(Received for publication, November 1, 1995; and in revised form, January 18, 1996)
From the
We have previously reported that albumin gene transcription is reduced in diabetes mellitus (DM). The present study explored the mechanism by which albumin gene transcription is down-regulated in DM. Deletional studies and displacement of factors binding to site B of the albumin promoter indicated that the repressive effects of DM are mediated by nuclear factors binding to this site. Since hepatocyte nuclear factor 1 (HNF1) activates albumin promoter activity and is the predominant factor binding to site B, we examined HNF1. The abundance and binding activity of HNF1 were reduced in hepatonuclear extracts from diabetic compared to control rats. However, HNF1 mRNA levels were unchanged, suggesting that the effect of DM on HNF1 is at the post-transcriptional level. Extracts from diabetic animals also contained another protein, distinct from HNF1 and vHNF1, which bound to site B in gel retardation studies. In summary, our studies demonstrate that the reduced abundance and binding activity of HNF1 correlates with decreased albumin gene transcription in DM.
Diabetes mellitus (DM) ()alters the transcription of
numerous genes in a variety of
tissues(1, 2, 3, 4, 5, 6) .
Although DM increases or decreases gene transcription, the mechanism
underlying these changes is largely unknown. We have shown previously
that transcription of the albumin gene is reduced in DM(7) .
Therefore, we are using the albumin gene as a model to understand how
DM down-regulates gene transcription.
Extensive studies have shown that control of albumin gene transcription resides primarily within the proximal 170 bp of the albumin promoter (8, 9) . At least six important cis-acting elements (A-F) have been defined within this promoter sequence(10) . Of these elements, site B plays a pivotal role in mediating liver-specific transcription of the gene(11, 12, 13) . Furthermore, mutations of this site decrease promoter activity to a greater extent than do changes to any of the other cis-acting elements(11) . Multimerization of site B also yields a strong artificial promoter active only in liver(11) . In addition, of these six elements, only site B is conserved from Xenopus to human(12, 14) .
Hepatocyte nuclear factor 1 (HNF1)
binds to site B and trans-activates albumin gene
transcription(14, 15, 16, 17) .
Albumin gene expression correlates closely with the presence of HNF1.
The association between HNF1 and albumin is exemplified by the
appearance of HNF1 in the transition from -fetoprotein to albumin
expression during hepatic ontogeny(18) . Similarly, HNF1
concentrations are high in differentiated hepatocyte cell lines (FAO
and H4II) that secrete albumin, and low in dedifferentiated hepatic
cell lines (C2 and H5) that do not produce appreciable levels of
albumin(19, 20) . In addition, there is a tight
correlation between the tissue-specific expression of albumin and the
expression of HNF1(21, 22, 23) . A variant
form of HNF1 termed vHNF1 also binds to site B of the albumin
promoter(16, 17, 24, 25, 26, 27) .
vHNF1 is, however, much less effective than HNF1 in transactivating the
albumin gene, and the presence of vHNF1 does not correlate well with
albumin expression. In fact, vHNF1 is abundant in cell lines (H5 and
C2) and kidney, where albumin expression is
low(16, 17, 24, 25, 27) .
Both HNF1 and vHNF1 bind DNA as a hetero-tetrameric complex comprising
two molecules each of HNF1 or vHNF1 and a dimerization cofactor, DCoH,
which enhances the transcriptional activity of its
partner(28) .
The critical role of HNF1 and site B in regulating albumin gene expression prompted us to postulate that decreased albumin gene transcription in DM is mediated through site B. Not only do our results support this hypothesis, they also show that HNF1 protein levels are reduced in the diabetic state. To our knowledge, this represents the first description in DM of a reduction in a nuclear factor essential for the transcription of an eukaryotic gene, albumin.
Figure 1: Transcriptional activity of albumin promoter deletions in hepatonuclear extracts. Panel A, schematic representation of the deletional constructs of the albumin promoter containing -130, and -78 to +22 bp attached to the GFC. Relative location of cis-acting elements A to D is shown. Panel B, autoradiographs of radiolabeled RNA products from the cell-free in vitro transcription assay with hepatonuclear extracts (100 µg) from control (Ctl) and diabetic (DM) animals. The top band originates from the pAlb-320 construct and the lower signal from the adenovirus major late promoter, the internal control. Transcriptional activity of the -130 construct appears in lanes 1 and 2. Lanes 3 and 4, transcriptional activity of the -78 promoter construct. Panel C, graph of albumin promoter activity in the two types of extracts. Values obtained for albumin are expressed relative to those of the internal control. Each bar represents the mean ± S.D. (control, n = 6; DM, n = 4; *, p < 0.02 by Student's t test).
Since site B (-72 to -58) is critical for albumin promoter activity (11, 12, 13, 35) , we asked whether decreased albumin gene transcription in DM was mediated through this site. To eliminate the effect mediated through site B, we displaced the binding of transcription factors to this site. Accordingly, we measured the transcriptional activity of an albumin promoter construct (-650 to +22) in the presence or absence of oligonucleotide homologous to site B (Fig. 2). The addition of site B oligomer to the reactions reduced transcriptional activity of the promoter in extracts from both euglycemic control (compare lanes 1 and 3) and diabetic (compare lanes 4 and 6) animals. Moreover, in the presence of site B oligomer, the level of albumin transcription supported by extracts from diabetic animals was the same as that from controls (compare lanes 3 and 6). Residual albumin transcription (11) is due to the effect of transcription factors other than HNF1, which interact with the promoter at other binding sites. In contrast, transcription of the construct in either extract was not significantly affected by adding nonspecific oligomer homologous to site C of the rat apolipoprotein A1 gene (compare lanes 1 to 2 and 4 to 5). These results suggest that site B of the albumin promoter mediates an inhibitory effect of DM on albumin gene transcription.
Figure 2: Albumin promoter activity in the presence of DNA competitor. Panel A, autoradiograph of an in vitro transcription assay using a -650 to +22 albumin promoter construct and 80 µg of hepatonuclear extracts. The activity of the promoter with extract from a euglycemic rat appears as follows: lane 1, by itself; lane 2, with a nonspecific oligomer; and lane 3, with DNA homologous to site B. The activity of the promoter with extract from a diabetic rat appears as follows: lane 4, by itself; lane 5, with a nonspecific oligomer; and lane 6, with DNA homologous to site B. Panel B, graph of the relative albumin promoter activity in the two types of extracts. Each bar represents the mean ± S.D. (control n = 5; diabetic, n = 4; *, p < 0.005;**, p < 0.001, by Student's t test).
Displacement of factors binding to site B of the albumin promoter allowed us to examine whether the suppressive effects of DM arise from a reduction in the abundance or activity of an activator of site B, the actions of an inhibitor(s), or a combination of both. If the effect of DM is due mainly to the action of an inhibitor, we would expect displacement of this factor to increase albumin gene transcription. Alternatively, if the effect of DM is due to reduced abundance or activity of a site B activator, we would anticipate displacement of factors binding to this site to further decrease albumin gene transcription. Our results are consistent with the latter possibility, suggesting that in DM the reduced abundance or activity of an activator of transcription is the principal mechanism responsible for decreasing albumin gene activity through site B.
Figure 3: HNF1 protein levels. Relative levels of HNF1 protein were determined by Western blot analysis using anti-HNF1 antiserum. Panel A: lanes 1 and 2 contain 60 µg of crude hepatonuclear preparations from control and diabetic animals, respectively. Lanes 3 and 4 contain 9 µg of partially purified extracts from control and diabetic animals, respectively. WGA, wheat germ agglutinin. Panel B: Western blot of COS-7 expressed HNF1 used as a control. Lanes 1 and 2 contain 50 µg of nontransfected COS-7 cells or cells transfected with an HNF1/pBJ5 expression vector, respectively. Lane 3 contains 60 µg of hepatonuclear extract from control euglycemic animals. Indicated to the left is the migration of protein markers (kDa), and to the right, the molecular mass of HNF1 estimated from the migration of those protein markers.
Figure 4: Site B binding activity in extracts from control and diabetic animals. Gel retardation assays were performed by incubating radiolabeled site B probe with partially purified preparations from control and diabetic animals. Panel A: lanes 1 and 3 show the retarded complexes in reactions containing hepatonuclear extracts from control animals. Lanes 2 and 4 show complexes with extracts from diabetic animals. Panel B shows binding activity in the presence of antisera against HNF1, vHNF1, or rat Apo A1, as indicated. Lanes 1-4 contain extracts from control animals, and lanes 5-8 contain extracts from diabetic animals.
Site B binds not only HNF1 (80-93 kDa)(16, 17) , but also vHNF1 (68-72 kDa)(15, 17, 36) , a nuclear protein that correlates with decreased albumin gene expression(24, 37) . We examined whether the faster migrating complex in extracts from diabetic animals might be due to the binding of vHNF1 to site B by adding specific antisera raised against HNF1 or vHNF1 to the gel retardation reaction (Fig. 4B). Rat Apo A1 antiserum (38) served as a negative control. HNF1 antiserum retarded the mobility of complexes formed using extracts from both control (lane 2) and diabetic (lane 6) rats. In contrast, vHNF1 antiserum had no effect on the mobility of these complexes (Fig. 4B, lanes 3 and 7). The functional integrity of the antisera against vHNF1 and Apo A1 was confirmed using control antigens (data not shown). In addition, the mobility of COS-7-expressed vHNF1 (see Fig. 6) was clearly different from that of the protein-site B complex in extracts from diabetic animals. These results suggest that the more rapidly migrating complex observed with extracts from diabetic animals arises from the binding of a protein to site B which is not vHNF1. This site B-binding protein is, however, recognized by antiserum against HNF1.
Figure 6: Gel retardation analysis in the presence or absence of DCoH. Electrophoretic mobility of the site B binding activity present in protein extracts from diabetic animals (lane 4), control animals (lane 3), and COS-7 expressed HNF1 (lane 2) or vHNF1 (lane 1).
Figure 5: Integrity of proteins in extracts from control and diabetic animals. Panel A, silver-stained SDS-polyacrylamide gel of partially purified protein preparations from control (lane 1) and diabetic (lane 2) animals. Panel B, gel retardation analysis of site B binding of hepatonuclear extracts from control (lanes 1 and 3) and diabetic (lanes 2 and 4) animals after 1 h (lanes 1 and 2) or overnight (lanes 3 and 4) incubation of the reactions at room temperature.
Figure 7: Estimated molecular mass of proteins bound to site B. Autoradiograph of protein-site B complexes formed by exposing gel retardation reactions to UV irradiation, followed by denaturing SDS-PAGE analysis. The migration of molecular mass markers (kDa) is indicated on the left. The estimated molecular mass of proteins bound to site B is indicated on the right. A discrepancy in size such as that obtained from UV-cross-linking and Western blot analysis of HNF1 has been reported previously(67) .
Since the molecular mass of DM-X (59 kDa) is different from
that of HNF1 and it is recognized by specific antiserum against HNF1,
one would expect to detect DM-X as a distinct band by Western blot
analysis. Although DM-X was recognized by anti-HNF1 antiserum in the
gel retardation assay, a
59-kDa band was not detected by Western
blot. This discrepancy could arise from the different conditions used
in the two experiments. There is documented evidence of differential
behavior of a single antibody in gel retardation and Western blot
analysis(39) .
Figure 8: Northern blot analysis of HNF1. Total RNA extracted from rat livers was analyzed by Northern blot, as described under ``Materials and Methods.'' Lanes 1 and 2 and lanes 3 and 4 contain 45 µg of total RNA from control and diabetic animals, respectively. To the right is indicated the migration position of the 3.6- and 3.2-kilobase HNF1 mRNA species. Means obtained by videodensitometry were analyzed by Student's t test (p > 0.05).
In addition, Northern blot analysis served to determine whether DM-X arises from translation of a mRNA that differs from that of HNF1, based on the following criteria. The anti-HNF1 antibodies used in gel retardation studies recognized both HNF1 and DM-X (see Fig. 4B). Therefore, since the HNF1 cDNA used as a probe in the Northern blot corresponds to the area of the molecule encoding the epitope recognized by the HNF1 antibodies, we might detect an extra band in RNA from diabetic animals if DM-X arises from translation of a mRNA distinct but similar to that of HNF1. The absence of any additional band in RNA from livers of diabetic animals (Fig. 8) suggests that DM-X does not arise from a distinct RNA.
In this study we examined whether the cis-acting element B mediates the suppressive effect of DM on albumin gene transcription. We show that this suppressive effect is retained by promoter fragments containing site B, and that it is abolished by displacing factors binding to this motif. Our results reveal that DM reduces the abundance and binding activity of the major site B-binding protein, HNF1. The reduced abundance of HNF1 may account for decreased albumin gene transcription in DM.
Our results suggest that suppression of albumin
gene transcription in DM is due to reduced transactivation through site
B. This mechanism of albumin gene suppression is supported by our
observation that DM decreases the abundance and binding activity of
HNF1, the major site B activator. The reduced abundance of HNF1 in DM
represents, to our knowledge, the first report of a transcription
factor essential for albumin gene expression being altered in this
disease. In contrast to HNF1, the mRNA levels of two other
transcription factors, which also enhance albumin transcription,
C/EBP and C/EBP
, are increased in
DM(40, 41) . If this increase in mRNA levels reflects
an enhanced abundance of C/EBP
and C/EBP
proteins, then it
should increase albumin gene activity in DM. However, transcription of
the albumin gene actually falls in DM, emphasizing the functional
importance of decreased HNF1 protein levels in this disease. Similar
mechanisms may regulate the expression of other genes in DM. For
example, the hormone binding activity of thyroid hormone receptor, a
ligand-dependent transcription factor, is decreased in DM and
correlates with reduced expression of
globulin (42) . The abundance of other transcription factors, such as
c-Jun and c-Fos(43, 44) , is altered by insulin,
suggesting that their levels may also be affected in DM. In addition,
other disorders appear to affect HNF1 expression. HNF1 mRNA has been
shown to decrease significantly in response to burns(45) ,
although HNF1 protein levels were not determined in this study.
Recently, oncotic pressure has been shown to reduce albumin gene
transcription in hepatoma cells through decreased HNF1 binding
activity(46) .
Although HNF1 protein levels are decreased in the diabetic state, the HNF1 mRNA levels are the same in control and diabetic animals. It therefore appears that the effect of DM on HNF1 expression is exerted at the translational or post-translational level. Control of HNF1 at the translational or post-translational level has been reported previously by others(25, 47) . Diabetes could potentially lower the translational efficiency of HNF1, increase the protein turnover, or a combination of both. In addition, sequestration of the HNF1 mRNA in translationally inaccessible messenger ribonucleoprotein particles is a possibility(48, 49, 50) . Effects of DM at the translational or post-translational level have been reported for other proteins. For example, the levels of apolipoprotein B and E drop in diabetic rats primarily as a result of slowed translation, with the levels of their respective mRNAs remaining unchanged(51) . In addition, translational regulation has been reported for several transcription factors, such as LAP and Pit-1/GHF-1(52, 53) . Regulating translation allows a cell to respond more rapidly to environmental cues than does de novo transcription(48) . This type of control is often seen for genes that play a role in development, as is the case for HNF1 (16, 17, 18) .
In addition to reduced HNF1
protein levels, our results indicate that the liver of diabetic animals
contains a 59-kDa protein (DM-X) which binds to site B of the albumin
promoter. Since displacement of factors binding to this site in DM
minimally decreases transcription of the albumin gene (Fig. 2),
the effect of DM-X through site B could at most be that of a weak
activator. Therefore, in contrast to the dominant role of decreased
HNF1 on albumin gene expression, our results suggest that DM-X plays a
minor, if any, role in lowering albumin gene transcription in DM. The
lack of a major function for this protein on albumin transcription is
also supported by other studies ()showing that correction of
HNF1 binding activity alone, without diminishing the binding of DM-X,
is sufficient to normalize albumin mRNA levels in diabetic animals.
These findings on the albumin gene, however, do not rule out
transcriptional regulation of other HNF1-regulated genes by this 59-kDa
protein. Although the identity of this protein is unknown, the fact
that it is recognized by anti-HNF1 antiserum (Fig. 4) suggests
that it is somehow related to HNF1. It is possible that DM-X arises
from post-translational modification of HNF1, or alternatively, from
the same message as HNF1 by mRNA editing, as has been described for
apolipoprotein B(54, 55) . In addition, we cannot
exclude the possibility that DM-X might arise from in vivo post-translational processing of HNF1. Since endogenous
proteolytic enzyme activity is altered in DM(56, 57) ,
this protein might be generated by truncation of the HNF1 protein.
Since levels of HNF1 are reduced in DM, one would expect this change
to affect the expression of not only albumin, but also other hepatic
genes that interact with this transcription factor. Two genes that
contain HNF1 binding sites in their promoter sequences are those
encoding -antitrypsin and
-fibrinogen. As the
hepatic expression of these genes is dependent on
HNF1(58, 59, 60) , one would anticipate
-fibrinogen and
-antitrypsin protein levels to
parallel those of albumin. Although the reduction of
-antitrypsin protein levels in DM (61) correlates with the reduced abundance of HNF1, increased
-fibrinogen protein levels (62, 63) do not. The
down-regulation of albumin gene expression in DM is regulated mainly at
the level of transcription. Whether the same is true for the
-fibrinogen gene remains unknown. It is conceivable that
transcription of the
-fibrinogen gene is low in DM, even though
its protein levels are increased. Changes in the rate of gene
transcription are not always accompanied by similar changes in protein
levels. For example, growth hormone up-regulates transcription of
albumin, even though protein levels remain unchanged due to a
compensatory increase in albumin mRNA degradation(64) .
The
expression of many hepatic genes other than albumin is decreased in DM.
Since some of these genes do not contain HNF1 binding sites in their
promoters, reduced levels of HNF1 cannot function as a general
mechanism to inhibit hepatic gene expression in DM. In addition, other
genes that are affected in DM contain HNF1 binding sites but are not
primarily regulated by this factor in the liver (e.g. PEPCK; (65) and (66) ). Therefore, changes in the abundance of
HNF1 in DM appear to affect the expression of a subset of hepatic genes
whose expression is predominantly regulated by this factor, such as
albumin and -antitrypsin.