The Effect of Suppressor of Cytokine Signaling 3 on GH Signaling in ß-Cells

Sif G. Rønn, Johnny A. Hansen, Karen Lindberg, Allan E. Karlsen and Nils Billestrup

Steno Diabetes Center (S.G.R., A.E.K., N.B.), DK-2820 Gentofte, Denmark; and Signal Transduction (S.G.R., J.A.H., K.L., N.B.), Novo Nordisk A/S, DK-2880 Bagsværd, Denmark

Address all correspondence and requests for reprints to: Nils Billestrup, Steno Diabetes Center, Niels Steensensvej 6, DK-2820 Gentofte, Denmark. E-mail: nbil{at}novonordisk.com.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH is an important regulator of cell growth and metabolism. In the pancreas, GH stimulates mitogenesis as well as insulin production in ß-cells. The cellular effects of GH are exerted mainly through activation of the Janus kinase-signal transducer and activator of transcription (STAT) pathway. Recently it has been found that suppressors of cytokine signaling (SOCS) proteins are able to inhibit GH-induced signal transduction. In the present study, the role of SOCS-3 in GH signaling was investigated in the pancreatic ß-cell lines RIN-5AH and INS-1 by means of inducible expression systems. Via stable transfection of the ß-cell lines with plasmids expressing SOCS-3 under the control of an inducible promoter, a time- and dose-dependent expression of SOCS-3 in the cells was obtained. EMSA showed that SOCS-3 is able to inhibit GH-induced DNA binding of both STAT3 and STAT5 in RIN-5AH cells. Furthermore, using Northern blot analysis it was shown that SOCS-3 can completely inhibit GH-induced insulin production in these cells. Finally, 5-bromodeoxyuridine incorporation followed by fluorescence-activated cell sorting analysis showed that SOCS-3 inhibits GH-induced proliferation of INS-1 cells. These findings support the hypothesis that SOCS-3 is a major regulator of GH signaling in insulin-producing cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DIABETES IS A disease characterized by insufficiency of the pancreatic ß-cells to produce appropriate amounts of insulin to maintain normal blood glucose levels. In type 1 diabetes this is due to an autoimmune response resulting in specific destruction of the pancreatic ß-cells, whereas in type 2 diabetes it is due to a combination of peripheral insulin resistance and ß-cell deficiency. Reduction in ß-cell mass and decreased responsiveness of the ß-cells to insulin secretory signals are key issues in the development of diabetes, and therefore it is important to study the mechanisms controlling ß-cell growth and differentiation.

Several different factors influence ß-cell proliferation (for review see Ref. 1), and among these, GH plays an important role. Studies of the endocrine pancreas using ß-cell lines, primary ß-cells grown in monolayer, or rat islets grown in culture have shown that GH stimulates proliferation of the pancreatic ß-cells (2, 3, 4, 5). Moreover, GH is able to induce biosynthesis of insulin in the ß-cells through a direct effect on the insulin promoter (6). GH exerts its effects via binding to the GH receptor, which is expressed in the ß-cell lines RIN-5AH and INS-1 as well as in rat islets (7, 8, 9). This, in turn, leads to activation of several intracellular signaling pathways (for review see Ref.(10), of which the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is the best characterized. Of the four currently known JAK proteins, JAK-2 is the one activated upon GH stimulation (11). JAK-2 activation results in tyrosine phosphorylation, dimerization, and nuclear translocation of the latent STAT proteins that subsequently bind to specific DNA-responsive elements to initiate gene transcription. STAT1, STAT3, and STAT5 have been shown to be activated upon GH stimulation (12, 13, 14, 15, 16).

Until recently the mechanisms controlling down-regulation of the GH signaling cascade were unknown. However, in 1997, a new group of proteins termed suppressors of cytokine signaling (SOCS), capable of inhibiting cytokine signaling in a classical negative feedback loop, were identified (17, 18, 19). Today, eight members of the SOCS family have been described, SOCS-1 to 7 and CIS. In general, the SOCS proteins are not constitutively expressed in cells but are induced upon stimulation with various cytokines, including GH (20, 21, 22, 23, 24, 25, 26, 27). Cytokine-induced SOCS expression is dependent upon STAT-induced transcriptional mechanisms (19, 22, 28, 29, 30, 31). Some cytokines induce the expression of several of the SOCS genes, whereas others induce only one or a few. Studies performed both in vitro and in vivo have shown that expression of SOCS-1 and in particular SOCS-3 is induced upon GH stimulation. When 3T3-F442A fibroblasts are stimulated with GH, a rapid, transient induction of SOCS-1 and SOCS-3 mRNA is seen. A preferential induction of SOCS-3 mRNA is observed in hepatic tissue isolated from mice treated with GH (20). Also, in the ß-cell line INS-1, GH is able to induce expression of SOCS-1 and SOCS-3 (26). Subsequently, SOCS-1 and SOCS-3 appear to inhibit GH signaling in different ways. SOCS-1 has been shown to bind directly to JAK, thereby inhibiting its activity. SOCS-3 also inhibits JAK, but in contrast to SOCS-1, this inhibition is receptor dependent (23, 32). In addition to inhibiting enzyme activity, the SOCS proteins can also down-regulate cytokine signaling by other mechanisms. One way is by masking tyrosine-phosphorylated docking sites in the cytoplasmic domain of the cytokine receptor (28, 33), whereas another is by promoting degradation of signaling molecules through linkage of these to the proteasome complex (34, 35, 36). Despite the different inhibitory mechanisms, the SOCS proteins are generally considered to regulate cytokine signal transduction through a classical negative feedback loop, as SOCS expression is induced by the same cytokine signaling pathway that is subsequently down-regulated by the SOCS proteins. However, increasing evidence suggest that cross-talk between different signaling pathways also can be mediated by SOCS proteins. For example, studies in liver cells have shown that endotoxin-, IL-1ß-, and TNF{alpha}-induced SOCS-3 expression might explain the GH resistance observed in these cells upon sepsis (25, 37). Furthermore, GH resistance is a major complication in chronic renal failure, and also in this condition the decreased responsiveness of the cells to GH seems to be caused by overexpression of SOCS proteins (38).

Upon GH stimulation, the SOCS-mediated inhibition of JAK activity results in an inhibition of GH- induced STAT activation and thereby a down- regulation of GH-induced gene transcription (20, 23, 32, 39, 40). Very little is known about the physiological role of SOCS-1/SOCS-3 on GH effects. SOCS-1 and SOCS-3 knockout mice are neonatally and embryonically lethal, respectively, and have not been informative in this regard (41, 42, 43).

Currently available data suggest that SOCS-3 is a major player in the down-regulation of GH signaling. However, most of the results have been obtained from studies in liver cells. Therefore, the aim of the present study was to investigate whether SOCS-3 also plays a role in GH-induced signaling in ß-cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inducible Expression of SOCS-3 in RIN-5AH and INS-1 Cells
To investigate the effect of SOCS-3 on GH signaling in ß-cells, two cell lines with inducible SOCS-3 expression were established as an alternative to cells with constant overexpression of SOCS-3. Previous attempts in the laboratory to generate cell lines with constant SOCS-3 overexpression have failed. Several studies have shown that the SOCS proteins are able to inhibit cell proliferation (33, 44, 45), and this might explain the failure to establish cell lines with stable overexpression of SOCS-3. Therefore, it was decided to establish a ß-cell line in which the expression of SOCS-3 could be controlled.

The ecdysone-inducible expression system was employed to generate stably transfected RIN-5AH cells with inducible SOCS-3 expression. First, RIN-5AH cells were stably transfected with the expression plasmid pVgRXR encoding the two nuclear receptors VgEcR and retinoid X receptor (RXR), which are activated upon stimulation with the steroid hormone ecdysone (or its analog ponasterone). A selected clone (EcR-RIN#10) was subsequently transfected stably with the expression plasmid pIND containing cDNA encoding FLAG-tagged SOCS-3 (FLAG-SOCS-3) downstream of five ecdysone response elements. Integration of VgEcR, RXR, and FLAG-SOCS-3 DNA was verified by PCR analysis (data not shown). The various clones were grown in the absence or presence of ponasterone, and by means of RT-PCR analysis the inducible level of SOCS-3 expression was investigated (data not shown). One clone with inducible SOCS-3 expression (EcR-RIN#10.6) was chosen for further studies.

Inducible expression of SOCS-3 mRNA in clone EcR-RIN#10.6 was investigated by ribonuclease (RNase) protection assay (RPA). The cells were grown for different time periods and ponasterone concentrations before the level of SOCS-3 mRNA was measured by means of a SOCS-3-specific probe. In the absence of the inducer ponasterone, no SOCS-3 mRNA could be detected in the cells (lane 1 in Fig. 1Go, A and B). However, when stimulated with 5 µM ponasterone, the expression level of SOCS-3 increased in a time- dependent manner, reaching a maximum after 8–24 h (Fig. 1AGo). The induction of SOCS-3 could also be induced in a dose-dependent manner, with the highest level of expression upon stimulation with 5 µM ponasterone (Fig. 1BGo). By Western blot analysis, no FLAG-SOCS-3 protein could be detected in the absence of ponasterone (lane 1 in Fig. 2Go, A and B). When the cells were stimulated with ponasterone, FLAG-SOCS-3 protein levels were increased in a time- and dose-dependent manner similar to the mRNA expression seen in Fig. 1Go. Ponasterone is used at concentrations of 0.5 to 10.0 µM in the following experiments.



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Figure 1. RPA Demonstrating Inducible Expression of SOCS-3 mRNA in RIN-5AH Cells

A, The RIN-5AH cell clone EcR-RIN#10.6 stably transfected with pVgRXR and pIND-FLAG-SOCS-3 was grown for the indicated time periods in the presence of 5 µM ponasterone. B, Cells were grown for 24 h in the presence of the indicated concentrations of ponasterone (Pona). SOCS-3 and cyclophilin mRNA expression levels were measured by RPA. The intensity of each SOCS-3 band was normalized to the corresponding cyclophilin band before calculation of the relative induction in SOCS-3 expression. The results shown are representative of two independent experiments.

 


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Figure 2. Western Blot Analysis Demonstrating Inducible Expression of FLAG-SOCS-3 Protein in RIN-5AH Cells

A, EcR-RIN#10.6 cells were grown for the indicated time periods in the presence of 5 µM ponasterone. B, Cells were grown for 24 h in the presence of the indicated concentrations of ponasterone. Total cell lysates were separated by SDS-PAGE and subsequently analyzed by Western blot analysis using anti-FLAG antibodies to detect FLAG-SOCS-3. As a control for FLAG-SOCS-3 expression, cell lysates from nontransfected 293 cells (-) and 293 cells transiently transfected with FLAG-SOCS-3 (+) were run on the right side of the gel. The results shown are representative of two independent experiments.

 
By using the Tet-On gene expression system, an INS-1 cell line with inducible expression of FLAG-SOCS-3, termed INS-r3#2, was also established (see Materials and Methods). Doxycycline was used to induce activation of the reverse tetracycline/doxycycline-dependent transactivator leading to FLAG-SOCS-3 expression in these cells. The expression level of FLAG-SOCS-3 obtained by the inducible expression systems was comparable to endogenous SOCS-3 levels obtained after stimulation with various cytokines and thereby physiologically relevant. Hence, when INS-r3#2 cells were stimulated with 200 U/ml interferon-{gamma} (INF-{gamma}), RT-PCR using SOCS-3-specific primers showed that the expression level of endogenous SOCS-3 was more than 50% of that obtained with a maximal induction by doxycycline (data not shown). In RIN-5AH cells, stimulation with 200 U/ml IFN-{gamma} resulted in an SOCS-3 expression level that was more than 75% of that obtained in the inducible system. In primary rat islets, stimulation with 150 or 1500 pg/ml IL-1ß resulted in expression levels that were more than 110% and 130%, respectively, of that obtained in the induced INS-r3#2 cells (data not shown). These results indicate that the two inducible cell lines express FLAG-SOCS-3 at levels comparable to that of insulin-producing cells stimulated with cytokines.

SOCS-3 Inhibits GH-Induced STAT5 and STAT3 DNA Binding Activity in ß-Cells
Because the JAK2-STAT5 pathway is the main signaling pathway activated by GH in ß-cells, we investigated whether SOCS-3 was able to influence the GH-induced activation of STAT5. This was performed by measuring the DNA binding activity of STAT5 by EMSA. EcR-RIN#10 and EcR-RIN#10.6 cells were incubated with the indicated concentrations of ponasterone (Fig. 3Go) for 20 h. Subsequently, cells were incubated with or without human GH (hGH) for 15 min. Nuclear extracts were isolated and analyzed using the STAT5 binding element from the Spi 2.1 promoter as a probe. In the inducible cell line (EcR-RIN#10.6), a clear STAT5 activation was seen in the absence of ponasterone (Fig. 3AGo, lane 2). However, in the presence of increasing concentrations of ponasterone, a dose- dependent inhibition of GH-induced STAT5 DNA binding activity was seen (Fig. 3AGo), suggesting that SOCS-3 inhibits GH-induced STAT5 activation in RIN cells. Competition and supershift analyses were included to confirm specific binding of the probe to STAT5. Equal loading of protein in the EcR-RIN#10.6 samples was verified by running an identical EMSA using a CREB-binding element from the {alpha}-chorionic gonadotropin promoter (Fig. 3AGo). In the EcR-RIN#10 cells, which were only transfected with the EcR and RXR receptors, stimulation with GH led to STAT5 activation both in the absence and presence of ponasterone (Fig. 3BGo).



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Figure 3. EMSA Demonstrating the Effect of SOCS-3 on GH-Induced STAT5 Activation

EMSA was performed using nuclear extracts from EcR-RIN #10 and #10.6 cells grown for 24 h in the presence of the indicated concentrations of ponasterone and 15 min in the absence or presence of 20 nM hGH. A, On the upper gel, the STAT5 binding element (GLE) from the Spi 2.1 promoter was used as a probe. On the lower gel, a CREB-binding probe was used to confirm the loading of equal amounts of protein in the different lanes. B, Nuclear extracts from the control cells were incubated with the STAT5 binding probe. To confirm specificity of the binding, supershift analysis was performed using an antibody recognizing STAT5. To verify specific binding of the probes, competition (comp.) was performed with the relevant oligonucleotides at 10- or 100-fold molar excess. The results shown are representative of three independent experiments.

 
We then investigated whether SOCS-3 expression could influence GH-induced STAT1 and STAT3 DNA binding activity. As a probe, the SIS-inducible element (SIE) from the c-fos gene (optimized for binding and termed M67), which binds both STAT1/STAT1 and STAT3/STAT3 homodimers as well as STAT1/STAT3 heterodimers (6), was used. In EcR-RIN#10.6, GH induced STAT3 activation in the absence of ponasterone (Fig. 4AGo, lane 2). However, in the presence of ponasterone, the GH-induced STAT activation was inhibited in a dose-dependent manner, with a complete inhibition seen upon stimulation with 2.5 µM ponasterone. Competition with a nonlabeled oligo was included to verify specific binding of the probe. Specificity of the STAT3 band was also confirmed by supershift analysis using a STAT3 antibody (data not shown). It should be noted that some basal DNA binding activity of STAT3 is seen in the absence of GH (Fig. 4AGo, lane 1) and that this activity is inhibited in the presence of ponasterone, indicating that SOCS-3 is also able to inhibit STAT activation that is not specifically induced by GH. Supershift analysis using an antibody recognizing STAT1 revealed that no GH-induced STAT1/STAT1 homodimers or STAT1/STAT3 heterodimers could be observed (data not shown), probably because the GH-induced STAT1 activation is very low in the RIN-5AH cells (6). GH-induced activation of STAT3 was observed both in the absence and presence of ponasterone in the control cell line (Fig. 4BGo). These results therefore indicate that SOCS-3 is able to inhibit GH-induced STAT3 and STAT5 activation in RIN cells.



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Figure 4. EMSA Demonstrating the Effect of SOCS-3 on GH-Induced STAT3 Activation

EMSA was performed using nuclear extracts from EcR-RIN #10 and #10.6 cells grown for 24 h in the presence of the indicated concentrations of ponasterone and 15 min in the absence or presence of 20 nM hGH. A, The modified STAT1/STAT3 binding element SIE from the c-fos promoter (M67) was used as a probe. B, Nuclear extracts from the control cells were incubated with the M67-probe. To verify specific binding of the probes, competition (comp.) was performed with the relevant oligonucleotides at 10- or 100-fold molar excess. The results shown are representative of three independent experiments.

 
SOCS-3 Inhibits GH-Induced Insulin mRNA Levels in RIN Cells
Previous work has demonstrated that GH is able to induce insulin synthesis in RIN-5AH cells via activation of STAT5 (6). Upon activation by GH, STAT5 molecules translocate to the nucleus where they bind to STAT5-specific elements in the insulin promoter (6). In the present study it was found that SOCS-3 is able to inhibit GH-induced STAT5 DNA-binding activity. We therefore investigated whether SOCS-3 was able to inhibit GH-induced insulin production in the RIN-5AH cells.

To examine the effect of GH on insulin expression in the RIN cells, the level of insulin mRNA was measured by Northern blot analysis. EcR-RIN#10 and EcR-RIN#10.6 cells were cultured in the presence or absence of ponasterone and subsequently incubated with hGH for 24 or 48 h. GH was found to induce a 1- to 2-fold increase in the insulin mRNA level in EcR-RIN#10 cells both in the absence and presence of ponasterone (Fig. 5Go). Likewise, hGH also increased insulin production in the inducible cell line by 1-fold in the absence of ponasterone. However, this induction was completely abolished when SOCS-3 expression was induced in the EcR-RIN#10.6 cells. Measurement of cyclophilin mRNA was included as an internal control, as previous studies have shown that the expression of cyclophilin is unaffected by GH (6). As can be seen from Fig. 5Go, ponasterone does not influence the cyclophilin expression either. The insulin bands were normalized to the corresponding cyclophilin bands before calculation of the relative induction in insulin production. These data indicate that SOCS-3 is able to inhibit GH-induced insulin mRNA levels in RIN cells.



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Figure 5. Northern Blot Analysis Showing the Effect of SOCS-3 on GH-Induced Insulin mRNA Levels

Total RNA isolated from EcR-RIN#10 and #10.6 cells cultured with or without ponasterone and stimulated for 24 or 48 h with 20 nM hGH was used for Northern blot analysis with radiolabeled probes specific for insulin and cyclophilin. The band intensities were determined from a gray-scale image by PhosphorImager analysis and quantitated using ImageQuant (Molecular Dynamics, Inc.). The intensity of each insulin band was normalized to the corresponding cyclophilin band, after which the relative induction in insulin production was calculated. The results shown are representative of two independent experiments.

 
SOCS-3 Inhibits GH-Induced Proliferation of INS-1 Cells
GH is known to induce proliferation of pancreatic ß-cells (2, 3, 4, 5). To investigate whether SOCS-3 could influence this GH effect, cells were stimulated with hGH, and 5-bromodeoxyuridine (BrdU) incorporation during the S phase of the cell cycle was measured by fluorescence-activated cell sorting (FACS) analysis. For these experiments, the ß-cell line INS-1 was used as it has been observed that these cells show a more pronounced proliferative response to GH stimulation than RIN-5AH cells (46, 47). Stimulation of the INS-r3#2 cells with hGH for 24 h increased the fraction of cells in S phase approximately 1-fold (Fig. 6AGo). Induction of SOCS-3 expression in the cells by stimulation with doxycycline resulted in a dose-dependent inhibition of the GH-induced proliferation, whereas basal BrdU incorporation was unaffected by the presence of doxycycline (Fig. 6AGo). Doxycycline did not affect the growth response to 10% fetal calf serum (FCS) (Fig. 6BGo). Likewise, doxycycline had no effect on GH- induced proliferation of the control cell line INS-r3, which was only transfected with the reverse transactivator (Fig. 6CGo). These experiments indicate that SOCS-3 inhibits GH-induced proliferation of INS-1 cells.



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Figure 6. FACS Analysis Showing the Effect of SOCS-3 on GH-Induced ß-Cell Proliferation

A, INS-r3#2 cells were grown for 24 h in the presence of the indicated concentrations of doxycycline and subsequently stimulated for 24 h with 20 nM hGH. Cells were exposed to 10 µM BrdU for 2 h, fixed, stained with an antibody recognizing BrdU, and analyzed by FACS cytometry. B, Cells were stimulated by 10% FCS and BrdU incorporation was measured by FACS. C, To ensure that doxycycline alone does not have any effect on cell proliferation, the experiment was repeated using the control cell line INS-r3, which does not have inducible SOCS-3 expression. Data are mean ± SE of three to five separate experiments. *, P <= 0.05 compared with GH-stimulated cells without doxycycline.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The effects of GH on pancreatic ß-cells include stimulation of ß-cell proliferation and insulin gene expression. During the last years, a much better understanding of the signaling pathways leading to GH effects in the cell has evolved. However, little is still known about the regulatory mechanisms controlling GH-induced signaling in pancreatic ß-cells.

In the present study, we investigated the influence of SOCS-3 on GH signaling in ß-cells. This was performed using ß-cell lines with inducible SOCS-3 expression, as this system allows a strict control of the SOCS-3 expression in the cells, thereby minimizing nonphysiological manipulation of the cells as seen with constant overexpression. Time- and concentration-dependent induction of SOCS-3 was observed at both the mRNA and protein level, and no basal activity of the inducible promoter was observed. After induction of SOCS-3, we found that GH activation of STAT5 DNA binding activity was greatly diminished, whereas binding of another transcription factor CREB was not affected. This observation is consistent with a number of other studies showing that SOCS-3 is able to inhibit STAT5 activation, an effect obtained through inhibition of JAK2 activity (23, 32). STAT5 is known to mediate mitogenic signals in various lymphoid cells (48, 49, 50). Recently it was shown that a dominant- negative form of STAT5 could abolish GH-stimulated proliferation of INS-1 cells, indicating a direct role of STAT5 in mediating the proliferative signaling stimulated by GH (47). On this background, it could be speculated that the SOCS-3-mediated inhibition of GH-induced STAT5 activation in the ß-cells, seen in this study, might be important for GH-induced ß-cell proliferation. This hypothesis is supported by the data obtained from the proliferation studies presented in Fig. 6Go. Here we show that SOCS-3 indeed is able to inhibit GH-induced proliferation of the INS-r3#2 cells. The finding that SOCS-3 can inhibit cytokine-induced cell proliferation is supported by another study showing that inducible SOCS-3 expression inhibits IL-2-mediated Ba/F3 cell proliferation (45). In addition to STAT5 inhibition, SOCS-3 was also able to inhibit GH-induced STAT3 activation. This might represent another mechanism by which SOCS-3 can suppress GH-induced ß-cell proliferation, as activation of STAT3 is involved in stimulation of proliferation of several cell types (14, 51, 52).

We have previously shown that STAT5 participates directly in insulin gene transcription stimulated by GH (6). A STAT5 DNA binding element is present in the rat 1 insulin promoter, and mutation of this element results in a promoter that is no longer responsive to GH. In the present study, the SOCS-3-mediated decrease in STAT5 activation was mirrored by a similar inhibition of GH-stimulated induction of insulin mRNA. Our data indicate that SOCS-3 is a potent inhibitor of GH action in insulin-producing cells and suggest that the expression level of SOCS-3 in ß-cells determines the responsiveness of these cells to GH.

The results obtained in this study are based on inducible expression of SOCS-3. The level of SOCS-3 expression in ß-cells is probably regulated by many factors. In INS-1 cells, both SOCS-1 and SOCS-3 are expressed after stimulation of the cells with GH (26). Furthermore, we have recently shown that IL-1 and interferon-{gamma} are able to induce expression of SOCS-3 in rat islets (53), supporting the fact that expression of the SOCS proteins can be induced by various cytokines in the same cell type. In addition to cytokines, developmentally regulated expression of SOCS genes has been reported in hematopoietic and neuronal tissue (42, 54). Finally, it has also been shown that various metabolic pathways might control the expression of SOCS genes (55). The complex regulation of SOCS expression in the cells is reflected by a complex effect of the SOCS proteins in the cells. In this study, SOCS-3 was shown to inhibit GH-induced proliferation and insulin gene expression of the ß-cells, but in addition, SOCS-3 has also been shown to inhibit signaling from other cytokines affecting ß-cells. It is well established that the proinflammatory cytokine interferon-{gamma}, together with IL-1ß and TNF{alpha}, induces apoptosis in ß-cells during the autoimmune process leading to type 1 diabetes (56). We have recently shown that expression of SOCS-3 in ß-cells can protect against apoptosis induced by these cytokines (53). Furthermore, leptin has been shown to regulate insulin gene expression (57) as well as protect ß-cells from free fatty acid-induced apoptosis (58), and as SOCS-3 inhibits leptin signaling (59, 60), it might be an important regulator of the ß-cell response to leptin stimulation. Taken together, it appears that the expression level of SOCS proteins is under complex regulation and that several diverse stimuli acting in a concerted manner determine the level of SOCS expression and thus the responsiveness of cells to cytokines such as GH. Thus, the inhibitory effect of SOCS-3 on, for example, GH signaling might represent negative feedback but might just as well represent an inhibitory effect of SOCS-3 induced by other cytokines. Our study concentrates on the effect of SOCS-3 on GH signaling. However, in cotransfection studies we have previously shown that SOCS-1 inhibits GH-induced transcription (20, 32), indicating that other SOCS proteins might also play a significant role in the regulation of GH signaling.

In summary, our data indicate that SOCS-3 is an important regulator of GH signaling in ß-cells. Further understanding of the mechanisms involved in the GH-mediated regulation of ß-cell proliferation and differentiation may lead to new ways of increasing the ß-cell mass. If, in the future, the ß-cell mass could be increased either in vivo in the patient or in vitro by propagation of ß-cells for transplantation, this could represent a decisive step toward a better treatment of diabetes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture, Antibodies, Chemicals, and Hormones
RIN-5AH and INS-1 cells were cultured in RPMI-1640 with glutamax supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified atmosphere containing 5% CO2. Furthermore, the INS-1 medium contained 50 µM ß-mercaptoethanol. In addition, medium for the different stable cell lines contained the following antibiotics: EcR-Rin#10, zeocin (400 µg/ml); EcR-RIN#10.6, zeocin (400 µg/ml) and G418 (150 µg/ml); INS-r3, G418 (100 µg/ml); and INS-r3#2, G418 (100 µg/ml) and hygromycin (100 µg/ml).

Mouse-{alpha}-FLAG antibody (no. F3165, Sigma-Aldrich Corp., St. Louis, MO) was used to detect SOCS-3. Mouse anti-BrdU antibody (no. M0744, DAKO Corp. A/S, Copenhagen, Denmark) was used to detect BrdU. Recombinant hGH was obtained from Novo Nordisk A/S (Gentofte, Denmark). Ponasterone was from Invitrogen (Carlsbad, CA), and doxycycline was from Sigma-Aldrich Corp.

Establishment of Cell Lines with Inducible SOCS-3 Expression
An RIN-5AH cell line with inducible SOCS-3 expression was established according to the instructions of the manufacturer, using the ecdysone-inducible expression system (Invitrogen). Briefly, RIN-5AH cells were grown to confluency. Using electroporation, 107 cells were stably transfected with 40 µg of the vector pVgRXR (Invitrogen), which expresses the two nuclear receptors VgEcR and RXR and the zeocin resistance gene. Cells were grown in complete medium for 3 d after which selection was performed using 750 µg/ml zeocin. Antibiotic-resistant cell clones were isolated after 2 wk. Expression of pVgRXR in resistant clones was checked by RT-PCR, using primers specific for VgEcR and RXR (Invitrogen). The positive clone used for the second round of transfection was grown in medium containing 400 µg/ml zeocin and named EcR-RIN#10. The plasmid used for the second round of stable transfection was constructed by subcloning FLAG-tagged, murine SOCS-3 cDNA from the pEF-BOS vector (kindly supplied by Dr. Douglas Hilton, Walter and Eliza Hall Institute, Melbourne, Australia) into the pIND vector (Invitrogen), which contains five ecdysone response elements upstream of a minimal heat shock protein promoter and a multiple cloning site. XbaI was used as a restriction site. The EcR-RIN#10 cells were transfected by electroporation and selection was performed in 250 µg/ml G418. Positive clones with inducible SOCS-3 expression were identified by RT-PCR using SOCS-3 specific primers. The clone chosen for this study was kept for long-term culture in 150 µg/ml G418 and 400 µg/ml zeocin and was termed EcR-RIN#10.6.

Establishment of INS-1 cells with inducible SOCS-3 expression was based on the Tet-On system (CLONTECH Laboratories, Inc., Palo Alto, CA).

INS-r3 cells were kindly supplied by Dr. P. B. Iynedjian (Geneva, Switzerland). These are INS-1 cells stably transfected with the reverse tetracycline-dependent transactivator. The plasmid used for the second round of stable transfection was constructed by subcloning murine SOCS-3 cDNA into the pTRE response plasmid (CLONTECH Laboratories, Inc.) downstream of the tetracycline operator-cytomegalovirus minimal promoter, using the XbaI site of the polylinker. INS-r3 cells were seeded (1 x 107 cells per 100 mm dish) and cultured overnight in complete medium containing 100 µg/ml G418. The next day, cells were cotransfected with 18.1 µg pTRE-SOCS-3 vector and 3.7 µg pTK-hyg Vector (encoding the selection marker providing hygromycin resistance) using LipofectAmine Plus Reagent (Life Technologies, Inc., Paisley, Scotland, UK) according to the description of the manufacturer. After incubation in Optimem overnight, medium was changed to complete medium containing 100 µg/ml G418. The following day, cells were trypsinized and reseeded in 100-mm dishes using complete medium containing 100 µg/ml G418 and 200 µg/ml hygromycin. Resistant cell clones were picked individually after 2–4 wk and maintained in long-term culture with 100 µg/ml hygromycin and 100 µg/ml G418. Clones with inducible SOCS-3 expression were detected by RT-PCR using primers specific to SOCS-3, and the clone used in this study was named INS-r3#2.

RPA
Cells were seeded in 100-mm dishes, cultured for 2 d in complete medium and subsequently incubated with the indicated concentrations of ponasterone for the specified periods of time. RNA was isolated from the cells using the RNeasy mini kit (QIAGEN GmbH, Hilden, Germany).

The gene sequence encoding murine SOCS-3 was excised from the pEF-BOS vector using XbaI and cloned into the pGEM vector (Promega Corp., Madison, WI). To determine the orientation of the insert, the recombinant construct was digested with KpnI and PflMI. To generate a 32P-labeled antisense probe, the construct was linearized with PstI and in vitro transcribed with T7 RNA polymerase and [{alpha}-32P]UTP, using a Riboprobe In Vitro Transcription System (Promega Corp.). Linearization with PstI results in a probe of 364 bp.

RPA was carried out as described by the manufacturer, using the RPA III kit (Ambion, Inc., Austin, TX). The antisense RNA probe (1 x 105 cpm) was hybridized overnight at 45 C with 1 µg RNA and then digested with RNase A/RNase T1 for 30 min at 37 C. As an internal control, hybridization was also performed with an in vitro transcribed probe complementary to cyclophilin transcripts. After precipitation, fragments protected from RNase digestion were separated on a 6% denaturing polyacrylamide gel. The sizes of the protected fragments were determined by running a labeled RNA marker (pBR322 MspI digested, New England Biolabs, Inc., Beverly, MA) on the gel.

Bands corresponding to probe fragments protected by hybridization to the SOCS-3/cyclophilin transcript were quantified by PhosphorImager analysis. The scanning results were analyzed by ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA) and the intensity of each SOCS-3 band was normalized to the corresponding cyclophilin band.

Western Blot Analysis
Cells were cultured in six-well plates for 2 d in complete medium. Medium was changed to complete medium containing the indicated concentrations of ponasterone and grown for the shown time periods. Cell lysates were prepared by addition of lysis buffer [50 mM HEPES (pH 7.2), 250 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 0.1% Nonidet P-40, 1 mM 4-(2-aminoethyl) benzene sulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate] to the cells, followed by 20 min incubation on ice. After centrifugation at 10,000 x g for 10 min at 4 C, 5 x sodium dodecyl sulfate loading buffer was added to the supernatants to a final concentration of 1x. Proteins were resolved by SDS-PAGE (4% stacking gel, 12% running gel) and transferred by electroblotting to enhanced chemiluminescence nitrocellulose membranes (Amersham Pharmacia Biotech, UK Ltd., Buckinghamshire, UK). Membranes were blocked for 1 h in TBST buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] containing 5% nonfat dry milk. Primary antibody diluted in TBST was added, and the blot was incubated overnight at 4 C. After three successive 20-min washes with TBST, the secondary antibody diluted in TBST was added and the membrane was incubated for 1 h at room temperature. Finally, protein was visualized by an enhanced chemiluminescence detection system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Nuclear Extracts and EMSA
Cells were cultured in 100-mm dishes for 2 d in complete medium. The medium was changed to RPMI-1640 containing 0.5% FCS with or without ponasterone. After 20 h the cells were incubated with or without 20 nM hGH for 15 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml buffer A [20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM MgCl2, 10 mM KCl, 20% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride/4-(2-aminoethyl) benzene sulfonyl fluoride, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 µg/ml aprotinin] containing 0.5% Triton X-100. After 5 min of incubation on ice, the cell lysates were scraped off, and the nuclei were collected by centrifugation at 2,500 x g for 5 min at 4 C. The nuclei were resuspended in 100 µl hypertonic buffer (buffer A containing 400 mM NaCl) and incubated on a rocking bench for 30 min at 4 C. The supernatants were collected after centrifugation at 20,000 x g for 30 min at 4 C, and aliquots were frozen in liquid nitrogen and stored at -80 C. After annealing, the double-stranded oligonucleotide Spi-GLE 2.1 (5'-agctATGTTCTGAGAAAATC-3') (Life Technologies, Inc., A/S, Roskilde, Denmark) and M67-SIE (5'-agctTCATTTCCCGTAAATCCCTA-3') (Life Technologies, Inc.) were 32P-labeled in a fill-in reaction using [{alpha}-32P]dATP and DNA polymerase (Klenow fragment). Nuclear extracts (5 µg protein) were incubated at 30 C for 30 min with 20 fmol probe in a 20 µl reaction containing EMSA buffer [20 mM HEPES (pH 7.9), 10 mM NaCl, 1 mM MgCl2, 1 mM EDTA, and 10% glycerol] and 0.1 µg/µl double-stranded poly(dI.dC)-poly(dI.dC). After the addition of loading buffer, the samples were separated on a 5% polyacrylamide gel containing 2% glycerol and 0.25% 25 mM Tris/HCl, 25 mM boric acid, and 0.25 mM EDTA (pH 7.9). The gel was dried and exposed to x-ray film. In competition experiments 200 or 2000 fmol of unlabeled Spi-GLE or M67-SIE were added to the binding reaction. As an internal control, nuclear extracts were incubated with an {alpha}-chorionic gonadotropin (5'-agctTTTTACCATGACGTCAATTTGATC-3') (Life Technologies, Inc.) promoter probe containing a cAMP response element that binds CREB. In supershift experiments, nuclear extracts were preincubated for 30 min at 4 C with 1 µl {alpha}STAT5 antibody.

Northern Blot Analysis
Cells were cultured in 100-mm dishes for 2 d in complete medium. The medium was changed to RPMI-1640 containing 0.5% FCS with or without ponasterone. After 20 h starvation, cells were stimulated for 24 or 48 h with 20 nM hGH. Subsequently, RNA was isolated from the cells using RNeasy (QIAGEN GmbH).

Ten micrograms of total RNA were separated by denaturing agarose gel electrophoresis in the presence of ethidium bromide to use 18S and 28S rRNA as molecular weight markers. The RNA was blotted onto a Duralon-UV membrane (Stratagene, La Jolla, CA) and immobilized by baking at 80 C. The membrane was prehybridized for 2–4 h at 65 C in the presence of 0.5 M NaPO4, 7% sodium dodecyl sulfate, 1 mM EDTA, and 50 µg/ml salmon sperm and calf thymus-DNA and hybridized at 65 C overnight in the same buffer containing 50 ng of each of the two 32P-labeled cDNA probes. The cDNA fragments, a 500-bp cDNA PstI fragment from the rat insulin gene and a 750-bp BamHI/HinDII fragment from the rat cyclophilin gene, were labeled using the multiprime DNA labeling system, RPN 1601Y, from Amersham Pharmacia Biotech, UK Ltd.). The membrane was washed in a 0.04 M NaPO4, 1% sodium dodecyl sulfate wash buffer at 65 C and examined by PphosphorImager analysis. The scanning results were analyzed by ImageQuant (Molecular Dynamics, Inc.) using a gray-scale image. Cyclophilin, whose expression has previously been shown to be unaffected by GH (6), was used as an internal control, and the intensity of each insulin band was normalized to the corresponding cyclophilin band.

FACS Analysis
Cells were seeded in six-well plates (500,000 cells per well) and grown in complete medium for 2 d. The medium was changed to one containing 0.25% BSA, and cells were incubated for 20 h in the presence or absence of doxycycline as indicated. The cells were stimulated for 24 h with either 20 nM hGH or 10% FCS and 2 h before harvesting 10 µM BrdU was added to the wells. The cells were harvested using 200 µl trypsin-EDTA per well and 1 ml complete medium to stop the trysinization. Cells were pelleted and to fix the cells, they were resuspended in 500 µl ice-cold 70% ethanol and stored at 4 C for up to 1 wk. To denature the DNA, 500 µl of 3 M HCl were added, and cells were incubated for 30 min at room temperature. Subsequently, cells were washed in PBS containing 0.1% human serum albumin and 0.3% Triton X-100, resuspended in the same buffer containing mouse anti-BrdU antibody (no. M0744, DAKO Corp. A/S) in a 1:100 dilution, and incubated under rotation at 4 C overnight. Cells were washed twice and resuspended in 1 ml buffer containing fluorescein isothiocyanate-conjugated goat-antimouse IgG (no.115–095-003, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in a 1:100 dilution and incubated under rotation at 4 C in the dark for 45 min. Cells were washed twice, resuspended in 500 µl PBS containing 5 µg/ml propidium iodide, and placed in the dark for 30 min. The number of BrdU-positive cells were analyzed by FACS analysis using CellQuest (Becton Dickinson and Co., San Jose, CA) as software.


    ACKNOWLEDGMENTS
 
We thank Jannie Rosendahl Christensen and Tina Kisbye for excellent technical assistance and Dr. Erica Nishimura for critical review of the manuscript. We also thank Dr. D. Hilton for providing the SOCS-3 cDNA and Dr. P. E. Iynedjian for providing the Ins-r3 cells.


    FOOTNOTES
 
S.G.R. was supported by a Ph.D. fellowship from the Health Sciences Faculty, The University of Copenhagen. K.L. was supported by the Danish Research Academy, and J.H. was supported by a grant from the Danish Cancer Foundation. Part of this work was supported by the Danish Cancer Foundation.

Abbreviations: BrdU, 5-Bromodeoxyuridine; CREB, cAMP response element binding protein; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; hGH, human GH; JAK, Janus kinase; RNase, ribonuclease; RPA, ribonuclease protection assay; RXR, retinoid X receptor; SOCS, suppressors of cytokine signaling; SIE, SIS-inducible element; STAT, signal transducer and activator of transcription.

Received for publication February 25, 2002. Accepted for publication June 5, 2002.


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