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Department of Medicine Banting and Best Diabetes Centre The Toronto Hospital University of Toronto Toronto, Ontario, M5G 2C4, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Experiments using transgenic mice and gene transfer in vitro have identified several regions within the first few kilobases of the rat proglucagon promoter that appear to be important for control of proglucagon gene transcription (2, 3). Expression of proglucagon gene-SV40 T antigen transgenes in mice led to the delineation of an upstream element, designated the proglucagon gene upstream enhancer, that is necessary for proglucagon gene transcription in enteroendocrine cells in vivo (4). A number of control elements in the more proximal proglucagon promoter have been identified that function as islet cell-specific enhancers (5). DNA sequences within the first 100 bp of the promoter are functionally important for restricting proglucagon gene transcription to islet and intestinal endocrine cell types (6). This proximal promoter element, termed G1, contains a number of AT-rich sequences characteristic of the binding sites for homeobox genes.
A combination of transfection and electrophoretic mobility shift assay experiments have suggested that the homeobox transcription factor isl-1 binds to the proglucagon G1 element and activates the proglucagon promoter (7). Reduction of isl-1 levels in InR1-G9 islet cells was associated with decreased levels of proglucagon mRNA transcripts, providing further support for the importance of isl-1 in the control of proglucagon gene expression (7). The caudal homeobox protein cdx-2/3, present in both islet and intestinal nuclear extracts, also binds to the AT-rich sequences in the proglucagon G1 element (8), and transfected cdx-2/3 activates the proglucagon promoter in BHK fibroblasts. To delineate a role for cdx-2/3 in the control of proglucagon gene transcription in endocrine cells, we have now examined the cdx-2/3-dependent regulation of proglucagon gene transcription in islet and enteroendocrine cell lines.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The cdx-2/3-dependent activation of the
proglucagon promoter plasmids containing the largest amounts of
5'-flanking sequences, such as (-2292)GLU-Luc, was somewhat greater in
InR1-G9 cells compared with GLUTag cells. In contrast, cdx-2/3 did not
function as an activator of the longer proglucagon-luciferase plasmids
in STC-1 cells until proglucagon gene 5'-flanking sequences upstream of
-155 were deleted. Further deletion of promoter sequences to -60
eliminated the cdx-2/3-dependent activation (data not shown),
consistent with the known location of the cdx-2/3-binding site in the
rat proglucagon promoter (8). Taken together, the differential
cdx-2/3-dependent activation of the proglucagon promoter in STC-1
vs. InR1-G9 and GLUTag cells suggests that the cell-specific
expression of DNA-binding proteins, as previously documented for the
upstream proglucagon enhancer sequences (4), may modify the
transcriptional activity of the proglucagon promoter, and its response
to cdx-2/3 in different cell types. Furthermore, the transfection data
shown here clearly establish that cdx-2/3 activates the proglucagon
promoter not only in fibroblasts (8), but also in islet and intestinal
cell lines.
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and 3. Increasing amounts of
cdx-2/3 transiently transfected into InR1-G9 cells resulted in a
corresponding increase in the levels of proglucagon mRNA transcripts.
In contrast, no increase in the levels of tubulin mRNA transcripts was
detected in the same experiment. The transcription factor isl-1 also
increased proglucagon gene expression in transfected InR1-G9 cells,
consistent with previous findings that depletion of isl-1 in InR1-G9
cells caused a reduction in the basal levels of proglucagon mRNA
(7).
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), suggesting that the activation of endogenous
proglucagon gene expression by cdx-2/3 is highly cell-specific.
Furthermore, we did not observe activation of proglucagon gene
transcription (as assessed by Northern analysis) after transfection of
cdx-2/3 into STC-1 enteroendocrine cells, or BHK fibroblasts (data not
shown).
To provide complementary evidence correlating increased expression of
cdx-2/3 with activation of endogenous proglucagon gene expression, we
transfected either isl-1 or cdx-2/3 [ligated in the plasmid pSR1neo
(7)] into InR1-G9 cells and selected transfectants with the antibiotic
G418. Analysis of a few randomly selected stably transfected
G418-resistant InR1-G9 clones that expressed cdx-2/3 displayed
increased basal levels of proglucagon mRNA transcripts in three of four
lines examined (data not shown). Accordingly, to correlate more
precisely the levels of cdx-2/3 expression with potential changes in
expression of the endogenous proglucagon gene, a larger number of
clones were isolated and examined (Fig. 3). Increased cdx-2/3
expression was generally associated with induction of proglucagon gene
expression in InR1-G9 cells. Of 21 clones examined, 18 had increased
levels of both cdx-2/3 and proglucagon mRNA, whereas three clones had
normal levels of cdx-2/3 and proglucagon (Fig. 3 and data not shown).
Nevertheless, we did not observe a perfect quantitative correlation
between cdx-2/3 and proglucagon gene expression, as some clones,
e.g. C4, had very high levels of cdx-2/3 mRNA, but
comparatively modest induction of proglucagon gene expression. In
contrast to the activation of the proglucagon gene detected in
association with increased cdx-2/3 expression, the levels of mRNA
transcripts for tubulin (Fig. 3) or the neuroendocrine gene
chromogranin (Fig. 3) were not induced by cdx-2/3 in the G418-resistant
cell lines. The results of these experiments strongly suggest that
cdx-2/3 activates the endogenous proglucagon gene in InR1-G9 islet
cells.
The increased levels of proglucagon mRNA transcripts were likely
attributable in part to an increase in proglucagon promoter activity.
To examine this directly, we transfected proglucagon-luciferase
plasmids into wild type InR1-G9 cells, and InR1-G9 clones C2 and C4,
that exhibited normal and increased levels of endogenous proglucagon
mRNA transcripts, respectively. Proglucagon promoter activity was
clearly increased in the C4 InR1-G9 clone with all three proglucagon
promoter plasmids that contained the cdx-2/3-binding site, consistent
with the increased levels of both cdx-2/3 and proglucagon mRNA
transcripts detected in the C4 cell line (Fig. 4). In
contrast, no increase in luciferase activity was observed after
transfection of the same plasmids in the C2 clone, which exhibited wild
type levels of proglucagon mRNA transcripts. Furthermore, the increased
activity of proglucagon promoter plasmids was not seen after deletion
of 5'-flanking sequences to -60, consistent with the elimination of
the cdx-2/3-binding site. Taken together, these experiments clearly
demonstrate a correlation between expression of the cdx-2/3 gene and
activation of both the endogenous proglucagon gene and the transfected
proglucagon promoter in InR1-G9 islet cells.
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NT-cdx-2/3,
an internally deleted cdx-2/3 cDNA (that we generated by PCR), which
lacks amino acids 8–180 (Fig. 5). Activation of the
proglucagon promoter by transfected cdx-2/3 in InR1-G9 cells was found
to be maximal at concentrations of transfected cdx-2/3 approximating
100 ng, and cotransfection with the NT-cdx-2/3 plasmid completely
abrogated the cdx-2/3 induction of proglucagon promoter activity,
despite increasing amounts of cdx-2/3 in the transfection experiments
(Fig. 5a). The NT-cdx-2/3 plasmid also attenuated the basal activity
of the proglucagon promoter in InR1-G9 cells (Fig. 5b), consistent with
the recently described competition of this protein with wild type
cdx-2/3 for binding to the G1 promoter site (9). To ascertain whether
the N-terminal cdx-2/3 deletion might also influence the levels of
endogenous proglucagon mRNA transcripts, the NT-cdx-2/3 plasmid was
transiently transfected into InR1-G9 cells alone or in the presence of
cdx-2/3, after which the levels of proglucagon mRNA transcripts were
examined (Fig. 5c). The results of this experiment demonstrate that the
NT-cdx-2/3 plasmid abrogates the cdx-2/3-dependent induction of
proglucagon mRNA transcripts in InR1-G9 islet cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Establishment of stable intestinal epithelial IEC-6 cell lines expressing high levels of mouse cdx-2 was followed by proliferation arrest and differentiation in association with expression of cdx-2. The proliferation arrest was released after several days, despite the continuing expression of cdx-2. Although no induction of the cdx-2-responsive SI gene was observed after 40 days of culture, IEC-6 cells expressing cdx-2 for more than 50 days did express SI gene transcripts (13). These observations suggest an indirect link between prolonged expression of cdx-2 and the activation of endogenous target gene expression.
The cdx-2/3-dependent activation of endogenous proglucagon gene
expression reported here was observed in InR1-G9 islet cells but not in
enteroendocrine or fibroblast cell lines. In contrast, cdx-2/3
activated the proglucagon promoter in transfection experiments in
fibroblasts and intestinal endocrine cells. The mechanisms determining
the cell-specific cdx-2/3 responsiveness of the endogenous proglucagon
gene promoter remain unknown. We have demonstrated by RT-PCR that
InR1-G9 and GLUTag cells express a different profile of homeobox genes
(data not shown), and it is possible that homeobox proteins with
different affinities for the G1-binding site may modify the effect of
transfected cdx-2/3 on activation of the endogenous proglucagon
promoter in GLUTag cells. Similar cell-specific differences in target
gene responsiveness have recently been reported for the IPF-1 induction
of insulin mRNA transcripts (14). The insulin gene homeobox
transcription factor IPF-1 induced the expression of the insulin and
amylin genes in stably transfected, glucagon-producing AN 697 islet
cells, but not in rat embryo fibroblasts. Furthermore, transient
transfection of IPF-1 into either 
TC1.9 or AN697 glucagon-producing
islet cell lines was not associated with induction of endogenous
insulin gene expression (14). These observations differ from our
demonstration that cdx-2/3 activates the proglucagon promoter and
endogenous proglucagon gene in both transient transfections and in
stable cell lines expressing increased levels of cdx-2/3. The
mechanisms for the inability of IPF-1 to induce insulin gene expression
in transient transfections remain unknown, but it has been suggested
that IPF-1 action may require the expression of other factors necessary
for activation of the insulin gene promoter, and induction of these
factors may require more than the 48–72 h duration of a transient
transfection (14).
The data reported here demonstrate that increased expression of cdx-2/3 correlates with the increased expression of a specific endogenous mRNA and not simply a transfected fusion gene. The LIM domain homeobox gene isl-1 was previously shown to bind to an adjacent site in the proglucagon gene G1 promoter region, and reduced levels of isl-1 were associated with a decrease in the levels of proglucagon mRNA transcripts (7). The results of our experiments demonstrate that transfected isl-1 is also associated with an increase in endogenous proglucagon mRNA transcripts, consistent with the postulated role for isl-1 as a positive activator of proglucagon gene transcription.
Although cdx-2/3 activated the expression of proglucagon-luciferase reporter genes in three different endocrine cell lines, considerable heterogeneity was observed with respect to the relative degree and pattern of reporter gene activation in the different cell types. We have previously documented that the relative profile of basal proglucagon promoter transcriptional activation differs after transfection of STC-1, InR1-G9, and GLUTag cell lines (4). Whereas the latter two cell types appear to more accurately represent the A and L cell phenotype, respectively, STC-1 cells are plurihormonal and much less representative of a pure population of differentiated glucagon-producing cells (15). Furthermore, we have recently shown that the pattern of DNA-protein interaction using STC-1 extracts and proglucagon promoter sequences clearly differs from the profile of binding events detected with extracts from InR1-G9 and GLUTag cells (4). Taken together, these observations suggest that cell heterogeneity and differential expression of transcription factors may account for the observed differences in proglucagon gene promoter activity in various cell types.
The recent report that an amino-terminally truncated cdx-2/3 RNA
transcript was detected in InR1-G9 cells by RT-PCR (9) prompted us to
examine the potential significance of such a molecule for the control
of proglucagon promoter activity. Although the relative degree of
inhibition of promoter activity varied somewhat with the various
proglucagon-promoter plasmids analyzed, we consistently observed an
inhibition of the cdx-2/3-dependent induction of proglucagon promoter
activity in the presence of the NT-cdx-2/3 plasmid. Furthermore, the
NT-cdx-2/3 plasmid diminished the ability of the wild type cdx-2/3
plasmid to activate endogenous proglucagon mRNA transcripts in InR1-G9
islets, but the NT-cdx-2/3 plasmid alone did not reduce the basal
levels of proglucagon mRNA. This may be due to competition for binding
to the proglucagon G1 promoter site (by other homeobox proteins), or to
other mechanisms that remain to be elucidated.
The results of our experiments with the NT-cdx-2/3 plasmid suggest
that such an N-terminally-truncated protein, if expressed in islet
cells, might possibly modulate the activity of cdx-2/3 on the
proglucagon promoter, and therefore the ratio of wild type and
truncated cdx-2/3 proteins could be an important determinant of cdx-2/3
activity. Nevertheless, we have not observed a smaller cdx-2/3 RNA
transcript by Northern blotting, and Western blot analysis of InR1-G9
cells using antisera detected against the carboxy-terminal region of
cdx-2/3 did not detect the presence of any smaller immunoreactive
cdx-2/3 proteins that might correspond in size to the predicted
N-terminal cdx-2/3 deletion (8). Accordingly, the biological
significance of the N-terminal cdx-2/3 deletion in the control of
proglucagon gene expression awaits the definitive detection of this
protein in islet and enteroendocrine cells and remains uncertain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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NT-cdx-2/3, which
contains a deletion from amino acids 8–180 in hamster cdx-3, was
constructed by PCR, and the sequence of the N-terminal-deleted cdx-3
cDNA was verified by DNA sequencing. For generation of stable
G418-resistant cell lines, hamster cdx-3 was ligated into the psR1neo
expression vector, which contains the neo resistance cassette (7).
Cell Lines, Northern Analysis, and Transfections
The InR1-G9, STC-1, and GLUTag cell lines were propagated and
transfected as previously described (15, 17, 18, 19). RNA isolation used
the acid phenol extraction method (20), and Northern blotting was
carried out using nylon membranes as described (21, 22). The luciferase
activity was analyzed by normalizing each transfection relative to the
protein concentration in each transfected extract, and luciferase
activity was measured as described (23). The reporter gene activity was
expressed relative to the activity obtained using the promoterless
luciferase plasmid pBluc in the same experiment. All transfections were
carried out in triplicate on at least three separate occasions, and
statistical significance was assessed by Student’s t
test.
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FOOTNOTES |
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This work was supported by an operating grant from the Medical Research Council of Canada. T.J. was supported by a fellowship award from the Ontario Ministry of Health. D.T. was supported by a fellowship award from the Banting and Best Diabetes Centre. D.J.D. is a Scientist of the Medical Research Council of Canada.
1 Present address: Oncology Research Laboratories, The Toronto Hospital,
University of Toronto, 67 College Street, Toronto, Ontario M5G 2M1,
Canada. 
Received for publication October 4, 1996. Revision received October 29, 1996. Accepted for publication November 4, 1996.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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