From the Departments of Biochemistry and
Molecular Biology, College of Medicine, University of South
Florida, Tampa, Florida 33612, § Isis Pharmaceuticals,
Carlsbad, California 92008, and ¶ The Research Service, James
A. Haley Veterans Hospital, Tampa, Florida 33612
Received for publication, July 8, 2002, and in revised form, October 16, 2002
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ABSTRACT |
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Previous studies showed that short term
exposure of cells to high glucose destabilized protein kinase C (PKC)
Protein kinase C (PKC),1
a serine/threonine kinase, comprises a family of 12 isozymes that have
been implicated in signaling pathways affecting cellular processes such
as cell proliferation and differentiation, apoptosis, tumor promotion,
transcriptional activation, and hormone production (1). The PKC
isozymes exhibit differential cellular distribution and substrate
specificity. The conventional PKC isozymes, which are
Ca2+-dependent and activated by phospholipid
and diacylglycerol, include PKCII mRNA, whereas PKC
I mRNA levels remained unaltered.
Because PKC
mRNAs share common sequences other than the PKC
II
exon encoding a different carboxyl terminus, we examined PKC
II
mRNA for a cis-acting region that could confer
glucose-induced destabilization. A
-globin/growth hormone reporter
con struct containing the PKC
II exon was transfected into human
aorta and rat vascular smooth muscle cells (A10) to follow
glucose-induced destabilization. Glucose (25 mM) exposure destabilized PKC
II chimeric mRNA but not control mRNA.
Deletion analysis and electrophoretic mobility shift assays followed by UV cross-linking experiments demonstrated that a region introduced by
inclusion of the
II exon was required to confer destabilization. Although a cis-acting element mapped to 38 nucleotides
within the
II exon was necessary to bestow destabilization, it was
not sufficient by itself to confer complete mRNA destabilization. Yet, in intact cells antisense oligonucleotides complementary to this
region blocked glucose-induced destabilization. These results suggest
that this region must function in context with other sequence elements
created by exon inclusion involved in affecting mRNA stability. In
summary, inclusion of an exon that encodes PKC
II mRNA introduces
a cis-acting region that confers destabilization to the
mRNA in response to glucose.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, PKC
I, PKC
II, and PKC
.
PKC
I and PKC
II are encoded by the same gene, and are translated
from alternatively spliced products of PKC
pre-mRNA. The
inclusion of the PKC
II exon in the 3'-region through alternative
splicing results in the PKC
II mRNA. This pattern of splicing
generates a stop codon at the
II-
I boundary such that the
I
exon, with its coding sequence and 3'-UTR, now becomes the 3'-UTR of
PKC
II mRNA (Fig. 1). As a result,
the PKC
I and PKC
II mRNAs differ only by the sequence of the
included PKC
II exon, and the proteins differ only by their
carboxyl-terminal 50-52 amino acids, respectively (1).
View larger version (11K):
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Fig. 1.
Schematic of alternative splicing of
PKC pre-mRNA. Splicing of the last
common domain C4 to PKC
IV5 domain results in mature PKC
I
mRNA whereas inclusion of the PKC
II-specific exon results in
PKC
II mRNA. A STOP codon is generated when
I exon splices
onto the
II exon to produce mature PKC
II mRNA in which the
I exon serves as an extended 3'-UTR.
We have previously shown that acute hyperglycemia down-regulates
PKCII, but not PKC
I, at the mRNA and protein levels in vascular smooth muscle cells (2). To understand the mechanism by
which elevated glucose down-regulates PKC
II expression in vascular
smooth muscle cells, earlier studies were carried out to determine at
what level suppression of PKC
II expression occurred. These studies
clearly demonstrated that high glucose (10-30 mM), at
levels that commonly occur in hyperglycemia associated with diabetes
mellitus, exerted some regulation at the level of transcription, but
that the most dominant level of regulation occurred
post-transcriptionally through increased destabilization of PKC
II
mRNA via a cytoplasmic nuclease activity (2). PKC
I and other
isozyme-specific PKC mRNAs were not destabilized. The effect of
elevated glucose (25 mM) was independent of osmotic
controls, because mannitol did not down-regulate PKC
II mRNA
expression (2, 3). We have also shown that PKC
I and PKC
II
enzymes have opposite signaling roles in cell division, where PKC
II
signaling suppresses vascular smooth muscle cell proliferation by
attenuating G1/S transition and high glucose treatment,
which down-regulates PKC
II mRNA and protein, and stimulates
vascular smooth muscle cell proliferation (4, 5). High glucose also
suppressed insulin effects on glucose uptake, another
PKC
II-dependent process (3).
The regulation of mRNA stability has emerged as an important mechanism for controlling gene expression. Depending on the system, half-lives of mRNA range from a few minutes to days. The decay rates of many eukaryotic mRNAs are regulated by developmental or environmental stimuli. Most of the mechanisms that control mRNA stability share common features, and determinants of mRNA stability have been shown to reside in the 5'-cap, 5'-UTR, coding region, poly(A)-tail, and the 3'-UTR (or the AU-rich elements) such that each may play some role in regulating mRNA decay rates (6-11).
Although it is now clear that the decreased stability of PKCII
mRNA correlates with cell exposure to high concentrations of
glucose, the molecular mechanism contributing to the regulation of
PKC
II expression by altering its mRNA stability remained to be
defined. Here, using deletion and competition analyses, we identify a
~38-nucleotide sequence within a region of secondary structure in the
alternatively spliced exon that binds factors in vitro in a
manner dependent on the sequence and the availability of factors
present only in the cytosol of glucose-treated cell extracts. The
degree to which protein-RNA complex was formed was dependent on the
presence of the 38-nt region. These data suggest that insertion of the
II exon specifies glucose responsive destabilization of PKC
II
mRNA. Because PKC
II and PKC
I mRNAs differ only by the
inclusion of the PKC
II exon through alternative splicing, we propose
that a cis-acting region introduced by inclusion of the
PKC
II exon defines glucose-mediated mRNA destabilization. To our
knowledge this is the first characterization of an element that is
introduced by alternative splicing that allows for metabolite regulation of stability of a specific mRNA.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- The vascular smooth muscle cell line (A10, ATCC CRL 1476), derived from rat aorta, was grown in Dulbecco's modified Eagle's medium (with 5.5 mM glucose) containing 10% fetal bovine serum, 100 units penicillin G, and 100 µg of streptomycin sulfate/ml, at 37 °C in a humidified 5% CO2, 95% air atmosphere in either 6-well or 100-mm plates. Cells were grown to >90% confluency and medium was changed every 4 days. Cell synchronization was achieved by serum deprivation (0.5% fetal bovine serum) for 48 h as demonstrated previously (12). Primary cultured human aortic smooth muscle cells (Clonetics, San Diego, CA) were grown in smooth muscle growth medium (Clonetics) containing 5.5 mM glucose, 5% fetal bovine serum, 10 ng/ml human recombinant epidermal growth factor, 390 ng/ml dexamethasone, 50 µg/ml gentamicin, and 50 ng/ml amphotericin-B at 37 °C in a humidified 5% CO2, 95% air atmosphere. Cells were grown to >90% confluency and medium was changed every 5 days.
Materials--
The isotopes [-32P]dCTP and
[
-32P]UTP (specific activity 3000 Ci/mmol) were
purchased from ICN Biochemicals. Oligo probe labeling kit
(Prime-a-Gene) was purchased from Promega. Riboscribe T7 RNA probe
synthesis kit and Ampliscribe T7 transcription kit were purchased from
Epicentre Technologies. RNase A, RNase T1, heparin, and RNase
inhibitors were purchased from Sigma.
Lipofectin Transfections--
A10 cells were grown and
synchronized by serum deprivation for 48 h in either 6-well or
100-mm plates. The pG or p
G-PKC
II expression vectors were
transfected in serum-free medium with the Lipofectin-DNA complex.
Lipofectin reagent was purchased from Invitrogen. Following
4 h of incubation, the Lipofectin-DNA complex was washed off with
1× Dulbecco's phosphate-buffered solution and replaced with
fresh medium containing 2% serum. For stably transfected A10 cell
selection, 0.7 mg/ml G-418 was added to the media. It was changed every
4 days, and 10-14 days later the colonies were pooled and maintained
in Dulbecco's modified Eagle's medium (10% fetal bovine serum) with
0.2 mg/ml G-418.
mRNA Half-life Determination--
pG-PKC
II stable
transfectants and p
G stable transfected A10 cells were plated into
100-mm dishes. 50 µg/ml DRB
(5,6-dichloro-1-
-D-ribofuranosylbenzimidazole) dissolved
in 95% ethanol was added to the plates 30 min prior to the treatments
and the 0-h RNA sample was isolated. 25 mM glucose was
added to the glucose-treated plates. RNA samples were isolated from
normal (5.5 mM glucose) and glucose-treated (25 mM glucose) p
G-PKC
II and p
G dishes at 2, 4, 6, 10, and 18 h. In a separate control, an equivalent amount of 95%
ethanol was added.
Isolation of RNA and Northern Blot Analysis--
Total cellular
RNA was isolated from 100-mm plates using Tri-Reagent (Molecular
Research Center, Inc.). RNA samples (10 µg) were prepared in
formamide, formaldehyde, and 1× MOPS, and fractionated on 1.2%
agarose-formaldehyde gels. Ethidium bromide was added in the loading
buffer for visualization and quantitation of 18 S and 28 S RNA. After
fractionation, the integrity and loading of RNA was assessed under UV
light (12-14). The size-fractionated RNA was then capillary
transferred to Hybond membranes (Amersham Biosciences), and
cross-linked to membranes by baking at 80 °C in a vacuum oven for
2 h. Membranes were hybridized overnight at 42 °C with 2 × 107 cpm of the -globin probe (labeled with
[
-32P]dCTP by nick translation as described (16)) per
ml of hybridization buffer. Membranes were washed with high stringency
conditions; label was detected and quantitated using a Amersham
Biosciences PhosphorImaging system.
Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)--
Total RNA was isolated from control or glucose-treated
A10 cells or aortic smooth muscle cells and 2 µg was used to
synthesize first strand cDNA using an oligo(dT) primer and
Superscript II reverse transcriptase (Invitrogen pre-amplification
kit). The upstream sense primer for -globin was
(5'-GCATCTGTCCAGTGAGGAGAA-3') and the downstream antisense primer was
(5'-AACCAGCACGTTGCCCAGGAG-3'). Sense and antisense primers for
-actin (number 5402-3) were obtained from
Clontech. To detect PKC
I and -
II, the
upstream sense primer corresponded to the C4 kinase domain common to
both PKC
I and PKC
II (5'-CGTATATGCGGCCGCGTTGTGGGCCTGAAGGGG-3') and
the downstream antisense primer was specific for PKC
I
(5'-GCATTCTAGTCGACAAGAGTTTGTCAGTGGGAG-3') (16). PCR was
performed using platinum Taq DNA polymerase (from Invitrogen) on 10% of the reverse transcriptase reaction product. Following amplification in a Biometra Trioblock thermocycler
(
-globin: 94 °C, 1 min; 58 °C, 1 min; and 72 °C, 3 min for
30 cycles; PKC
I and -
II: 95 °C, 30 s; 68 °C, 2 min for
35 cycles), 20% of the amplified products were resolved on a 1.2%
agarose gel or 5% of products were resolved on 6% PAGE gels and
detected by silver staining. The PCR reaction was optimized for linear
range amplification to allow for quantification of products.
cDNA Probe Preparation--
The pRSV-G vector (obtained
from Dr. Norman C. Curthoys, Colorado State University) was digested
with HindIII and BglII to obtain a 507-bp
fragment, which was isolated from agarose gel using the QIAquick gel
extraction kit (Qiagen). The
-globin probe was labeled with
[
-32P]dCTP using Prime-a-Gene (Promega) to a specific
activity of 109 disintegrations/min.
Synthesis of pG-PKC
II Chimeric Minigene--
The
parent vector p
G contains the strong viral promoter derived from the
long terminal repeat of the Rous sarcoma virus followed by the
transcriptional start site, the 5'-nontranslated region, the entire
coding sequence, and two introns from the rabbit
-globin gene, a
multicloning site containing four unique restriction sites, and the
3'-nontranslated region and polyadenylation site of the bovine growth
hormone (17). It carries neomycin and ampicillin resistance. The 404-bp
PKC
II product corresponding to the 216-bp
II exon and
flanking regions was obtained by PCR amplification using sense primer
to the upstream PKC
common C4 domain containing the
SpeI site (5'-CGTATATACTAGTGTTGTGGGCCTGAAGGGGAACG-3') and antisense primer to the
IV5 domain containing the XbaI
site (5'-TGCCTGGTGAACTCTTTGTCGAGAAGCTCT-3') such that the
exon-included PKC
II mRNA was amplified. The insert contained 70 bp of the C4 exon and 118 bp of the
I exon common to both PKC
I
and PKC
II in addition to the 216-bp exon specific for PKC
II.
After size fractionation it was extracted from the gel (Qiagen QIAquick
gel extraction kit), digested with SpeI and XbaI,
and purified. The PKC
II cDNA was ligated into the
SpeI and XbaI sites of the multicloning region of
p
G vector. The construct, p
G-PKC
II, was verified by
restriction mapping and dideoxynucleotide sequencing.
Using PCR primers for C4 (last common domain) as sense primer
with the SpeI site synthesized upstream
(5'-CGTATATACTAGTGTTGTGGGCCTGAAGGGGAACG-3') and for II exon
antisense primer with the XbaI site synthesized downstream
(5'-CGGAGGTCTACACATCTACTTTCTAGAAGCTCT-3'), PKC
II exon without
the -
I exon (286 bp product) was amplified. This insert contained 70 bp corresponding to the C4 exon and the entire PKC
II exon. The
product was gel purified and ligated into the SpeI and XbaI sites of the multicloning region of p
G vector. The
construct, p
G-PKC
II
I, was verified by restriction mapping.
The region of instability comprising 38 nucleotides was synthesized
with the SpeI and XbaI restriction enzyme
sites to facilitate cloning into the pG vector:
5'-AACTCTACTAGTGAATTTTTAAAACCCGAAGTCAAGAGCTCTAGATAGTA-3'. The
construct, p
G-PKC
II38, was verified by restriction mapping.
As a control, the analogous carboxyl-terminal region of PKC
(C4-V5/
) was digested using BstXI and XbaI
(450 bp), purified, and ligated into the multicloning region of the
G vector. The construct, p
G-PKC
, was verified by restriction mapping.
In Vitro Transcription Vectors--
The 404-bp PKCII
product corresponding to the 216-bp
II exon and flanking
regions described above was obtained by PCR amplification using
sense primer to the upstream PKC
common C4 domain
(5'-CGTATATGCGGCCGCGTTGTGGGCCTGAAGGGG-3') and antisense primer to
I
exon (5'-GCATTCTAGTCGACAAGAGTTTGTCAGTGGGAG-3') such that the
exon-included PKC
II mRNA was amplified. This PKC
II cDNA
piece was cloned into the pCR-Blunt vector (Invitrogen) such that
transcripts could be generated from the upstream T7 RNA
polymerase promoter.
In Vitro Transcript Preparation--
The RNA probes were
generated by consecutive restriction digestion of the
pCR-Blunt-PKCII vector. Riboprobe A (RpA) was the full-length
PKC
II insert described above, linearized with BamHI; riboprobe B (RpB) was the PKC
II insert linearized at 175 bp with BglII within the PKC
II exon such that the
PKC
I-specific exon was eliminated; riboprobe C (RpC) was linearized
at 137 bp with HpaI, which cut within the PKC
II-specific
exon; riboprobe D (RpD) was linearized at 102 bp with SspI,
which cut within the PKC
II-specific exon. After digestion, the
riboprobes were purified and their sizes and linearity were confirmed
following size fractionation on agarose gels. One µg of each
linearized plasmid DNA was further used for in vitro
transcription with the Ampliscribe kit (Epicentre) for competitor
unlabeled probes or with Riboscribe kit (Epicentre) for transcribing
labeled RNA probes using T7 RNA polymerase at 37 °C for 2 h in
the presence of nucleotides, RNase inhibitor, and buffer according to
the manufacturer's instructions. The nonspecific unlabeled probes were
from analogous fragments of PKC
(described above), PKC
I (15), and
-actin, and were prepared in a similar manner. For labeled RNA
probes, 5 µl of [
-32P]CTP (3000 Ci/mmol) was used in
the reaction and 1 µl of RNase-free DNase I was then added and
incubated for 15 min at 37 °C to remove the template DNA. The
transcripts were precipitated using 5 M ammonium acetate
and incubated for 15 min on ice followed by a 70% ethanol wash. The
pellet was resuspended in RNase-free water. The integrity of RNA probes
was confirmed and probes were purified by 6% native polyacrylamide gel electrophoresis.
Cytoplasmic Extract (S100) Preparation--
In brief, A10
cells incubated in 5.5 mM glucose (low) or 25 mM glucose (high glucose) for 4 h were washed with 1×
phosphate-buffered saline, gently scraped, collected, and centrifuged
at 1850 × g for 10 min. The packed cells were
re-suspended in hypotonic buffer (10 mM HEPES, pH 7.9, at
4 °C, 1.5 mM MgCl2, 10 mM KCl,
0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol, 10 µM leupeptin, 10 µM
antipain), allowed to swell for 10 min on ice, and homogenized in a
Dounce homogenizer with 60 strokes using type B pestle. The nuclei were
pelleted by centrifuging at 3300 × g for 15 min, and the cytoplasmic extract was mixed with 0.11 volume of cytoplasmic buffer (0.3 M HEPES, pH 7.9 at 4 °C, 1.4 M
KCl, 0.03 M MgCl2). After centrifugation for
1 h in a Beckman type 50 rotor at 40,000 rpm, the supernatant
(S100) was aliquoted, frozen in liquid nitrogen, and stored at
80 °C. An aliquot was used to determine the protein concentrations
using the Bradford protein assay (18).
RNA Electrophoretic Mobility Shift Assay-- S100 extracts from A10 cells exposed to low (5.5 mM) glucose and high (25 mM) glucose concentrations containing 3 µg of protein were incubated with 3 µg of yeast tRNA and 10 units of RNase inhibitor in a final volume of 10 µl of RNA shift buffer (12 mM HEPES, pH 7.9, 10 mM KCl, 10% glycerol, 5 mM EDTA, 5 mM dithiothreitol, 5 mM MgCl2) for 10 min at room temperature. 100-300-Fold excess specific cold competitors or 100-fold excess nonspecific cold competitors quantified spectrophotometrically using a Pharmacia Gene Quant were added to the binding reactions and incubated for 5 min at room temperature. Labeled RNA probes RpA, RpB, RpC, or RpD (described above) to an activity of ~1 × 106 cpm were added and incubated for 20 min at room temperature. RNase T1 was added to the binding reaction to digest the unbound RNA. Because the cytoplasmic extracts may contain proteins that bind to negatively charged polyanions, like nucleic acids, heparin was added to the reaction to suppress nonspecific binding (19, 20). 100 Units of RNase T1 was added and further incubated for 15 min at room temperature, followed by the addition of 5 mg/ml heparin to reduce nonspecific binding, for 10 min on ice. Samples were separated on a 10% polyacrylamide gel in 0.5× TBE buffer. Gels were dried and exposed to Amersham Biosciences PhosphorImaging screen.
UV Cross-linking of RNA-Protein Complexes--
RNA-protein
binding reactions were carried out as described above for the RNA
electrophoretic mobility shift assay. To demonstrate specificity, the
38-nucleotide sequence (50 nM) described above, or 50 nM of a 20-nucleotide antisense
2'-O-(2-methoxy)ethyl (MOE) oligonucleotide (AS 25647)
targeting a portion of the 38-nucleotide region,
5'-CTTGACTTCGGGTTTTAAAA-3', were added to binding reactions. As a
control, another 20-nucleotide antisense MOE oligonucleotide (AS
25649), 5'-GAAGTTGGAGGTGTCTCGCT-3', upstream of this region or a
scrambled 20-mer MOE oligonucleotide was used. Following heparin
addition, the samples were transferred to a 96-well plate, and
irradiated for 10 min in Stratalinker (Stratagene) on ice. Laemmli's
buffer was added to the sample, which was then boiled for 5 min and
separated on a 10% SDS-polyacrylamide gel. Gels were dried and exposed
to Amersham Biosciences PhosphorImaging screens. To control for
nonspecific protein/RNA interactions, 1 µg of micrococcal nuclease
(Sigma) was added to control and glucose-treated cell extracts with 70 mM EDTA in addition to the labeled probe. There was no
difference in the UV cross-linking in the presence of micrococcal
nuclease to either control or high glucose-treated cytosolic extracts.
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RESULTS |
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Effects of the PKCII Exon Insertion on the Stability of
-Globin Reporter mRNA--
Previous experiments had suggested
that insertion of the 3'-PKC
II exon via alternative splicing
introduced a glucose-responsive instability region that was not present
in the PKC
I mRNA (2). To test this, a corresponding region
consisting of 70 nucleotides in the last domain common to both PKC
I
and PKC
II (C4), the PKC
II-specific exon (V5) (216 nucleotides),
and 119 nucleotides of the flanking
I exon were recloned into an
expression vector (p
G) to form a vector expressing
-globin-PKC
II mRNA (see Fig. 2,
a and b). The resulting chimeric vector,
p
G-PKC
II was under the regulation of a viral promoter and
contained a polyadenylation sequence, eliminating regulatory effects on
transcription and nuclear decay. The p
G-PKC
II vector was
transiently transfected into A10 cells, a rat clonal vascular smooth
muscle cell line, and also into human aorta smooth muscle cells. Aorta
smooth muscle cells respond to glucose-induced destabilization of
PKC
II mRNA in an analogous manner to A10 cells and provide a
primary cell model for corroborating glucose effects in human cells
(2). As demonstrated by RT-PCR analysis (Fig. 2c), acute
exposure (2 h) to high glucose (25 mM) resulted in a
decrease (>80%) in the chimeric
-globin-PKC
II mRNA, when
compared with the same cells exposed to control levels of low glucose
(5.5 mM). In A10 cells transfected with the parent vector,
p
G,
-globin mRNA levels remained unaltered after exposure to
high glucose concentrations. Levels of
-actin mRNA remained unchanged in low glucose and high glucose-treated p
G-PKC
II and p
G-transfected cells.
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To further rule out the possibility of nonspecific effects caused
by inserting the fragment of PKCII cDNA into the p
G vector, an analogous fragment corresponding to the exon for the COOH-terminal domain of PKC
(C4-V5/
) was subcloned into the parent p
G vector creating a chimeric reporter expressing
-globin-PKC
mRNA
(Fig. 3a). This chimeric
plasmid was transiently transfected into A10 cells. As shown by RT-PCR
analysis (Fig. 3b), exposure of cells to high glucose had no
effect on the stability of
-globin-PKC
mRNA.
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The Effect of High Glucose on the Half-life of -Globin-PKC
II
mRNA--
Half-life determinations of
-globin-PKC
II mRNA
were carried out in the presence of the transcriptional inhibitor,
DRB. DRB was used rather than actinomycin D, because actinomycin
D, commonly used to inhibit RNA polymerase II, has also been reported
to inhibit translation (21), and in some instances to also inhibit
mRNA degradation (22, 23). Stable A10 cell transfectants of
p
G-PKC
II or p
G were pretreated with DRB, and then incubated
with low (5.5 mM) or high (25 mM) glucose for
various times up to 6 h. Northern blot analysis of total RNA
showed that only 20% of the
-globin-PKC
II mRNA remained
after exposure to high (25 mM) glucose concentrations, compared with
-globin-PKC
II mRNA in the presence of control (5.5 mM) glucose concentrations within 2 h (Fig.
4). This is in agreement with the results
presented in Fig. 2. High glucose concentrations reduced the half-life
of the
-globin-PKC
II mRNA to 45 min, effectively decreasing
the amount of the mRNA.
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The Effect of Glucose-induced Destabilization by the II Exon in
the Absence of the
I Exon--
Inclusion of the PKC
II-specific
exon in many cell types results in mature PKC
II mRNA in which
PKC
I exon functions as an extended 3'-UTR (Fig. 1). Exon inclusion
produces new C4-
II and
II-
I exon junctions in the PKC
II
mRNA. Hence, to examine the influence of glucose on mRNA
stability that may be mediated by insertion of the
II exon, a new
construct without the 3'-
I exon was examined. The
-globin-PKC
II
I vector was transiently transfected into A10
cells, and cells were exposed to high or low glucose (control). As
shown (Fig. 5), high glucose also
destabilized the
-globin-PKC
II
I mRNA. The extent of
destabilization was less than that observed for the
-globin-PKC
II
chimeric mRNA shown in Fig. 2c. Approximately 50% of
the
-globin-PKC
II
I mRNA was degraded compared with
>80% degradation of the
-globin-PKC
II chimeric mRNA in
cells treated with high glucose. These results suggested that inclusion
of the
II exon was necessary to confer full glucose-sensitive
mRNA degradation, but the C4-
II exon junction alone was not
sufficient to produce the same level of glucose-induced destabilization
as observed for the complete 3' PKC
II mRNA sequence, including
both the C4-
II and
II-
I exon junctions as shown in Fig.
2c.
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Cytoplasmic Extracts from A10 Cells Treated with High Glucose
Concentrations Specifically Retard a Labeled PKCII Exon
Transcript--
Initial studies demonstrated that a nuclease activity
present in the cytosolic extracts from glucose-treated cells mediated PKC
II mRNA destabilization (2). To further investigate regions important for destabilization within the PKC
II exon, cytoplasmic extracts from A10 cells were examined for transacting components that
specifically interact with a glucose-sensitive element within the
II
exon using electrophoretic mobility shift assays. As shown in Fig.
6a, only the extract from A10
cells exposed to high glucose retained the labeled probe, suggesting a
large protein-RNA complex was forming in these extracts. No complex
formation was observed with the control (5.5 mM glucose)
cytoplasmic extracts. To further demonstrate specificity of the
complex, excess cold competitor RNA (corresponding unlabeled
transcript) was added to the incubation and shown to eliminate binding
whereas a nonspecific RNA competitor (an analogous region of PKC
)
did not (Fig. 6a). The bound probe observed here may
represent cooperative assembly of large complexes as a result of
protein-RNA interactions on this element. It is also noteworthy that
the complexes are observed only in glucose-treated cytosolic
extracts.
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To identify the boundaries of sequence contributed by the PKCII exon
required for retention of probe, deletion constructs were generated as
shown in Fig. 6e. Probes RpB (BglII digestion), RpC (HpaI digestion), and RpD (SspI digestion)
were transcribed and analyzed with cytoplasmic extracts from control
(5.5 mM glucose) and glucose (25 mM)-treated
cells using RNA gel shift analysis. Excess unlabeled RNA competitors
and nonspecific competitors were added for each transcript as shown in
Fig. 6b. Shifted material was observed for transcript RpB,
where the restriction deletion removed all the sequence(s) encoding the
PKC
I-specific exon. Complexes were also noted as with RpA, but the
intensity of the interaction was diminished with RpB. No complexes were
observed using transcripts RpC or RpD, both transcripts resulting from deletion of coding sequence within the PKC
II-specific exon (Fig. 6,
c and d). From the deletion analysis, a region of
38 nucleotides positioned between the HpaI and
BglII sites, within the PKC
II exon, was shown to form
complexes with cytosolic extracts from glucose-treated cells. To
further confirm that this area was responsible for the shift, excess
cold RpC and cold PKC
I (C4-
I) probe were added to the labeled RpB
reaction but failed to compete the shift (data not shown).
-Globin-PKC
II-38 Chimeric Vector Lacks Glucose
Sensitivity--
To determine whether this 38-nucleotide region could,
by itself, confer glucose-sensitive instability when taken out of the region of secondary structure provided by exon inclusion, it was cloned
into the p
G vector reporter system. The new construct encoding a
-globin-PKC
II-38 nucleotide region was transfected into A10
cells. As shown in Fig. 7, high glucose
failed to destabilize
-globin-PKC
II-38 mRNA suggesting that
the region of 38 nucleotides was not sufficient by itself to elicit the
glucose response.
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MOE Antisense Oligonucleotides Block Glucose-induced
Destabilization of PKCII mRNA--
Because of the limitations
of the
-globin system to map glucose-responsive elements within the
PKC
II mRNA, a third approach was taken to further demonstrate
that this 38-nt region was associated with destabilization. Antisense
(AS) MOE oligonucleotides (20-mers) were designed to "walk"
complementary to this putative 38-nt PKC
II mRNA instability
region. MOE modifications are resistant to exo- and endonuclease
degradation and do not support cleavage of hybridized mRNA by RNase
H. Furthermore, these oligonucleotides bind with high affinity to the
complementary mRNA sequences (24). AS oligonucleotides 25646 and
25648 spanned consecutive regions while AS 25647 overlapped these
sequences (Fig. 8a). AS 25645 and AS 25649 corresponding to upstream (not shown) and downstream
sequences, served as controls because they are outside the 38-nt
region. To evaluate specific binding and targeting of the antisense, as
a separate control, a scrambled sequence (AS 25581) was used. A10 cells
were transfected with the AS oligonucleotides (50 nM) and
then exposed to 25 or 5.5 mM glucose. As shown in Fig.
8b, AS 25647 blocked high glucose-induced destabilization.
AS 25646 and AS 25648 blocked destabilization to a lesser extent. The
downstream AS 25649 did not block glucose effects. Scrambled
control sequences did not block destabilization (data not shown). This
approach further mapped the relevant element to 20 nucleotides within
this 38-nt region and suggested that the sequence was important for
protein interaction.
|
UV Cross-linking Detected Association of Proteins--
To provide
insight into the basis for the mobility shift and nature of the
components that bind to the PKCII mRNA in response to high
glucose exposure, UV cross-linking experiments were performed using
transcripts RpA and RpB, which demonstrated specific binding by
mobility shift assays. Binding assays were carried out in parallel with
the RNA shift analyses, but were further subjected to UV light to
generate covalent bonds between the 32P-labeled RNA
transcript and associated proteins, digested with RNase A, and
separated by SDS-polyacrylamide gel electrophoresis. Under these
conditions a single band was observed at 10-14 kDa for the control
extracts (Fig. 9), and extracts from high
glucose-treated cells showed a 5-fold increase in intensity over that
observed for extracts from control cells exposed to low glucose. Longer exposure times did not elucidate any other bands. An excess
38-nucleotide sequence, corresponding to the
HpaI-BglII region identified in mobility shift
assays, competed for protein binding with RpB (Fig. 9). Unlabeled
probes RpA and RpB competed for protein binding, respectively, with
labeled RpA and RpB, but unlabeled RpC, RpD, and PKC
I probes did not
compete for protein binding following UV cross-linking (data not
shown). To control for possible nonspecific protein interactions,
micrococcal nuclease (1 µg) was added to cell extracts in the
presence of excess EGTA. The affinity of this exogenous protein binding
to labeled RpB in control and high glucose-treated cell extracts
remained unaffected (data not shown). Addition of proteinase K to the
reactions abolished complex formation (data not shown). These results
were taken to demonstrate that cell extracts from A10 cells contain a
small molecular weight protein (10,000-14,000) that
specifically binds to a PKC
II mRNA region containing the
38-nucleotide HpaI-BglII sequence, and that the
efficiency of binding, as measured by UV cross-linking, increases in
response to high glucose.
|
Antisense Corresponding to a Portion of the
HpaI-BglII Region Blocked Protein Interaction and
Glucose-induced Destabilization--
To demonstrate whether the protein
binding to the mRNA was involved in its destabilization, antisense
MOE oligonucleotide (Fig. 8) complementary to a portion of the
HpaI-BglII region and shown to block
destabilization, was used as a competitor in the UV cross-linking
experiments. The 20-mer, AS 25647, was shown to compete for protein
binding with RpB (Fig. 9). Another antisense oligonucleotide upstream
of this region, AS 25649, did not block the protein interaction with
RpB or glucose-induced destabilization. Hence, the association of a low
molecular weight protein with a specific sequence in the PKCII
carboxyl terminus exon was required for glucose-induced destabilization
of the mRNA.
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DISCUSSION |
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The previously reported observation that only PKCII and
not PKC
I was subject to glucose-induced mRNA destabilization by a cytoplasmic nuclease activity suggested that the sequence within the
PKC
II exon was responsible. The results of this investigation indicate that when this region is inserted into a
-globin reporter gene, glucose-dependent instability is introduced and the
mRNA half-life is markedly reduced.
Deletion analysis of the PKCII exon monitored by RNA electrophoretic
mobility assay showed that a 38-nt region near the middle of the exon
was required for this interaction. Furthermore, the addition of an
RNase H-resistant antisense MOE oligonucleotide that targeted 20 nt
within the 38-nt region abolished the interaction. However, when
inserted into the
-globin reporter gene, the 38-nt region by itself
was not sufficient to confer glucose-dependent destabilization, indicating that other sequences within the PKC
II exon are necessary. In fact, the destabilization was maximal when a
portion of the PKC
I exon was present in addition to the PKC
II exon. This difference highlights the importance of the context in which
the
II exon is placed and may reflect differences in factors such as
translation rate, secondary structure, and intracellular localization,
which may also influence mRNA stability (25). Computer modeling of
the RNA sequence encoded by the C4-
II-specific exon indicates that
the 38-nt region would likely form a stem-loop structure. The
complementary antisense that spanned an AU-rich element and the
sequence at the base of a putative stem-loop structure blocked
glucose-induced destabilization of PKC
II mRNA as well as protein
interaction. This possible secondary structure may, in part, explain
some structural features of the context necessary for the
cis-acting element to function.
The antisense oligonucleotide that blocked destabilization also
targeted an AU motif. One group of cis elements that may
mediate mRNA instability are the AU-rich elements (AREs or AUREs)
(see Ref. 25 for review). These elements have been associated
with AU-rich element-binding proteins. Analysis of the PKCII
complete mRNA indicates 46% AU content. However, the RpB probe
containing the PKC
II exon contains 58% AU and the 38-nt fragment
contains 66% AU. Furthermore, the 38-nt fragment contains an extended
pentamer sequence AUUUUUA that has been identified as a putative
AU-rich element-binding protein target. It has also been identified as conferring mRNA instability in the epidermal growth factor receptor transcript (26). Other U-rich sequences identified by Levine et
al. (27) as possible AU-rich element-binding protein targets are
also present in the PKC
II exon. It is interesting to note that the
c-fos mRNA contains two domains: an AUUUA pentamer
region and a U-rich region (28). Both regions appear to be necessary for maximal RNA destabilization. In the case of PKC
II, however, these elements occur in the coding region and not the 3'-UTR. They are
also introduced in a regulated fashion via exon inclusion.
Our results support the proposal that the PKCII exon, introduced by
hormone-regulated alternative splicing of a common pre-mRNA (16),
not only specifies a different 52-amino acid carboxyl-terminal end in
the protein, but also confers glucose responsive instability to the
PKC
II mRNA in vascular smooth muscle cells. High glucose concentrations increased the levels of a low molecular weight protein
that binds to this region and may target the mRNA molecule for
degradation. To our knowledge, this is the first report that defines an
mRNA instability element present within an exon that responds to
regulation by an external stimulus, acute high glucose concentrations
in a mammalian cell. The scheme shown in Fig.
10 illustrates how hormone-regulated
alternative splicing inserts a region into the mRNA that under
conditions of high glucose concentrations, results in the
destabilization of the mRNA encoding an important regulatory
protein.
|
Taken together with previous reports by this laboratory showing that
the alternative splicing of PKC mRNA is regulated by insulin (5,
16, 29), this study highlights the possible integration of metabolic
regulation mediated between nutrient (glucose) and endocrine (insulin)
controls on vascular smooth muscle cell gene expression. Significantly,
it is known that hyperglycemia can further increase smooth muscle cell
proliferation that may contribute to the development of atherosclerotic
lesions in diabetic subjects (30). In view of the results presented
here, hyperglycemic episodes could result in the rapid destabilization
of PKC
II mRNA. As a consequence, destabilization would remove
PKC
II signaling that has been shown previously to repress smooth
muscle cell proliferation (31-33), and may therefore help explain the
contribution of acute hyperglycemic incidences to the increased risk of
vascular disease in diabetes.
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ACKNOWLEDGEMENTS |
---|
The -globin construct was provided by Dr.
Norman Curthoys, Colorado State University. We thank
Christopher Esposito, Daniel Mancu, Hercules S. Apostolatos, Suresha
Rajaguru, and Laura Miller for excellent technical assistance, and Dr.
Huntington Potter, Department of Biochemical and Molecular Biology,
University of South Florida, for helpful discussions and critical
reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by American Heart Association, Florida Affiliate Research, Grant-in-aid 0050991B (to D. R. C.), Merit Review funds from the Medical Research Service of the Veterans Administration (to D. R. C.), and National Institutes of Health DK54393 (to D. R. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: J. A. Haley
Veterans Hospital (VAR 151), 13000 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-972-2000 (ext. 7017); Fax: 813-972-7623; E-mail: dcooper@hsc.usf.edu.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M206797200
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ABBREVIATIONS |
---|
The abbreviations used are:
PKC, protein kinase
C;
UTR, untranslated region;
nt, nucleotide;
DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole;
MOPS, 4-morpholinepropanesulfonic acid;
RT, reverse transcriptase;
MOE, 2'-O-(2-methoxy)ethyl;
RpA, riboprobe A;
AS, antisense.
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