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Endocrinology Vol. 142, No. 9 3909-3916
Copyright © 2001 by The Endocrine Society

ARTICLES

GH Inhibits Interferon-{gamma}-Induced Signal Transducer and Activator of Transcription-1 Activation and Expression of the Inducible Isoform of Nitric Oxide Synthase in INS-1 Cells

Nobuo Sekine, Seiji Fukumoto, Toshio Ishikawa, Tomoki Okazaki and Toshiro Fujita

Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo 113-0033, Japan

Address all correspondence and requests for reprints to: Dr. Nobuo Sekine, Department of Internal Medicine, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: nobuosek-tky{at}umin.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} and TNF{alpha} synergistically induce the inducible isoform of nitric oxide synthase and elicit severe cytotoxicity in pancreatic ß-cells. We demonstrate here that GH, the well known ß-cell mitogen, inhibits nitric oxide production by reducing inducible nitric oxide synthase gene induction by the two cytokines and counteracts their cytotoxic effect in insulin-secreting INS-1 cells. To elucidate the underlying mechanism, we examined activation of the transcription factors implicated in the induction of inducible nitric oxide synthase, signal transducer and activator of transcription-1, and nuclear factor-{kappa}B. GH inhibited tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription-1 promoted by interferon-{gamma}, whereas nuclear factor-{kappa}B activation by TNF{alpha} was not affected by GH. GH was found to induce suppressor of cytokine signaling-1 and -3, both of which are able to inhibit interferon-{gamma} activation of signal transducer and activator of transcription-1, suggesting that they are likely to mediate the inhibitory action of GH. Finally, exposure of INS-1 cells to interferon-{gamma} resulted in the impairment of insulin secretion in response to glucose, which was restored by the addition of GH. These results indicate that GH counteracts the effect of interferon-{gamma} through the inhibition of signal transducer and activator of transcription-1. This action of GH may be sufficient to suppress the synergistic induction of inducible nitric oxide synthase by interferon-{gamma} and TNF{alpha}, thereby preventing the cytotoxicity to ß-cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS KNOWN to exert a variety of effects on the growth and the function of insulin-secreting cells (1, 2, 3, 4, 5, 6, 7, 8, 9). GH promotes the growth and survival of adult (2) and neonatal (3) pancreatic ß-cells. GH also stimulates the biosynthesis of insulin and IGF-I in various types of insulin-secreting cells (4, 5, 6, 7). Moreover, a defined serum-free medium containing GH has been reported to maintain the function of rat pancreatic islet cells (8, 9), indicating that GH plays an essential role in ß-cell culture. Similar effects on ß-cells have been observed by the peptides that are structurally related to GH, such as PRL and placental lactogen (2, 9). It is thus speculated that the GH-related hormones or the signaling events activated by these peptides may play a major role in the development and maturation of pancreatic ß-cells.

Recent investigations on cytokine signaling have established the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway as the major signaling paradigm for a number of cytokines, including GH and PRL (10, 11). It has been shown that GH activates JAK2 followed by the activation of some STAT proteins (STAT1, STAT3, and STAT5) according to its target cell type (11). On the other hand, interferon-{gamma} (IFN{gamma}), an inhibitory cytokine for ß-cells (12), activates both JAK1 and JAK2, followed by the activation of STAT1 (10). It is well known that IFN{gamma}, especially in combination with other cytokines, such as IL-1ß and TNF{alpha}, elicits severe cytotoxicity in ß-cells (13, 14, 15). These cytokines are capable of producing nitric oxide (NO) by stimulating the expression of the inducible isoform of NO synthase (iNOS) in ß-cells, the event implicated in the ß-cell damage (16).

Considering the similarity in the signaling mechanisms, despite the opposing biological effects of GH and IFN{gamma} on ß-cells, we hypothesize that there may be an interaction between their signaling pathways. Thus in the present study we examined whether GH is capable of preventing the deleterious effect of IFN{gamma}, using a rat insulin-secreting cell line. INS-1 (17), the differentiated features of which have been well characterized (17, 18, 19). We have previously shown that GH and PRL, both of which stimulate cell growth and insulin biosynthesis, activate JAK2 in this cell line (9). Moreover, the effects of IFN{gamma} in INS-1 cells (20, 21) are consistent with those reported in other insulin-secreting cells, especially human islet cells (14, 22), in that IFN{gamma} alone inhibits glucose-induced insulin secretion and, in combination with TNF{alpha}, elicits ß-cell cytotoxicity.

We demonstrate here that GH counteracts the cytotoxic effect by IFN{gamma} and TNF{alpha} through the inhibition of NO production as well as of iNOS induction by these cytokines. Our results suggest that this effect is probably mediated by the inhibition of IFN{gamma}-activated STAT1 by GH, and that this inhibitory action of GH may be sufficient to abolish the synergism by IFN{gamma} and TNF{alpha}. We also show that GH induces the gene expression of suppressor of cytokine signaling (SOCS) proteins, the negative regulators of STAT proteins, which might mediate the inhibitory effect of GH on IFN{gamma}-induced STAT1 activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
RPMI 1640 and 2-mercaptoethanol were purchased from Life Technologies, Inc. (Gaithersburg, MD), and the other components of the culture medium, ovine PRL, NG-monomethyl-L-arginine (NMMA), and herbimycin A, were from Sigma-Aldrich Corp. (St. Louis, MO). Bovine GH, which binds specifically to GH receptors in ß-cells (23), was obtained from Biogenesis (Poole, UK). Recombinant murine IFN{gamma} was purchased from Genzyme Diagnostics (Cambridge, MA), and recombinant murine TNF{alpha} was obtained from R & D Systems (Funakoshi, Tokyo, Japan). Monoclonal antibodies against phosphorylated tyrosine (4G10) and STAT1 were from Upstate Biotechnology, Inc." (Lake Placid, NY), and specific cDNA probe for mouse macrophage iNOS was purchased from Cayman Chemical Co. (Ann Arbor, MI). [{alpha}-32P]Deoxy-CTP, [{gamma}-32P]ATP, and [methyl-3H]thymidine were obtained from Amersham International (Little Chalfont, UK). The One Step RT-PCR kit (avian myeloblastosis virus) was purchased from TaKaRa (Otsu, Japan).

Cell culture
INS-1 cells were cultured in the complete medium (CM) composed of RPMI 1640 medium supplemented with 10 mM HEPES, 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol as previously reported (17).

[3H]Thymidine incorporation
Cells (2 x 104 cells/microwell) were cultured in CM for 2 d and incubated for 72 h with 100 U/ml IFN{gamma} plus 50 ng/ml TNF{alpha} in the absence or presence of 5 nM GH. During the final 24 h, 0.5 µCi/well [methyl-3H]thymidine was added to the medium. The labeled cells were lysed in 0.1% SDS and precipitated in 20% trichloroacetic acid containing 0.8 mg/ml BSA. The pellet was resuspended in 0.2 M NaOH, and radioactivity was measured with a liquid scintillation counter.

Measurement of nitrite production
INS-1 cells were incubated for 48 h in a defined serum-free medium (SFM) composed of RPMI 1640 supplemented with 0.65 nM IGF-I, T3, ethanolamine, phosphoethanolamine, and 0.1% human albumin (19) in the absence or presence of 5 nM GH. Nitrite production in the culture medium was determined colorimetrically using a commercial Griess reagent assay kit (Dojindo Laboratories, Kumamoto, Japan). Optical density was measured using a microplate reader (model 3550, Bio-Rad Laboratories, Inc., Hercules, CA), and the absolute values were calculated according to the manufacturer’s instructions (Dojindo).

Northern blotting
INS-1 cells (107 cells/dish) were cultured in CM for 5 d before an overnight culture in a serum-free RPMI 1640 medium supplemented with 1% BSA. Cells were then incubated in SFM for 6 h with 100 U recombinant murine IFN{gamma} and/or 50 ng/ml recombinant murine TNF{alpha} in the absence or presence of 5 nM GH. Total RNA was extracted by the acid guanidium thiocyanate-phenol-chloroform method (24). Northern blotting was performed by a standard protocol using a specific cDNA probe for mouse macrophage iNOS labeled by Megaprime kit (Amersham Pharmacia Biotech). The blots were reprobed with 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as an internal standard to ensure equal loading of RNA.

Immunoprecipitation and Western blotting
Cells (107 cells/dish) were cultured as described for Northern blotting. After removal of the culture medium, cells were washed twice with the modified Krebs-Ringer bicarbonate-HEPES buffer (KRBH) containing 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM HEPES (pH 7.4), 2 mM NaHCO3, 0.1% BSA, and 3 mM glucose. Cells were then preincubated in the same buffer for 30 min and stimulated with the cytokines for 15 min at 37 C in the absence or presence of 5 nM GH. After two washes with PBS, whole cell lysates were prepared in the lysis buffer composed of 20 mM Tris-HCl (pH 7.4), 30 mM sodium fluoride, 30 mM sodium pyrophosphate, 2 mM EDTA, 2 mM EGTA, 10 µg/ml leupeptin, 2 µg/ml aprotinin, 1% Triton X-100, 1% deoxycholate, 1 mM phenylmethylsulfonylfluoride, and 2 mM sodium orthovanadate. Cell lysates were sonicated briefly and centrifuged at 12,000 x g for 60 min. For immunoprecipitation, supernatants were incubated with the monoclonal anti-STAT1 antibody by rotating end over end overnight at 4 C. The immune complex was then precipitated by incubation for 60 min at 4 C with protein G-Sepharose beads (Pharmacia Biotech Europe, Brussels, Belgium), and eluted by boiling in SDS-sample buffer for 5 min. All samples were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell, Inc., Dassel, Germany). After blocking with Tris-buffered saline containing 1% BSA for 120 min, the nitrocellulose membrane was incubated at 4 C overnight in the same buffer with monoclonal antibodies against phosphorylated tyrosine (4G10). Blots were washed, exposed to horseradish peroxidase-conjugated goat antimouse IgG (Sigma) for 90 min, and detected by the enhanced chemiluminescence kit according to the manufacturer’s instructions (Amersham Life Science, Little Chalfont, UK).

EMSA
INS-1 cells were cultured and stimulated as described for immunoprecipitation and Western blotting. Nuclear extracts were prepared from the cells as previously described (25). Two synthetic double-strand oligonucleotides were end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase (Takara, Japan) and used as probes. These represent the IFN{gamma}-activated site (GAS) in the iNOS gene promoter, 5'-CTTTTCCCCT-AACAC-3' (26), and the consensus nuclear factor-{kappa}B (NF-{kappa}B) binding sequence ({kappa}B site), 5'-GATCCC-AACGGCAGGGGAATTCCCCTCTCC-3' (27). EMSA was performed according to a method described previously (28). The probe (104 cpm) was incubated with 10 µg of the nuclear protein along with 1 µg poly(dI-dC)·poly(dI-dC) (Pharmacia Biotech) in each reaction for 30 min at room temperature. DNA-protein complexes were resolved on 4% nondenaturing acrylamide gels, dried, and visualized by autoradiography.

RT-PCR for the detection of SOCS mRNA
INS-1 cells were incubated and total RNA was extracted as described for Northern blotting. Total RNA (1 µg), which was treated by deoxyribonuclease I for 2 h, was reverse transcribed with avian myeloblastosis virus-derived reverse transcriptase. Sequences of the forward and reverse primers to detect rat SOCS proteins were: SOCS1, 5'-CCGCTCCCACTCTGATTACCG-3' and 5'-AGTGCTCCAGCAGCTCGAAGA-3'; SOCS2, 5'-GCGAGAGACTTTGCCATACCA-3' and 5'-AGAATCCAATCTGAATTTCCCG-3'; and SOCS3, 5'-ACCGTTGACAGTCTTCCGACA-3' and 5'-GCCTCAAGACCTTCAGCTCCA-3'. In addition, the expression of GAPDH was examined as a control using the following primers: GAP1, 5'-GGAGCCAAAAGGGTCATCATC-3'; and GAP2, 5'-AGAGGCAGGGATGATGTTCTG-3'. The PCR condition was one cycle at 94 C for 2 min; 27 cycles at 94 C for 30 sec, at 63 C for 30 sec, and at 72 C for 30 sec; and one cycle at 72 C for 7 min. PCR products were analyzed by 1% agarose gel electrophoresis.

Static incubation for determination of insulin secretion and the 3-(4,5-dimethylthiazol-2-yl)-2,5 tetrazolium bromide colorimetric assay
INS-1 cells (~5 x 104 cells/well) were seeded in 96-well microtiter plates coated with poly-L-ornithine (Sigma), cultured for 3 d in CM, and incubated for 24 h in the absence or presence of 100 U/ml IFN{gamma} and/or 5 nM GH. The culture medium was removed, and cells were washed twice with KRBH containing 3 mM glucose. Cells were then preincubated in the same buffer for 30 min at 37 C, followed by a 30-min incubation in KRBH containing 3 or 15 mM glucose. After the incubation, the 3-(4,5-dimethylthiazol-2-yl)-2,5 tetrazolium bromide colorimetric assay (18) was performed by incubating the cells for an additional 30 min. Cellular insulin content was measured by RIA after acid-ethanol extraction using rat insulin as standard.

Statistics
Results are presented as the mean ± SEM for the number of preparations given in parentheses. Statistical significance was determined by unpaired t test. In case of multiple comparisons, data were evaluated by one-way ANOVA followed by post-hoc Scheffé analysis. Differences between experimental and control groups were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of GH on DNA synthesis of INS-1 cells exposed to IFN{gamma} and TNF{alpha}
DNA synthesis was measured by the [3H]thymidine incorporation assay in INS-1 cells exposed to GH in the absence or presence of the combination of two cytotoxic cytokines, IFN{gamma} and TNF{alpha}. Addition of 5 nM GH to SFM resulted in an increase in [3H]thymidine incorporation by approximately 70% (Fig. 1Go). By contrast, the combination of 100 IU/ml IFN{gamma} and 50 ng/ml TNF{alpha} markedly decreased [3H]thymidine incorporation, whereas either cytokine alone had no effect (Fig. 1Go). When INS-1 cells were incubated with 5 nM GH in addition to the two cytokines, the inhibition of [3H]thymidine incorporation was partially (~60%) restored (Fig. 1Go).



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  		Figure 1. Effects of cytokines and GH on DNA synthesis in INS-1 cells. INS-1 cells were cultured for 72 h in SFM in the absence or presence of 5 nM GH with 100 U/ml IFN{gamma} and/or 50 ng/ml TNF{alpha} as indicated. During the final 24 h, 0.5 µCi/well [methyl-3H]thymidine was added, and radioactivity incorporated in the cells was measured with a liquid scintillation counter. Values are the mean ± SEM (with the mean value without cytokine as 100%) from three independent experiments performed in quadruplicate. Statistical analysis by ANOVA: *, P < 0.01 vs. no addition; #, P < 0.01 vs. IFN{gamma} plus TNF{alpha}; $, P < 0.01 vs. GH alone.

 
Effects of GH on NO production and iNOS mRNA expression in INS-1 cells induced by IFN{gamma} and TNF{alpha}
Exposure of INS-1 cells to the combination of 100 U/ml IFN{gamma} and 50 ng/ml TNF{alpha} for 48 h resulted in a marked increase in nitrite production, whereas neither cytokine alone affected nitrite production from INS-1 cells (Fig. 2Go). This effect was considered to be due to the synergistic induction of iNOS gene by the two cytokines (Fig. 3Go, lane b) (21). As neither IFN{gamma} nor TNF{alpha} alone induces the iNOS gene (21), nitrite production appeared to occur in parallel with the induction of iNOS mRNA. Addition of 5 nM GH significantly inhibited nitrite production stimulated by the two cytokines (Fig. 2Go). Similarly, GH, which by itself did not induce iNOS gene (Fig. 3Go, lane e), reduced the iNOS mRNA expression induced by the combination of IFN{gamma} and TNF{alpha} (Fig. 3Go, lane c). The cytokine-induced iNOS gene expression was also inhibited by a tyrosine kinase inhibitor, herbimycin A, indicating that protein tyrosine phosphorylation is involved in this effect (Fig. 3Go, lane d).



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  		Figure 2. Effects of GH on cytokine-induced nitrite production in INS-1 cells. INS-1 cells were incubated for 48 h with the combination of 100 U/ml IFN{gamma} plus 50 ng/ml TNF{alpha} in the absence or presence of 5 nM GH. Nitrite (NO2) accumulated in culture medium during the incubation period was determined colorimetrically using Griess agent. Note that nitrite production was remarkably inhibited by NMMA, the iNOS inhibitor. Values are the mean ± SEM from three independent experiments performed in quadruplicate. Statistical analysis by ANOVA: *, P < 0.01 vs. no addition; #, P < 0.01 vs. IFN{gamma} plus TNF{alpha}.

 


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  		Figure 3. Effects of GH on iNOS mRNA expression induced by the cytokines in INS-1 cells. INS-1 cells were incubated for 6 h with 100 U/ml IFN{gamma} plus 50 ng/ml TNF{alpha} in the absence (lane b) or presence of 5 nM GH (lane c) or 500 nM herbimycin A (lane d). Expression of iNOS mRNA was examined by Northern blotting using a specific cDNA probe for mouse macrophage iNOS. The expression of GAPDH mRNA is shown as an internal control. The effect of GH alone is shown in lane e. Similar results were obtained in at least three independent experiments.

 
Effects of GH on IFN{gamma}-induced tyrosine phosphorylation and DNA binding of STAT1 in INS-1 cells
STAT1 is believed to play a major role in the activation of IFN{gamma}-inducible genes (29), including iNOS (30). We therefore examined the effects of GH on the IFN{gamma}-induced activation of STAT1 in INS-1 cells by both immunoblotting as well as EMSA. GH itself did not induce tyrosine phosphorylation of STAT1 (not shown), but inhibited the STAT1 phosphorylation promoted by IFN{gamma} after a 15-min exposure (Fig. 4Go, lanes b and c). The inhibitory effect of GH appeared somewhat reversed by the addition of the JAK2 inhibitor tyrphostin B42 (31) (Fig. 4Go, lane d). The activation of STAT1 by IFN{gamma} was confirmed by EMSA, which showed the binding of nuclear proteins to iNOS GAS (Fig. 5AGo, lane b). Consistent with the alterations of STAT1 phosphorylation, GH reduced the DNA binding of STAT1 stimulated by IFN{gamma} in both the absence (Fig. 5AGo, lanes b and c) and presence (Fig. 5AGo, lanes d and e) of TNF{alpha}.



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  		Figure 4. Effects of GH on IFN{gamma}-induced tyrosine phosphorylation of STAT1 in INS-1 cells. Quiescent cells were stimulated in KRBH for 20 min with 100 U/ml IFN{gamma} in the absence or presence of 5 nM GH. Whole cell lysates were immunoprecipitated with monoclonal anti-STAT1 antibody, followed by immunoblotting with the monoclonal antiphosphotyrosine antibody (A, upper panel). Note that the effect of GH was reduced by the addition of a specific JAK2 inhibitor, tyrphostin B42 (TyrB42). The same nitrocellulose membrane was reblotted with the anti-STAT1 antibody (A, lower panel). B, The density of each signal was calculated using ImageQuant (bersion 1.1, Molecular Dynamics, Inc., Sunnyvale, CA), and was expressed as the fold increase compared with the control. Statistical analysis by ANOVA for IFN{gamma} and IFN{gamma} plus GH, P < 0.01. Similar results were obtained in three (for IFN{gamma} and IFN{gamma} plus GH) or two (for IFN{gamma}, GH, and TyrB42) independent experiments.

 


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  		Figure 5. Effects of GH on the cytokine-induced DNA binding of STAT1 (A) and NF-{kappa}B (B) in INS-1 cells. INS-1 cells were incubated for 15 min with 100 U/ml IFN{gamma} alone or together with 50 ng/ml TNF{alpha} in the absence or presence of 5 nM GH as indicated. DNA binding of nuclear extracts obtained from the treated cells was examined by EMSA using synthetic oligonucleotides representing iNOS GAS (A) or the consensus binding sequences for NF-{kappa}B (B). A, The STAT1 signal was confirmed by the presence of supershift complex containing anti-STAT1 antibody ({alpha}STAT1). B, Competition analysis was performed in the identical condition as lane b with a 100-fold excess of the unlabeled probes. The irrelevant oligonucleotides used as a negative control for the competition analysis was iNOS GAS. Representative results from four independent experiments are shown. NSB, Nonspecific band; FP, free probe.

 
Effect of GH on TNF{alpha}-induced DNA binding of NF-{kappa}B in INS-1 cells
We next examined by EMSA the effect of GH on the activation of NF-{kappa}B, another transcription factor that has been implicated in the iNOS induction (30). TNF{alpha} promoted the DNA binding of nuclear proteins to the consensus NF-{kappa}B binding site (Fig. 5BGo, lane b). In contrast to the DNA binding of STAT1, NF-{kappa}B binding was not affected by an addition of GH (Fig. 5BGo, lane c).

Effects of GH and IFN{gamma} on SOCS mRNA expression in INS-1 cells
Recent investigations have identified a new family of proteins that inhibit cytokine signaling (32, 33, 34). SOCS1 and SOCS3 are such proteins; their expression has been reported to be induced by GH in 3T3-F442 fibroblasts as well as in murine hepatocytes in vivo (35). Moreover, both SOCS1 and SOCS3 have been shown to inhibit IFN{gamma} signaling pathways in cells (36). We therefore examined the expression of the SOCS proteins, including SOCS2 in INS-1 cells, by RT-PCR to investigate whether the inhibitory effect of GH on the IFN{gamma}-induced STAT1 activation could be mediated by any of these proteins. After 30 min of incubation, GH induced the mRNA expression of all three SOCS proteins (Fig. 6Go, lane b). Interestingly, as shown by this semiquantitative analysis with RT-PCR, GH seemed to augment the SOCS1 induction by IFN{gamma} (Fig. 6Go, upper panel, lanes c and d). There was a weak induction of the SOCS2 mRNA both by GH (Fig. 6Go, middle panel, lane b) and IFN{gamma} (Fig. 6Go, middle panel, lane d); again, their effects appeared to be additive. On the contrary, GH (Fig. 6Go, lower panel, lane b) as well as IFN{gamma} (Fig. 6Go, lower panel, lane d) clearly increased the expression of SOCS3 mRNA, although, in contrast to SOCS1and SOCS2, there was no additive effect when cells were stimulated simultaneously with GH and IFN{gamma} (Fig. 6Go, lower panel, lane c).



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  		Figure 6. Induction of the SOCS gene by GH and IFN{gamma} in INS-1 cells. INS-1 cells were incubated for 30 min in the presence of 5 nM GH and/or 100 IU/ml IFN{gamma}. Total RNA was isolated and examined by RT-PCR for rat SOCS1, SOCS2, and SOCS3 genes as indicated. The results of experiments performed without RT [RT(-)] are also shown for each SOCS gene. Expression of GAPDH gene is shown as an internal control in the lowest panel. Representative results from two (for SOCS1 and SOCS2) or four (for SOCS3) independent experiments are shown. a, Control; b, GH; c, GH plus IFN{gamma}; d, IFN{gamma}.

 
Effect of GH on glucose-induced insulin secretion in INS-1 cells exposed to IFN{gamma}
The above findings suggest that GH counteracts the effect of IFN{gamma}, but not that of TNF{alpha}, in inhibiting NO production and DNA synthesis by these cytokines. We therefore examined the effect of GH on glucose-induced insulin secretion in INS-1 cells, which has been shown to be impaired by exposure to IFN{gamma} (21). Exposure of INS-1 cells to 100 U/ml IFN{gamma} for 24 h indeed resulted in approximately 50% reduction of insulin secretion in response to glucose (Fig. 7Go). Addition of 5 nM GH during the exposure to IFN{gamma} maintained the glucose responsiveness of INS-1 cells to secrete insulin (Fig. 7Go). The absolute values of released insulin were higher both basally (3 mM) and at the stimulating (15 mM) concentration of glucose compared with values with IFN{gamma} alone (Fig. 7Go). This might be due to the increase in cellular insulin content and/or to the enhanced glucose metabolism in the cell by the exposure to GH (9).



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  		Figure 7. Effect of GH on glucose-induced insulin secretion from INS-1 cells exposed to IFN{gamma}. INS-1 cells were cultured for 24 h with 100 U/ml IFN{gamma} in the absence () or presence ({blacksquare}) of 5 nM GH. After a 30-min preincubation in KRBH, cells were stimulated with 15 mM glucose. Values are the mean ± SEM (with a control value at 3 mM glucose without cytokine treatment as 100%) from three independent experiments performed in quadruplicate. Statistical analysis by ANOVA: *, P < 0.01 vs. 3 mM glucose without cytokine treatment; #, P < 0.01 vs. 15 mM glucose without cytokine treatment; $, P < 0.01 vs. 15 mM glucose with exposure to IFN{gamma}.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates for the first time that GH is capable of inhibiting the cytokine-induced NO production by reducing the expression level of iNOS mRNA in the insulin-secreting cell. The production of NO has been implicated in the cytotoxic effects of cytokines, such as IL-1ß, TNF{alpha}, and IFN{gamma}, on pancreatic ß-cells (12, 16). NO generated through the induction of iNOS in the cell may cause inhibition of mitochondrial metabolism, protein modification, and DNA cleavage (16, 37, 38), all of which could lead to ß-cell death. We show here that exposure of INS-1 cells to IFN{gamma} and TNF{alpha} resulted in a severe impairment of DNA synthesis in parallel with NO production, both of which were partially restored by the addition of GH. Thus, the protective effect of GH on INS-1 cells exposed to the cytokines could be explained at least partly by the inhibition of NO production.

It is well known that IFN{gamma}, which by itself has little effect on the iNOS expression, synergistically induces iNOS in combination with TNF{alpha} in various cell types, including ß-cells (13, 14, 15, 22). The induction of iNOS gene is regulated by cytokine-activated transcription factors that bind to each specific site in the promoter of the iNOS gene (30, 39). Activation of NF-{kappa}B is believed to be an essential step for the TNF{alpha}-induced iNOS expression (40, 41). It has been shown that the inhibition of NF-{kappa}B decreases the cytokine-induced expression of iNOS in insulin-secreting cells (21, 40). Similarly, the requirement of IFN{gamma}-activated transcription factors, interferon regulatory factor-1 (IRF-1) (42) as well as STAT1 (21), for the induction of iNOS by IFN{gamma} has been reported. The promoter of the mouse iNOS gene, in fact, contains binding sites for both NF-{kappa}B and IFN{gamma}-responsive transcription factors, including STAT1 (39). Interestingly, the activation of either NF-{kappa}B or STAT1 alone is insufficient, but that of both factors is required, for full induction of iNOS gene (21), suggesting that there may be interaction between their activation pathways. In this study we have shown that GH inhibited both tyrosine phosphorylation and DNA binding of STAT1, whereas DNA binding of NF-{kappa}B was not affected. It is therefore likely that the inhibitory effect of GH on iNOS gene expression is via the inhibition of STAT1 activation by IFN{gamma}, but not via that of NF-{kappa}B by TNF{alpha}.

To elucidate the mechanism mediating the inhibition of STAT1 by GH, we investigated the expression of SOCS proteins, the negative regulator of the JAK-STAT pathway (35, 36). A number of related proteins have recently been identified and also referred to as cytokine-inducible SH2-containing proteins (43), JAK2 binding protein (33), or STAT-induced STAT inhibitors (34). These proteins have been shown to bind directly to JAKs or to interact with tyrosine-phosphorylated STATs or cytokine receptors. Our results indicate that GH indeed up-regulates the expression of SOCS1, SOCS2, and SOCS3. Of these SOCS members, both SOCS1 and SOCS3, but not SOCS2, are capable of inhibiting STAT1 phosphorylation by IFN{gamma} (36, 44). It has been shown that SOCS1 efficiently inhibits IFN{gamma} activation of JAK1 and Tyk2 by directly binding these tyrosine kinases (36). On the contrary, SOCS3, which binds to receptor-specific sequences, does not strongly inhibit IFN{gamma} signaling (36). Moreover, the present study shows that GH appears to augment the SOCS1 mRNA expression induced by IFN{gamma}. It should therefore be reasonable to suppose that GH may inhibit IFN{gamma} activation of STAT1 mainly by enhancing the induction of SOCS1.

With respect to SOCS3, similar findings have recently been reported in monocytes, where IL-10 inhibits the expression of IFN{gamma}-induced genes by suppressing tyrosine phosphorylation of STAT1 (44). Expression of the SOCS3 gene, which is induced in response to IL-10, is considered to mediate this action. Moreover, lipopolysaccharide (LPS) also induces SOCS3, thereby inhibiting STAT1 activation by IFN{gamma} in Bac1.2F5 macrophages (45). In this context, the effect of LPS is interesting, because LPS decreases tyrosine phosphorylation of JAK1, but has no effect on JAK2. We have previously shown that GH clearly promotes JAK2 tyrosine phosphorylation, whereas IFN{gamma} fails to phosphorylate JAK2, but induces JAK1 phosphorylation in INS-1 cells (21). Taken together, it is also possible that SOCS3 activated by GH inhibits IFN{gamma}-induced JAK1 activation in INS-1 cells.

The counteraction of GH against the effect of IFN{gamma} is also demonstrated in this study for the restoration of glucose-induced insulin secretion. After a 24-h exposure to IFN{gamma}, INS-1 cells show blunted insulin secretion. This can be ascribed to impairment of glucose metabolism in the mitochondria (21), which generates metabolic coupling factors to elicit exocytosis of insulin granules (46). The underlying mechanism for this effect of IFN{gamma} remains unknown. The results of this study suggest that STAT1 may mediate this effect, and that the inhibition of STAT1 by GH could lead to the restoration of insulin secretory function. Alternatively, the action of GH that retains cellular metabolism in the ß-cell (9) might be related.

In conclusion, we demonstrate in this study a novel anticytokine effect of GH, which counteracts the induction of iNOS as well as the inhibition of insulin secretion by IFN{gamma} in ß-cells. This effect is probably mediated by the inhibition of IFN{gamma}-induced STAT1 activation through the induction of the SOCS proteins, especially SOCS1.


   Acknowledgments
 
We thank Prof. Claes B. Wollheim (University of Geneva, Geneva, Switzerland) for kindly providing INS-1 cells. The authors are indebted to R. Odajima for expert technical assistance and editorial help.


   Footnotes
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan 09671032 (to N.S.).

Abbreviations: CM, Complete medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAS, interferon-{gamma}-activated site; IFN{gamma}, interferon-{gamma}; iNOS, inducible isoform of nitric oxide synthase; IRF-1, interferon regulatory factor-1; JAK, Janus kinase; KRBH, Krebs-Ringer bicarbonate-HEPES buffer; LPS, lipopolysaccharide; NMMA, NG-monomethyl-L-arginine; NF-{kappa}B, nuclear factor-{kappa}B; NO, nitric oxide; SFM, defined serum-free medium; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

Received January 19, 2001.

Accepted for publication May 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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