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(Received for publication, October 5, 1995; and in revised form, November 15,
1995) From the
Expression in mice of transgenes directed by regulatory regions
of the rat aldolase B gene requires the presence of a B element located
in the first intron, while constructs devoid of this intronic enhancer
are silent. Histo- and immunochemical staining of transgenic tissue
sections showed that the longer transgene was expressed in the proximal
tubular cells of the kidney, enterocytes located in small intestine
villi and liver parenchymal cells. In the liver, a maximal expression
was observed in perivenous hepatocytes, while the transgene was weakly
active in periportal hepatocytes, which reproduced the pattern of
functional zonation already reported for other glycolytic and
gluconeogenic genes in the liver. We also established that the
transgene retained the necessary elements for a correct chronological
expression during development but was lacking elements necessary for
activation by high carbohydrate diet. Instead, transgene expression was
paradoxically stimulated in fasted animals, suggesting that the
endogenous gene, which must be active under both glycolytic and
gluconeogenic conditions, could possess distinct elements activating it
in fasted as well as in carbohydrate-fed animals; the former element
might be conserved in the transgene and the latter one might be lost. Aldolase B is the isoform of fructose 1,6-bisphosphate aldolase,
which is specific to hepatocytes, proximal tubular cells of the kidney,
and enterocytes, where it plays an essential role in fructose
metabolism. Indeed, aldolase B is much more active on fructose
1-phosphate resulting from fructose phosphorylation by fructokinase
than the other two aldolase isoforms, aldolase C (specific to the
brain) and aldolase A (ubiquitous and very active in the muscle). In
addition, aldolase B activity is required in gluconeogenic organs for
both glycolysis (hydrolysis of fructose 1-phosphate and fructose
1,6-bisphosphate into trioses) and gluconeogenesis (condensation of
triosephosphates into fructose 1,6-bisphosphate). In humans, aldolase B
deficiency is responsible for hereditary fructose intolerance, a
recessive autosomal disease characterized by hypoglycemia and clotting
disorders upon fructose feeding. In the liver, aldolase B progressively
replaces aldolase A (and, to a lesser extent, aldolase C) during fetal
development, becoming practically the only isoform in postnatal
hepatocytes (Schapira et al., 1975; Numazaki et al.,
1984). Aldolase B gene expression is regulated at the transcriptional
level during development and cell differentiation and is also subjected
to a transcriptional regulation by diet and hormones; transcription is
activated about 4-fold by fasting rats fed a high glucose/fructose diet
and is inhibited by fasting, glucagon, and cyclic AMP (Munnich et
al., 1985; Weber et al., 1984). The 200-base pair
proximal promoter fragment of the aldolase B gene contains binding
sites for ubiquitous and liver-enriched transcriptional factors,
especially for proteins of the CAAT/enhancer binding protein family,
hepatocyte nuclear factors 1 and 3 (HNF1 ( In
the present paper, we show that this element is absolutely required for
the expression in transgenic mice of constructs in which the
chloramphenicol acetyltransferase (CAT) gene is put under the control
of aldolase B regulatory regions (aldolase B/CAT constructs). This
tissue-specific expression has been more precisely analyzed by in
situ detection of the CAT protein using immunological and
enzymatic methods whose performances are compared. The expression of
the transgene was localized to proximal tubules of the kidney,
enterocytes in the upper region of intestine villi, and in the liver to
pericentrolobular hepatocytes.
Figure 1:
Maps of the different transgenes.
Elements A, B, and C of the first intron are
represented by shaded boxes. In the ``232A100''
transgene series, the 600-base pair element A was deleted from
its 500 more 3`-nucleotides. The 5`-flanking sequence, upstream of the
start site of transcription (arrow indicating position
+1), is represented by a thin line. The CAT gene is
represented by an open box.
RNAs were
isolated from tissues by lysis in guanidium chloride, followed by
phenol extraction (Chomczynski and Sacchi, 1987). Northern blot
analysis was performed as described by Cuif et al. (1992). The
aldolase B probe used was a 379-base pair mouse aldolase B cDNA
fragment amplified by reverse transcriptase polymerase chain reaction,
and then cloned in pBluescript plasmid. The CAT probe was isolated by ClaI-EcoRI digestion of the PeCAT vector
(Grégori et al., 1991). The 18 S R45
ribosomal probe was used as a standard for RNA quantification
(Concordet et al., 1993).
However, the element B is clearly insufficient to
confer on the transgenes an expression dependent on the number of
integration copies and independent of site, which is to say to behave
as a locus control region (Grosveld et al., 1987; Fraser et al., 1990). Indeed, the CAT activity directed by a same
transgene is highly variable in different lines and is not dependent on
the transgene copy number. In two lines, the expression of the
232ABC/CAT (line 14) and 232A100B/CAT (line 52) transgenes is even nil
in the liver and ectopically stimulated in the spleen. Therefore, even
in the presence of the element B, expression of the transgenes is
highly influenced by the integration site, which indicates that they
contain neither locus control region nor so-called
``insulators'' (Bode and Maas, 1988; Kalos and Fournier,
1995; Phi-Van et al., 1990) able to protect transgenes from
the influence of neighbor regulatory elements. Nevertheless, except for
the two lines mentioned above, the transgenes with a B element are at
least 100-fold more expressed in the liver and kidney than in the brain
and spleen. The CAT activity in small intestine extracts seemed to be
in the nonspecific range, but this could be due to artifacts, for
instance to proteolytic degradation of the CAT protein in extracts
contaminated with pancreatic secretions or to dilution of expressing
cells by abundant non-expressing tissue, because the CAT protein was
well detected in some enterocytes by immunohistochemical and
histological techniques (see below).
Both immunohistochemical and histochemical techniques showed exactly
the same cellular and tissue pattern of positivity. In the liver (Fig. 2, a and b), positive hepatocytes were
localized in the central region with a perivenular ring distribution.
Very rare positive hepatocytes were also seen in the periportal area.
No positivity was observed in vascular cells,
Küpfer cells, epithelial bile duct cells, and
portal connective tissue.
Figure 2:
In situ detection of the CAT protein from
1600ABC/CAT transgenic mice line 7. Visualization is shown of CAT gene
product, either with rabbit polyclonal antibody to CAT (a, c, e, f) or with histochemical staining for
CAT activity (b, d). Primary antibody was revealed
with DIG-labeled goat antibody to rabbit followed by peroxidase- (a, c, e) or fluorescein (f)-labeled antibodies to DIG. CAT enzymatic activity was
detected by brown Fe
In the kidney (Fig. 2, c and d), the epithelial cells of the first two convoluted
parts of the proximal tubule (S1 and S2) and the capsular epithelium
(Bowman's parietal cells) of the glomerulus were positive for CAT
with either histochemistry or immunohistochemistry. Nuclei staining was
particularly strong in the S2 segment. No other kidney cell was
positive for CAT. Jejunum immunostaining revealed a 50% positivity
of villi enterocytes, with no staining of
Lieberkühn crypts (Fig. 2, e and f). There was no positivity in the lamina propria. CAT
histochemistry disclosed the same pattern of positivity in a weak
manner. These patterns of immuno- and histochemical labeling of the
aldolase B/CAT transgene product are consistent with previous results
using anti-aldolase B specific antibodies for detecting the enzyme in
various tissues (Schapira et al., 1975). However, the pattern
of extreme zonation of transgene expression, detected in perivenous
hepatocytes and in rare periportal hepatocytes, was not observed before
with anti-aldolase B antibodies. ( As mentioned before,
aldolase B activity is reversible, acting in both glycolysis and
gluconeogenesis. As a glycolytic enzyme, it could be expected to be
mainly expressed in the centrolobular region, as is glucokinase (Trus et al., 1980), pyruvate kinase (Miethke et al.,
1985), glucose 6-phosphate dehydrogenase (Welsh, 1972), and
6-phosphogluconate dehydrogenase (Hildebrand, 1980). However, as a
gluconeogenic enzyme, it could also be present in periportal
hepatocytes synthesizing phosphoenolpyruvate carboxykinase (Miethke et al., 1985), fructose 1,6- bisphosphatase (Katz et
al., 1977), and glucose 6-phosphatase (Miethke et al.,
1985). The molecular bases for the metabolic zonation of liver
parenchyma are not well known. They could include transcriptional and
post-transcriptional events controlled by hormone and oxygen gradients,
innervation, cell-cell interactions etc. (Jungerman, 1988; Kuo and
Darnell, 1991). The apparent discrepancy between zonation of
1600ABC/CAT transgene expression and the inapparent zonation of in
vivo aldolase B activity could result from the in vivo stability of aldolase B contrasting with the lower stability of
the CAT enzyme. Alternatively, we cannot exclude that the special
pattern of expression of the transgene in the liver lobule reflects the
lack of some important cis-acting element(s) in the transgene, as
evidenced by the strong dependence on the site of integration. In
any case, further analyses of the aldolase B promoter and enhancers
will be required to determine the role of these regulatory regions in
the observed restriction of transgene expression to perivenous
hepatocytes.
Figure 3:
Northern blot analysis of aldolase B and
CAT transcripts in livers of transgenic mice under different
nutritional conditions. 20 µg of total liver RNAs were
electrophoresed in formaldehyde-agarose gel and then blotted and
hybridized with 2
In other words, it seems that the
transgene lacks a positive glucose response element, perhaps similar to
that characterized in the promoter of the L-type pyruvate kinase gene
(Bergot et al., 1992; Diaz-Guerra et al., 1993), or
in a distal upstream region of the spot 14 gene (Shih and Towle, 1994).
However, it could retain an element whose role is to ensure a
persistence of aldolase B synthesis under gluconeogenic dietary
conditions, while purely glycolytic genes, such as the L-type pyruvate
kinase gene, are totally extinguished (Weber et al., 1984;
Vaulont et al., 1986). This hypothesis is in line with the
observation that the L-pyruvate kinase glucose response element (L-PK
GlRE) behaved, in transfected hepatocytes, as a positive element in the
presence of glucose but also as a negative element in the absence of
glucose or in the presence of glucagon (Bergot et al., 1992).
If such an element exists in the endogenous aldolase B gene but not in
the transgene, this could account for both absence of positive response
to glucose and sustained expression in fasted animals, while a
different element could account for the stimulation of transgene
expression during fasting. The aldolase B gene needs to be expressed in vivo with an intronic activator cooperating with its tissue-specific
promoter. These elements are sufficient to confer on a CAT reporter
transgene a correct tissue-specific expression in the kidney, small
intestine, and liver plus (in the liver) a drastic restriction of the
expression to hepatocytes around the centrolobular vein. However, the
physiological stimulation of the gene by a carbohydrate-rich diet was
replaced by the opposite phenomenon, that is to say a stimulation in
fasted animals. This paradoxical dietary response of the transgene
suggests that the aldolase B gene, which must be expressed under both
glycolytic and gluconeogenic conditions, could contain different
elements stimulating transcription in either conditions. The glucose
response element would be lacking in the transgene while the element
enhancing transcription during fasting would be present.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3469-3473
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)and HNF3). The
HNF1 and HNF3 binding sites overlap each other, and their occupancy is
mutually exclusive, HNF1 being a transcriptional activator whose effect
is counteracted by HNF3 (Grégori et al.,
1993, 1994). This competition between HNF1 and HNF3 could explain why
the activity of this promoter tested by transient expression is very
weak in hepatocytes and hepatoma cells (Grégori et al., 1991, 1994). In HepG2 hepatoma cells, activity of the
aldolase B promoter is stimulated about 50-fold by an intronic element,
termed element B, located in a fragment spanning from nucleotides 650
to 2448 (Grégori et al., 1991).
DNA Constructs
The maps of the different
transgenes analyzed are shown in Fig. 1. The 232ABC/CAT and
232A600/CAT transgenes were derived from previously described
constructs (2200ABC/CAT and 2200A/CAT) (Grégori et al., 1991) by XbaI digestion at position
-232. The transgene 232A100/CAT was derived from 232A600/CAT by PstI digestion cutting at position +197 and subcloning
into the VB8 CAT vector. The transgene 232A100B/CAT was derived from
232A100/CAT by cloning blunt-ended HindIII fragment B into the SmaI site of this construct.
Production and Detection of Transgenic Mice
The
1600ABC/CAT, 1600AB/CAT and 1600 AC/CAT fragments were isolated from
the previously described (Grégori et
al., 1991) 2200/ABC/CAT, 2200/AB/CAT and 2200/AC/CAT constructs,
respectively, by a ClaI digestion (Fig. 1). The
232ABC/CAT, 232A600/CAT, 232A100/CAT, and 232A100B/CAT fragments were
isolated from 232ABC, 232A600, 232A100, and 232A100B constructs,
respectively, using the AflIII and ClaI sites in the
vector. After electrophoresis in 0.8% (w/v) GTG-seakem agarose gel
(Life Technologies, Inc.), fragments were electroeluted and purified by
using elutip-d columns (Schleicher & Schuell). 3-10 ng of
fragment diluted in a 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA buffer was microinjected into fertilized B6D2 mouse eggs
according to (Gordon and Ruddle, 1983). Transgenic founders and
offsprings were identified by Southern blot, and transgene copy numbers
were estimated by quantitative analysis on a phosphorimager system
(Molecular Dynamics). CAT assays were performed with 0.001-100
µg of F1 animal tissue extract proteins (liver, kidney, small
intestine, spleen, and brain) according to Gorman et
al.(1982).Nutritional Treatment and Northern Blot
Analysis
The animals were fasted for 24 h and then either fasted
for an additional 18-h period or refed during this period with a high
carbohydrate diet (Weber et al., 1984). Mice were sacrificed,
and livers were stored at -80 °C until use.Analysis of Regional and Cellular Patterns of Transgenic
Expression
Transgenic and non-transgenic (wild type) mice of
line 7 were perfused under anesthesia using cardiac puncture with 2%
(w/v) paraformaldehyde in phosphate-buffered saline (PBS), pH 7.2
(Biomérieux, Marcy l'Etoile, France) (Kim et al., 1993). The intestinal tract (proximal and distal
jejunum), liver, and kidneys were subsequently removed immediately
after sacrifice and fixed by immersion in 2% paraformaldehyde in PBS,
pH 7, for 1 h at +4 °C. Fixed tissues were then rinsed in PBS,
cryoprotected by immersion in sucrose (10% (wt/vol) sucrose in PBS for
1 h, then 30% sucrose in PBS overnight, both at 4 °C), and then
frozen and stored in liquid nitrogen until use. Tissue blocks were
cross-sectioned at 7 µm in a cryostat, and sections were mounted on
slides (Super Frost/Plus, CML, Nemours, France), air dried, and
processed for immunochemical or histochemical staining without any
further fixation.Immunohistochemical Staining for CAT
Rabbit
polyclonal affinity-purified antibody to CAT (Miskimins et
al., 1992) were purchased from 5 Prime 3 Prime, Inc.
(Paoli, PA). Anti-rabbit IgG-digoxigenin (DIG), F(ab`)2 fragments from
sheep, anti DIG-peroxidase, F(ab) fragments, and anti-DIG-fluorescein
isothiocyanate were purchased from Boehringer Mannheim (Meylan,
France). Tissue sections were exposed to a 1/1000 dilution for anti-CAT
antibodies at room temperature for 1 h and rinsed three times in PBS.
Sections were then treated with anti-rabbit-DIG antibody (1/400
dilution) for 30 min and rinsed three times in PBS; this was then
followed by either anti-DIG-fluorescein isothiocyanate or
anti-DIG-peroxidase (1/400 dilution). Sections labeled with fluorescein
isothiocyanate were mounted with Vectashield (Vector, Biosys,
Compiègne, France) and immediately observed with
an epifluorescence microscope. Anti-DIG-peroxidase antibody was
revealed with diaminobenzidine solution (10 mg/15 ml in presence of
H
O (1/10000)) for 15 min, rinsed in PBS, and
mounted after dehydration and clarification. Control sections were
obtained by omission of the anti-CAT antibody and by using complete
technique on wild type tissues.Histochemical Staining for CAT
CAT activity was
revealed using the Donoghue et al. technique(1991, 1992).
Briefly, sections were incubated for 4-24 h with a mixture that
contained 0.3 mM acetyl-CoA (lithium salt; Pharmacia Biotech,
Paris), 4 mM chloramphenicol (Sigma, Saint Quentin Fallavier,
France), 5 mM potassium ferricyanide, 5 mM sodium
citrate, 3 mM copper sulfate, and 63 mM of
Sorensen's phosphate buffer, pH 6.0. They were then rinsed in
distilled water, dehydrated in alcohol, cleared in xylene, and mounted.
Control slides were obtained by omission of acetyl-CoA or
chloramphenicol in the mixture or by using the complete mixture on
non-transgenic tissues.
Expression of the Different Aldolase B-CAT Transgenes
in Vivo
Between two and four lines of each of the seven aldolase
B/CAT transgenes tested were obtained, except for the 232A100/CAT
transgene present in one line only (Table 1). CAT activity was
determined in tissue extracts from 3-month-old transgenic mice
harboring between 2 and 100 copies of the transgenes per cell. The main
result of these studies in transgenic mice is that the intronic element
B is indispensable to the expression of the transgenes; 1600AB/CAT,
232ABC/CAT, 1600AB/CAT, and 232A100B/CAT transgenes are expressed in
the liver and kidney of most transgenic lines while no 1600AC/CAT,
232A600/CAT, and 232A100/CAT transgenes were active in any line. These
latter results confirmed previous, non-published data from our
laboratory, namely that different types of transgenes directed by up to
2000 base pairs of 5`-flanking sequence of the aldolase B gene were
totally inactive in transgenic mice. Therefore, the intronic element B,
which is able to stimulate about 50-fold the activity of the promoter
in transiently transfected HepG2 cells (Grégori et al., 1991) is also an indispensable element for
tissue-specific and high level expression of the aldolase B transgene in vivo. Such a cooperation between a proximal promoter and a
distal enhancer to ensure a correct expression of transgenes has been
described for other genes, e.g. the albumin gene (Pinkert et al., 1987) and the apolipoprotein gene (Brooks et
al., 1994).
Developmental Regulation of the Aldolase B 1600ABC/CAT
Transgene in the Liver
Expression of the aldolase B gene in the
liver is developmentally regulated; the mRNA is undetectable before day
14 of gestation, and from that stage to adult it was estimated to
increase about 9-fold (Numazaki et al., 1984; Schapira et
al., 1975). Using the line 7 expressing the 1600ABC/CAT transgene
at a high level, we observed that developmental regulation of transgene
expression mimics that of the endogenous aldolase B gene (Table 2). Therefore, the transgene contains the elements
required for a correct regulation of aldolase B gene expression during
development.
In Situ Detection of the CAT Protein in Transgenic Mice
Harboring the 1600ABC/CAT Transgene
Since the 1600ABC/CAT
transgene was that containing most potentially regulatory sequences of
the aldolase B gene, we used mice of line 7, expressing this transgene
at a high level, for an immunohistochemical analysis of CAT transgene
expression.CAT Immunohistochemical Staining
Fluorescence or
diaminobenzidine staining was equivalent on all transgenic tissues
examined with a cytoplasmic and occasionally nuclear positivity. No
positivity was noted in all controls.Histochemical Stain for CAT
Positivity of
transgenic tissues consisted of a cytoplasmic granular brown
precipitate. In addition, some nuclei were strongly stained in
transgenic tissue. Nevertheless, in transgenic and wild type tissues,
some nuclei were occasionally weakly stained, indicating that this
nuclear staining is not specific, as already described (Donoghue et
al., 1991, 1992). No cytoplasmic positivity was noted in controls.
- Cu
precipitate with no counterstain. a, CAT
immunoperoxidase staining on frozen liver tissue; hepatocytes near to
the central vein (cv) disclose strong immunostaining for CAT.
Periportal hepatocytes near to a portal vein (pv) are not
labeled or disclose very weak staining. b, histochemical stain
for CAT activity on frozen liver tissue from the same animal shows a
very closely related pattern of positivity with strong staining of the
pericentrolobular area (central vein (cv)) and no positivity
of the periportal area. c, CAT immunoperoxidase staining on
frozen renal cortex; CAT expression is strictly restricted to proximal
tubular cells. Glomerular tufts (arrow heads) and distal
tubules are negative. d, histochemical stain for CAT on frozen
renal cortex shows the same distribution. Note obvious nuclear
positivity (Glomerular tufts, arrow heads). e, CAT
immunoperoxidase staining on frozen jejunal section; differentiated
enterocytes at margins and top of villi are stained. Germinal
epithelial cells inside the Lieberkühn crypt and
Paneth cells (
) are negative. f, CAT immunofluorescence
staining of frozen transversal jejunal villi; approximately half of the
enterocytes are stained with variable intensity. Intravillous
macrophagic cells disclose spontaneous brown autofluorescence. Bar, 100 µm.
)Paradoxical Response of the Aldolase B/CAT Transgene to
Fasting and Carbohydrate-rich Diets
Fig. 3shows that the
response of endogenous aldolase B mRNA and transgenic CAT mRNA to
fasting and refeeding glucose were practically inverse; while, as
expected (Weber et al., 1984; Munnich et al., 1985),
the abundance of the aldolase B messenger increased when fasted rats
were fed a high carbohydrate diet, the abundance of the transgenic CAT
mRNA decreased under the same conditions. The first hypothesis for
explaining this paradoxical response of the transgene relies on the
absence of some cis-acting element(s) in the transgene and on the
duality of the aldolase B functions in the opposite pathways,
glycolysis, and gluconeogenesis.
10
cpm/ml mouse aldolase B or CAT
probes labeled by random priming. F48 and F24,
animals fasted for 24 and 48 h. G, animals refed a 75%
carbohydrate diet for 24 h. R45, ribosomal 18 S RNA revealed
with 2 10
cpm/ml R45 probe, used as an internal
standard.
))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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