(Received for publication, March 5, 1997, and in revised form, June 3, 1997)
From the Department of Biochemistry, School of Medical Sciences,
University Walk, University of Bristol, Bristol BS8 1TD, United Kingdom
and the Institut Cochin de Génétique
Moléculaire, U. 129 INSERM, Université René
Descartes, 24, rue du Faubourg Saint-Jacques,
75 014 Paris, France
Elevated glucose concentrations stimulate
L-pyruvate kinase (L-PK) gene transcription in liver and islet
-cells. A glucose response element termed the L4 box (two
noncanonical E-boxes located
165 and
154 base pairs upstream of the
transcriptional start point) has previously been defined within the
proximal promoter region of the gene. However, the identity of the
transacting factor(s) which binds to this site remains unclear. We have
used photon counting digital imaging of firefly luciferase activity to
monitor promoter activity continuously in single living islet
and
derived INS-1 cells, and to analyze the molecular basis of the
regulation by glucose. L-PK promoter activity, normalized to
cytomegalovirus promoter activity using the distinct Renilla
reniformis luciferase, was
6-fold higher in cells cultured at
16 mM glucose or above compared with cells cultured at 3 mM glucose. Microinjection of antibodies against the
ubiquitous transcription factor USF2 inhibited L-PK promoter activity
in
- and INS-1 cells incubated at 30 mM glucose by
71-87%. Anti-USF2 antibodies had a much smaller effect on promoter
activity in INS-1 cells cultured at 3 mM glucose, and on
the activity of a modified promoter construct lacking an L4 box. These
data support the view that glucose enhances L-PK gene transcription in
-cells by modifying the transactivational capacity of USF2 bound to
the upstream L4 box.
Increases in extracellular glucose concentration stimulate
transcription of the L-pyruvate kinase
(L-PK)1 gene in liver and
islet -cells (1, 2). This effect is mediated by a
cis-acting DNA sequence termed the L4 box centered 160 base
pairs upstream of the transcriptional start site (3-5) and consisting
of a tandem repeat of non-canonical E-boxes (underlined) as follows:
5
-CACGGGGCACTCCCGTG-3
. This site appears also
to confer glucose responsiveness on the L-PK promoter in islet
-cells (6). Oligonucleotides derived from this sequence bind to
major-late transcription factor or USF (upstream
stimulatory factor), a ubiquitous member of the
basic helix-loop-helix and leucine zipper (LZ) family (7). Members of
this family form dimers through the interaction of helix-loop-helix and
leucine zipper motifs, and then bind to cognate sites on DNA via their
basic regions. Two forms of USF, USF1 (43 kDa) and USF2 (44 kDa), were
first identified in HeLa cells (8, 9) and are encoded by separate genes
(9-12). Furthermore, alternative splice variants of USF2, USF2a, and
USF2b have recently been identified, while heterodimers of USF1 and
USF2a appear to represent the predominant form in vivo (13).
Consistent with a role for USF in the response of the L-PK promoter to
glucose in hepatocytes, the expression of truncated USF proteins
lacking the DNA-binding domain inhibits the stimulation of promoter
activity by glucose, probably by dimerizing non-productively with
endogenous USF (14).
To provide a more specific assay of the role of USF in the
transcriptional activation of the L-PK gene in the -cell type, we
have developed a technique whereby the activity of the L-PK promoter
can be constantly imaged at the single cell level using firefly
luciferase. Simultaneous measurement of the activity of a second
reporter protein, luciferase from the sea flower Renilla reniformis, then allows L-PK promoter activity to be normalized to
the activity of a constitutive viral promoter. We report here that
inhibition of USF2 function with specific antibodies microinjected directly into the nucleus of living clonal
-cells (INS-1)
substantially prevents the induction of L-PK gene transcription by
glucose. This combination of techniques should provide a general means with which to investigate the molecular mechanisms underlying the
control of transcription in living cells.
Beetle luciferin (K+ salt) was obtained from Promega Ltd., United Kingdom, and coelenterazine from Molecular Probes, or was a gift from Dr. B. Sherf (Promega). Bovine serum albumin solutions of predefined density were obtained from Dr. R. F. L. James (Leicester, UK). Collagenase (Pan Plus) was from Serva (Wokingham, UK). Rabbit polyclonal antisera to USF2 was raised as described earlier (13), or was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies were lyophilized and microdialyzed versus 10 mM Tris, pH 8.0, 0.2 mM EDTA, and quantitated by Bradford assay (15) with bovine serum albumin as standard. Plasmid pRL.CMV, containing cDNA encoding Renilla luciferase, was purchased from Promega.
Plasmid ConstructionA fragment of the L-PK gene
corresponding to the region 183 to +10 nucleotides with respect to
the transcriptional start site was subcloned from plasmid 183PKCAT (16)
via flanking KpnI and XhoI sites into plasmid
pGL3.basic (Promega). The resulting plasmid (p.LPK.LucFF)
contained a modified version of the firefly luciferase cDNA gene,
lacking the three amino acid C-terminal peroxisomal targeting sequence,
and engineered for optimal codon usage in mammalian cells (17). Use of
the modified construct provides 10-50-fold greater firefly luciferase
synthesis compared with the unmodified
cDNA.2 A plasmid which
lacked the L4 box containing the putative USF-binding sites
(p
L4-LPK.LucFF) was generated by polymerase chain
reaction amplification of base pairs
148 to +10 from plasmid
183PKCAT, using primers:
5
-TTTTAGATCTGTTCCTGGACTCTGGCCC-3
(BglII
site underlined) and 5
-TTTTAAGCTTGTTGCTTACCTGCTGTGT-3
(HindIII site underlined). Standard molecular biology
cloning procedures were followed (18) and all plasmids were purified on
CsCl gradients.
Rat islets were
isolated from adult males (220-250 g) by in situ
collagenase digestion, followed by purification on a discontinuous bovine serum albumin gradient (19). Islets were dissociated into single
cells by incubation for 2 min, at 20-22 °C in modified, nominally
Ca2+-free Krebs-Ringer-bicarbonate medium (20) containing
trypsin (Type XI, Sigma; 0.5 mg·ml1). Cells (0.5 × 106/ml) were then resuspended in serum-free modified
Eagle's medium (Life Technologies, Inc.) and seeded as a 50-µl
droplet onto plastic Petri dishes (35 mm diameter, Corning) coated with
poly-L-ornithine (0.05 mg·ml
1) and
poly-L-lysine (0.012 mg·ml
1) or Cell-Tak
228 (1:5 dilution; Becton-Dickinson, Bedford, MA). After allowing 5-30
min for cell adherence, culture was continued for 16 h at 37 °C
in 2 ml of modified Eagle's medium plus 10% (v/v) fetal calf serum,
100 IU penicillin and 100 µg of streptomycin·ml
1, in
the presence of 5% CO2.
Prior to microinjection, cells were cultured for 48 h on 24-mm diameter poly-L-lysine-coated coverslips as described previously (21).
Microinjection and Cell ImagingIntranuclear pressure microinjection was performed using an Eppendorf 5172 transjector as described before (20, 22). IgG and anti-USF2 antibodies were always microinjected into cells on adjacent areas in the same coverslip to ensure identical culture and imaging conditions.
Cell imaging was performed at 37 °C using intensified charge-coupled device cameras (Hamamatsu C2400-40; Hamamatsu Photonics, Welwyn, Herts, UK; Photek ICCD216; Photek Ltd., East Sussex, St. Leonards-on-Sea, Sussex, UK) attached to the lower port of a Zeiss Axiovert 100 microscope with 10 × 0.5 NA objective (Hamamatsu) (22) or an Olympus IX-70 microscope with 10 × 0.4 NA objective (Photek). Other details are given in the figure legends.
To assess the dependence upon extracellular glucose concentration of L-PK promoter activity we microinjected cells with a mixture of the pLPK.LucFF construct and a second plasmid (pRL.CMV) providing constitutive expression of R. reniformis luciferase. We chose microinjection over other transfection approaches since this (i) allows a large number (100-1000) of copies of each plasmid to be introduced directly into cell nuclei, providing sufficient synthesis of reporter protein to allow its detection in single cells (22) and (ii) permits the introduction of other molecules such as antibodies at the same time.
Whereas firefly luciferase uses luciferin as cofactor,
Renilla luciferase oxidizes only coelenterazine (23). These
specificities were retained in living cells, with neither enzyme, when
expressed alone, showing any activity except in the presence of its
cognate substrate (data not shown). When -cells were microinjected
with pLPK.LucFF plus pRL.CMV and incubated 16 h at
either 3 or 16 mM glucose, cells maintained at the higher
glucose concentration displayed a 6-fold higher ratio of firefly to
Renilla luciferase activity compared with those at 3 mM glucose (Fig. 1). This
change can be ascribed entirely to enhanced transcription of the L-PK promoter, since any nonspecific alterations in basal transcriptional or
translational efficiency are normalized (with Renilla
luciferase). Furthermore, this effect of glucose was mediated by the L4
box of the L-PK promoter, since glucose was ineffective in altering the
transcriptional activity of a truncated promoter lacking this element
(p
L4-LPK.LucFF; firefly:Renilla luciferase
activity ratio 0.034 ± 0.013, n = 10 cells, and
0.020 ± 0.01, n = 11, after incubation for
24 h at 3 and 16 mM glucose, respectively).
Effects of USF1/2 Inhibition on L-PK Promoter Activity in Primary
To determine whether isoforms of USF may be involved in
the response to glucose we comicroinjected either an antibody (USF-G) raised to the 49-amino acid N-terminal domain of USF2a/b or control IgG. This domain may be involved in transactivation by USF (24). In
three separate experiments, the ratio of firefly:Renilla
luciferase activity was lowered 3 h after injection from 0.63 ± 0.009 × 102 (n = 12 cells) to
0.08 ± 0.0001 × 10
2 (n = 16)
in the presence of the microinjected antibody (86.7% inhibition).
These experiments demonstrated that monitoring glucose-regulated gene
expression with single microinjected -cells was feasible and
implicated USF1/2 in the effects of glucose. To achieve a detailed
analysis of the regulation of the L-PK promoter in a large and
homogeneous cell population we turned to highly differentiated INS-1
-cells (21, 25) whose flattened morphology (Figs. 2 and 3)
facilitated microinjection.
L-PK Promoter Activity Can Be followed in Real Time in Single INS-1 Cells
Using photon counting digital imaging in the presence of luciferin alone it was possible to follow the kinetics of the increase in luciferase luminescence from individual living INS-1 cells immediately after their microinjection with pLPK.LucFF (Fig. 2A). Quantitation of photon release at different times after microinjection demonstrated a biphasic increase in photon production, with the majority of cells displaying a peak in luciferase expression 4-8 h after microinjection (Fig. 2B). This biphasic pattern may reflect a balance between the synthesis of luciferase, and the degradation of the injected plasmid. Supporting this view, the kinetics of expression of Renilla luciferase under constitutive CMV promoter control were broadly similar, reaching a peak 8 h after injection (data not shown). The rate of appearance of either luciferase, and the maximum luminescence achieved, varied considerably between different cells, probably reflecting variations in: (i) the amount of the plasmid introduced, (ii) responsiveness to glucose (pLPK.LucFF only), and (iii) basal transcriptional activity. Subsequent experiments were therefore performed after comicroinjection with plasmid pRL.CMV to allow these intercellular variations to be normalized.
Increases in Extracellular Glucose Concentration Activate the L-PK Promoter Activity in INS-1 CellsIn line with the responses
observed in primary -cells, INS-1 cells microinjected with
pLPK.LucFF plus pRL.CMV, and subsequently incubated for
16 h at 30 mM glucose, exhibited an 11-fold higher ratio of firefly:Renilla luciferase than cells maintained at
3 mM (Fig. 3).
To investigate the role of
USF2 in the effect of glucose on L-PK promoter activity, we used two
antibodies raised against the leucine zipper domain of USF2 proteins.
The antibody termed LZ2 (see Ref. 13, Fig. 2A) was raised
against amino acids 298-346 of human USF2a corresponding to the entire
leucine zipper motif and the antibody termed USF2-SC, developed by
Santa Cruz, was directed against amino acids 327-346 including 2 of
the 4 leucine residues of the leucine repeat of mouse USF2. This latter
antibody also recognizes the analogous epitope in USF1. We analyzed the effects of comicroinjecting these antibodies at the earliest possible time point after microinjection, i.e. 3 h. As shown in
Table I, antibodies LZ2 and USF2-SC
caused a decrease in the ratio of firefly:Renilla luciferase
activity of 85.1 and 70.9%, respectively. Microinjection of these
antibodies had no significant effect on L-PK promoter activity in cells
maintained at 3 mM glucose (Table I). In addition, antibody
USF-G substantially inhibited (74.8%) glucose-dependent L-PK promoter activity. By contrast, an antibody (USF-F) raised to a
central epitope of USF2 (residues 143-193 of USF2a, corresponding to
residues 76-126 of USF2b) was without effect on apparent activity. However, this antibody inhibited by 77.6% the expression of a luciferase construct (pCol.Luc), bearing the fragment 517 to +63 of
the human collagenase promoter (22), which possesses a non-canonical
E-box (
408CAGGTG). No significant change in
firefly:Renilla luciferase activity was observed in INS-1
cells microinjected with LPK.LucFF using an irrelevant
antibody, anti-c-Fos (Table I), nor after microinjection of
anti-USF2 antibodies into the cell cytosol (data not shown).
|
To confirm that the effect of USF2a/b inhibition was the result of
disrupting an interaction between this transcription factor and the L4
box, we examined the effect of the antibody on the expression of
luciferase in cells microinjected with the truncated plasmid
pL4-LPK.LucFF (see above). As observed after
transfection of the analogous CAT construct into liver cells (5), the
L4-LPK promoter in this plasmid was unable to mediate a response to
glucose (Table I). Indeed, in cells microinjected with the construct and incubated at 30 mM glucose, the ratio of
firefly:Renilla luciferase activity was lower than in
identically injected cells maintained at 3 mM glucose
(Table I). In addition, comicroinjection with antibody USF-2 (SC) had a
greatly diminished inhibitory effect (41.9%) on the activity of the L4
box-less promoter at 30 mM glucose compared with the
effects of this antibody on the full-length promoter (85.1%; see Table
I). Similarly, antibodies LZ and G failed to provoke any significant
inhibition in
L4-LPK promoter activity in cells maintained at 30 mM glucose, or a small effect in cells maintained at 3 mM glucose (antibody G).
The
microinjection/imaging technique we have developed here provides a
rapid, non-invasive and highly quantitative assay of gene expression in
single living cells (26). The technology we describe extends our
earlier studies (22), by allowing the easy assay of the activity of two
reporter proteins in the same living cell, simply by switching between
two cofactors (luciferin and coelenterazine). In this way, the activity
of a regulated promoter (L-PK) can be normalized to a promoter which is
constitutively active (CMV). Extremely high sensitivity is achieved due
to the absence of significant background signals and the high
sensitivity of the intensified imaging camera. Typically, firefly
luciferase activities corresponded to 50-500 photon·cell·15
min1 (intact L-PK promoter, 30 mM glucose)
versus a background of about 20 photons (i.e.
signal:noise, S:N, ratio between 2.5:1 and 25:1); Renilla
luciferase activity produced 1000-10,000 photon·cell·15 min, with
a background of 50-100 photons·cell·15 min
1
(S:N = 20-200:1). These S:N ratios are at least 1 order of
magnitude higher than we have been able to achieve using enhanced
mutants of green fluorescent protein (22, 26, 27).
Previous studies have established that an intact L4 box
is crucial for the induction of the L-PK gene in the -cell type (6) as well as in liver cells (5, 28). Whereas a role for USF proteins,
interacting with this site, is implicated in liver cells (29), no data
has been available up to now on the role of this family in regulating
expression in islet
-cells. Here we provide evidence that USF2,
probably as a heterodimer with USF1, is responsible for the activation
by glucose of L-PK gene transcription in
-cells. Thus, in
preliminary experiments, an antibody to the N terminus of USF2a/b
inhibited L-PK promoter activity in primary
-cells. In INS-1 cells,
antibody USF-G and two further antibodies against the leucine zipper
motif (LZ2, USF2-SC) each inhibited L-PK promoter activity. However, an
antibody (USF-F) to the central portion of the protein was without
effect. Although the reason for this difference is unclear, it might be
speculated that the three inhibitory antibodies, but not USF-F, are
able to: (i) disrupt the interaction of USF2 with the L4 box; (ii) to
prevent the transactivation function of the factor; or (iii) to block
an interaction with an ancillary activating protein. Of these, it might
be noted that option (i) would seem the least likely since the
antibodies are able to supershift complexes between USF2 and L4
box-derived oligonucleotides (13).
How might glucose control the transactivational capacity of USF proteins? Since in the liver, glucose has no effect on the ability of USF2 to interact with L4-derived oligonucleotides, nor on the occupancy of the L4 site as determined by in vivo footprinting3 this suggests that a glucose-derived second messenger alters the interaction of L4-bound USF2, or USF1/USF2 dimer, with a binding protein. One possibility is that the activity of this binding protein is modulated by reversible phosphorylation. What might cause this alteration in the phosphorylation state/activity of a USF2-interacting protein? One hypothesis is that glucose acts through the pentose phosphate shunt (29) via xylulose 5-phosphate. An alternative hypothesis suggests that increases in intracellular glucose 6-phosphate are critical (30). Future studies will be required to identify the signals which lead to the activation of USF, and in particular the in vivo binding partners of USF2.
We thank Professor C. B. Wollheim (University of Geneva) for the provision of INS-1 cells.