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Endocrine Institute, Sheba Medical Center (R.S., O.B-D., Y.C., A.K., M.B., S.F.), Tel-Hashomer 52621; Tel-Aviv University Sackler School of Medicine (R.S., O.B.-D., A.K., M.B.), Tel-Aviv; and Bar-Ilan University (Y.C.), Ramat-Gan 52900, Israel; and the Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center (C.B.N.), Dallas, Texas 75235
Address all correspondence and requests for reprints to: Sarah Ferber, Ph.D., Endocrine Institute, Sheba Medical Center, Tel-Hashomer, Israel. E-mail: berezin{at}post.tau.ac.il
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transcription factor PDX-1 has an important role in pancreatic islet differentiation (6, 7, 8, 9, 10, 11). Cooperativity between PDX-1 and bHLH proteins in trans-activation of the insulin gene was demonstrated (12). The ubiquitously expressed E47 protein creates heterodimers with the ß-cell-specific HLH protein BETA2 (13). In terminally differentiated ß-cells, PDX-1 participates in mediating glucose stimulation of insulin promoter activity and trans-activation of other important ß-cell-specific genes, such as GK, GLUT-2, and IAPP (14, 15, 16, 17). These findings suggest that alterations in PDX-1 levels could influence the expression of a host of islet cell genes that are important for normal function. Long term exposure to glucose reduces PDX-1 dosage or binding activity in pancreatic islets, cell lines, and islets of an in vivo model of noninsulin-dependent diabetes mellitus (NIDDM), the 90% pancreatectomized rat. These changes in PDX-1 activity have been correlated with reduced insulin and GLUT-2 gene expression (3, 18, 19, 20, 21, 22, 23, 24, 25). In addition, dexamethasone induces a rapid decrease in PDX-1 expression in HIT-T15 cells, which correlates with a decrease in insulin mRNA levels, whereas fatty acids induce a decrease in PDX-1 binding activity and a commensurate decrease in insulin, GLUT-2, GK, and somatostatin mRNA levels in pancreatic islets (3, 4 25A ). On the other hand, Kajimoto et al. (26) documented that suppression of the transcription factor PDX-1 by administration of antisense oligonucleotides to MIN6 insulinoma cells caused no decrease in insulin mRNA levels. Moreover, in islets from an alternate rodent model of NIDDM, the db/db mouse, loss of GLUT-2 expression was associated with a 3-fold increase in PDX-1 binding to this promoter (27).
The seemingly controversial data regarding the effect of PDX-1 dosage and activity on islet cell function motivated us to analyze the role of PDX-1 in regulating insulin gene expression in the rat insulinoma cell line RIN-38. An important issue is whether this functional deterioration process is reflected in and can be explained by alterations in ß-cell-specific transcription factors. We demonstrate that the decrease in insulin production in RIN-38 cells that occurs with aging in culture is associated with a similar decrease in basal rat insulin promoter (RIP) activity, and a decline in the protein levels of the insulin gene trans-activators, E47 and BETA2 (12, 13), but is also associated with a paradoxical 3-fold increase in PDX-1 levels. Systematic titration of PDX-1 levels in RIN-38 and HIT-T15 cells demonstrates that overexpression of PDX-1 alone, without reducing BETA2 and E47 levels, suppresses insulin promoter activity in a dose-dependent manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of recombinant adenoviruses
All recombinant adenoviruses were constructed according to the
procedure reported by Becker et al. (28). The gene of
interest was ligated into the pACCMV.pLpA plasmid followed by
cotransfection with the adenovirus plasmid pJM17 and harvesting the
recombinant virions as previously described (28). The recombinant
adenovirus termed AdCMV-CAT was prepared by ligation of a
BamHI/EcoRI restriction fragment containing the
intact bacterial chloramphenicol acetyltransferase (CAT) gene distal to
the cytomegalovirus (CMV) promoter in the pACCMV.pLpA plasmid. The
AdRIP-CAT recombinant adenovirus was prepared by insertion of 410
nucleotides of the 5'-flanking region of the rat insulin-1 gene
(supplied by Dr. Larry Moss) in place of the viral CMV promoter in the
pACCMV.pLpA plasmid and ligation of the
BamHI/EcoRI CAT gene insert distal to the insulin
promoter fragment. The AdCMV-PDX-1 recombinant adenovirus contains a
HindIII/BamHI fragment encompassing a complete
coding region of the mouse homolog of PDX-1 [insulin promoter factor-1
(IPF-1)] (6) [PDX-1 complementary DNA (cDNA), a gift from
Dr. Christopher V. E. Wright]. AdRIP-PDX-1 recombinant adenovirus
contains a HindIII/BamHI fragment encompassing a
complete coding region of the mouse homolog of PDX-1 (IPF-1), which
replaced the CAT fragment in pACRIP.pLpA plasmid used for the AdRIP-CAT
recombinant adenovirus.
Preparation of viral stocks
293 cells were cultured in 145/20 plates (Greiner Friekenhausen,
Germany) and treated with recombinant adenoviruses at
multiplicity of infection (moi) of 10 for 90 min in minimal volume (8
ml). Forty-eight hours later, cells and media were collected, cells
were pelleted by centrifugation in 800 x g, and the
virus in the supernatant fraction was precipitated overnight in 20%
polyethylene glycol (PEG 8000, Sigma Chemical Co., St.
Louis, MO) and 2.5 M NaCl at 4 C. The virus-containing
medium was centrifuged at 10,000 x g, and the pellet
was resuspended in physiological saline [137 mM NaCl, 5
mM KCl, 10 mM Tris-HCl (pH 7.4), and 1
mM MgCl2]. Viral stocks were stored at
concentrations of 109–1012 plaque-forming
units/ml at 4 C.
Determination of insulin promoter (RIP) activity
Cells were plated in 12-well dishes at a density of
106 cells/well. Twenty-four to 48 h after plating,
cells were incubated with 4 moi AdRIP-CAT or 1 moi AdCMV-CAT for 90
min. Thereafter, the virus was removed, cells were washed with PBS, and
fresh medium was applied on the plates. Forty-eight hours after
infection, cells were extracted in lysis buffer [luciferase assay
system with reporter lysis buffer from Promega Corp.
(Madison, WI) suitable for the use in CAT and protein assays] and
analyzed for CAT activity and protein concentrations.
CAT and luciferase activity assays
CAT activity was measured with a Promega Corp. kit
in the presence of butyryl coenzyme A (Sigma Chemical Co.)
and [14C]chloramphenicol (ICN, Irvine, CA), according to
the instructions provided by the manufacturer. Luciferase activity was
measured with a Promega Corp. kit.
Western blot analysis of nuclear proteins
Preparation of nuclear extracts from mammalian cells.
Nuclear extracts were prepared according to the method of Leonard
et al. (29). Approximately 107 cells were washed
in ice-cold PBS and collected in 400 µl ice-cold hypotonic buffer
[10 mM HEPES (pH 7.9), 10 mM KCl, 1
mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 0.1 mM p-amino benzoic acid, 1
mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 1
µg/ml pepstatin, and 50 µg/ml aprotinin] and left to swell on ice
for 15 min. Twenty-five microliters of 10% Nonidet P-40 (Sigma Chemical Co.) were added to the suspension, the cells were
vortexed vigorously for 10 sec and centrifuged at 12,000 x
g for 30 sec at 4 C, and the supernatant (cytoplasmic
fraction) was separated from the pellet (nuclear fraction). Nuclear
proteins were extracted in 200 µl high salt buffer [20
mM HEPES (pH 7.9), 400 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 0.1 mM p-amino benzoic acid, 1
mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 1
µg/ml pepstatin, and 1 mg/ml aprotinin] for 15 min with gentle
rotation. The nuclear extracts were then centrifuged for 5 min at 4 C,
and the pellet was discarded. The extracts were kept in 50-µl
aliquots at -80 C. Protein concentration was determined using the
Bradford method (30).
Western blot analysis. Forty micrograms of nuclear extract proteins were resolved on 12% SDS-PAGE and electroblotted onto 0.2-µm nitrocellulose membranes (PROTAN BA 83, Schleicher & Schuell, Inc., Dassel, Germany) (31). The membranes were blocked for 1 h in a 10% solution of 1% fat liquid milk (Tnuva, Tel-Aviv, Israel), and 0.05% Tween-20 in PBS and incubated with a 1:10,000 dilution of a rabbit polyclonal antibody against the N-terminal region of mouse PDX-1 (11) (contributed by Christopher V. E. Wright) in blocking buffer for 1 h, followed by horseradish peroxidase-conjugated goat antirabbit secondary antibody (Amersham, Arlington Heights, IL). Membranes were stripped and rehybridized with anti-BETA2 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), E47 (1:500; Santa Cruz Biotechnology, Inc.) and anti-ß-thymopoietin (1:10,000; contributed by A. Simon and G. Goldstein) antibodies. Immunoreactive proteins were visualized using the enhanced chemiluminescence detection system (Amersham).
Statistical analysis
Data are presented as the mean ± the SE.
Differences among means were analyzed using one-way ANOVA, followed by
Bonferroni’s method for pairwise multiple comparison.
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Cells
treated with the AdCMV-CAT control virus exhibited no significant
reduction in CAT activity compared with cells of passage 20. Therefore,
CAT activity under control of the RIP promoter was normalized to that
under control of the CMV promoter for each set of conditions. As the
profound decrease in promoter activity was specific to RIP, we
postulated that the decrease in RIP activity with time in culture was a
function of alterations in the activity or the levels of transcription
factors interacting with this promoter.
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). Densitometric
analysis of several experiments and exposures revealed that cells at
intermediate and high passages (passages 30 and 40) contained 35.3
± 13% and 309 ± 68% more PDX-1 protein, respectively, in their
nuclear fraction than RIN-38 cells at passage 20 (Fig. 2B). In
contrast, levels of immunodetectable E47 and BETA2, two additional
transcription factors that have been implicated in control of insulin
promoter activity, decreased in aging RIN-38 cells to 25–35% of their
levels at passage 20 (Fig. 2, A and C).
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and 4,
nuclear extracts from control RIN-38 (passage 20) and HIT-T15 cells
contained immunodetectable PDX-1. Treatment of cells with increasing
amounts of AdCMV-PDX-1 revealed that the transcription factor is
correctly expressed in the nucleus in increasing quantities that
correlate with the moi in both cell lines tested. Binding to the
cognate locus on RIP was elevated in proportion to the extent of
overexpression (data not presented). Moreover, AdCMV-PDX-1 treatment
did not reduce BETA2 and E47 nuclear proteins levels in RIN-38 (Fig. 3)
or HIT-T15 (Fig. 4) cells, which allows us to dissect the separate role
of PDX-1 dosage on insulin promoter activity (the slight increase in
BETA2 or E47 protein levels in RIN and HIT cells, respectively, was not
consistent).
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) and HIT-T15
(Fig. 5B) cells were treated with constant concentrations of AdRIP-CAT
(4 moi), or as a control with AdCMV-CAT (1 moi), plus increasing
amounts of AdCMV-PDX-1. CAT activity was determined 48 h after
infection. PDX-1 expression dramatically inhibited RIP activity in low
passage RIN-38 cells. One and 10 moi of AdCMV-PDX-1 inhibited RIP
activity to 66 ± 3% and 14 ± 7% of its activity in
untreated cells, respectively (Fig. 5A). The fact that PDX-1
participates in trans-activation of insulin promoter
activity is demonstrated in high passage HIT-T15 cells cultured in 5.5
mM glucose (Fig. 5B). One and 2 moi of
AdCMV-PDX-1 increased RIP activity to 130 ± 10.3% and 114.4
± 8%, respectively, but higher doses suppressed RIP activity in these
cells in a manner similar to that observed in RIN-38 cells. Treatment
of HIT-T15 cells with 20 moi of AdCMV-PDX-1 inhibited insulin promoter
activity to 20% of its maximal activity (at 1 moi treatment). CMV
promoter activity was not significantly altered by PDX-1 overexpression
(Fig. 5, A and B). Similar results were obtained when RIN-38 cells were
treated with an adenovirus containing the cDNA encoding the rat homolog
of PDX-1 (somatostatin transcription factor-1; data not
presented). To demonstrate that high titers of adenovirus per
se were not responsible for altered RIP activity, we treated the
same cells with increasing multiplicities of infection of AdCMV-Luc
instead of AdCMV-PDX-1. As shown in Fig. 5C, this manipulation
proportionally increased luciferase activity (Fig. 5C, lower
panel), but did not affect RIP activity (Fig. 5C, upper
panel).
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), we decided to construct an
additional adenovirus (AdRIP-PDX-1) that could direct a moderate
increase in PDX-1 nuclear protein levels. Three moi of this adenovirus
resulted in a 3-fold increase in the homeobox protein levels (Fig. 6A).
This manipulation did not inhibit insulin promoter activity in RIN-38
cells (Fig. 6C). Ten moi of AdRIP-PDX-1 resulted in an additional
2.4-fold increase in these protein levels above those
achieved with the 3 moi treatment. The 2.4-fold increase in
PDX-1 protein levels between 3–10 moi of AdRIP-PDX-1 treatment
decreased RIP-CAT activity by 44% (Fig. 6C). PDX-1 overexpression in
this experiment, as in the above experiments, did not reduce the E47
and BETA2 protein levels (data not presented). Importantly, 10 moi of
AdRIP-ß-gal did not affect RIP-CAT activity, meaning that the effect
is specific to PDX-1 overexpression and not to an increase in the
insulin-1 promoter copy number (data not presented).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most studies correlate reduced insulin gene expression with a decrease in PDX-1 levels (3, 20, 23, 24, 34). When viewed in this context, our results with RIN-38 cells may seem paradoxical. Nevertheless, our findings are in accord with other observations that relate PDX-1 dosage to the level of insulin expression. 1) PDX-1 levels are generally higher in transformed ß-cell lines than in pancreatic islets, yet all such lines contain substantially less insulin than found in primary cells (2, 14). 2) Using transient transfection methods, it was demonstrated that overexpression of PDX-1 in pancreatic islets affects insulin promoter activity with a bell-shaped curve, such that low levels activated and high levels suppressed RIP activity (14). 3) In the rodent model of NIDDM, the db/db mouse, loss of GLUT-2 expression was associated with a 3-fold increase in PDX-1 binding to the GLUT-2 promoter (27). 4) In addition, in MIN6 cells, decreasing PDX-1 dosage had no effect on insulin or GK mRNA levels (26).
The correlation between decreased RIP activation and low E47 protein levels visualized in high passage RIN-38 cells is in agreement with previous data (35). Moreover, E47 and BETA2 overexpression in HIT-T15, ßTC-6, and RIN 5AH cells resulted in trans-activation of the insulin promoter (12, 13, 33, 35, 36).
The value of the current study is that it presents a cellular model in which a decrease in insulin gene expression is associated with a 3-fold increase in PDX-1 dosage, whereas E47 and BETA2 levels decrease. Moreover, it clearly demonstrates that increased PDX-1 levels repress insulin gene expression and provides a systematic analysis of the relationship between PDX-1, E47, and BETA2 expression levels and insulin promoter activity in two ß-cell lines. Our experimental system is able to dissect between the effect of increased PDX-1 dosage from that of decreased BETA2 and E47 levels on insulin gene transcription. PDX-1 overexpression did not alter the levels of these two insulin gene trans-activators. In addition, it should be taken into consideration that the decrease in BETA2 and E47 levels (and possibly additional transcription factors) may further potentiate the decrease in RIP activation demonstrated in high passage RIN-38 cells.
Our data suggest that PDX-1 may play a dual role in insulin gene expression, and that low as well as high doses may be deleterious. The idea that homeobox genes have multiple roles depending on their relative dosage is not unique to PDX-1. The paired box Pax proteins are crucial regulators of organogenesis in thymus, kidney, thyroid, pancreas, and eye. Thus, overexpression of these genes in tissues in which they are normally expressed may lead to tumorigenesis and abnormal development, suggesting that doses of Pax proteins are critical for their normal function (37), and that squelching of transcription factors can naturally occur when inappropriately high levels of such factors are reached. The dosage of the homeobox genes and the cellular context of the available nuclear factors are two of the most important parameters responsible for specificity of HOX action (38).
The increased PDX-1 levels could directly induce nonproductive cooperation between insulin gene trans-activators, which may compete for binding to relevant regulatory loci on the insulin promoter. In addition, its increased levels could induce an increase in levels of transcription factors, such as C/EBPß or Ids, which may indirectly mediate dysregulation of RIP activation (39, 40, 41). C/EBPß was identified as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels in HIT-T15 and INS-1 cells. Inhibition of RIP activation occurred by direct protein-protein interaction with the basic helix-loop-helix transcription factor E47. This interaction was suggested to lead to the inhibition of E47 binding to the E elements of the insulin promoter, thereby reducing the trans-activation potential of E47 on insulin gene transcription (39).
We conclude that PDX-1 is sufficient to affect the insulin gene rate of transcription in a dose-dependent manner. However, our results also suggest that the relative quantities of cooperating insulin gene trans-activators may play an important role in regulating the expression of this gene. Moreover, the perturbation of the balance between these interacting factors could contribute to ß-cell dysfunction in islet cell lines or in animal models of diabetes. The exact mechanism by which PDX-1 converts from a trans-activator of the insulin promoter to its repressor in a dose-dependent fashion requires further analysis.
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Acknowledgments |
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Footnotes |
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Received September 10, 1998.
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73-kDa band; (c),
goat antimouse BETA2 (50- to 60-kDa band) (both b and c at dilution of
1:500, Santa Cruz Biotechnologies, Inc., Santa Cruz, CA),
and (d) mouse antihuman ß-thymopoetin, nuclear factor (53 kDa band) at dilution of
1:10,000. Lanes 1–3 RIN-38 cells of passage 20, 30, and 40,
respectively. B, Densitometric analysis of eight separate PDX-1
immunoblot experiments was performed using the Bio-Rad
Multi-Analyst/PC version 1.1. Endogenous PDX-1 protein levels were
normalized to ß-thymopoetin and were all statistically different from
each other. Data are mean ± SE, n = 8, *P< 0.05. C, Densitometric analysis of four separate immunoblots
for E47 (black bars) and Beta2 (open bars),
normalized to ß-thymopoetin. The levels of both proteins at passage
30 and 40 are statistically different than that of passage 20. Data are
mean ± SE, n = 4, *P < 0.05.
