From the
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
activity and mRNA levels were significantly reduced in FRTL-5 cells
transformed with the Kirsten-Moloney sarcoma virus (KiMol); these cells
have lost thyrotropin dependence and express high levels of
p21
The effect of v-K-ras could be mimicked in FRTL-5
cells by tetradecanoyl phorbol acetate and reverted in KiMol and Ats
cells, expressing active Ras protein, by increasing intracellular cAMP
and/or by protein kinase C inhibition.
The data are consistent with
the contention that v-K-ras, through protein kinase C and
depletion of intracellular cAMP, is inhibitory for the protein kinase A
pathway. This is the first demonstration that active v-K-ras down-regulates HMG-CoA reductase expression.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
FRTL-5
cells depend on thyrotropin (TSH) and cAMP for growth and induction of
housekeeping genes such as malic enzyme and HMG-CoA
reductase(9, 10) , and thyroid-specific functions such
as I
FRTL-5 cells
transformed with the Kirsten-Moloney sarcoma virus (KiMol), express
active v-K-ras protein and lose TSH-dependence for growth and
cell-specific functions(15, 16) . These effects are
accompanied by cAMP depletion, down-regulation of the thyroid-specific
trans-acting factor TTF-1(17) , and predominance of protein
kinase C(17, 18) .
Since we have identified and
characterized a cAMP-responsive element (CRE) in the reductase
promoter(19) , it was hypothesized that HMG-CoA reductase gene
expression should be down-regulated by active ras. In the
present study, we investigate this hypothesis. To this end, we have
measured reductase activity, mRNA levels, and gene expression in FRTL-5
and KiMol cells and in FRTL-5 cells transformed with a Kirsten-murine
sarcoma virus variant (Ats cells), which expresses a
temperature-sensitive Ras protein, active at 33 °C and inactive at
39 °C(20) .
We show that v-K-ras down-regulates
HMG-CoA reductase gene expression; we also demonstrate that in KiMol
cells CREB binds to reductase CRE and suggest that impairment of
reductase transcriptional activity, in cells expressing active ras, is the result of reduced CREB phosphorylation by protein
kinase A.
These findings strongly suggest that active Ras
protein down-regulates HMG-CoA reductase expression by inhibiting
cAMP-dependent protein kinase A activity, and by preventing protein
kinase A-dependent CREB phosphorylation.
The data presented show that expression of v-K-ras in thyroid cells decreases significantly the level of HMG-CoA
reductase mRNA and quenches enzyme activity. We believe that ras-dependent repression of the reductase gene is exerted at
transcriptional level for two reasons. First, in normal thyroid cells
cAMP increases the rate of reductase gene transcription (10, 19);
second, in this study, we show that reductase promoter activity is
inhibited in cells expressing active v-K-ras, and the
inhibition can be released by cAMP. These data further qualify the
reductase promoter as a cAMP-dependent
promoter(19, 33) , a finding that has not been reported
so far.
The cAMP signaling pathway is severely impaired in thyroid
cells transformed by v-K-ras; indeed, constitutive levels of
cAMP are drastically reduced in KiMol cells, in comparison with the
parent FRTL-5 cells. This results from loss of TSH
receptor(34) , reduced adenylyl cyclase(35) , and
increased cAMP phosphodiesterase activities
Depletion of cAMP caused by v-K-ras in thyroid cells has
also a stimulatory effect on the activity of membrane-associated
protein kinase C; thus, in thyroid cells, and in other cell strains,
protein kinase C activation appears to mediate the effects of ras(36, 40) . The data presented in this study
are consistent with these observations, since protein kinase C
stimulation in FRTL-5 cells, and in Ats cells carrying an inactive
p21
Regulation of HMG-CoA reductase is
most complex. It is exerted at different levels and affected by
different factors(1, 41, 42) . We believe that
the data presented in this study are consistent with the interpretation
that down-regulation by active Ras protein is exerted largely, if not
exclusively, at transcriptional level through depletion of
intracellular cAMP, redistribution and augmentation of protein kinase C
activity, and failure of CREB phosphorylation by inactive protein
kinase A. It is also possible that, in addition, low cAMP may induce
the uptake or reduce the efflux of cholesterol(10, 43) .
We do not know yet what is the role played by HMG-CoA reductase in ras transforming activity; therefore, the interpretation of
the significance of its down-regulation remains speculative. It could
be hypothesized that active ras may turn on a mechanism of
negative feed-back by one of the end-products of the isoprenoid
pathway. Indeed, we have found an increased level of farnesylation of
p21
We thank Prof. V. E. Avvedimento for critical review
of the manuscript and M. Berardone, F. D'Agnello, and F. Moscato
for the art work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. FRTL-5 cells, transformed with a
temperature-sensitive mutant of the v-K-ras oncogene (Ats
cells: 33 °C, permissive; 39 °C, nonpermissive), showed
significant reduction of HMG-CoA reductase expression when exposed to
33 °C. In KiMol cells, as well as in Ats cells at 33 °C, the
transcription driven by cAMP-responsive element was probed by measuring
chloramphenicol acetyl transferase (CAT) levels after transfection with
a chimeric plasmid containing the reporter gene linked to the rat
reductase promoter. Basal CAT activity in KiMol cells transfected with
wild-type promoter was lower than in FRTL-5 cells but was increased by
forskolin to the levels attained in thyrotropin-stimulated FRTL-5
cells. Forskolin failed to increase CAT activity in KiMol cells
transfected with the plasmid harboring a reductase promoter in which
the cAMP-responsive element octamer was mutated to a nonpalindromic
sequence.
(
)reductase is the rate-limiting enzyme in the
biosynthesis of mevalonate, the precursor of several biologically
relevant isoprenoids such as cholesterol, and a number of nonsterol
compounds, most notably farnesyl residues, which are covalently linked
to growth-regulating proteins(1, 2) . Prenylation, a
covalent post-translational modification, allows protein anchorage at
the plasma membrane(2, 3) . Activation of
p21
requires attachment of isoprenoid units
(farnesylation)(4, 5, 6) , whose availability
depends on the activity of HMG-CoA reductase. Ras proteins
(H-Ras, N-Ras, K-Ras4A, and
K-Ras4B) are generally considered as molecular switches in
signal transduction pathways leading to cell proliferation. The
involvement of ras in uncontrolled growth is relevant, since
an increased expression of normal or mutated ras has been
found in 40% of the human cancers(7) , including human
anaplastic and follicular thyroid carcinomas(8) .
transport and thyroglobulin
synthesis(11, 12, 13) . The induction of malic
enzyme and HMG-CoA reductase precedes DNA synthesis and progression of
quiescent FRTL-5 cells into the cell cycle(9, 10) .
Competitive inhibition of HMG-CoA reductase by lovastatin results in
the arrest of cell proliferation(14) .
Materials
TSH was a highly purified preparation
from bovine pituitary extracts(21) .
[C]Acetic acid (53 mCi/mmol) and
[
H]mevalonolactone (35 Ci/mmol) were purchased
from DuPont NEN. Thin-layer chromatographic plates were from E. Merck,
Darmstad, Germany. All other materials were obtained as described in
the accompanying paper(19) .
Cells
FRTL-5 cells (ATCC CRL-8305) are a strain of
rat thyroid cells grown at 37 °C in a humidified atmosphere
composed of 95% air and 5% CO, in Coon's modified
Ham's F-12 medium supplemented with 5% calf serum and a mixture
of six hormones and growth factors (referred to as
6H)(11, 19, 22) . KiMol cells, derived from
FRTL-5 cells upon infection and transformation with a wild-type strain
of KiMSV-MolMuLV (15, 16) and Ats cells, derived from
FRTL-5 cells transformed with a temperature-sensitive mutant (33
°C, permissive; 39 °C, nonpermissive) of Kirsten-murine sarcoma
virus (20), were kindly provided by Dr. G. Vecchio, Naples, Italy.
KiMol cells are grown at 37 °C, and Ats cells were grown at 39
°C, both in Coon's modified Ham's F-12 medium,
supplemented with 5% calf serum. Where indicated, Ats cells were
shifted to 33 °C for different length of times, in different
experiments.
Total RNA Preparation and Slot-Blot Analysis
Total
RNA was isolated by the guanidinium thiocyanate-acid phenol
procedure(23) . Total RNA slot-blots were performed using a
Schleicher & Schuell Minifold II apparatus following the
manufacturer's recommendations. RNA samples were mixed with 3
volumes of a denaturing solution (50% formamide, 2.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate,
0.5 mM EDTA), heated 5 min at 65 °C and diluted 2-fold in
19 SSC(10) . The samples were then applied to a nylon
membrane equilibrated in 10
SSC. Prehybridization (5 h at 42
°C), hybridization (18 h at 42 °C), and high stringency washes
(15 min at 37 °C, 1 h at 60 °C, 1 h at 55 °C) were carried
out as already reported(10) . A XbaI fragment of
HMG-CoA reductase cDNA from plasmid pRED-227 (24) was used as
radiolabeled probe. Quantitative analysis of the autoradiograms was
performed by laser densitometry on a PDI laser scanner. Normalization
was accomplished using radiolabeled glyceraldehyde-phosphate
dehydrogenase cDNA as reference probe.
[
Proliferating (6H) FRTL-5 cells and KiMol cells
were incubated for 2 h with [C]Acetate and
[
H]Mevalonate Incorporation into
Cholesterol
C]acetic acid (53
mCi/mmol) or for 18 h with [
H]mevalonolactone (35
Ci/mmol). At the end of the incubation period, medium was aspirated,
cells were washed with ice-cold Hanks' balanced salt solution,
collected in 3 ml of Hanks' balanced salt solution, and pelleted
by centrifugation for 5 min at 1,000 rpm in a Beckman J-6 centrifuge.
Pellets were suspended in 1 ml of 2-propanol, sonicated at 4 °C
with 5 bursts of 10 s each, and recentrifuged for 15 min at 2,500 rpm
(Beckman J-6 centrifuge). The supernatant was aspirated, and the pellet
was re-extracted as above. Both extracts were combined, dried under a
stream of N
, and dissolved in 0.5-1 ml of chloroform.
Aliquots of 10 µl were applied to Silica Gel-60 thin-layer
chromatographic plates and developed in hexane/diethylether/acetic acid
(70:30:1). Each lipid fraction was located by comparison to high purity
standards, scraped off the plate, and radioassayed(10) .
Cholesterol values were normalized to cell proteins measured on the
2-propanol insoluble pellet, dissolved in 0.1 N KOH.
Nuclear Extracts
Nuclear extracts from
TSH-challenged FRTL-5 cells or KiMol cells were prepared as described
previously (19, 25, 26) prior to use in
electrophoretic mobility shift assays.
Electrophoretic Mobility Shift
Assays
Electrophoretic mobility shift assays were performed as
described previously(19, 27) . Where indicated,
antiserum to CREB or ATF-2 or preimmune serum were added to nuclear
extracts in the same buffer for 20 min on ice before the addition of
the labeled probe and processing as above. Gels were dried and
autoradiographed.
Promoter-Chloramphenicol Acetyltransferase Chimeric
Plasmids
The 765-base pair (bp) PstI fragment (from
-323 to +442) containing the rat HMG-CoA reductase promoter (19) was cloned into the pEMBL-8-CAT vector (19, 28) upstream to the bacterial gene for CAT.
Mutations of the CRE sequence present in the reductase promoter were
created by two-step, recombinant polymerase chain reaction methods, as
described previously(19, 29) . Similar to the wild-type
reductase promoter, the CRE-mutated 765-bp fragment (19) was
cloned into the same vector, 5` to the CAT gene. Luciferase expression
vector (pRSV-Luc) was used as positive control to evaluate transfection
efficiency(30) .
Transient Expression Analysis
Normal and
transformed FRTL-5 cells were grown to 50-60% confluence.
Transfection was by the DEAE-dextran protocol as described
previously(19, 31) . Cells were co-transfected with 20
µg of pEMBL-8-CAT under the control of wild-type or CRE-mutated
765-bp PstI fragment from HMG-CoA reductase promoter and 2
µg of pRSV-Luc/dish and then incubated 48 h before harvesting.
Where indicated, forskolin (10 µM) was added to the cells
during the last 6 h. CAT assays were performed as described
previously(32) . Luciferase activity was measured using a
luminometer.
Other Assays
Protein concentration was determined
using a Bio-Rad kit; recrystallized bovine serum albumin was the
standard.
HMG-CoA Reductase mRNA and Activity
We have
compared reductase mRNA levels and the rates of
[C]acetate incorporation into cholesterol in
normal cells and in KiMol cells, which express constitutively active
Ras protein. In FRTL-5 cells, HMG-CoA reductase mRNA levels are 3-fold
higher than in the virally transformed counterpart. Higher reductase
message yields a higher rate of [
C]acetate
incorporation into cholesterol in normal cells (the relative rates
varying between 15- and 20-fold in different experiments) (Fig. 1). Under the conditions detailed under ``Experimental
Procedures,'' when cells were exposed to
[
C]acetate, the yield of
[
C]cholesterol was 50,000 ± 5,000 cpm/mg
of cell protein in FRTL-5 cells and 3,000 ± 300 cpm/mg of cell
protein in KiMol cells. When cells were incubated with
[
H]mevalonate, the yield of
[
H]cholesterol was 100,000 ± 15,000 cpm/mg
of cell protein in FRTL-5 cells and 20,000 ± 3,000 cpm/mg of
cell protein in KiMol cells. Thus, also the rate of
[
H]mevalonate incorporation into cholesterol was
lower in cells expressing active Ras; this is not surprising since
preliminary data show that also mevalonate kinase is down-regulated by
v-K-ras (data not shown). However, the difference in the
relative rates of acetate versus mevalonate incorporation into
cholesterol were consistent with the magnitude of reductase
down-regulation (3-fold) in cells expressing active ras.
Figure 1:
[C]Acetate
incorporation into cholesterol and HMG-CoA reductase mRNA levels in
FRTL-5 cells and in KiMol cells. Cholesterol values are normalized to
cell proteins measured on 2-propanol insoluble cell pellets after
solubilization in 0.1 N KOH. Total RNAs were extracted as
reported under ``Experimental Procedures,'' brought to
identical concentration based on optical density at 260 nm, denatured
and blotted onto nylon filters as a series of 2-fold dilutions starting
at 5 µg of RNA, and hybridized to
P-labeled HMG-CoA
reductase or glyceraldehyde-phosphate dehydrogenase probes, as
described under ``Experimental Procedures.'' Filters were
exposed to x-ray film and mRNAs quantitated by densitometry. The
densitometric values of reductase mRNA from KiMol cells, after
normalization to glyceraldehyde-phosphate dehydrogenase mRNA values,
were set arbitrarily to 1; all other values are relative to this. Data
are representative of three different determinations in which the
variation was less than 10%. The errorbars indicate
the standard error of three separate experiments performed in
duplicate.
In
FRTL-5 cells transformed with a Kirsten murine sarcoma virus variant
producing a temperature-sensitive mutant Ras protein (Ats), the levels
of reductase mRNA and enzyme activity depend upon the incubation
temperature. Thus, when exposed to 39 °C, the nonpermissive
temperature for ras activation, HMG-CoA reductase mRNA levels,
and [C]acetate incorporation into cholesterol
are comparable with those exhibited by normal FRTL-5 cells (Fig. 2). Shifting the cells to 33 °C caused a time-dependent
fall of both HMG-CoA reductase mRNA levels and rate of cholesterol
synthesis. Reduction was significant after 3 days, and maximal after 7
days (Fig. 2). HMG-CoA reductase activity was also decreased
after 3 days at 33 °C (data not shown). Changing temperature from
33 to 39 °C resulted in recovery of the normal phenotype within 1
day only (Fig. 2), suggesting that the rate of inactivation of
the mutant Ras protein is much faster than its turnover rate. Exposure
of FRTL-5 cells to either 39 or 33 °C had no appreciable effect on
HMG-CoA reductase mRNA and activity (data not shown).
Figure 2:
The effect of exposing FRTL-5 cells
transformed with the temperature sensitive mutant v-K-ras, for
the noted time, at 33 °C or 39 °C, on the incorporation of
[C]acetate into cholesterol and on the levels of
HMG-CoA reductase mRNA, measured and normalized as detailed in the
legend to Fig. 1 and under ``Experimental Procedures.'' The
reductase mRNA level present in Ats cells incubated at 33 °C for 3
days has been given the arbitrary value of 1. The data indicate the
mean of three separate experiments, performed in duplicate, in which
the variation was less than 15%.
The Effects of Protein Kinase A and Protein Kinase C
Modulation
Since TSH and cAMP induce HMG-CoA reductase gene
expression in FRTL-5 cells, we tested whether the down-regulatory
effect of active ras could be reverted by raising
intracellular cAMP. In the presence of forskolin (10 µM),
reductase mRNA levels in Ats cells at 33 °C were increased by
2-fold (Fig. 3). Nearly identical effects were obtained by
treating Ats cells exposed to 33 °C with sphingosine (10
µM) (Fig. 3). The combination of forskolin and
sphingosine did not cause any additive effect (Fig. 3). These
observations strongly suggest that the effects of ras activation on reductase expression are mediated by cAMP depletion
and protein kinase C activation. The involvement of protein kinase C
was confirmed by the finding that tetradecanoyl phorbol acetate (10
ng/ml) mimicked the effect of ras activation in Ats cells at
39 °C (Fig. 3).
Figure 3:
The individual and combined effects of
protein kinase A and protein kinase C activation or inhibition on
HMG-CoA reductase mRNA levels in Ats cells maintained either at 39
°C or shifted from 39 °C to 33 °C for 7 days. Forskolin (10
µM) and sphingosine (10 µM) were added
separately or in combination for 9 h before cells harvesting.
Tetradecanoyl phorbol acetate (TPA) (10 ng/ml) was added 9 h
prior to RNA preparation. HMG-CoA reductase mRNA levels were estimated
and normalized as detailed in the legend to Fig. 1 and under
``Experimental Procedures.'' The upperpart of the figure shows the fluorograph of one of the slot-blots
performed; in the lowerpart is shown the
corresponding densitometric analysis (the errorbars indicate the standard error of three separate experiments; to the
mRNA level of Ats cells incubated at 33 °C has been given the
arbitrary value of 1).
Transcriptional Regulation
We have investigated
cAMP-dependent DNA binding factors in nuclear extracts from KiMol
cells. Fig. 4shows retarded protein-DNA complexes formed with an
oligonucleotide containing the CRE identified in the reductase
promoter. The retardation pattern is identical to the one generated
with FRTL-5 nuclear extracts, as we have characterized in the
accompanying paper(19) . The KiMol protein-DNA complex, like the
one from FRTL-5 cells, was supershifted by antibodies to CREB, whereas
it was not affected by anti-ATF-2 antibodies or preimmune serum (Fig. 4). This observation indicates that CREB, as we have shown
in the accompanying paper(19) , is the nuclear trans-acting
factor that binds the reductase CRE.
Figure 4:
The effect of anti CREB and anti ATF-2
antibodies and of preimmune serum on the mobility of protein-DNA
complexes in EMS assays. End-labeled RED CRE (5`-CGTTCGTGACGTAGGCCG-3`)
was incubated with nuclear extracts as indicated on top of
each lane. Experimental details are under ``Experimental
Procedures.''
Analysis of reductase promoter
activity in normal FRTL-5 cells and in KiMol and Ats cells was
approached by transfecting the different cell strains with a chimeric
plasmid containing the rat HMG-CoA reductase promoter (the 765-bp PstI fragment, from -323 to +442) linked to the CAT
reporter gene. Assays for CAT activity performed in these cells (Fig. 5) showed a significant reduction (3-fold) in KiMol and in
Ats cells exposed to 33 °C, compared with FRTL-5 cells and Ats
cells exposed to 39 °C.
Figure 5:
The effect of 6-h incubation with
forskolin (10 µM) on CAT activity in FRTL-5 cells, in
KiMol cells or Ats cells at 39 and 33 °C. Each cell strain (3
dishes) was co-transfected with 20 µg of a chimeric plasmid
containing either wild-type (wt) or nonpalindromic (np) CRE of HMG-CoA reductase promoter ligated to the 5` end
of the bacterial CAT gene and 2 µg of pRSV-Luc. To the CAT activity
of KiMol cells, transfected with the wild-type reductase promoter and
not exposed to forskolin, is given the arbitrary value of 1, after
normalization with pRSV-Luc for transfection efficiency. The errorbars indicate the standard error of three separate
experiments.
Adenylyl cyclase stimulation gained
through the incubation of the cells with forskolin (10 µM)
for 6 h, was able to restore HMG-CoA reductase promoter activity, in
KiMol cells, to levels comparable with those observed in TSH-stimulated
FRTL-5 cells, in which forskolin failed to cause further increase. In
KiMol cells transfected with a chimeric construct in which the CAT
reporter gene was under the control of the CRE-mutated 765-bp fragment
of the reductase promoter, forskolin failed to increase CAT activity (Fig. 5).
(
)in
cells expressing active ras. Low cAMP levels are inhibitory
for protein kinase A activity; under these conditions, the transfer of
cytoplasmic protein kinase A to the nucleus is hampered(36) .
This has been shown to impair phosphorylation of trans-acting factors
such as CREB and TTF-1(36, 37) which in the
nonphosphorylated state fail to bind and activate their respective
targets. On the contrary, high levels of cAMP and active protein kinase
A inhibit ras pathway as they interfere with Raf-1 activation (38) interrupting the link between the oncogene product
(Ras-Raf) and the mitogen-activated protein kinases cascade (39).
, mimics a v-K-ras like effect on
reductase mRNA. In contrast, protein kinase C inhibition abolishes the
effects of active ras.
in v-K-ras-transformed FRTL-5 cells
in comparison with normal FRTL-5 cells.
(
)This
should be clarified when the role of isoprenoid metabolism in cell
transformation by ras oncogenes will be further elucidated.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.