BSF-3 in the folliculostellate cell line TtT/GF. TtT/GF is an agranular folliculostellate cell line, established from a murine thyrotropic pituitary tumor (17). TtT/GF cells possess most characteristics of folliculostellate cells, like stellate appearance, phagocytotic activity, and localization of GFAP and S-100 protein (17). Studies have previously suggested that folliculostellate cells may have a paracrine influence on hormone production of endocrine pituitary cells; several agents like LPS (18), calcitonin (19), and cytokines like IL-1 (20) and TNF{alpha} (21) enhance IL-6 production from folliculostellate cells, which controls the secretion of several hormones from the anterior pituitary (22, 23). RT-PCR revealed constitutive NNT-1/BSF-3 expression in TtT/GF cells and normal pituitary (Fig. 1Go). The NNT-1/BSF-3 cDNA product derived by RT-PCR was found to be consistently more prominent in mRNA samples derived from TtT/GF cells in comparison with murine pituitary tissue. Although RT-PCR is not a quantitative method, this finding indicates folliculostellate cells to be a major source of NNT-1/BSF-3 in the pituitary. Furthermore, we were able to detect constitutive NNT-1/BSF-3 expression in TtT/GF cells by quantitative Northern blot technique and consecutively tested the regulation pattern of this cytokine. Increasing concentrations of fetal calf serum were able to induce NNT-1/BSF-3 expression in a dose-dependent manner (Fig. 2Go), thus implicating that NNT-1/BSF-3 expression can be up-regulated by distinct factors. Preliminary studies (time-course experiments with 0.5, 1, 2, 4, 6, and 24 h) suggested no significant effects on folliculostellate NNT-1/BSF-3 expression for IL-1, interferon-{alpha}, leukemia inhibitory factor, OSM, 17ß-estradiol, CRH, vasopressin, and angiotensin II (data not shown). Only minor effects on folliculostellate NNT-1/BSF-3 expression were observed for TNF{alpha}, LPS, and IL-6 (data not shown). Biological activity of IL-6 was proven by reblotting the membranes with a specific c-fos probe, which showed maximal c-fos expression at 30 min (data not shown). Although IL-6 is an important mediator of folliculostellate function and both PACAP and IL-6 have been shown to regulate vascular endothelial growth factor release in pituitary folliculostellate cells (24), our data demonstrate IL-6 to be not significantly involved in the regulation of NNT-1/BSF-3 expression.

The PKA stimulus Bu2cAMP (Fig. 3Go) and the PKC stimulus PMA (Fig. 4Go) potently induced NNT-1/BSF-3 mRNA expression in TtT/GF cells. The effects of these two stimuli could be decreased by the PKA inhibitor H-89 and the PKC inhibitor GF109203X to 64 and 20%, respectively (Fig. 6AGo). The only partial inhibition of Bu2cAMP-induced NNT-1/BSF-3 expression by H-89 might be due to the experimental setup, although a partial action of Bu2cAMP being mediated by the butyrate rather than the cAMP component cannot be totally excluded.

PACAP and VIP are pleiotropic neuropeptides of the glucagon/secretin/VIP family (25, 26, 27). Both PACAP-38 and VIP are potent inducers of folliculostellate NNT-1/BSF-3 mRNA expression (Figs. 5Go and 7Go). PACAP- and VIP-induced NNT-1/BSF-3 expression is mediated by PKA- and PKC-dependent pathways, as could be demonstrated by specific inhibitors (Figs. 6Go and 8Go).

There exist several types of PACAP and VIP receptors. The PACAP type 1 receptor PAC1-R has been reported to exhibit a 2- to 3-fold higher affinity for PACAP than for VIP, whereas the PACAP type 2 receptors VPAC1-R and VPAC2-R have been reported to exhibit similar affinity for both neuropeptides (27). TtT/GF cells have been shown to express type 1 PACAP receptors (28), and PACAP has been demonstrated to regulate IL-6 transcription and enhance IL-6 secretion from these cells (29, 30).

However, the PAC1-R exists in several splice variants (presence or absence of amino acid cassettes in the third intracellular loop), which couple to different signal transduction pathways (27, 31, 32). The short PAC1-Rs and the hop variants (PAC1-R-hop1 and PAC1-R-hop2) are coupled to both PKA and PKC signaling with similar EC50 values for stimulation of intracellular cAMP and inositol phosphate production. In contrast, the hip cassette (PAC1-R-hip) exhibits inhibitory properties and reduces adenylate cyclase activity and phospholipase C activation (31). Therefore, we examined the expression of all PAC1-R variants by RT-PCR with the use of specific primer pairs.

Expression of the short and the hop variant but not the hip variant of the PAC1-R was found in folliculostellate TtT/GF cells. In contrast, all three variants including the inhibitory hip variant were found in murine pituitary tissue (Fig. 11Go). Expression of VPAC1-R and VPAC2-R, which activate primarily the adenylate cyclase system (31), was also confirmed in pituitary tissue, whereas in TtT/GF cells we consistently detected only the VPAC2-R (Fig. 11Go).

Preincubation of TtT/GF cells with the PACAP and VIP receptor antagonist PACAP 6–38 inhibited only the effect of VIP on NNT-1/BSF-3 mRNA expression (Fig. 7CGo). In contrast, we could not observe a significant effect of PACAP 6–38 on PACAP-38 stimulated NNT-1/BSF-3 expression (not shown). Although PACAP 6–38 is mainly considered a PAC1 and VPAC2 receptor antagonist (33), studies have demonstrated PACAP 6–38 to more potently inhibit VIP, compared with PACAP-induced effects (34, 35), possibly due to incomplete inhibition in particular cellular systems.

Involvement of PKA and PKC pathways in both PACAP- and VIP-induced NNT-1/BSF-3 mRNA expression was demonstrated by the use of the PKA inhibitor H-89, the PKC inhibitor GF109203X, and the PKA and PKC inhibitor H-7 (Figs. 6Go and 8Go).

In our study, PMA was a major stimulus of NNT-1/BSF-3 expression (Figs. 4Go and 6AGo). The potency of the PKC inhibitor GF109203X (Figs. 6Go and 8Go) and the expression of the hop but not the hip variant of the PACAP type I receptor (Fig. 11Go) suggest PKC to be a major signaling modus involved in PACAP- and VIP-induced NNT-1/BSF-3 expression of TtT/GF cells. Whether the involvement of PKC in VIP-induced NNT-1/BSF-3 is based on particular G protein subunit expression or on a more distant adenylate cyclase-phospholipase C pathway interconnection still remains to be elucidated.

Because PACAP and VIP signaling involves the Raf-MEK-ERK1/2 cascade in the pituitary cell line GH4C1 (36), we examined ERK1/2 phosphorylation and the effects of the MEK inhibitor U0126 in PACAP- and VIP-induced NNT-1/BSF-3 expression in pituitary folliculostellate TtT/GF cells. Both neuropeptides rapidly induced ERK1/2 phosphorylation, peaking at 5 min (Figs. 9AGo and 10AGo) and pretreatment of TtT/GF cells with the MEK inhibitor U0126 significantly reduced the stimulatory effect of PACAP and VIP (Figs. 9BGo and 10BGo). These data suggest ERK1/2 to be another important signaling modus in PACAP- and VIP-induced NNT-1/BSF-3 expression in TtT/GF cells.

It has been demonstrated that PACAP-induced p38 MAPK activation in PC12 cells is dependent on mobilization of intracellular Ca2+ stores and Ca2+ influx through voltage-dependent Ca2+ channels (37). To examine a putative role of Ca2+ influx on folliculostellate NNT-1/BSF-3 expression, we tested the effect of the Ca2+ channel blocker D-600 on PACAP and VIP-induced NNT-1/BSF-3 expression. Preliminary data (not shown) did not suggest a significant involvement of Ca2+ influx on NNT-1/BSF-3 expression; however, putative direct effects of mobilization of intracellular Ca2+ on ERK1/2 phosphorylation and/or NNT-1/BSF-3 mRNA expression still remain to be elucidated.

Because folliculostellate TtT/GF cells express functional glucocorticoid receptors (38), we examined the effect of the synthetic glucocorticoid dexamethasone on PMA-induced NNT-1/BSF-3 mRNA expression. Dexamethasone treatment almost completely abolished PMA-induced NNT-1/BSF-3 mRNA expression from folliculostellate cells (Fig. 4CGo). Similarly, dexamethasone has been reported to inhibit the release of vascular endothelial growth factor or IL-6 by folliculostellate cells in vitro (18, 24, 39). NNT-1/BSF-3 derived from folliculostellate cells might contribute to hypothalamus-pituitary-adrenal axis activation as a paracrine neuroimmunoendocrine modulator (12). Glucocorticoids, however, might down-regulate folliculostellate NNT-1/BSF-3 expression in a negative feedback loop.

CLF-1 was described as a novel member of the cytokine type I receptor family (40). Cellular secretion of NNT-1/BSF-3 has been suggested to require complex formation with either CLF-1 or sCNTFR{alpha} (5, 6, 7, 8). However, NNT-1/BSF-3 transgenic mice secrete NNT-1/BSF-3 without requirement of co-overexpression of CLF-1 or sCNTFR, respectively (16). We observed abundant CLF-1 mRNA expression in TtT/GF cells using Northern blot technique (Figs. 2–5GoGoGoGo), but no regulated expression pattern was observed under the stimuli used. Hence, in TtT/GF cells, CLF-1 may be constitutively expressed in high levels and might complex with NNT-1/BSF-3 when necessary to facilitate its cellular secretion.

Both PACAP and VIP have been reported to be expressed in the hypothalamus as well as the pituitary and both peptides can be transported from the hypothalamus through the hypophyseal portal vein system to the pituitary (27, 41, 42). For both peptides, stimulation of pituitary hormone secretion from various pituitary cell types has been demonstrated (27, 41, 42). PACAP and VIP have a direct effect on proopiomelanocortin expression and ACTH secretion in the murine corticotroph AtT20 cell line (43, 44, 45). On the other hand, our data and the findings of others (9) also propose indirect effects of PACAP/VIP on corticotroph function. For example, PACAP- and VIP-induced expression of NNT-1/BSF-3 or IL-6 (9) by pituitary folliculostellate cells may contribute to the regulation of corticotroph function in a paracrine manner.

In summary, the novel gp130 cytokine NNT-1/BSF-3 is expressed and differentially regulated in pituitary folliculostellate TtT/GF cells. The neuropeptides PACAP and VIP induce folliculostellate NNT-1/BSF-3 expression, mediated by PKA-, PKC-, and ERK1/2-dependent mechanisms. The glucocorticoid dexamethasone inhibits PMA-induced NNT-1/BSF-3 expression. NNT-1/BSF-3 is a potent modulator of pituitary corticotroph function (12). Due to these properties, NNT-1/BSF-3 appears as a novel paracrine modulator of pituitary function, derived from folliculostellate cells.


   Acknowledgments
 
We gratefully acknowledge skillful technical support by G. Spoettl and J. Meinecke.


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

This work contains parts of the unpublished doctoral thesis of K. Zitzmann at the Ludwig-Maximilians-University of Munich, Germany.

Abbreviations: Bu2cAMP, N6,2'-O-dibutyryladenosine-3',5'-cyclic-monophosphate; CLF-1, cytokine-like factor-1; CNTF, ciliary neurotrophic factor; LPS, lipopolysaccharide; MEK, MAPK kinase/ERK kinase; NNT-1/BSF-3, novel neurotrophin-1/B cell stimulating factor-3; OSM, oncostatin M; PACAP-38, pituitary adenylate cyclase-activating polypeptide; PAC1-R, type I PACAP receptor; pERK, phosphorylated ERK; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; RT, reverse transcription; sCNTFR{alpha}, soluble CNTF receptor-{alpha}; SDS, sodium dodecyl sulfate; STAT, signal transducer and activator of transcription; VIP, vasoactive intestinal peptide.

Received June 30, 2003.

Accepted for publication October 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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