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The Hagedorn Research Institute Department of Cell Biology 2820 Gentofte, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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749, was expressed in INS-1 cells under the control of a
doxycycline- inducible promoter by stable transfection. Two clones
were found to exhibit dose-dependent, doxycycline-inducible expression
of STAT5749 and suppression of hGH-stimulated transcriptional
activation of a STAT5-regulated PRL receptor (PRLR) promoter-reporter
construct. Furthermore, induction of STAT5749 expression completely
inhibited hGH-induced DNA synthesis. Analysis of endogenous gene
expression revealed a doxycycline-dependent inhibition of
hGH-stimulated PRLR and cyclin D2 mRNA levels. Our results suggest that
GH/PRL-induced ß-cell proliferation is dependent on the Janus Kinase2
(JAK2)/STAT5 signaling pathway but not the MAPK, PI3K, and PKC
signaling pathways. Furthermore, the cell cycle regulator cyclin D2 may
be a crucial target gene for STAT5 in this process. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), and BrdU incorporation into primary
ß-cells was increased by 2.7 ± 0.4-fold over the basal level
(Fig. 1B). Human GH-induced DNA synthesis was completely inhibited in
both INS-1 and primary ß-cells (1.1 ± 0.1-fold and 0.9 ±
0.1-fold, respectively, over the basal levels) in the presence of 25
µM genistein, an inhibitor of protein tyrosine kinase
activity, as would be expected from inhibition of JAK2. In the presence
of the MEK inhibitor, PD98059 (20 µM), and the p38 MAPK
inhibitor, SB203580 (10 µM), hGH-induced DNA synthesis
was partially reduced in INS-1 cells (1.4 ± 0.1-fold and 1.4
± 0.1-fold, respectively, over basal levels), whereas these two
inhibitors had no significant effect in primary ß-cells. Wortmannin
(10 nM), a specific PI3K inhibitor, had no effect on
hGH-induced DNA synthesis in either INS-1 or in primary ß-cells,
whereas staurosporine (20 nM), an inhibitor of PKC, protein
kinase A (PKA), and protein kinase G (PKG), blocked both basal (not
shown) and hGH- induced proliferation of INS-1 cells (0.2 ±
0.1-fold of basal) but did not inhibit proliferation of primary
ß-cells. On the contrary, staurosporine was found to potentiate DNA
synthesis in primary cells. Addition of the various inhibitors in the
absence of hGH (data not shown) had no effect on the basal
proliferation level in any of the cell types with the exception of
staurosporine, as described above.
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). INS-1 cells and islets cultured in
0.5% serum were either left untreated or stimulated with 0.5 µg/ml
hGH for 15 min to 24 h. Nuclear extracts were prepared and
incubated with radiolabeled, double-stranded oligonucleotide (1A-GLE)
representing the previously identified STAT5-binding element of the
PRLR 1A promoter (10). Free and bound probe was separated by
nondenaturing PAGE and visualized by autoradiography. Nuclear extracts
from INS-1 cells that had been stimulated with hGH for 15 min
(upper panel, lane 2) produced a strong shifted band, which
was also present in incubates with nuclear extracts from INS-1 cells
treated for 1, 4, and 24 h with hGH (upper panel, lanes
3, 4, and 5, respectively). In contrast, this complex was absent in
incubates with nuclear extracts from untreated INS-1 cells (upper
panel, lane 1). A similar pattern was observed with nuclear
extracts from newborn rat islets (lower panel, lanes 1–5),
although the intensity of the complex was somewhat reduced after
prolonged hGH treatment (
30–50% after 4 and 24 h).
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749) in INS-1 Cells with Dominant Negative Activity749, which has been reported to exert dominant
negative activity (29). An INS-1 clone, INS-r3, stably transfected with
an expression plasmid encoding the reverse
tetracycline/doxycycline-dependent transactivator (30), was transfected
with either the expression vector, pTRE, without insert or the
pTRE-vector containing a cDNA encoding STAT5a749 under the control
of the tetracycline operator. Cotransfection of an expression vector
containing a hygromycin resistance gene allowed for selection of stably
transfected clones, which were tested for DNA integration of the pTRE
plasmids by PCR analysis. Twenty-five of 38 clones examined were found
to contain integrated pTRE vector without insert, and 2 of 69 clones
examined contained a full-length insert of STAT5a749.
Expression of STAT5a749 in the two transfected clones (termed BB32
and EB03) was determined by Western blot analysis (Fig. 3A). The cells were cultured for 20
h in the absence or presence of 0.05–1.0 µg/ml doxycycline, and
total cell lysates were examined for STAT5 expression using an antibody
raised against the amino acids (aa) 451–649 of STAT5. In the absence
of doxycycline (lanes 1 and 6) only wild-type STAT5 protein was
detectable, which in this experiment probably represents STAT5b, since
Western blot analysis, using a more potent antibody recognizing the C
terminus part of STAT5, indicates that STAT5b is approximately 4–5
times more abundant than STAT5a in these cells (data not shown). In
both cell lines, addition of 0.05 µg/ml doxycycline induced the
expression of a protein of faster mobility than wild-type STAT5, likely
to represent STAT5a749 (lanes 2 and 7). The effect of doxycycline
was found to be dose dependent with maximal expression observed at a
concentration of 0.1 µg/ml doxycycline for both BB32 and EB03 (lanes
2–5 and 7–10, respectively). Doxycycline had no effect on STAT5
protein expression in a control clone (termed BA15) stably transfected
with pTRE without insert (data not shown).
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749, EMSA
was performed as described above. The stably transfected clones, BA15,
BB32, and EB03, were cultured for 24 h in the absence or presence
of doxycycline and stimulated the last 15 min of the culture period
with 0.5 µg/ml hGH. Nuclear extracts of the cells were prepared and
incubated with radiolabeled 1A-GLE oligonucleotide probe, the mobility
of which was examined by gel electrophoresis. hGH treatment alone was
found to induce 1A GLE binding of nuclear protein in all the cell lines
(Fig. 3B
, lanes 2, 6, and 10), and this binding was unaffected by the
addition of doxycycline in BA15 cells (Fig. 3B, lane
4). In BB32 and EB03 cells, a doxycycline-dependent
increase of DNA complex formation was observed (Fig. 3B, lanes 8 and
12–16) likely to represent binding of STAT5a749. A
promoter-reporter gene assay (10) was used to address whether
overexpression of STAT5a749 would suppress signaling via
endogenously expressed STAT5 (Fig. 3C). The parental and the three
transfected lines were transiently transfected with the firefly
luciferase reporter gene under the transcriptional control of the
5'-flanking region of the PRLR gene containing a STAT5-responsive
element. hGH stimulation induced transcriptional activity of the PRLR
promoter in INS-1 cells by 2.9 ± 0.1-fold and in BA15 cells by
5.5 ± 0.8-fold (upper panel), in BB32 cells by
4.6 ± 0.4-fold (middle panel) and in EB03 cells by
17.7 ± 0.3-fold (lower panel) over the basal levels.
The addition of 0.5 µg/ml doxycycline affected neither the basal nor
the hGH-induced transcriptional activity in INS-1 and BA15 cells.
However, addition of doxycycline to BB32 and EB03 cells increased the
basal transcriptional level by 1.8 ± 0.3-fold and a 4.3 ±
1.3-fold, respectively (middle and lower panel).
The reason for this stimulatory activity is not known. However,
hGH-induced transcriptional activity in both BB32 and EB03 was
inhibited by the induction of STAT5a749 in a doxycycline
dose-dependent manner, which was significant in BB32 cells at a
concentration of 0.25 µg/ml and in EB03 cells at a concentration of
0.1 µg/ml.
Induction of STAT5a749 Results in Inhibition of hGH-Stimulated
DNA Synthesis in INS-1 Cells
To determine the influence of dominant negative STAT5 activity on
cell proliferation, 3H-thymidine incorporation
assay was carried out, as described above, on the transfected clones
(Fig. 4). hGH stimulated
3H-thymidine incorporation by approximately
2-fold in the two transfected clones (1.9 ± 0.1 and 2.4 ±
0.4 fold over the basal levels in BB32 and EB03 cells, respectively)
similar to what was found in INS-1 cells and BA15 cells (data not
shown). The addition of 1 µg/ml doxycycline had no effect on either
the basal or the hGH-induced DNA synthesis in INS-1 and BA15 cells
(data not shown). Neither was the basal level in BB32 (upper
panel) and EB03 (lower panel) affected by doxycycline
addition alone. However, induction of STAT5a749 by doxycycline in
BB32 and EB03 blocked hGH-induced 3H-thymidine
incorporation in a dose-dependent manner, whereas no effect of
doxycycline was observed on the growth response to 10% FCS. The
inhibition of 3H-thymidine incorporation was
maximal at a doxycycline concentration of 0.5 µg/ml for both the BB32
cells and the EB03 cells, at which close to basal levels of DNA
synthesis were detected (1.25 ± 0.03-fold and 1.1 ±
0.2-fold over basal in BB32 and EB03 cells, respectively).
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) using BrdU incorporation and
DNA staining with propidium iodide. Stimulation with hGH for 24 h
was found to increase the percentage of cells in S-phase by
approximately 2-fold in both INS-1 cells and the transfected clones
(INS-1, 2.5 ± 0.2; BA15, 1.9 ± 0.3; BB32, 1.8 ± 0.1;
and EB03, 2.5 ± 0.3) (Table 1, middle column). The
percentage of cells in the G2/M phase was not
affected by hGH stimulation (Table 1, third column), whereas
the percentage of cells in the
G0/G1 phase was found to
decrease upon hGH stimulation, indicating that hGH primarily in these
experiments increases the
G0/G1-to-S phase transition
frequency. Neither the basal nor the hGH-induced cell cycle profiles in
INS-1 and BA15 cells were affected by the addition of 0.5 µg/ml
doxycycline. Furthermore, addition of doxycycline alone did not affect
the cell cycle profiles in either BB32 or EB03 cells. However, the
hGH-induced increase of cells in S-phase was totally blocked by the
induction of STAT5a749 expression in both BB32 and EB03 cells.
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749749 overexpression on the endogenous mRNA
levels of the PRLR 1A and the cell cycle regulators, cyclin D1, D2, and
D3, were investigated by quantitative RT-PCR in BA15, BB32, and EB03
cells (Fig. 5). The PRLR mRNA levels were
increased by hGH in BA15 cells by 7.4 ± 0.4-fold (upper
panel) in BB32 by 4.1 ± 0.5-fold (middle panel),
and in EB03 cells by 4.4 ± 0.4-fold (lower panel),
respectively, in accordance with previous results in INS-1 cells (10).
Addition of doxycycline alone had no effect on either the basal or the
hGH-induced PRLR mRNA level in BA15 cells. Neither was the basal level
affected by doxycycline in BB32 and EB03 cells. However,
doxycyline-induced STAT5a749 expression in BB32 and EB03
significantly suppressed the hGH-induced PRLR mRNA levels in a
dose-dependent manner at a concentration of 0.05 µg/ml for both BB32
and EB03. At 0.5 µg/ml doxycycline, the induction by hGH was reduced
to 0.8 ± 0.03-fold in BB32 cells and to 1.6 ± 0.2-fold in
EB03 cells, supporting the previous finding of a primary role of STAT5
in the regulation of the PRLR 1A promoter.
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Immunocytochemical Detection of Cyclin D2 in INS-1 Cells and
Primary ß-Cells
Cyclin D2 protein expression was examined by peroxidase staining
in INS-1 cells (Fig. 6, panel A) and
primary ß-cells cultured in monolayer (Fig. 6, panel B). The INS-1
cells were cultured 4 days in the presence of 10% FCS, and the primary
ß-cells were stimulated with hGH for 24 h before fixation and
staining using a monoclonal anticyclin D2 antibody. Cyclin D2 protein
was detected in the nucleus of a subpopulation of INS-1 cells and
primary ß-cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present study we examined the signaling pathways that may play a
role in hGH-induced mitogenic signaling in INS-1 cells as well as in
primary neonatal rat ß-cells. At 11 mM glucose, hGH was
found to increase DNA synthesis by 2- to 3-fold over a 24-h stimulation
period in both cell types in accordance with previous findings (1, 28).
Inhibition of tyrosine protein kinase activity by coincubation with
genistein resulted in a complete inhibition of hGH-induced
proliferation in both cell types, as would be expected from inhibition
of JAK2 and, thereby, most downstream signaling from both the GHR and
the PRLR. PKC is involved in cell cycle progression in mammalian cells
during the G1-phase and
G2/M phase, and both positive and negative
regulatory effects of PKC on cell growth have been reported (33).
Staurosporine, an inhibitor of PKC, PKA, and protein kinase G, was
found to inhibit basal as well as hGH-induced proliferation in INS-1
cells, but not in the primary ß-cells in which, on the contrary, a
tendency for a growth-potentiating effect was observed. This
paradoxical effect may reflect an important difference in the
growth-signaling pathway between the primary ß-cells and the tumor
cell line, INS-1. Inhibition of MAPK signaling pathways using the MEK
inhibitor PD98059 and the p38 MAPK inhibitor SB203580 was only
partially inhibitory to hGH-induced mitogenic signaling in INS-1 cells
but did not affect primary ß-cells. Furthermore, we found no
indications of hGH-stimulated activity of these enzymes by Western blot
analysis of protein extracts using antiphosphothreonine-phosphotyrosine
(anti-pTpY)-specific antibodies against these proteins (data not
shown). These data confirm previous negative effects of this pathway in
GH/PRL-stimulated INS-1 cell proliferation as mentioned above. The
partial effect of the MAPK inhibitors may relate to a role of p42 and
p38 MAPK in the glucose-induced proliferation of these cells (13, 34)
and/or the glucose dependence of GH-induced INS-1 cell proliferation
reported by Cousin et al. (14). The PI3K pathway seems to
play an essential role in glucose- and IGF-I-stimulated INS-1
proliferation (31). However, inhibition of this pathway using
coincubation with wortmannin did not influence hGH-induced
proliferation in either INS-1 cells or primary ß-cells cultured in 11
mM glucose. This correlates well with the finding
that rGH is unable to mediate the association of IRS-1, IRS-2, Grb2, or
mSOS with PI3K (14) and the previous report that the mitogenic effects
of IGF-I and hGH are additive, indicating distinct signaling mechanisms
of these growth factors. Intriguingly, the study by Cousin et
al. found an inhibitory effect of wortmannin on both glucose- and
rGH-stimulated INS-1 cell proliferation in assays in which the cells
had been precultured in the absence of glucose (14). Using these
culture conditions, we have also seen a partial wortmannin-sensitive
component of the hGH response (50% inhibition, data not shown).
However, in our hands considerable cell death occurs in the absence of
glucose, which may obscure the interpretation of the data. A cautious
conclusion from our inhibitor experiments is that the MAPK, PI3K, and
the PKC signaling pathways, in contrast to findings in other cell
types, are probably not essential for hGH-stimulated ß-cell
replication, and we therefore turned our focus to the STAT5
pathway.
We have previously shown that activation of either GHRs or PRLRs in INS-1 cells induces binding of both STAT5a and STAT5b to the GLE of the PRLR 1A promoter (10). Stimulation of both GHRs and PRLRs using hGH led to a similar STAT5 activation. In the present study the kinetics of STAT5 activation in hGH-stimulated INS-1 and primary islet cells was examined. Pronounced STAT5 binding was observed after 15 min that persisted up to 24 h in both cell types. In INS-1 cells, the STAT5 activity after 24 h was comparable to that of 15 min hGH stimulation, whereas in islets a partial reduction was observed, indicating an influence of counterregulatory mechanisms operating in the primary cells, e.g. phosphatases or members of the suppressors of cytokine signaling (SOCS) family (35, 36). The long-term activation of STAT5 DNA binding in hGH-stimulated INS-1 cells correlates with the prolonged nuclear translocation of STAT5 in these cells in response to PRL as reported by Sorenson and co-workers (37). Interestingly, hGH-induced STAT5 DNA binding in the insulin-producing RIN-5AH cells was found to be only transient (9) with no detectable changes in STAT5 localization (37). The effects of GH and PRL on proliferation and PRLR gene expression in RIN-5AH cells are small compared with the effect of these hormones in INS-1 cells and cultured newborn rat islets (5, 10, 37, 38). Thus, the potency of GH and PRL action in insulin-producing cells may be correlated to the kinetics of GH- and PRL-induced STAT5 activation.
To address the role of STAT5 in GH/PRL-stimulated mitogenic signaling
in ß-cells, we took advantage of a mutant of STAT5a that is deleted
in its C-terminal transactivation domain and reportedly exerts dominant
negative activity by its ability to inhibit the effect of both
wild-type STAT5a and STAT5b (29). This mutant, STAT5a749, was stably
expressed in INS-1 cells using the inducible Tet-On gene expression
system, and we showed in two different clones (BB32 and EB03) that
STAT5a749 expression and activation were doxycycline inducible.
Furthermore, dominant negative activity of STAT5a749 in the two
clones was verified by analysis of the transcriptional effect on
STAT5-regulated PRLR promoter, as previously described (10). The
expression of STAT5a749 was highly controllable, and there were no
signs of leakage, as determined by Western blotting, EMSA, and reporter
gene assay. Although the STAT5a749 expressing cells do not have
increased expression of PRLRs, they should still express the initial
amount of GHRs (these are not regulated by STAT5) and the initial
amount of PRLRs, as only one of the three characterized PRLR promoters
contain a STAT5 response element (10). The expression of functional
receptors in the presence of doxycycline was confirmed by the
hGH-induced activation and binding of STAT5a749 to DNA. Analysis of
the effect of STAT5a749 expression on DNA synthesis measured by
3H-thymidine incorporation and FACS analysis
revealed a close correlation between STAT5 activity and hGH-stimulated
S-phase entry, and we conclude that STAT5 activation is essential for
GH/PRL-induced proliferation of INS-1 cells. Since previous experiments
show that hGH, rat GH, and rat PRL have the same maximal mitogenic
effect in neonatal rat ß-cells (3), the STAT5-mediated up-regulation
of PRLRs is probably not rate limiting in short-term experiments.
Whether this up-regulation after prolonged stimulation could be
important for the extent of the ß-cell response remains to be
addressed.
D-type cyclins, which regulate the activity of the cyclin-dependent kinases and S-phase entry, exhibit both cell type-specific and differential regulation in their expression, suggesting that the biological functions of these cyclins are not fully redundant (39). In this study we found that cyclin D2 mRNA was abundant in the INS-1 cells, whereas cyclin D3 and cyclin D1 mRNA were detectable at lower levels. The role of STAT5 in the growth of lymphoma cells has been discovered quite recently and, importantly, revealed a direct transcriptional effect of STAT5 on the human cyclin D1 gene (27). Furthermore, STAT5-deficient mice lacked peripheral T cell expression of cyclin D2, cyclin D3, cyclin A, and the cyclin-dependent kinase (cdk)-6 in response to interleukin-2 (IL-2) (21). In mouse and rat, no STAT5 binding element is present in the cyclin D1 promoter, whereas the cyclin D2 and D3 promoters contain PRL-responsive elements (40, 41), and a potential STAT5 binding sequence is present in the cyclin D2 promoter (Ref. 21 and our unpublished observations). In the present study we found that hGH increased the mRNA level of cyclin D2 and PRLR in the stably transfected cell lines, and the doxycycline- inducible inhibition of this effect indicates that STAT5 is involved in this regulation. However, whereas expression of the PRLR 1A mRNA was totally inhibited at the maximal doxycycline dose, only partial inhibition was observed for cyclin D2, indicating that other hGH-stimulated signaling pathways play a role in the transcriptional regulation of cyclin D2. Interestingly, an inhibitory effect of hGH on cyclin D1 mRNA levels was observed, which was augmented dose-dependently by doxycycline treatment. Our finding may indicate that STAT5 is involved in negative regulation of this cyclin through a mechanism that may involve competition of cofactors in transcription factor complexes (42). In a recent study a reciprocal regulation of cyclin D1 and D2 was identified in mouse myeloid leukemia cells in response to granulocyte-colony-stimulating-factor (G-CSF), indicating a correlation between G-CSF-stimulated up-regulation of cyclin D1 mRNA levels and G-CSF-induced apoptosis (43). One may speculate that the inverse effect of hGH on these cyclins in insulin-producing cells is related to a combined effect on antiapoptosis and replication.
In conclusion, our study has shown a requirement for STAT5 signaling in hGH-stimulated proliferation of INS-1 cells, which may involve a direct transcriptional effect on the cyclin D2 promoter. Future studies using adenoviral transfer of STAT5 mutants and establishment of transgenic animals expressing STAT5 mutants under the control of the insulin promoter will hopefully reveal whether these findings are relevant to primary ß-cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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3H-Thymidine Incorporation
Cells were seeded in 96-wells plates (50,000 cells per well) and
cultured for 2 days in 200 µl/well complete medium. The medium was
changed to RPMI 1640 containing 0.25% BSA (Fraction V,
Sigma) and culture proceeded for 24 h. For the
inhibitor assay, various inhibitors (see cells, hormones, and
chemicals) were added followed by stimulation with 0.5 µg/ml hGH for
24 h. For the stable clones, the cells were cultured in the
absence or presence of doxycycline for 20–24 h followed by stimulation
with either 0.5 µg/ml hGH or 10% FCS containing medium for an
additional 24-h culture period. The last 4 h before harvesting 0.5
µCi [methyl, 1',2'-3H]Thymidine (no. TRK.565,
Amersham Pharmacia Biotech, Buckinghamshire, UK) was
additionally added per well. Cells were harvested onto a filter paper
(Filtermate 196, Packard Instruments, Meriden, CT) by a cell harvester
(Wallac, Inc., Gaithersburg, MD) using
H2O for lysis. The filter paper was dried for
1 h at 37 C and transferred to a bag containing 5 ml of Optiscent
scintillation fluid (Wallac, Inc.). The filter was counted
in a 1450 Microbeta Plus counter (Wallac, Inc.).
Preparation of Monolayers of ß-Cells and 5-BrdU
Labeling
Monolayer cultures of islet cells were prepared essentially as
previously described (1). Briefly, islets were precultured for 5–7
days in RPMI 1640, containing 0.5% human serum (HS) and were then
dispersed into single cells by trypsin-EDTA treatment. The cells
(50–100,000) were plated in plastic cell culture
9-cm2 slideflasks (Nunc, Roskilde, Denmark) in
RPMI 1640 medium containing 2% HS and 0.5 µg/ml hGH. The cells were
allowed to attach and establish a monolayer for 5–7 days, after which
they were washed twice in medium without hGH and then cultured for
24 h in RPMI 1640 containing 2% HS. The medium was changed, and
the cells were cultured further for 24 h in the presence of 10
µM BrdU, and in the absence or presence of the various
protein kinase inhibitors, which were added before the addition of 0.5
µg/ml of hGH. The cells were washed twice in RPMI 1640 without serum
before fixation in 1% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4. The cells were double stained for BrdU and insulin as
described previously (1). Briefly, the cells were exposed to 1.5
M HCl for 30 min and washed. They were stained with a
monoclonal mouse antibody to BrdU (no. M 0744, DAKO Corp.,
Glostrup, Denmark) diluted 1:50 and with guinea pig antiinsulin
antibody (Novo Nordisk A/S) diluted 1:500. The antibodies
were visualized by a Texas red-conjugated goat antimouse-IgG (no.
115–075-100, Jackson ImmunoResearch Laboratories, Inc.
West Grove, PA) and fluorescein isothiocyanate
(FITC)-conjugated goat antiguinea pig-IgG (no. 106–095-003,
Jackson ImmunoResearch Laboratories, Inc.) both diluted
1:100. Dilution of the antibodies was performed in PBS with 0.3%
Triton X-100 and 0.1% human serum albumin (HSA). The slides were
mounted in 20% glycerol/0.05 M Trisma base adjusted to pH
8.4 and stored at 4 C.
Stable Transfection
The establishment of the stable clone, INS-r3, expressing the
reverse tetracycline-dependent transactivator, has been described
previously (30) and was kindly provided by Dr. P. B. Iynedjian
(Geneva, Switzerland). A vector containing the cDNA encoding the STAT5a
mutant, STAT5a749, which lacks the C-terminal transactivation
domain, was provided by Dr. B. Groner (Freiburg, Germany). This mutant
cDNA was subcloned into the Tet-On gene expression vector, pTRE
(CLONTECH Laboratories, Inc. Palo Alto, CA) using the
EcoRI restriction 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
following day the medium was changed to Opti-MEM, and transfection was
carried out using the LipofectAMINE PLUS Reagent (Life Technologies, Inc., Paisley, UK) essentially as described
by the manufacturer. The cells were transfected overnight with 3.7 µg
pTK-Hyg Vector (CLONTECH Laboratories, Inc.) and 18.1 µg
pTRE vector with or with out STAT5a749 insert. The cells were
cultured for 2 days in complete medium and were then trypsinized and
reseeded in 5 x 100 mm dishes using complete medium containing
100 µg/ml G418 and hygromycin. Medium was changed every fourth day,
and antibiotic-resistant colonies of cells were isolated after 2–4
weeks, transferred to 24-well plates, and split to single cells by
trypsination. Hygromycin-resistant clones were tested for integration
of pTRE by PCR amplification on purified DNA. The DNeasy Tissue kit
(QIAGEN, Valencia, CA) was used for DNA purification. The
PCR reaction was run with 250 ng of total DNA as a template, and 50
pmol per specific pTRE primers (CLONTECH Laboratories, Inc.), 1.25 U Taq polymerase (Promega Corp., Madison, WI), thermophilic DNA 10x buffer (Promega Corp.), 0.2 mM deoxynucleoside
triphosphate (dNTP) (Amersham Pharmacia Biotech),
1.5 mM MgCl2 (Promega Corp.), and H2O to 50 µl. A single
denaturing step at 94 C/1 min was followed by 25 cycles as given: 94
C/15 sec; 63 C/1 min; 68 C/3 min. The products were detected on 1%
agarose gel.
Western Blot Analysis
Cells were seeded in 100-mm dishes (4 x
106 cells per dish) and cultured for 2 days in 10
ml complete medium per dish. The medium was changed to medium
containing 0.25% BSA, and cells were cultured approximately 20 h
in the presence and absence of doxycyline as indicated. The cells were
washed once in cold PBS, scraped off in 1 ml PBS, transferred to
microfuge tubes, and pelleted. The cells were resuspended in 500 µl
PBS, containing 1% NP40, 0.1% SDS, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, and 0.5 mM AEBSF (Calbiochem) and
allowed to lyse on ice for 30 min. Cell debris was removed by
centrifugation at 15,000 x g for 20 min, and the
supernatants were stored at -80 C after addition of 125 µl 5x
sample buffer. Proteins were denatured by boiling for 2 min and
separated by electrophoresis on a 7.5% SDS polyacrylamide gel. For
protein size determination, High-Range Rainbow Marker (Amersham Pharmacia Biotech) was used. The proteins were transferred to a
nitrocellulose membrane by Western blotting for 2 h at 200 mA. The
membrane was blocked for 15 min and washed once in PBS before probing
with antibodies. The primary antibody was either no. S21520
(Transduction Laboratories, Inc., Lexington, KY) raised
against aa 451–649 of STAT5 or (C-17)-G no. SC835 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) raised against the carboxy
terminus of STAT5b diluted 1:1000. The secondary antibody was antimouse
IgG horseradish peroxidase-linked whole antibody (Amersham Pharmacia Biotech) diluted 1:5,000. Proteins were visualized
using the ECL Western blotting detection reagents (Amersham Pharmacia Biotech).
Nuclear Extracts
Isolated newborn rat islets (5,000 islets per dish), which had
been precultured for 1 week in 15 ml of RPMI 1640 supplemented with
0.5% normal HS, were stimulated with 0.5 µg/ml hGH for 15 min before
harvest. INS-1 cells and the three INS-r3 clones (BA15, BB32, and EB03)
were seeded in 100-mm dishes (4 x 106 cells
per dish) and cultured for 2 days in 10 ml complete medium per dish.
The medium was changed to medium containing 0.25% BSA, and cells were
cultured for 20 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 (9, 10). 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 Bio-Rad protein assay
(Bio-Rad Laboratories, Inc. Hercules, CA).
EMSA
EMSA was performed essentially as described previously (9, 10).
Briefly, the double-stranded oligonucleotide 1A-GLE
(5'-agctAGTTCTAGGAATAagct) containing a STAT5 binding element derived
from the rat PRLR 1A promoter (45) was radiolabeled in a fill-in
reaction using [
-32P] dATP (Amersham Pharmacia Biotech) and DNA polymerase (Klenow fragment) and used
as probe (10). Nuclear extracts (10 µg) were incubated at 30 C with
20 fmol of probe in a 20 µl reaction. Free and bound probe were
separated by nondenaturing PAGE and visualized by autoradiography.
Transient Transfection and Dual Luciferase Reporter (DLR)
Assay
Cells were seeded in 24-well plates (300,000 cells per well) in
500 µl/well complete medium. The cells were transfected as described
previously (10) with 1.5 µl LipofectAMINE PLUS Reagent (Life Technologies, Inc.) and 0.5 µg DNA [250 ng of pGL2–1A, 10 ng
of pRL-SV40 plasmid (internal control) and 240 ng of pUC18 plasmid].
The cells were transfected overnight in Opti-Mem (240 µl/well). The
medium was changed to RPMI 1640 containing 0.5% FCS (500 µl/well)
and incubated for 24 h in the presence or absence of doxycycline.
Seven hours 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 (supplied with DLR Assay System no. E1910,
Promega Corp.) followed by shaking the plate for 15 min at
room temperature. The cell extracts were stored in the plate at -80 C
until measuring was performed as described previously (10). The
pRL-SV40 vector contains the coding region of the Renilla
luciferase gene under the transcriptional control of the SV40 early
enhancer/promoter (Promega Corp.). The pGL2–1A is
generated by the insertion of the 5'-flanking region of PRLR exon 1A
(-462/+81) into the pGL2-basic vector that contains the coding region
of the firefly luciferase gene (45). The pUC18 vector was used as
carrier plasmid.
FACS
Cells were seeded in six-well plates (500,000 cells per well)
and cultured for 2 days in 3 ml complete medium per well. The medium
was changed to medium containing 0.25% BSA, and cells were cultured
approximately 20 h in the presence and absence of doxycyline as
indicated. Respective wells were stimulated 24 h with 0.5 µg/ml
hGH and 2 h before harvesting, 10 µM BrdU was added
per well. The cells were harvested by adding 100 µl 0.5%
trypsin-EDTA per well. Trypsination was stopped by adding 900 µl
serum-containing medium, and cells were transferred to
Eppendorf (Madison, WI) tubes, pelleted, and resuspended
in 500 µl -20 C 70% ethanol. Cells were stored at 4 C up to 1 week.
For denaturation of DNA, 500 µl of 3 M HCl were added and
the cells were incubated at room temperature for 30 min. The cells were
washed once in PBS containing 0.1% HSA and 0.3% Triton X-100 and were
resuspended in 1 ml of this buffer containing mouse anti-BrdU antibody
(DAKO Corp.) in a 1:100 dilution. Incubation was carried
out overnight at 4 C with rotation. Cells were washed twice,
resuspended in 1 ml buffer containing FITC-conjugated goat-antimouse
IgG no. 115–095-003 (Jackson ImmunoResearch Laboratories, Inc.), and incubated for 45 min at 4 C in the dark. The cells
were washed twice, resuspended in 500 µl PBS containing 5 µg/ml
propidium iodide, and placed in the dark. FACS analysis was carried out
using Cell Quest (Becton Dickinson and Co., San Jose, CA)
as software.
RT-PCR
Cells were seeded in 60-mm dishes (2 x
106 cells per dish). The cells were cultured for
2 days in 4 ml complete medium per dish. The medium was changed to
medium containing 0.25% BSA, and cells were cultured approximately
20 h in the presence and absence of doxycyline as indicated.
Respective wells were stimulated 24 h with 0.5 µg/ml hGH. Total
RNA was extracted using the RNeasy method from QIAGEN
(Chatsworth, CA). cDNA was synthesized from 1 µg RNA using AMV
Reverse Transcriptase and dNTP mix from Stratagene (La
Jolla, CA) and random primers from Life Technologies, Inc.
The reaction was run at 42 C for 1 h, and the sample was diluted
in 40 µl of 0.1% Triton-X-100 and stored at -20 C. PCR was carried
out in 12.5 µl reactions using 0.75 µl of cDNA as template. The
primer sequences were: PRLR, 5'-TTG TGG ATC TCA GGT TTC CCT GGT G
(forward); 5'-AGC GAG CTG GAT TCT AGG GAA ACA T (reverse); cyclin D1,
5'-TCT ACA CTG ACA ACT CTA TCC G (forward); 5'-TAG CAG GAG AGG AAG TTG
TTG G (reverse); cyclin D2, 5'- AGA CCT TCA TCG CTC TGT GT (forward);
5'- TAG CAGATG ACG AAC ACG CC (reverse); cyclin D3, 5'-CTG CTG GCG GGA
ATC ACA (forward); 5'-GGC CCC TCC TCT GCT TGG T (reverse); G6PDH,
5'-GAC CTG CAG AGC TCC AAT CAA C (forward); 5'-CAC GAC CCT CAG TAC CAA
AGG G (reverse); TBP, 5'-ACC CTT CAC CAA TGA CTC CTA TG (forward);
5'-ATG ATG ACT GCA GCA AAT CGC (reverse). The expected lengths of the
various PCR products were as follows: PRLR, 329 bp; cyclin D1, 304 bp;
cyclin D2, 372 bp; cyclin D3, 246 bp; G6PDH, 214 bp; TBP, 192 bp. The
PCR incubates contained 50 mM KCl; 10 mM
Tris-HCl; 1.5 mM MgCl2; 40
µM dATP; dGTP; and dTTP; 20 µM dCTP; 2.5
mCi of 3,000 Ci/mmol [-33P]dCTP; 10 pmol of
each primer and 2.5 U Ampli Tag Gold polymerase. A single denaturing
step at 94 C/10 min was followed by either 20 cycles (cyclin D2) or 25
cycles (PRLR, cyclin D1/D3) as given: 94 C/30 sec; 55 C/1 min; 72 C/1.5
min. PCR products were separated on 6% denaturing polyacrylamide gels
(GEL-MIX 6, Life Technologies, Inc.), dried, and exposed
to Phosphorimage storage screens that were scanned by Phosphorimager
series 400 (Molecular Dynamics, Inc., Sunnyvale, CA), and
band intensities were calculated using the program Image Quant
(Molecular Dynamics, Inc.). The PRLR and the cyclin D1 and
D3 were quantified relative to the internal standard TBP and cyclin D2
was quantified relative to the internal standard G6PDH.
Immunocytochemistry
INS-1 cells were seeded in slide flasks (100,000 cells per
flask) and cultured 4 days in complete medium. Newborn rat islets were
dispersed into single ß-cells and cultured as described above. The
cells were fixed in 1% paraformaldehyde and stained with peroxidase
using the HISTOSTAIN-PLUS KIT (Zymed Laboratories, Inc.
South San Francisco, CA) according to the instructions provided by the
manufacturer. The primary antibody monoclonal cyclin D2 antibody no.
MS-221 (NeoMarkers, Union City, CA) diluted 1:100 in PBS + 0.1% HSA +
0.3% Triton-X-100 was incubated for 1 h.
Statistical Analysis
Statistical analysis was performed using SAS 6.12
software (SAS Institute, Cary, NC). Two-way ANOVA with
Dunnett’s method for adjustment of multiple comparisons was carried
out.
|
ACKNOWLEDGMENTS |
|---|
749
cDNA; to Dr. P. B. Iynedjian, University of Geneva School of
Medicine, Geneva, Switzerland, for supplying INS-r3 cells; and Dr.
C. B. Wollheim, Centre Medical Universitaire, Geneva, Switzerland,
for the supply of INS-1 cells. We thank Dr. S. N. Jakobsen and Dr.
N. Billestrup, Novo Nordisk, Bagsværd, Denmark, and Dr. K. Seedorf,
Lilly Research Laboratories, Hamburg, Germany, for helpful advice and
discussion. |
FOOTNOTES |
|---|
Received for publication March 30, 2000. Revision received September 11, 2000. Accepted for publication September 20, 2000.
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