(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 ()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).
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- 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.
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 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.
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.