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Molecular Endocrinology, doi:10.1210/me.2002-0356
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Molecular Endocrinology 17 (5): 945-958
Copyright © 2003 by The Endocrine Society

Signal Transducer and Activator of Transcription 5 Activation Is Sufficient to Drive Transcriptional Induction of Cyclin D2 Gene and Proliferation of Rat Pancreatic ß-Cells

Birgitte N. Friedrichsen, Henrijette E. Richter, Johnny A. Hansen, Christopher J. Rhodes, Jens H. Nielsen, Nils Billestrup and Annette Møldrup

Department of Islet Discovery Research (B.N.F., A.M.) and Department of Signal Transduction (H.E.R., J.A.H., N.B.), Novo Nordisk A/S, 2820 Bagsværd, Denmark; Pacific Northwest Research Institute and Department of Pharmacology (C.J.R.), University of Washington, Seattle, Washington 98122; and Institute for Medical Biochemistry and Genetics (J.H.N.), University of Copenhagen, 2200 Copenhagen, Denmark

Address all correspondence and requests for reprints to: Birgitte Nissen Friedrichsen, Novo Nordisk A/S, Mammalian Cell Technology, Smørmosevej 6AI.64, 2880 Bagsværd, Denmark. E-mail: bttm{at}novonordisk.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic ß-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and ß-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5a{Delta}749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary ß-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the ß-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE INSULIN-PRODUCING ß-CELLS of the pancreas are highly differentiated cells with a low mitotic index. However, increasing evidence suggests that adaptive changes of the ß-cell mass may be critical for the maintenance of normoglycemia in conditions such as pregnancy and obesity (for review see Refs. 1 and 2). The hormones of the GH family, placental lactogen (PL), prolactin (PRL), and GH are potent growth factors for the ß-cells both in vitro and in vivo (3), and appear to stimulate replication without compromising the ability of the ß-cell to produce insulin. In fact, both GH and PRL have been shown to stimulate insulin gene transcription, biosynthesis, and secretion (4, 5). Their effects are mediated through GH and PRL receptors (GHRs and PRLRs) that are both expressed in ß-cell lines as well as in primary ß-cells (6, 7, 8). The relative potencies of PL, PRL, and GH on ß-cell proliferation depend on the respective receptor expression levels, which are prone to differential hormonal regulation. PRLR expression is stimulated by GH, PRL, and estrogens whereas GHR expression is stimulated by glucocorticoids (7).

The physiological significance of hormones of the GH family is mainly indicated by the increased islet mass during pregnancy in both rodents and humans (9, 10, 11). In rats the increased ß-cell mass correlates with secretion of PL (12), and the expression of the GHR and the PRLR is markedly increased in pancreas from pregnant rats (7, 10, 13). Novel studies in the PRLR-deficient mice show that the islet size and ß-cell mass are reduced (14) and, in addition, reduced ß-cell proliferation in diabetic GK rats correlates with impaired PRL secretion (15). Furthermore, overexpression of PL in ß-cells both in vitro and in vivo results in a marked increase of ß-cell growth (16, 17).

GHR and PRLR are members of the cytokine receptor superfamily (18), which upon activation associates with the Janus kinases (JAKs). The activated receptor/JAK complex is capable of activating the latent transcription factors, signal transducers and activators of transcription (STATs). We have shown previously that human (h)GH-induced ß-cell proliferation is dependent on the JAK/STAT5 pathway (19). Among seven members of the STAT family, two isoforms of STAT5 (a and b) encoded by different genes are highly homologous and share 96% sequence similarity at the protein level (20). They are activated by phosphorylation of a conserved tyrosine residue in the C-terminal region (Y694 for STAT5a and Y699 for STAT5b) (20, 21). Upon activation, the two isoforms of STAT5 (a and b) homo- or heterodimerize, translocate to the nucleus, and bind to the response element TTCNNNGAA termed GAS site ({gamma}-interferon-activated sequence) (20). Even though STAT5a and STAT5b can form heterodimers upon tyrosine phosphorylation (22) and bind to the same DNA sequences, individual transcriptional regulation of genes has been detected (23, 24, 25). The STATs can furthermore bind as tetramers through dimer-dimer interaction to tandem-linked GAS motifs (26), which further increase the opportunities to diverge in different systems. In the pancreatic ß-cell, PRL- and GH-induced STAT5 activation has been shown to stimulate the PRLR, insulin, and glucokinase expression of genes that contain GAS motifs in their promoters (27).

We demonstrated recently that hGH-induced S-phase entry of the ß-cells was blocked by the expression of the dominant negative STAT5 mutant, STAT5a{Delta}749 (19). Furthermore, we showed that the mRNA level of the cell cycle-regulatory factor cyclin D2 was up-regulated by hGH in INS-1 cells and that expression of STAT5{Delta}749 suppressed 50% of this induction. These findings indicated an important role of cyclin D2 in GH/PRL-stimulated ß-cell proliferation. The D-type cyclins, D1–D3, act as growth factor sensors for the transition of cells from G1 phase to S-phase depending on extracellular mitogenic signals (28, 29). Cyclin D1–D3 are closely related proteins that are differently expressed in a wide variety of organs in a tissue-specific manner (29). During G1 phase of the cell cycle, these cyclins associate with catalytic subunits, cyclin-dependent-kinase (CDK) 4 or 6, leading to phosphorylation and inactivation of the key substrate retinoblastoma protein, pRb, followed by release and activation of the transcription factor family, E2F, important for S-phase activity (28, 30). Recent studies using CDK4 knock-in and knockout mice have shown that this catalytic subunit is important in regulation of the ß-cell mass (31). In the present study, we have investigated whether STAT5 directly regulates cyclin D2 transcription and whether STAT5 alone is sufficient to drive proliferation of both INS-1 cells and primary ß-cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Analysis of hGH-Induced Cyclin D Expression in INS-1 Cells and in Primary ß-Cells
The kinetics of hGH-stimulated cyclin D expression were investigated at mRNA level by quantitative RT-PCR (Fig. 1AGo) and at protein level by Western blot analysis (Fig. 1BGo). The cyclin D2 mRNA level was increased in a time-dependent manner by hGH in INS-1 cells. The mRNA expression was increased approximately 1.7-fold over the basal level after 4 h and reached a maximum after 24 h (~3.5-fold over basal). The cyclin D1 and D3 mRNA levels were unaffected by hGH stimulation (Fig. 1AGo). At the protein level, cyclin D2 was induced by hGH in a time-dependent manner with maximal induction after 12 h (2.95 ± 0.79-fold induction, mean ± SEM, n = 4; Fig. 1BGo, lane 4). hGH had no significant effect on cyclin D1 and D3 expression (0.85 ± 0.15 and 0.94 ± 0.13, respectively, mean ± SEM, n = 4). Furthermore, we investigated the expression of the catalytic partners of cyclin D, CDK4 and 6, and found that CDK4 was not appreciably affected by hGH (1.02 ± 0.09, mean ± SEM, n = 4) but was highly expressed (Fig. 1BGo, bottom panel), whereas CDK6 was undetectable in INS-1 cells (data not shown).



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  		Figure 1. The Effect of hGH on Cyclin D Expression in INS-1 Cells

A, INS-1 cells were cultured in the absence or presence of 500 ng/ml hGH. The INS-1 cells were harvested at the indicated time points, and cDNA was synthesized from total RNA extracts. RT-PCR was performed with primer sets specific for cyclin D1, D2, and D3 and primer sets specific for glucose-6-phosphate dehydrogenase (G6PDH) or TATA-binding protein (TBP) were included as internal controls. The PCR products were separated by denaturing PAGE and visualized and quantified by phosphoimager analysis. The cyclin D1 and D3 PCR products were quantified relative to the internal standard TATA-binding protein (TBP). The cyclin D2 products were quantified relative to G6PDH. The results are expressed as fold induction (mean ± SEM, n = 3) compared with control levels and represents the mean of three independent cDNA preparations. B, Western blot analysis was performed on total protein extracts prepared from INS-1 cells that had been cultured in the absence (lanes 1 and 8, for 0 h and 24 h, respectively) or in the presence of 500 ng/ml hGH (lanes 2–7). Twenty microliters of each extract were separated on a 4–12% Bis/Tris polyacrylamide gel; proteins were transferred to nitrocellulose incubated with antibodies, and proteins were visualized using ECL Western blotting detection reagents. The data are representative of three independent experiments.

 
In previous studies using a dominant negative STAT5 approach, we have shown that hGH-induced cyclin D2 mRNA is partially dependent on STAT5 activation (19). To determine whether hGH-induced cyclin D2 mRNA expression is dependent on protein synthesis, hGH was added in the presence or absence of 50 µg/ml cycloheximide (CX). Addition of CX did not affect induction of cyclin D2 mRNA at early time points (up to 4 h; Fig. 2AGo), and the expression was only partially (~25%) inhibited after 8 h, indicating that de novo protein synthesis is not required for hGH-stimulated cyclin D2 expression. At the protein level, cyclin D2 expression was decreased as early as 5 min after addition of CX (Fig. 2BGo, lane 2) and was undetectable after 4 h (lane 4), indicating a rapid turnover of cyclin D2 in INS-1 cells.



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  		Figure 2. The Effect of CX on Cyclin D2 Expression

INS-1 cells were cultured in the absence or presence of 50 µg/ml CX and 500 ng/ml hGH and total RNA was extracted at the indicated time points. A, Quantitative RT-PCR detection of cyclin D2 mRNA expression in the extracts was performed as in Fig. 1Go. The results are expressed as fold induction (mean ± SEM, n = 3) compared with control levels and represent the mean of three independent cDNA preparations. B, Western blot analysis was performed on total protein extracts. Twenty microliters of each extract were separated on a 4–12% Bis/Tris gel, and proteins were transferred to nitrocellulose, incubated with cyclin D2-specific antibodies, and visualized using ECL Western blotting detection reagents. The data are representative of two independent experiments.

 
To determine whether hGH-induced cyclin D2 protein expression is dependent on STAT5 activation, the cell line, EB03 (a ß-cell line derived from INS-1) inducibly expressing the dominant negative STAT5a variant, STAT5a{Delta}749, was grown in the presence or absence of doxycycline and/or hGH for 12 h, and total cell extracts were subjected to Western blot analysis. The cyclin D2 expression was increased by hGH (Fig. 3Go, lane 2), and induction of STAT5a{Delta}749 by doxycycline resulted in a partial inhibition (38% ± 4.4, mean ± SEM, n = 6) of hGH-induced cyclin D2 expression (lane 3). No effect of STAT5a{Delta}749 was observed on cyclin D1 and D3 (lane 3). However, a certain decrease (16% ± 8.9, mean ± SEM, n = 6) of CDK4 protein was observed upon doxycycline treatment (lane 3) compared with both basal and hGH-stimulated levels. The reason for this down-regulation is not known. Similar results (data not shown) were obtained in another clone (ß-cell-derived line expressing STAT5a{Delta}749), BB32 (19).



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  		Figure 3. The Effect of STAT5a{Delta}749 Expression on hGH-Stimulated Cyclin D Expression

Western blot analysis was performed as in Fig. 2Go on total protein extracts prepared from the stably transfected clone (EB03) that had been cultured for 24 h in the absence (lanes 1, 2, and 4) or presence (lane 3) of doxycycline (Dox) followed by stimulation with 500 ng/ml hGH (lanes 2 and 3). Similar results were obtained in BB32 cells.

 
Expression of STAT5 Mutants Affects the Transactivation of the Cyclin D2 Promoter
To address the role of STAT5 in cyclin D2 transcriptional control, we performed promoter-reporter gene analysis. The INS-r3 cells were transiently transfected with the firefly luciferase gene under the control of a 2.3-kb promoter fragment of the cyclin D2 promoter (33). The cells were cotransfected with the doxycycline-inducible expression vector, tetracycline response element (pTRE), containing cDNA encoding either wild-type (WT)-STAT5a or b or constitutive active STAT5 mutants, CA-STAT5a and CA-STAT5b. Stimulation by hGH for 7 h of INS-r3 transfected with only the cyclin D2 construct resulted in a significant increase of transcriptional activity (4.4 ± 1.2-fold over the basal level) (Fig. 4Go, white columns). In the absence of doxycycline and hGH, cotransfection with either of the pTRE-STAT5 constructs had no effect on the basal transcriptional levels. However, in the presence of hGH the transcriptional activities were somewhat increased, which may indicate a certain leak from the pTRE plasmid. In the absence of hGH, doxycycline-induced expression of WT-STAT5a and b moderately increased the transcriptional activity by 1.8 ± 1.1- and 2.8 ± 1.6-fold, respectively. However, doxycycline-induced expression of the CA-STAT5a and b mutants profoundly increased the transcriptional activities (by 6.5 ± 1.6- and 4.8 ± 1.2-fold over basal levels, respectively) that were comparable to the hGH-induced levels (5.8 ± 0.7- and 5.5 ± 1.0-fold over basal levels, respectively). Moreover, the combination of hGH and doxycycline significantly increased the transcriptional level for both CA-STAT5a and b by 3.0 ± 0.9-fold and 2.3 ± 0.7-fold, respectively, compared with the respective hGH-induced levels. The combination of doxycycline and hGH increased the hGH induced level only slightly and not significantly in the WT-STAT5a and b transfection experiments.



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  		Figure 4. The Effects of STAT5 Mutants on the Transactivation of the Cyclin D2 Promoter

INS-r3 cells were transiently cotransfected with firefly luciferase reporter gene construct containing the cyclin D2 promoter and pTRE-STAT5 constructs and the internal standard, pRL-SV40, which contains the Renilla luciferase gene. The cells were cultured for 24 h in the absence or presence of 500 ng/ml doxycycline (Dox). Seven hours before harvesting, the cells were cultured in the absence or presence of 500 ng/ml hGH. White columns represent transfections without pTRE-STAT5 constructs; hatched gray columns, pTRE-WT-STAT5a; solid gray columns, pTRE-WT-STAT5b; hatched black columns, pTRE-CA-STAT5a; solid black columns, pTRE-CA-STAT5b. The results are expressed as the ratios between the firefly and the Renilla luciferase activities (means ± SEM, n = 4). *, P <= 0.05; ***, P <= 0.0001.

 
Inducible Expression of CA-STAT5b in INS-1 Cells
To address whether STAT5 activation alone is sufficient to drive the mitogenic response of ß-cells, we generated stably transfected INS-1 cells that, in an inducible manner, express the CA-STAT5b. The INS-r9 cell line, which stably expresses the reverse tetracycline/doxycycline-dependent transactivator (37), was transfected with the pTRE-CA-STAT5b construct, and stably transfected clones were selected by hygromycin resistance. Clones were tested for DNA integration of the pTRE plasmids by reporter-gene assay and PCR. The expression of CA-STAT5b in the clone, 1*6–18, was further determined by Western blot analysis. The cells were cultured for 48 h in the absence or presence of doxycycline, and immunoprecipitates from total cell extracts were examined for activated STAT5 expression using an antibody against phosphotyrosines. Weak tyrosine phosphorylation of CA-STAT5b was detected in doxycycline-stimulated 1*6–18 cells (Fig. 5Go, lanes 5 and 6). Expression of CA-STAT5b was further examined in 1*6–18 cells by immunocytochemical staining. There was no detectable increase of nuclear STAT5 after 4 h of doxycycline stimulation whereas at 24 h the level was highly increased compared with control and hGH-stimulated cells (data not shown).



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  		Figure 5. Expression of CA-STAT5b

Cells were cultured in the absence (lanes 1, 3, and 4) or presence (lanes 2, 5, and 6) of doxycycline (Dox) for 48 h. Fifteen minutes before harvesting, control cells were stimulated with hGH (lane 4). The cells were lysed and total protein extracts were immunoprecipitated (IP) with anti-STAT5b (C-17) antibody. Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with phosphotyrosine antibody followed by reprobing with STAT5b antibody. IB, Immunoblotting.

 
To determine the DNA-binding activity of the doxycycline-inducible CA-STAT5b, we performed EMSA. 1*6–18 cells were cultured in 0.5% serum with or without various concentrations of doxycycline for 24 h. As positive control we used cells stimulated for 15 min with hGH. Nuclear extracts were incubated with a radiolabeled double-stranded oligonucleotide representing a putative STAT5 binding element of the murine cyclin D2 promoter located at position -1129 to -1121 (39). hGH treatment was found to induce strong DNA binding activity of endogenous STAT5 (Fig. 6AGo, lane 4) whereas CA-STAT5b exhibited weak but significant DNA binding activity in a doxycycline-inducible manner (Fig. 6AGo, lanes 5–9), reaching a maximum level at 1.0 µg/ml. To verify the binding of STAT5 to the probe, supershift experiments were carried out. The complex formation was inhibited with a STAT5 antibody (lane 10) but not with an irrelevant control antibody (CDK4 antibody; lane 11). Furthermore, STAT5 binding was competitively inhibited by incubation with a nonradioactive WT-probe (lane 12) but not with a mutated probe (lane 13), and no STAT5 binding was observed to a radiolabeled mutated probe (lane 14).



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  		Figure 6. DNA-Binding and Transcriptional Activity of CA-STAT5b

A, The radiolabeled oligonucleotide probes, D2-WT and D2-MUT, were incubated with nuclear extracts from 1*6–18 that had been cultured in the absence (lanes 3, 4, and 10–14) or presence (lanes 5–9) of indicated amounts of doxycycline (Dox) and were stimulated with 500 ng/ml hGH (lanes 4 and 10–14) 15 min before harvesting. Free (lanes 1–2) and bound probe were separated by nondenaturing gel electrophoresis and visualized by autoradiopgraphy. Lane 10, Addition of STAT5 antibody; lane 11, addition of CDK4 antibody; lane 12, addition of cold WT-probe (1:100); lane 13, addition of cold MUT-probe (1:100); lane 14, hot MUT-probe. The band indicates the migration of the cyclin D2*STAT5/CA-STAT5b complex. B, INS-r9 (minimized figure) and 1*6–18 were transiently transfected with the firefly luciferase reporter gene construct containing either the wild-type (solid bars) or the mutated (hatched bars) cyclin D2 promoter and the internal control construct, pRL-SV40, which contain the Renilla luciferase gene. The cells were cultured for 24 h in the absence or presence of the indicated amounts of doxycycline. Twenty-four hours before harvest, the cells were cultured in the absence (left panel) or presence (right panel) of 0.5 µg/ml hGH. The results are expressed fold induction compared with control levels (mean ± SEM, n = 4). *, P <= 0.05; **, P <= 0.001.

 
To address whether CA-STAT5b is sufficient to drive transactivation of the cyclin D2 promoter in our stable clone, we performed promoter-reporter gene assay as above. The INS-r9 and the stably transfected clone were transiently transfected with the firefly luciferase reporter gene containing the cyclin D2 promoter with the STAT5-responsive element. Human GH stimulation significantly increased the transcriptional activity of the cyclin D2 promoter in INS-r9 cells by 3.8 ± 0.9 (Fig. 6BGo, inset) and in 1*6–18 cells by 9.6-fold ± 3.3 over basal levels (Fig. 6BGo, right panel), respectively. Addition of doxycycline alone did not affect the transcriptional activity of the INS-r9 cells (inset) but dose-dependently increased the transcriptional activity in a dose-dependent manner in 1*6–18 reaching a significant induction at 0.5 µg/ml and maximal level (3.0 ± 1.0-fold over basal) at 1 µg/ml doxycycline (left panel). The combination of hGH and doxycycline addition further increased the transcriptional activity of the cyclin D2 promoter (39.3 ± 18.7-fold induction, right panel).

To examine the functional role of the STAT5-binding element in the cyclin D2 promoter, we performed site-directed mutagenesis on the cyclin D2 construct using the primers described in Materials and Methods. Transfection of 1*6–18 cells with this mutated construct resulted in an approximately 80% decrease of hGH-induced transcriptional activity, and the activity induced by the combination of hGH and doxycycline was significantly reduced by 73% (Fig. 6BGo, right panel, hatched bars). The doxycycline-induced activity was completely blocked using 1 µg/ml doxycycline (left panel, hatched bar).

Expression of CA-STAT5b in INS-1 Cells and in Primary ß-Cells Results in Increased Cell Proliferation
The effect of induced CA-STAT5b on ß-cell proliferation was measured in the stable clone, 1*6–18, by viability study and in primary ß-cells by virus transduction followed by bromodeoxyuridine (BrdU) incorporation assay. Cells (INS-r9 and 1*6–18) were grown for 5 d in 0.5% fetal calf serum (FCS) and in the presence or absence of either 0.5 µg/ml hGH or 1.0 µg/ml doxycycline or a combination, and viable cells were counted using trypan blue staining (Fig. 7Go). Stimulation with hGH significantly increased the cell number in both parental cells (white columns) and 1*6–18 cells (black columns) by 115% and 129%, respectively. Doxycycline alone did not affect the basal cell number in the parental cells whereas the cell number was significantly increased in the 1*6–18 cell line by 76% compared with basal level. The combination of hGH and doxycycline did not affect the hGH-induced level in the parental cells but significantly decreased the proliferation in the 1*6–18 cell line by 82% compared with the hGH-induced level. This might be due to an apoptotic effect caused by overstimulation and overexpression of activated STAT5, which has also been reported for Ba/F3 cells (40).



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  		Figure 7. The Effect of Doxycycline (Dox)-Induced CA-STAT5b Expression on Proliferation

A, Cells (50,000 cells per well) were seeded in a 24-well plate and cultured for 5 d in the absence or presence of either 1 µg/ml doxycycline or 500 ng/ml hGH. After incubation, the cells were detached in trypsin-EDTA and counted using trypan blue for viability (mean ± SEM, n = 3). Results are represented as cells per well. *, P <= 0.05; **, P <= 0.001.

 
To be able to examine the effect of STAT5 on ß-cell proliferation in primary ß-cells, adenovirus expressing WT- and CA-STAT5b were generated. The expression levels of the transduced constructs were investigated by Western blot analysis in INS-1 cells (Fig. 8Go). The expression levels for both WT- and CA-STAT5b were increased with increasing adenovirus titer, and for both constructs a near-maximal expression level was obtained at 1.25 x 109 plaque-forming units (pfu) or 625 multiplicity of infection (MOI; lanes 3 and 9). To verify constitutive activity of the CA-STAT5b construct, primary ß-cells cultured in monolayer were transduced with the appropriate amount of the CA-STAT5b adenovirus construct, and nuclear translocation was visualized by immunocytochemistry. The staining showed that CA-STAT5b was highly expressed in cells that had been transduced for 2 d corresponding to a subpopulation of approximately 60–70%. In addition, nuclear localization of CA-STAT5b was detectable in all transduced cells in the absence of growth factor (data not shown).



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  		Figure 8. Expression of STAT5b Adenovirus Constructs

Western blot analysis was performed on total protein extracts prepared from INS-1 cells that had been transduced with the indicated amounts of WT-STAT5b or CA-STAT5b adenovirus constructs for 2 h. Ten microliters of each extract were separated by SDS-PAGE on a 7.5% gel. Proteins were transferred to nitrocellulose, incubated with antibodies, and visualized using ECL Western blotting detection reagents.

 
The mitotic activity was examined in the primary ß-cells in monolayer culture by BrdU incorporation assay, as previously described (38). Stimulation of mock cells by 0.5 µg/ml hGH for 24 h resulted in a 1.6 ± 0.1-fold increase of BrdU incorporation (Fig. 9AGo). Transduction with WT-STAT5b-adenovirus moderately increased the DNA synthesis, but additional stimulation by hGH did not affect the BrdU incorporation significantly compared with control. However, transduction of CA-STAT5b-adenovirus profoundly increased ß-cell replication (3.4 ± 0.5-fold compared with the hGH-induced level). Addition of hGH resulted in a lowering of the response [2.4 ± 0.5 compared with the hGH-induced level; similar observations with INS-1 cells transduced with CA-STAT5b-adenovirus (data not shown)]. Transduction with adenovirus containing a luciferase construct and culturing in the absence or presence of hGH did not affect the DNA synthesis (data not shown). Figure 9BGo shows a representative double immunostaining for insulin [fluorescein isothiocyanate (FITC)-green fluorescence] and BrdU (Texas red-red fluorescence) in ß-cells transduced with CA-STAT5b-adenovirus.



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  		Figure 9. The Effect of Activated STAT5b on BrdU Incorporation into Primary ß-Cells

Islet monolayer cultures were cultured in the absence (mock) or presence of STAT5 adenovirus (5 x 108 pfu/ml) for 2 d followed by a 24-h culture period in the absence or presence of 0.5 µg/ml hGH and in the presence of 10 µM BrdU for 24 h. The cells were fixed and immunocytochemically double-stained for BrdU and insulin. A, A total of 1500 cells were counted in each preparation, and the percentage of BrdU-positive ß-cells was determined by counting under the microscope. The results are expressed as fold induction compared with control levels (mean ± SEM, n = 4). B, Example of immunocytochemical double staining for BrdU (red fluorescence) and insulin (green fluorescence) in untransduced (control) and in CA-STAT5b-transduced islet cells both cultured in the absence of hGH stimulation. ***, P <= 0.0001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Growth of the pancreatic ß-cell can be induced by multiple factors such as metabolites (glucose, amino acids) (41, 42), insulin (43), IGF-I (44), incretins (glucagon-like peptide-1 and glucose-dependent insulinotropic peptide) (45, 46, 47), and hormones of the GH family (PL, PRL, and GH) (9). Several distinct, although in some cases partially overlapping, signaling pathways are probably involved in the action of these diverse growth factors. Studies of knockout mice have indicated that insulin receptor substrate 2, protein kinase Bß, and CDK4 are critical for a normal postnatal ß-cell replication capacity (31, 48, 49, 50). Furthermore, several in vitro studies have implicated cAMP, cytosolic Ca2+, p38 and p42/44 MAPK, phosphatidylinositol 3-kinase, protein kinase B{alpha}, and recently protein kinase C{zeta} in the regulation of ß-cell replication (47, 51, 52, 53, 54, 55, 56, 57). Our studies involving overexpression of a dominant negative STAT5 mutant in INS-1 cells indicated that the STAT5 pathway is essential for GH/PRL/PL-induced ß-cell replication and that the cell cycle-regulatory factor, cyclin D2, was induced by hGH in a STAT5-dependent manner.

It is well known that the cyclin Ds are transcriptionally regulated in response to growth factors and that they are responsible for controlling entry into the S phase of the cell cycle. Most cell types express all three D-type cyclins but at different levels depending on the cell lineage and mitogens. Studies have indicated redundant as well as specific functions of these proteins (58, 59, 60). For instance, the cyclin D1- and D2-deficient mice are indistinguishable phenotypically from their wild-type littermates, but whereas the cyclin D1-deficient mice showed severe retinopathy and impaired breast development, the cyclin D2-deficient mice showed impaired gonadal cell proliferation.

In accordance with our previous finding of hGH-stimulated mRNA expression in INS-1 cells of cyclin D2, but not D1 and D3, after a 24-h stimulation period, we have shown in this study that the cyclin D2 mRNA and protein expression were up-regulated by hGH in a time-dependent manner with maximal induction at 24 and 12 h, respectively. No effect of hGH was seen on cyclin D1 and D3 expression during the time course studied. The regulation of cyclin D2 protein expression was furthermore found to be STAT5 dependent. Thus, the expression was partially inhibited by overexpression of STAT5a{Delta}749 in accordance with the partial inhibition of the cyclin D2 mRNA expression shown previously (19). The catalytic subunit of cyclin D, CDK4, which is normally constitutively expressed during the cell cycle (61), was unaffected by hGH stimulation in the INS-1 cells. CDK4 and 6 are coexpressed in many cell types and appear, to some extent, to have redundant functions (62). Interestingly, expression of CDK6 in INS-1 was undetectable, which may explain the dramatic effects on the ß-cell mass in CDK4 knockout mice (31), because there would be no compensatory effect of CDK6 present in the ß-cells of these mice.

The CX experiments indicated that the transcriptional induction by hGH of cyclin D2 is independent of de novo protein synthesis. Cyclin D2 protein was undetectable after 4 h of CX treatment of INS-1 cells, indicating a rapid turnover consistent with observations in other cell types where cyclin Ds are found to be transiently induced and degraded during the cell cycle with a half-life of 10–30 min, depending on the cell type (61). Studies in human hematopoietic cells have shown that cyclin D1 can be a direct target gene for STAT5 in response to cytokine treatment (63, 64), and it was shown recently that PRL-induced activation of the human cyclin D1 promoter (which contains two consensus sequences for PRL-induced STAT binding) depend on STAT5 binding to the distal GAS site (65). This site appears to be mutated in the rodent cyclin D1 genes (Ref. 66 and data not shown), probably explaining the lack of hGH effect on this gene in rat ß-cells. An IL-2-regulated functional STAT-5 binding site was recently identified at position -1199 to -1191 of the human cyclin D2 promoter (67).

To further characterize the functional role of STAT5 and cyclin D2 in ß-cells, we took advantage of the CA mutants developed by Onishi and co-workers in 1998 (34). These mutants were reported to induce cell growth and survival of the IL-3-dependent cell line, BA/F3, in the absence of cytokine. The mechanism behind the constitutive activity of these mutants is not known, but it was found that they are constitutively tyrosine phosphorylated and can bind to GAS sequences. Using reporter-gene assay we showed that both the CA-STAT5a and CA-STAT5b mutants were capable of activating the cyclin D2 promoter in INS-1 cells in the absence of hGH, and the stimulated transcriptional activity was comparable to that induced by hGH. Stimulation with hGH further increased the cyclin D2 promoter activity. The enhanced activity of the CA-STAT5 mutant compared with wt STAT5 may reflect the prolonged DNA binding of the mutants as previously described (34), a phenomenon that we also observed in INS-1 cells (data not shown). Another possibility might be that other GH-dependent pathways play a role in cyclin D2 expression.

To investigate the role of the putative STAT5 binding sequence located at position -1129 to -1121 of the murine cyclin D2 promoter (39), we performed EMSA using a labeled double-stranded oligonuclotide probe representing the GAS motif of the cyclin D2 promoter. We stably transfected INS-r9 cells with the CA-STAT5b using the inducible Tet-On expression system. Endogenous hGH-activated STAT5 from these cells was found to bind to the GAS motif of the cyclin D2 promoter, but not to the mutated sequence (described in Materials and Methods). Furthermore, in the absence of hGH stimulation, CA-STAT5b in these cells exhibited binding to the GAS site of the cyclin D2 promoter and conferred stimulation of the cyclin D2 promoter-reporter in transcriptional reporter assay. The binding to the GAS site in vitro was quite weak as observed by Onishi et al. (34) in Ba/F3 cells. Whether this reflects that CA-STAT exerts its transcriptional effects mainly due to sustained DNA binding and a strong transactivation capacity or that the in vitro DNA binding to a relatively short probe does not reflect the binding to the endogenous promoter in which adjacent cis elements may contribute to stabilize the complex is not known. When using a cyclin D2 promoter-luciferase construct containing a mutated GAS motif, no effect of CA-STAT5b in the 1*6–18 clone was observed, and hGH-induced transcriptional activity was partially inhibited. These results provide evidence that this GAS site of the cyclin D2 promoter is a functional binding site for STAT5 in pancreatic ß-cells and that this site is conserved between the rodent and human species. The partial inhibition of hGH-stimulated cyclin D2 transcription obtained with the GAS-mutated promoter construct and the data described above showing partial inhibition by the STAT5a{Delta}749 mutant of cyclin D2 mRNA and protein expression may indicate that STAT5-independent signaling pathways also contribute to the hGH-induced up-regulation of the cyclin D2 gene in INS-1 cells.

STAT5a and b are coexpressed in many tissues; however, their relative expression levels differ, and their knock-out phenotypes indicate that they exert redundant as well as nonredundant functions. STAT5a is highly expressed in the mammary gland relative to STAT5b (68, 69), which may explain why STAT5a-deficient mice show incomplete mammopoiesis and failure of lactogenesis but remain indistinguishable from wild-type mice in size, weight, and fertility (68). This phenotype is similar to that observed in the PRLR-deficient mice (70). On the other hand, the STAT5b isoform is highly expressed in the liver relative to STAT5a (71), which may explain the defects in sexual dimorphism of liver gene expression in STAT5b-deficient mice (72). These mice showed characteristics similar to those of Laron-type dwarfism, a human GH-resistance disease due to a defective GHR. Furthermore, STAT5b-deficient mice are small and resistant to GH and have elevated plasma GH and low plasma IGF-I, indicating a major role for STAT5b in growth and proliferation (69, 72). In the pancreatic ß-cells the relative abundance of STAT5b is higher than of STAT5a (36). We addressed the role of STAT5b in the regulation of proliferation in both INS-1 cells, using our stably transfected clone expressing CA-STAT5b in a doxycycline-inducible manner, and in primary rat ß-cells by adenovirus-mediated transduction of WT-STATb and CA-STAT5b. In INS-1 cells we found that doxycycline-induced CA-STAT5b expression was sufficient to enhance proliferation correlating with the previous findings in Ba/F3 cells (34). Interestingly, hGH stimulation in the presence of doxycycline severely reduced proliferation/survival of these cells. This could be due to an inhibition of JAK activity by members of the suppressor of cytokine signaling/cytokine-inducible inhibitor of signaling (CIS) family of genes that has also been shown for the Ba/F3-expressing CA-STAT5 cells, where enforced JAK-binding protein/suppressor of cytokine signaling-1 (SOCS-1), but not CIS, expression induced apoptosis (40). Another possibility could be accelerated apoptosis due to an overexpression of mitogens. Recently, the oncoprotein c-Myc, which is implicated in ß-cell proliferation, has been reported to induce apoptosis when it is continuously activated in ß-cells by a regulatable system in transgenic mice. However, when c-Myc is only intermittently activated, it leads to islet hyperplasia (73).

In primary ß-cells we observed a markedly enhanced proliferation in cells transduced with CA-STAT5b. These results confirmed that an activated STAT5 pathway is sufficient to drive ß-cell proliferation and furthermore indicates that STAT5 activation may be the limiting factor in hGH-induced ß-cell proliferation, since the effect of CA-STAT5b compared with the effect of hGH on cells transduced with WT-STAT5b is increased. Limiting components in STAT5 activation may be the GHR and/or PRLR, which previously have been shown to be heterogeneously expressed within the islets (74). The combination of CA-STAT5b expression and hGH stimulation did not further enhance the mitogenic activity of the ß-cells but rather resulted in a decrease of the proliferation level compared with CA-STAT5b expression alone. As discussed above, this could be due to hGH-promoted activation of CIS family members or a certain toxicity when the ß-cells are overstimulated with mitogen.

In conclusion, our studies have shown that activated STAT5b is sufficient to drive proliferation efficiently in both INS-1 cells and primary ß-cells and that the cell cycle regulator, cyclin D2, is a direct target gene for STAT5. Further studies are however required to establish whether cyclin D2 is the major target in regulating ß-cell proliferation and whether STAT5 activity is the primary determinant of cyclin D2 expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Antibodies
Recombinant hGH was obtained from Novo Nordisk A/S (Gentofte, Denmark). Hygromycin B was obtained from Calbiochem (La Jolla, CA). CX (no. C-7698), doxycycline, and G418 were from Sigma (St. Louis, MO). Mouse anti-cyclin D1 (no. MS-210), cyclin D2 (no. MS 221), and cyclin D3 (no. MS-215) monoclonal antibodies were purchased from Neo Markers (Fremont, CA). Rabbit anti-CDK4 (no. sc-260), -CDK6 (no. sc-177), and anti-STAT5b (C-17) (no. sc-835) polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse antiphosphotyrosine-horseradish peroxidase (HRP) monoclonal antibody (no. 03–7720) was from Zymed Laboratories, Inc. (South San Francisco, CA). HRP-linked mouse IgG (no. NA 931) and rabbit IgG (no. NA 934) were from Amersham Pharmacia Biotech (Buckinghamshire, UK). FITC-conjugated goat antirabbit IgG (no. 111-095-003) was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Cells and Culture
The INS-1-derived cell lines, INS-r3 and INS-r9 cells, kindly provided by Dr. P. B. Iynedjian (Geneva, Switzerland), were cultured in RPMI 1640 supplemented with Glutamax and 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM ß-mercaptoethanol (complete medium) and 100 µg/ml G418. The INS-1 cell clones, EB03, BB32 [described previously, (21)], and 1*6–18 were cultured in the media described above plus 100 µg/ml hygromycin B. Islets were isolated from 3- to 5-d-old Wistar rats by the collagenase method (32) and cultured in RPMI 1640 supplemented with 10% newborn calf serum (NCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 0.0375% NaHCO3, and 20 mM HEPES at 37 C in a humidified atmosphere. Before use, the islets were precultured for 1 wk in 15 ml RPMI 1640 supplemented with 0.5% NuSerum (no. 355100, Becton Dickinson Labware, Bedford, MA). Animal research complied with all relevant federal guidelines and institutional policies.

Plasmids
The pGL2-cyclin D2 promoter construct was provided to us by Dr. M. Eilers (Marburg, Germany) and is generated by insertion of 2.3 kb of the mouse cyclin D2 promoter into the SacI site of the pGL2-basic vector (33). The cDNA encoding the WT mouse STAT5a and STAT5b and the constitutively active mouse STAT5a and STAT5b mutants, CA-STAT5a and CA-STAT5b (also known as STAT5a 1*6 and STAT5b 1*6), respectively, which contain two amino acid substitutions (H299R and S711F) that make them constitutively tyrosine phosphorylated (34) were kindly provided by Dr. T. Kitamura (Minato-ku, Tokyo, Japan). To generate inducible STAT5 expression vectors, each of the STAT5 cDNAs was subcloned into the Tet-On gene expression vector, pTRE (CLONTECH Laboratories, Inc., Palo Alto, CA) using the EcoRI and XbaI restriction site of the polylinker. The pGL2–1A is generated by the insertion of the 5'-flanking region of PRLR exon 1A (-462/+81) into the pGL2-basic vector (35). The pRL-SV40 vector, used as internal standard, contains the coding region of the Renilla luciferase gene under the transcriptional control of the SV40 early enhancer/promoter (Promega Corp., Madison, WI). The pUC18 vector was used as carrier plasmid.

Adenoviral Constructs
Generation of recombinant adenovirus was carried out using the AdEasy kit from Q-BIOgene (AES1000B, Carlsbad, CA) according to the manufacturer’s instructions. Briefly, mouse WT-STAT5b cDNA was subcloned into a HindIII–XbaI site, and mouse CA-STAT5b cDNA was subcloned into a KpnI–NotI site in the multiple cloning site of the pShuttle-cytomegalovirus transfer vector. One microgram of recombinant linearized pShuttle-cytomegalovirus and 200 ng Ad5{Delta}E1/{Delta}E3 were cotransformed in BJ5183 electro-competent cells using general guidelines for Bio-Rad Laboratories, Inc. instruments (200 {Omega}, 25 µF, 2.5 kV). Recombinants were identified by plasmid size and digestion with PacI. Positive clones were transformed in DH5{alpha}-competent cells using the same conditions as for BJ5183 electro-competent cells. Plasmids were checked by restriction enzyme analysis and sequencing. Five micrograms of recombinant adenoviral DNA were completely digested with PacI and used for transfection of 293A cells. After viral plaque formation, small-scale virus amplification was carried out. Recombinant viruses were screened using Western blot analysis. Large-scale virus amplification was performed and recombinant viruses were purified on CsCl gradients. Virus titers were measured at A260.

RT-PCR
Cells were seeded in 60-mm dishes (2 x 106 cells per dish). The cells were cultured for 2 d in 4 ml/dish in their respective mediums. The medium was changed to medium containing 0.25% fatty acid-free BSA for BB32/EB03 and to medium containing 0.5% FCS for 1*6–18, and cells were stimulated as indicated in the figure legends. Islets were seeded in 100-mm dishes (~4000 islet per dish). The islets were cultured in 15 ml medium per dish containing 0.5% human serum, and islets were stimulated with 0.5 µg/ml hGH for 24 h and harvested. RNA extraction, cDNA synthesis, primer designs, and PCR were performed as described previously (19).

Protein Extraction, Immunoprecipitation, and Western Blot Analysis
Cells were seeded in 100-mm dishes (4 x 106 cells per dish) and cultured for 2 d in 10 ml medium/dish. The medium was changed as described above and cells were stimulated as indicated in the figure legends. Islets were seeded in 100-mm dishes (~3000 islets per dish) and cultured for 1 d in 10 ml medium containing 0.5% human serum. The medium was changed, and the islets were stimulated with 0.5 µg/ml hGH and harvested to the time points indicated in the figure. The cells or islets were washed once in cold PBS and transferred to Eppendorf tubes (Madison, WI) and pelleted. The cells/islets used for cyclin D detection and immunoprecipitation were resuspended in 500 µl lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween-20, 1% Triton X-100, 1 mM NaF, 10 mM ß-glycerophosphate), and the cells used for STAT5 detection were resuspended in 500 µl PBS containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS). The lysis buffers were supplemented with 1 µg/ml leupeptin, 1 µg/ml aprotonin, 0.5 mM [4-(2-aminoethyl)benzenesulfonylfluoride, HCl], 0.5 mM sodium orthovanadate, and 1 mM dithiothreitol just before use. The cells/islets were lysed on ice for 30 min and cell debris was removed by centrifugation at 15,000 x g for 20 min. The supernatants were stored at -80 C. Proteins were denatured by boiling for 2 min in 5x sample buffer (50 mM Tris-HCl, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol). For immunoprecipitations, equal amounts of proteins were incubated with antibodies at 4 C for 1.5 h. Immune complexes were precipitated by adding protein G-sepharose (no. 17–0618-01, Amersham Pharmacia Biotech) overnight at 4 C. The precipitates were washed three times in lysis buffer, and proteins were eluted by boiling in 1x sample buffer.

Proteins were separated by electrophoresis on a NuPAGE 4–12% Bis/Tris Gel (no. NP0322, Invitrogen Corp., Carlsbad, CA) with NuPAGE 3-(N-morpholino)propanesulfonic acid SDS running buffer (no. NP0001, Invitrogen) using the Novex electrophoresis and blotting system (Invitrogen). The nitrocellulose membrane was blocked for 1 h in 5% skimmed milk powder PBST (PBS and 0.05% Tween-20) and washed. The membrane was incubated with primary antibodies diluted in PBST overnight at 4 C, washed, and incubated for 1 h at room temperature with secondary HRP-linked antibodies (1:5000). The cyclin D antibodies were diluted 1:200; CDK 4/6, 1:500; phosphotyrosine-HRP, 1:1000; and STAT5b (C-17), 1:500. Proteins were visualized using enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Pharmacia Biotech).

Transient Transfection and Dual Luciferase Reporter (DLR) Assay
Cells were seeded in 24-well plates (~300,000 cells per well) in 1 ml/well in their respective medium and cultured overnight. The cells were transfected with 2 µl/well LipofectAMINE 2000 Reagent (no. 11668–019, Invitrogen) in 50 µl/well Opti-MEM and 0.8 µg DNA/50 µl Opti-MEM/well (500 ng of reporter plasmid, 10 ng of internal standard, and 290 ng of carrier plasmid). The cells were transfected overnight in 500 µl/well Opti-MEM added to 100 µl/well DNA/Lipo-mix. The medium was changed to RPMI 1640 containing 0.5% FCS (500 µl/well) and incubated 24 h in the presence or absence of doxycycline. Seven hours or 24 h before harvesting, 0.5 µg/ml hGH was added to the respective wells. The cells were lysed by adding 100 µl/well of 1x passive lysis buffer (Promega Corp.) followed by shaking the plate 15 min at room temperature (RT). The cell extracts were stored in the plate at -20 C. Luciferase activity was measured as described previously (36) using a luminometer (Lumat LB 9507, EG & G, Berthold) and DLR Assay System E1910 (Promega Corp.).

Stable Transfection
Establishment of stable clones, INS-r3 and INS-r9, expressing the reverse tetracycline-dependent transactivator has been described previously (37) and kindly provided by Dr. P. B. Iynedjian (Geneva, Switzerland). The CA-STAT5b mutant cDNA was subcloned into the Tet-On gene expression vector, pTRE (CLONTECH Laboratories, Inc.) using the EcoRI and XbaI restriction sites of the polylinker. INS-r9 cells were seeded (1 x 107 cells per 100-mm dish) and cultured overnight in complete medium containing 100 µg/ml G418. The following day the medium was changed to Opti-MEM, and transfection, further culturing of the cells, and selection of clones was performed as described previously (19). Hygromycin-resistant clones were tested for pTRE-CA-STAT5b integration by DLR assay and PCR. pGL2–1A was used as reporter plasmid, pRL-SV40 was used as internal control, and pUC18 was used as carrier plasmid. The cells were transfected as described above. The cells were cultured for 24 h with 0.5 µg/ml doxycycline and then for an additional 7 h with 0.5 µg/ml hGH before harvesting.

Mutagenesis
The pGL2-cyclin D2 plasmid was subjected to site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA). Primers used for cyclin D2-MUT were: 5'-TCC CCG AGC CAT TTC CTA TCA AGC TGT ATC AAT GTG GCA AGT C (forward); 5'-GAC TTG CCA CAT TGA TAC AGC TTG ATA GGA AAT GGC TCG GGG A (reverse). The mutated cyclin D2 promoter construct was checked by restriction cleavage and sequencing.

Immunofluorescence
The cells (200,000) were seeded in 9-cm2 plastic cell culture slide flasks (NUNC, Roskilde, Denmark) and cultured 2 d in their respective mediums. The medium was changed to RPMI 1640 containing 0.5% FCS, and cells were cultured overnight. The medium was changed and cells were cultured in the absence or presence of either 0.5 µg/ml hGH or 1.0 µg/ml doxycycline for the indicated time. The cells were washed twice in RPMI 1640 without serum and fixed in 1% paraformaldehyde. The cells were stained overnight at 4 C with the anti-STAT5 antibody diluted 1:100 in PBS with 0.3% Triton-X-100 and 0.1% human serum albumin. The antibody was visualized by the FITC-conjugated secondary antibody, and the slides were mounted in 20% glycerol/0.05 M Trisma base adjusted to pH 8.4 and stored at 4 C.

Nuclear Extracts
Cells were seeded in 100-mm dishes (4 x 106 cells per dish) and cultured for 2 d in 10 ml/dish in their respective mediums. Medium was changed to medium containing 0.5% FCS, and cells were cultured for 24 h in the presence or absence of doxycyline. When indicated, the cells were stimulated with 0.5 µg/ml hGH for 15 min. Nuclear extracts were prepared essentially as described previously (27, 36). Briefly, cells were lysed in hypotonic buffer containing 1% Triton X-100. Nuclei were collected by centrifugation, and nuclear proteins were extracted in hypertonic buffer containing 400 mM NaCl. After centrifugation, aliquots of the supernatants were frozen in liquid nitrogen and stored at -80 C. Protein concentrations were measured using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL).

EMSA
EMSA was performed essentially as described previously (27, 36). Briefly, the double-stranded oligonucleotides D2-WT (5'-agctCATTTCCTAGAAAGC) containing a STAT5 binding element derived from the mouse cyclin D2 promoter and the D2-MUT (5'-agctCATTTCCTATCAAGC) were radiolabeled in a fill-in reaction using [{alpha}-32P] dGTP (Amersham Pharmacia Biotech) and DNA polymerase (Klenow fragment; United States Biochemical Corp., Cleveland, OH) and used as probes (36). Nuclear extracts (5 µg) were incubated at 30 C with 20 fmol of probe in a 20 µl reaction. Free and bound probe were separated on a 6% DNA Retardation Gel (EC63652, Invitrogen) by gel electrophoresis and visualized by autoradiography.

Cell Proliferation Assay (Cell Counting)
Cells were seeded in 24-well plates (~50,000 cells per well) and cultured for 2 d in 1 ml/well of their respective mediums. The medium was changed to RPMI containing 0.5% FCS, and 1 µg/ml doxycycline and 0.5 µg/ml hGH were added to respective wells. After 5 d of culture, the cells were treated by trypsin-EDTA (100 µl/well) followed by addition of 10% FCS containing medium (300 µl). The cell number was quantitated by counting the viable cells using trypan blue solution (0.4%) (Sigma).

Adenovirus Transduction and BrdU Labeling
The murine WT-STAT5b and CA-STAT5b adenoviruses were generated using the Q-BIOgene AdEasy vector system as described above. The appropriate titer for each recombinant adenovirus was determined by the addition of various dilutions of each adenovirus to INS-1 cells cultured in six-well plates to 60–70% confluence (~2 x 106 cells per well). The cells were transduced with increasing amounts 0, 0.25 x 109, 1.25 x 109, 2.5 x 109, 5 x 109, and 1 x 1010 pfu/well as measured by A260. The adenovirus-containing medium (2 ml/well) was removed 2 h post infection by washing twice with PBS, and culture medium was added before incubation for an additional 16 h. Cells were washed twice with ice-cold PBS and lysed in ice-cold lysis buffer (50 mM HEPES, pH 7.5, 1% Nonidet P-40, 2 mM sodium orthovanadate, 4 mM EDTA, 1 mM [4-(2-aminoethyl)benzenesulfonylfluoride, HCl], 1 µg/ml aprotinin, and leupeptin). After sonication, insoluble material was removed by centrifugation, and samples were stored at -80 C. Cell lysates were normalized for total protein concentration (protein assay of Bio-Rad Laboratories, Inc., Hercules, CA), and 10 µg protein was used for immunoblot analysis. Proteins were separated by SDS-PAGE (4% stacking gel and 7.5% separating gel) and transferred by electroblotting to ECL nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were blocked for 1 h in TBST buffer (50 mM Tris/HCl, pH 7.4; 150 mM NaCl; 0.1% Tween 20) containing 5% skimmed milk powder. Membranes were washed and incubated with STAT5 antibody S21520 from Transduction Laboratories, Inc. (Lexington, KY) diluted 1:1000 in TBST for 1 h at room temperature, washed, and incubated with a diluted HRP-linked secondary antibody for additional 1 h. Proteins were visualized by the ECL detection system according to the manufacturer’s instructions (Amersham Pharmacia Biotech). A rainbow-colored protein molecular weight marker was used to determine molecular weight (Amersham Pharmacia Biotech).

Primary ß-cells cultured in monolayer were used for determination of DNA synthesis as described previously (38). Cells (150, 000) were seeded in plastic cell culture 9-cm2 slide flasks (NUNC) and cultured in 2 ml medium as previously described (38). Cells were transduced with WT-STAT5b or CA-STAT5b adenovirus 109 pfu/slide flask (based on Western blot and area of the slide flask) for 2 d. BrdU (10 µM) was added and culture was continued for a further 24-h period in the absence or presence of 0.5 µg/ml hGH. The slides were fixed in 1% paraformaldehyde and double-immunostained for BrdU and insulin as described previously (38). The mitotic index was determined by counting a total of 1,500 insulin-positive cells per flask and the fraction (expressed as percentage) of these positive for both insulin and BrdU.

Statistical Analysis
Statistical analysis was validated relative to Procedure General Linear Model of the SAS system. Two-way ANOVA with specified t tests for adjustment of multiple comparisons was carried out.


   ACKNOWLEDGMENTS
 
We thank Dagny Jensen (Novo Nordisk A/S), Iben Jonassen (Novo Nordisk A/S), and Jill McCuaig (University of Washington, Seattle, WA) for excellent technical assistance. We are indebted to Dr. T. Kitamura (University of Tokyo, Minato-ku, Tokyo) for supplying STAT5 cDNA, to Dr. M. Eilers (Phillips-Universität Marburg, Marburg, Germany) for supplying the cyclin D2-luciferase construct, to Dr. P. B. Iynedjian (University of Geneva School of Medicine, Geneva, Switzerland) for supplying INS-r3 and INS-r9 cells, and Dr. C. B. Wollheim (Centre Medical Universitaire, Geneve, Switzerland) for the supply of INS-1 cells.


   FOOTNOTES
 
This work was supported by the Danish Research Academy (B.N.F.), the Danish Academy of Technical Sciences (H.E.R.), and by a grant from the Danish Cancer Foundation (J.A.H.). J.H.N. was supported by Juvenile Diabetes Research Foundation, Danish Research Council, Danish Diabetes Association, and Novo Nordisk Foundation.

Abbreviations: BrdU, Bromodeoxyuridine; CA-, constitutionally active; CDK, cyclin-dependent kinase; CIS, cytokine-inducible inhibitor of signaling; CX, cycloheximide; DLR, dual luciferase reporter; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GAS, {gamma}-interferon-activated sequence; GHR, GH receptor; hGH, human GH; HRP, horseradish peroxidase; JAK, Janus kinase; pfu, plaque-forming unit; PL, placental lactogen; PRL, prolactin; PRLR, PRL receptor; pTRE, tetracycline response element; SDS, sodium dodecyl sulfate; STAT, signal transducer and activator of transcription; WT, wild-type.

Received for publication October 22, 2002. Accepted for publication February 7, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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