From the
The role of protein kinase C (PKC) in the regulation of the
Catecholamines play a central role in the regulation of lipid
metabolism in adipose cells. Through activation of
Catecholamine
responsiveness of adipocytes can be controlled through several
mechanisms, including the modulation of
Recently, we demonstrated that insulin specifically
inhibits
The aim of the present study was to determine
whether prolonged activation of PKC by phorbol esters modulates
Adenylyl cyclase activity (EC 4.6.1.1) was measured as described
(6) . Unless when tested in the presence of GTP
Binding assays were carried out as previously mentioned
(12) . Saturation experiments were performed with
(-)-[
Data
from saturation and competition binding experiments were analyzed with
the EBDA and LIGAND programs (Biosoft-Elsevier, Cambridge, United
Kingdom).
For RT-PCR
analysis of
For
quantitation, autoradiograms of the Northern blots or ethidium
bromide-stained gels of PCR products were analyzed by video
densitometric scanning (Vilber Lourmat Imaging).
In our study, several observations support the proposed role
of PKC in the mediation of the phorbol ester-induced decrease in
Earlier studies have
reported that in several cell types, phorbol esters cause an
attenuation of
Transcriptional
and/or post-transcriptional mechanisms are involved in the control of
gene expression by PKC. In our study, the absence of any significant
effect of PMA on
We have previously
reported
(13) that insulin can down-regulate
In summary, the results of the
present study indicates that sustained PKC stimulation specifically
down-regulates
Membranes were obtained from 3T3-F442A adipocytes treated or
not with PMA (300 nM) for 12 h. Adenylyl cyclase activity in
response to an optimal concentration (100 µM) of each
indicated effector was determined. Results are expressed as
effector-stimulated over basal adenylyl cyclase activity and
represented the mean ± S.E. of 4-13 independent
experiments. Basal adenylyl cyclase activity was 12.7 ± 1.0 and
14.6 ± 1.0 pmol of cAMP/min/mg of protein in control and
PMA-treated cells, respectively.
Membranes from
control and PMA-treated (300 nM for 12 h) 3T3-F442A adipocytes
were tested in (-)-[
Membranes were prepared from control and PMA-exposed (300
nM for 12 h) 3T3-F442A adipocytes. Competition binding
experiments were performed at 250 pM (-)-[
We thank J. L. Guillaume, Dr. D. Lacasa, Dr. S.
Cazaubon, Dr. S. Marullo, and Dr. B. Manning for helpful discussions
and for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic receptor (
-AR) gene was
examined in murine 3T3-F442A adipocytes, which express this receptor
subtype at a high level. We also investigated the involvement of this
kinase in the modulation of
-AR gene expression by
insulin. Long term exposure of 3T3-F442A adipocytes to phorbol
12-myristate 13-acetate (PMA) decreased
-AR mRNA
content in a time- and concentration-dependent manner, with maximal
changes observed at 6 h (6.5-fold decrease) and at 100 nM PMA.
This inhibition was selective for
-AR transcripts,
since
- and
-AR mRNA content remained
unchanged. Also, (-)-[
I]cyanopindolol
saturation and competition binding experiments on adipocyte membranes
indicated that PMA induced an
2-fold decrease in
-AR expression, while that of the two other subtypes
was not affected. This correlated with a lower efficacy of
-AR agonists to stimulate adenylyl cyclase.
Conversely, long term exposure to PMA did not alter adenylyl cyclase
activity in response to guanosine 5`- O-(3-thiotriphosphate) or
forskolin. The inactive phorbol ester 4
-phorbol 12,13-didecanoate
did not repress
-AR mRNA levels. Inhibition of
-AR mRNA by PMA was suppressed by the PKC-selective
inhibitor bisindolylmaleimide, and was not observed in PKC-depleted
cells, indicating that PKC was involved in this response. mRNA turnover
experiments showed that the half-life of
-AR
transcripts was not affected by long term PMA exposure. When 3T3-F442A
adipocytes were pretreated with PMA for 24 h to down-regulate PKC, or
with bisindolylmaleimide, the insulin-induced inhibition of
-AR mRNA levels was reduced by 44-67%. These
findings demonstrate that sustained PKC activation exerts a specific
control of
-AR gene expression and is involved, at
least in part, in the modulation by insulin of this adrenergic receptor
subtype.
-ARs,
(
)
they modulate cAMP-dependent
processes, such as lipolysis, and the genetic control of the lipogenic
or thermogenic pathways. The
-AR, whose gene and cDNA
have been characterized in human
(1) and rodents
(2, 3, 4) , appears to be the main functional
subtype in rodent adipocytes
(5) . The murine 3T3-F442A cell
line, which undergoes adipose conversion in vitro, presents a
coordinated expression of the three
-AR subtypes. Overall, the
-AR subtype, which is absent in 3T3 preadipocytes,
represents more than 90% of total
-ARs in mature adipocytes
(6) . The 3T3-F442A cell line thus constitutes an appropriate
in vitro model to investigate the physiology of the
-AR in rodent adipocytes.
-AR number on the cell
surface. Through heterologous regulation, a number of other hormone
systems can influence, at the genetic level,
-AR density and
adrenergic sensitivity
(7) . The complexity of these regulatory
mechanisms is emphasized by the
-AR subtype-selective modulation
by several effectors. Thus glucocorticoids exert at a transcriptional
level a differential regulation of the three
-ARs in 3T3
adipocytes: while they enhance
-AR expression, they
strongly repress that of
- and
-ARs
(8, 9, 10, 11) . Butyric acid, a short
chain fatty acid, up-regulates
- and
-AR gene expression, but potently decreases that of
the
-AR
(12) . These heterologous regulations
are likely to contribute to adipocyte adaptation to environmental
conditions.
-AR gene expression, while that of
- and
-ARs remains unchanged
(13) . This regulation is exerted at a transcriptional level and
is followed by a decrease in
-AR sites and coupling to
the adenylyl cyclase system. It represents a mechanism for the long
term regulation by insulin of cAMP-dependent biological processes in
adipocytes and may be involved in the pathogenesis of nutritional
disorders associated with hyperinsulinemia. So far, the intracellular
signaling pathways leading to the heterologous regulation of the
-AR by insulin are still unknown. One possibility is
that insulin activates one or several isotypes of PKC
(14) . PKC
activation by insulin is involved in the stimulation of glucose
transport
(15) , lipogenesis
(16) , protein synthesis
(17) , c-Fos expression
(18, 19) , and
mitogen-activated protein kinase and protein phosphatase-1 activation
(20) . However, due to the persistance of a normal insulin
effect after PKC down-regulation, several authors have questioned the
involvement of PKC in insulin action (for review, see Ref. 21). The
role of PKC in mediating insulin effects may depend on a specific
cellular environment or phenotype, on the diversity of PKC isotypes in
a given cell
(22) , or on the nature of the insulin-regulated
process
(23) .
-AR subtype expression in 3T3-F442A adipocytes. In addition, we
addressed the issue of the contribution of PKC to the specific
transcriptional regulation of the
-AR mRNA by insulin.
Cell Culture
3T3-F442A cells were grown and
differentiated as described
(6) . At day 7 after confluence,
more than 90% of the cells had the morphology of mature adipocytes.
After two washes, cells were kept for 24 h in a defined medium
consisting of Dulbecco's modified Eagle's
medium/Ham's F-12 medium (2:1, v/v) and 0.1% bovine serum
albumin. From there on the cells were maintained either in the absence
or presence of PMA and/or the indicated drugs.
Pharmacological Experiments
Cell extracts were
prepared as described previously
(6) . Protein content was
assayed
(24) using bovine serum albumin as a standard.
S or
forskolin, assays were performed in the presence of 100 µM
GTP.
I]CYP concentrations ranging from 5
to 4000 pM. Competition experiments were carried out at 250
pM (-)-[
I]CYP. Nonspecific
binding was determined in the presence of 10 µM
(±)-bupranolol and was usually 15 ± 4% of total binding
at 250 pM (-)-[
I]CYP.
Chemicals
(-)-[I]CYP
was obtained from Amersham and [
-
P]ATP from
ICN Radiochemicals. CGP12177 and CGP20712A were generous gifts from
Ciba-Geigy (Basel, Switzerland). ICI118551 and ICI201651 were provided
by Imperial Chemical Industries (Macclesfield, United Kingdom),
BRL37344 by Smith Kline Beecham Pharmaceuticals (Epsom, United
Kingdom), and cyanopindolol by Sandoz (Basel, Switzerland).
(±)-Bupranolol was donated by Schwarz Pharma (Monheim, Germany).
ISO, forskolin, GTP
S, PMA, dioctanoyl- sn-glycerol,
4
-PDD, and insulin were Sigma products. GTP, bisindolylmaleimide,
and actinomycin D were from Boehringer Mannheim.
RNA Analysis
Total RNA was extracted from
3T3-F442A adipocytes by the method of Cathala et al.(25) . RNA samples were electrophoresed through a 1.5%, 2.2
M formaldehyde gel, and transferred to nylon Nitran-plus
membranes (Schleicher and Schuell). After RNA fixation, RNA content in
each lane was assessed by methylene blue staining of ribosomal RNAs.
Prehybridization was carried out at 60 °C for 30 min in the
presence of 0.5 M sodium phosphate (pH 6.8), 7% SDS, 1% bovine
serum albumin, and 1 mM EDTA
(26) . Hybridization was
performed in the same buffer in the presence of the heat-denaturated
probe (2-3 10
cpm/ml). Membranes were washed
twice for 30 min at 60 °C in 2
SSC (1
SSC: 150
mM NaCl, 15 mM sodium citrate), 0.1% SDS, then once
in 0.1
SSC, 0.1% SDS for 15 min at 60 °C. Probes were
labeled by random priming with [
-
P]dCTP
(ICN Radiochemicals). The
-AR probe is a 305-base pair
amplification product of the cloned murine
-AR gene
(2) between a sense 5`-GCATGCTCCGTGGCCTCACGAGAA-3` and an
antisense primer 5`-CCCAACGGCCAGTGGCCAGTCAGCG-3`.
-AR expression, total RNA was digested for 15 min at
37 °C with 0.1 unit of RNase-free DNase I (RQ1 DNase, Promega) per
µg of nucleic acid in 40 mM Tris-HCl (pH 7.9), 10
mM NaCl, 6 mM MgCl
, 10 mM
CaCl
in the presence of 1 unit/µl ribonuclease
inhibitor (RNAguard, Pharmacia). After phenol/chloroform extraction and
ethanol precipitation, RNA (0.25-2 µg) was
reverse-transcribed with MMLV RT (200 units/µg) (Life Technologies,
Inc.) in the presence of 10 µM random hexanucleotides
(Pharmacia), 1 unit/µg ribonuclease inhibitor, 400 µM
of each dNTP in a final volume of 20 µl consisting of 50
mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl
, and 10 mM dithiothreitol. After a 1-h
incubation at 42 °C, MMLV RT was heat-inactivated. To ensure that
subsequent amplification did not derive from contaminant genomic DNA, a
control without MMLV RT was included for each RNA sample. cDNAs were
denatured for 5 min at 94 °C and submitted to 25
(
-AR and
-actin) or 33 (
- and
-AR) cycles of amplification (1 cycle: 94 °C, 1
min; 60 °C, 2 min; 72 °C, 2 min) followed by a final extension
of 7 min at 72 °C in a DNA thermal cycler 480 (Perkin Elmer). PCR
was performed in a 25-µl reaction containing 1 unit of Taq polymerase (Bioprobe, France), 125 µM each dNTP, 5%
formamide, 125 nM both sense and antisense oligonucleotides.
The buffer consisted of 20 mM Tris-HCl (pH 8.55), 16
mM (NH
)
SO
, 2.5 mM
MgCl
, and 150 µg/ml bovine serum albumin. Sequences of
the sense and antisense oligonucleotides were:
5`-GGATCCAAGCTTTCGTGTGCACCGTGTGGGCC-3` and
5`-GGATCCAAGCTTAGGA-AACGGCGCTCGCAGCTGTCG-3` for the
-AR; 5`-GCCTGCTGACCAAGAATAAG-GCC-3` and
5`-CCCATCCTGCTCCACCT-3` for the
-AR;
5`-ATGGCTCCGTGGCCT-CAC-3` and 5`-CCCAACGGCCAGTGGCCAGTCAGCG-3` for the
-AR; 5`-GAGACC-TTCAACACCCC-3` and
5`-GTGGTGGTGAAGCTGTAGCC-3` for
-actin. These oligonucleotides were
derived from the sequences of the corresponding genes and cDNAs
(2, 27, 28, 29) . Amplification products
had expected sizes of 286, 329, 308, and 236 base pairs for
-,
-, and
-ARs and
-actin, respectively. They were separated on a 2% agarose gel and
visualized by ethidium bromide staining. In mature adipocytes,
amplification of
-AR and
-actin cDNAs with 25
cycles was linear up to 100 and 50 ng of RNA, respectively.
- and
-AR cDNA amplification with 33
cycles was linear up to 200 and 400 ng of RNA, respectively. With this
prerequisite, we were thus able to compare the relative levels of each
transcript between the different culture conditions.
Statistical Analysis
Results are presented as
means ± S.E. The level of significance between groups was
assessed using either paired or unpaired Student's t test.
Effects of Long Term PMA Treatment on
To evaluate the effects of
activation of PKC on -AR Expression
-AR gene expression, 3T3-F442A
adipocytes were treated with 300 nM PMA for 1-72 h, and
-AR mRNA abundance was examined by RT-PCR analysis.
While no significant change in the level of
-actin amplification
product could be detected during PMA exposure, we observed a decrease
in
-AR mRNA transcript as early as 2 h (Fig. 1)
after the onset of phorbol ester treatment, with a maximal effect
occurring by 6 h (6.5 ± 1.0-fold decrease, p <
0.005). Thereafter, with regards to PKC down-regulation induced by a
prolonged exposure to a high dose of PMA
(30) ,
-AR mRNA levels began to increase and returned almost
to control levels within 24 h. In addition, after an initial 30-min
treatment by PMA, cells were extensively washed and maintained for a
further 6-h period without the phorbol ester. This procedure had no
effect on
-AR mRNA levels (not shown), indicating that
a sustained PKC activation was required for
-AR mRNA
regulation. We also tested the time course of
-AR mRNA
content during exposure to dioctanoyl- sn-glycerol, a cell
permeable analog of diacylglycerol (100 µg/ml every 30 min). No
effect of this compound was detectable after a 60- or 90-min treatment,
but
-AR transcripts were decreased by 30 ± 5%
at 2 h ( p < 0.002, n = 6).
Figure 1:
Time-dependent down-regulation by PMA
of -AR mRNA expression in 3T3-F442A adipocytes.
3T3-F442A adipocytes maintained in a serum-free medium were exposed or
not to PMA (300 nM) for the indicated times. Total RNA from
control and PMA-treated cells were prepared at each time point. Samples
were digested by DNase I and treated or not with reverse transcriptase
to verify that subsequent PCR amplification performed with a mixture of
-AR and
-actin specific primers did not derive
from contaminating genomic DNA.
-AR and
-actin
cDNA amplification was carried out in nonsaturating conditions (25
cycles, 25 ng of RNA). PCR products were resolved on a 2% agarose gel
stained with ethidium bromide, and analyzed by video densitometric
scanning.
-Actin mRNA levels were used to standardize
-AR mRNA content. Results for PMA-treated cells are
expressed as the percentage of the level detected in control adipocytes
and represented the mean ± S.E. of three independent
experiments. *, p < 0.005, PMA-treated versus control adipocytes.
The dose
dependence of -AR mRNA repression by PMA was studied
by both Northern blotting and RT-PCR analyses. With these two
approaches, down-regulation of the
-AR mRNA was
detectable at a PMA concentration of 10 nM (Fig. 2).
Repression of the
-AR transcript was maximal between
0.1 and 1 µM, the half-maximal action being between 15 and
20 nM. The major mRNA species of 2.3 kilobases in size and the
two minor species of 2.8 and 4.4 kilobases appeared equally affected by
PMA treatment.
Figure 2:
Effect of PMA concentration on
-AR mRNA levels. 3T3-F442A adipocytes were exposed for
6 h to the indicated concentrations of PMA. Total RNA was extracted and
analyzed by Northern hybridization with a specific
-AR
probe, or by RT-PCR as described in the legend to Fig. 1. Panel A shows a representative autoradiogram of a Northern blot analysis.
The sizes (in kilobases) of
-AR transcripts are
indicated in the left margin, and positions of 28 and 18 S
ribosomal RNAs are shown in the right margin. Panel B corresponds to a typical RT-PCR experiment. Positions of
-AR and
-actin PCR products are given on the
left. The sizes of molecular weight markers (in base pairs)
are indicated on the right. In panel C is represented
the mean ± S.E. of three independent experiments of Northern
(
) or RT-PCR (
) analysis. Results are represented as the
percentage of the level detected in control adipocytes. EC
values for PMA-induced down-regulation of
-AR
mRNA were 18.3 ± 3.0 and 15.5 ± 9.4 nM PMA in
Northern and RT-PCR analyses, respectively. *, p < 0.01;
**, p < 0.001, PMA-treated versus control
adipocytes.
Analysis of -AR subtype gene expression by
RT-PCR showed that PMA selectively inhibited
-AR mRNA
levels (Fig. 3). For this purpose we ensured that
-,
-, and
-AR cDNA
amplification was performed in nonsaturating conditions, as indicated
by the stoichiometry between the amount of RNA and the level of the
corresponding amplification product. While a 6-h exposure of the cells
to 300 nM PMA caused a 3.3-4-fold decline in
-AR mRNA content, this treatment did not influence the
abundance of
- and
-AR transcripts.
Figure 3:
Selective down-regulation of
-AR mRNA levels by PMA. 3T3-F442A were treated
( filled bars) or not ( empty bars) with PMA (300
nM) for 6 h. Total RNA was extracted, digested with DNase I,
then treated or not with MMLV RT. cDNA derived from various amounts of
total RNA (indicated in ng under each column) were then amplified in
the presence of Taq polymerase and primers specific for
-,
-, or
-ARs. The
resulting PCR products were analyzed as described in legend to Fig. 1.
Results are expressed as the percentage of the
-,
-, or
-AR mRNA content observed in
control adipocytes with the maximal indicated amount of RNA (200, 400,
and 100 ng of RNA for
-,
- and
-ARs, respectively). They represented the mean
± S.E. of three to five separate experiments. *, p <
0.05; **, p < 0.01, PMA-treated versus control
adipocytes.
To determine the functional consequences of the specific
down-regulation of -AR mRNA levels by PMA, adenylyl
cyclase activity in response to CGP12177 was measured on membranes from
control or PMA-treated cells. CGP12177 is a
/
-AR antagonist, but has agonistic
properties at the
-AR site
(2, 31) , so
that this compound allows to address specifically
-AR
coupling. Exposure of 3T3-F442A adipocytes to PMA provoked a time- and
dose-dependent decline in adenylyl cyclase activity in response to a
maximal dose (100 µM) of CGP12177. As compared to the
kinetics of the PMA-induced decrease in
-AR mRNA
levels, the inhibitory effect of the phorbol ester on
-AR responsiveness was slightly delayed. A significant
decline in CGP12177-stimulated adenylyl cyclase activity was detectable
after a 6-h exposure to PMA (Fig. 4 A) and was maximal
after a 12-h treatment (2.9-fold decrease, p < 0.02).
Thereafter, we observed an increased adenylyl cyclase responsiveness to
CGP12177, with a restoration of the control adenylyl cyclase
sensitivity within 48 h. Inhibition of CGP12177-stimulated adenylyl
cyclase activity was observed at 30 nM PMA and was maximal at
300 nM, giving a half-maximal concentration of 44 ± 11
nM (Fig. 4 B). In control and PMA-exposed (300
nM for 12 h) adipocytes, adenylyl cyclase activity was also
measured in response to maximal concentrations of various effectors:
forskolin, GTP
S, ISO, and the
-AR agonists
CGP12177, ICI201651, and CYP. As illustrated in , chronic
PMA exposure specifically altered
-AR responsiveness.
By contrast, GTP
S- and forskolin-stimulated adenylyl cyclase
activities were not affected by phorbol ester treatment. This result
strongly suggested that the PMA-induced decrease in
-AR
sensitivity was a receptor- rather than a G-protein- or adenylyl
cyclase-mediated event.
Figure 4:
Time and dose dependence of PMA-induced
decrease in CGP12177-stimulated adenylyl cyclase activity. A, adenylyl cyclase activity in response to a maximal concentration
(100 µM) of CGP12177 was determined in membranes from
adipocytes treated or not by PMA (300 nM) for the indicated
times. Results of PMA-treated cells are expressed as the percentage of
CGP12177 stimulated over basal adenylyl cyclase activity measured in
control cells. B, adenylyl cyclase activity in response to a
maximal concentration (100 µM) of the -AR
agonist CGP12177 was measured in membranes from adipocytes exposed for
12 h to the mentioned concentrations of PMA. Results are expressed as
CGP12177-stimulated over basal adenylyl cyclase activity (in pmol
cAMP/min/mg of protein). The figures represent the mean ± S.E.
of three independent experiments performed in triplicate. *, p < 0.02; **, p < 0.01, PMA-treated versus control adipocytes.
We also confirmed that the decrease in
-AR mRNA and
-AR coupling to the
adenylyl cyclase system corresponded to specific regulation of
-AR sites. (-)-[
I]CYP
saturation binding experiments were performed in membranes from control
and PMA-treated (300 nM for 12 h) 3T3-F442A adipocytes. In
control cells, the density of
-ARs (low-affinity
class) was 544.2 ± 59.4 fmol/mg, while that of
- and
-ARs (high-affinity class) was
only 11.3 ± 2.3 fmol/mg (). In PMA-exposed cells,
we observed an
2-fold reduction in
-AR population
(278.9 ± 50.6 fmol/mg), whereas the number of high-affinity
binding sites remained unaffected. No significant difference in the
K
values of the two binding classes for
(-)-[
I]CYP could be detected between
treated and untreated cells. Furthermore, competition curves of
-,
- and
-AR
selective ligands against (-)-[
I]CYP
allowed estimation of the relative proportions of the three
-AR
subtypes in control and PMA-treated adipocytes (I). These
experiments confirmed that PMA induced a
2-fold decrease in the
-AR population which corresponds to the high affinity
sites for the
-AR selective compound BRL37344. In
agreement with RT-PCR analysis of
- and
-AR mRNA levels, analysis of the displacement curves
of (-)-[
I]CYP by the
-AR
selective antagonist CGP20712A or by the
-AR selective
antagonist ICI118551 indicated that the density of
-
and
-ARs was not modified by PMA treatment.
PKC Involvement in the Effect of PMA on
A series of experiments were
designed to determine whether the effects of PMA on
-AR mRNA
-AR gene expression were mediated by activation of
PKC. The biologically inactive phorbol ester 4
-PDD did not modify
-AR mRNA content (Fig. 5 A), thereby
excluding a nonspecific effect of the phorbol moiety. Also, 3T3-F442A
adipocytes were exposed for 24 h to a high dose (1 µM) of
PMA to down-regulate PKC; after this initial treatment, a subsequent
exposure to PMA (100 nM) for 6 h did not modify
-AR mRNA levels (Fig. 5 B). We also
studied the reversibility of the PMA-induced decrease in
-AR mRNA content by a recently characterized PKC
inhibitor, bisindolylmaleimide, which acts as a competitive inhibitor
with respect to ATP binding site on the catalytic moiety of the kinase
(32) . In contrast with other PKC inhibitors, this novel
compound displays a high selectivity for classical isoforms of PKC as
compared to several other protein kinases. Pretreatment of 3T3-F442A
adipocytes with increasing concentrations of bisindolylmaleimide
completely inhibited the PMA-induced down-regulation of
-AR mRNA (Fig. 6), with an IC
value
of 52.1 ± 11.9 nM. Cell exposure to bisindolylmaleimide
alone did not affect
-AR gene expression.
Figure 5:
Effects of 4-PDD and PKC
down-regulation on
-AR mRNA levels. 3T3-F442A
adipocytes were exposed or not (control, C) for 6 h to PMA
(100 nM) or to the biologically inactive phorbol ester
4
-PDD (100 nM) ( panel A). Adipocytes pretreated
for 24 h by 1 µM PMA to down-regulate PKC (desensitized,
DSTZ) were then treated ( PMA) or not (control,
C) for 6 h with 100 nM PMA ( panel B). Total
RNA was prepared from 3T3-F442A adipocytes cultured under these various
conditions. Samples were digested with DNase I, then treated or not by
MMLV RT. cDNAs were amplified (25 cycles) in the presence of Taq polymerase and specific primers for
-AR and
-actin. cDNA content in the PCR assay corresponded to initial
amounts of DNase I-treated RNA of 25 ng. PCR products (one-fifth
volume) were separated on a 2% agarose gel, visualized by ethidium
bromide staining, and analyzed by video densitometric scanning.
-AR mRNA levels were normalized to
-actin mRNA
content and are expressed as the percentage of the level detected in
control adipocytes. The values represent the mean ± S.E. of
three to four independent experiments. *, p < 0.001,
PMA-treated versus control
adipocytes.
Figure 6:
Effect of the selective PKC inhibitor
bisindolylmaleimide on PMA-induced decrease in -AR
mRNA levels. 3T3-F442A adipocytes were pretreated or not for 30 min
with the indicated concentrations of bisindolylmaleimide. Thereafter,
cells were treated for 6 h with or without 100 nM PMA. Total
RNA was prepared, digested with DNase I and treated or not with MMLV
RT. cDNA amplification, analysis of PCR products, and expression of the
results were carried out as described in the legend to Fig. 5. The
figure shows the mean ± S.E. of three independent experiments
performed in cells exposed (
) or not (
) to PMA. *, p < 0.005; **, p < 0.02, PMA-treated versus control cells.
Effect of PMA on
The PMA-induced decrease in -AR mRNA
Turnover
-AR mRNA
levels resulted either from decreased rate of gene transcription,
accelerated rate of RNA degradation, or a combination of both. We
therefore examined the turnover of the
-AR mRNA in
adipocytes exposed to an inhibitor of transcription (actinomycin D) in
the absence or presence of PMA. The disappearance of the
-AR transcripts was then assayed by Northern analysis
over a 4-h period (Fig. 7). The rate of receptor mRNA decay in
PMA-treated adipocytes ( t
= 84 ± 10 min)
was not different from that observed in control cells
( t
= 86 ± 6 min), strongly suggesting
that PMA caused a decrease in
-AR gene expression by
inhibiting gene transcription, rather than by a decrease in mRNA
stability.
Figure 7:
Effect of PMA on -AR mRNA
stability. 3T3-F442A adipocytes were treated (
, dashed
line) or not (
, solid line) by 300 nM PMA
for 2 h, then actinomycin D (5 µg/ml) was added to control or
PMA-exposed cell cultures. At the indicated times of actinomycin
treatment, total RNA was extracted and Northern analysis was performed
as described under ``Materials and Methods.'' Autoradiograms
were analyzed by video densitometric scanning. The half-life of
-AR mRNA was calculated by linear estimation from the
best fit of each line on the logarithmic plot. The figure represents
the mean ± S.E. of three separate
experiments.
Evaluation of the Role of PKC in Insulin Action on
We have recently shown that
insulin selectively down-regulates -AR mRNA
-AR transcripts in
3T3-F442A adipocytes by a transcriptional mechanism
(13) . To
further test the hypothesis that insulin could modulate
-AR gene expression through the PKC signaling pathway,
3T3-F442A adipocytes were maintained with or without the hormone in the
absence or presence of the selective PKC inhibitor bisindolylmaleimide.
RT-PCR analysis indicated that bisindolylmaleimide partially inhibited
the decline in
-AR mRNA levels caused by insulin
(Fig. 8). When cells were exposed to the PKC-selective inhibitor
prior to insulin addition, the decrease in
-AR mRNA
content induced by 1 or 5 nM of the hormone was reduced by 44
and 49%, respectively. In addition, after initial down-regulation of
PKC by a 24-h exposure to a high dose (1 µM) of PMA, we
evaluated the effect of insulin on
-AR mRNA content in
these desensitized cells. While insulin provoked a clear decrease in
-AR gene expression in nondesensitized cells, the
initial down-regulation of PKC partially prevented its modulating
action which was reduced by 67% (Fig. 9), thus providing
additional evidence for the role of PKC in
-AR mRNA
regulation by insulin.
Figure 8:
Effect of bisindolylmaleimide on
insulin-induced decrease in -AR mRNA levels. 3T3-F442A
adipocytes were treated or not with 1 or 5 nM insulin
( INS) for 6 h in the absence or the presence of the PKC
inhibitor bisindolylmaleimide ( BIM) (0.5 µM added
30 min prior to insulin). RNA extraction, RT-PCR, and expression of the
results were performed as described in the legend to Fig. 5. The
results represent the mean ± S.E. of five independent
experiments. **, p < 0.001, insulin-treated cells
versus control cells. *, p < 0.01, insulin- and
bisindolylmaleimide-treated cells versus control cells. ,
p < 0.005, insulin- and bisindolylmaleimide-treated cells
versus insulin-(alone) treated
cells.
Figure 9:
Effect
of insulin on -AR mRNA expression after chronic
incubation with PMA. 3T3-F442A adipocytes were untreated or treated
(desensitized, DSTZ) for 24 h with 1 µM PMA to
down-regulate PKC. Thereafter, desensitized or nondesensitized cells
were exposed or not for 4 h to 10 nM insulin ( INS).
The control ( C) condition corresponded to cells neither
treated by insulin nor PMA. Total RNA was isolated, digested with DNase
I, and treated (+) or not (-) with MMLV RT. cDNA
amplification and analysis of the results were carried out as described
in the legend to Fig. 5. Panel A shows a typical ethidium
bromide-stained gel of PCR products. Sizes (in base pairs) of the
molecular weight markers are indicated in the right margin,
while the positions of
-AR and
-actin PCR
products are shown in the left margin. Panel B represents the mean ± S.E. of seven separate experiments.
*, p < 0.001, insulin-treated versus control
adipocytes. #, p < 0.001, desensitized cells treated with
insulin ( DSTZ + INS) versus insulin-treated
cells. , p < 0.05, desensitized cells treated with insulin
( DSTZ + INS) versus desensitized cells
( DSTZ).
-AR mRNA content: (i) the high sensitivity to PMA
contrasts with the lack of effect of the inactive phorbol ester
4
-PDD, in agreement with recently published results
(33) .
(ii) PKC down-regulation abolishes the subsequent inhibitory action of
PMA on
-AR mRNA levels. (iii) The effect of PMA is
suppressed by a selective PKC inhibitor, bisindolylmaleimide, with
IC
values consistent with its potency to inhibit classical
PKC subtypes
(32) . In contrast to most reported PKC inhibitors
that are poorly selective, such as H-7
(34) or staurosporine
(35) , bisindolylmaleimide displays high selectivity for PKC, as
compared to other kinases
(32) .
-AR-mediated cAMP production (Ref. 36 and
references herein), while others have described a potentiation of this
response (Refs. 37 and 38, and references herein). Phosphorylation of
the
-AR is at the molecular basis of the phorbol ester-provoked
attenuation in catecholamine responsiveness
(39, 40) .
The PKA/PKC consensus phosphorylation site, located in the third
intracellular loop, is involved in rapid desensitization by receptor
uncoupling from Gs
(41, 42, 43) . Nakada et
al.(36) have also suggested that the nature of the
-AR subtype may determine the ability of PKC activation to
uncouple the receptor from adenylyl cyclase activation. Interestingly,
the PKA/PKC phosphorylation sites are lacking in the
-AR, and this absence is likely to contribute to the
resistance of this subtype to undergo rapid agonist-promoted
desensitization
(44, 45) . The present study establishes
the fact that PKC activation allows a long term desensitization of the
-AR through a process of down-regulation. A sustained
activation of PKC provokes a specific inhibition in
-AR gene expression, whereas
- and
-AR mRNA levels remain unchanged. An accelerated
receptor turnover may also be involved in
-AR
down-regulation, although this seems unlikely in view of our previous
results
(46) . In 3T3 adipocytes, the regulation of
-AR
responsiveness by activation of PKC could involve the complex and
sequential contribution of both transcriptional and post-translational
processes that differentially affect the three
-AR subtypes.
Acutely, phosphorylation of
- and
-ARs
(36) by PKC uncouples these subtypes from
G-protein, while a more prolonged PKC activation decreases
-AR gene and protein expression.
-AR mRNA stability indicates that PKC
activity regulates the transcription rate of the
-AR
gene. Several mechanisms at the basis of this transcriptional
modulation can be considered. Studies performed in various cell types,
including adipocytes
(30) , have shown that PKC activation leads
to the transcriptional induction of the cellular immediate-early
response gene c- fos. c-Fos and c-Jun proteins heterodimers, or
c-Jun homodimers are components of the transcription factor AP-1. AP-1,
alone or in combination with other transcription factors
(47, 48) , modulates gene activity through interaction
with a specific DNA recognition sequence. Alternatively, the
PMA-induced increase in AP-1 activity could involve post-translational
modifications of pre-existing AP-1. Boyle et al.(49) have demonstrated that activation of PKC results in
dephosphorylation of c-Jun and coincides with increased AP-1 binding
and trans-activating activity. Furthermore, the PKC signaling
pathway controls the nuclear translocation of transcription factors.
The transcription factor NF-
B provides a prototypical example of
an ``anchorage-release'' signal transduction mechanism to the
nucleus. In a resting state, NF-
B is present in an inactive form
in the cytoplasm by association with an inhibitory protein known as
I
B. I
B phosphorylation by a variety of kinases, including
PKC, leads to the dissociation of the complex and nuclear translocation
of NF-
B that can bind to its target elements
(48) .
Specific consensus sequences for AP-1 and NF-
B are present in the
promoter of the human, mouse, and rat
-AR genes
(50, 51) , but no data are currently available to
suggest a functional role for these sequences. It is conceivable that
these and other mechanisms controlling gene transcription do not
function independently of one another. Signal cross-talk could occur at
every level of the transduction pathways. In this regard it has been
reported that NF-
B and AP-1 can physically interact to synergize
DNA-binding and biological function
(52) .
-AR gene expression, primarily through a
transcriptional mechanism. This sharp insulin-induced decrease of the
main
-AR subtype of rodent adipocytes inhibits
-AR responsiveness and has potential consequences on
all cAMP-dependent biological processes of adipocytes. Thus it is of
considerable interest to identify the intracellular transduction
pathways responsible for such a regulation. Our results suggest that
modulation of
-AR mRNA levels by insulin could involve
PKC-dependent and -independent mechanisms. Several of our data indicate
that the down-regulation of
-AR gene expression by
insulin occurs partially through the PKC signaling pathway. PMA
provokes a decrease in
-AR mRNA amounts comparable to
that caused by insulin
(13) . Both the phorbol ester and insulin
induce a specific modulation of
-AR gene expression.
Depletion of cellular PKC levels by prolonged exposure to PMA, or
pretreatment with a PKC selective inhibitor alter the effects of
insulin on
-AR transcripts. Also, PMA and insulin
appear to control
-AR gene expression primarily at the
transcriptional level. Taken together, these data confirm that PKC is
involved in the repression of the
-AR gene by insulin.
However, other results suggest that PKC is not a unique and obligatory
step in the control by the peptidic hormone of this adrenergic
receptor. While insulin induces a rapid decrease in
-AR mRNA content
(13) , PMA- or diacylglycerol
analog-induced down-regulation of these transcripts was significantly
detectable only after 2 h. Moreover, PKC depletion or pretreatment with
bisindolylmaleimide reversed only
50% of the insulin effect on
-AR transcripts. Different explanations could account
for this partial reversibility of insulin action after PKC depletion or
blockade. One possibility is that the extent of PKC down-regulation
after a chronic exposure to phorbol esters varies with cell type. In
this regard, it is noteworthy that in 3T3-L1 adipocytes, there is a
relative lack of effectiveness of this procedure of PKC depletion
(21) . However, the absence of PMA effect on
-AR transcripts in PKC-depleted adipocytes does not
favor this hypothesis. Alternatively, insulin could regulate gene
expression by activation of PKC isotypes which are less responsive to
PMA. More recently discovered PKC subtypes, such as the
and
isoforms, appear to be more or completely resistant to down-regulation
by phorbol esters
(53, 54, 55) and might be
preferentially activated by insulin in some cell types
(56, 57) . In rat adipocytes, it is thus documented that
chronic phorbol ester treatment differentially affects PKC isoform
depletion
(58) . Whatever the phenotype of PKC isozymes in
mature 3T3 adipocytes, our study leaves open the possibility that
activation of atypical PKC isotypes by non-diacylglycerol activating
ligands plays a role in the control by insulin of
-AR
gene expression. Thus, it has been reported that phosphatidylinositol
3,4,5-trisphosphate produced by phosphatidylinositol 3-kinase, an
enzyme of the insulin receptor signaling system, is able to activate
the atypical PKC
(59) . Finally, the persistance of a
significant insulin effect on
-AR mRNA after PKC
depletion or selective blockade could also reflect the dual involvement
of PKC-dependent and -independent pathways. The similarity between the
effects of insulin and PMA may be related to the convergence at a
common point of cellular events caused by these two effectors, for
example, at the level of transcription. This hypothesis is supported by
the characterization in the phosphoenolpyruvate carboxykinase promoter
of an insulin-responsive sequence that coincides with the phorbol ester
recognition site
(60) .
-AR gene expression in 3T3-F442A
adipocytes. In addition, the effect of insulin on this adrenergic
receptor subtype could be mediated, at least in part, through this
intracellular signaling pathway. The decrease of the main
-AR
subtype of rodent adipocytes and of catecholamine responsiveness
induced by a prolonged PKC activation may have important consequences
on cAMP-dependent biological processes of adipocytes, such as the
positive control of lipolysis or thermogenesis, or the negative
modulation of lipogenesis.
Table: PMA selectively inhibits adenylyl cyclase
activity stimulated by -adrenergic agonists
Table:
Characterization of
(-)-[I]CYP binding sites in membranes
from control and PMA-treated 3T3-F442A adipocytes
I]CYP saturation
binding experiments using a wide range of concentrations (5-4000
pM) of the radioligand. Scatchard analysis of the data with
the EBDA/LIGAND program was used to calculate the
K
and B
values of
the high (
- and
-ARs) and the low
(
-AR) affinity sites for
(-)-[
I]CYP. Results are expressed as mean
± S.E. of five separate experiments. The percentage of each
affinity binding class is indicated in parentheses after the
B
value.
Table:
Competition of
(-)-[I]CYP against
-AR
subtype-selective ligands in membranes from control and PMA-exposed
cells
I]CYP in the absence or the
presence of various concentrations of CGP20712A, ICI118551, or
BRL37344. Data from displacement of
(-)-[
I]CYP binding by these
subtype-selective ligands were used to calculate the
K
values for each affinity component. The
corresponding B
values were derived from total
-AR density drawn from (-)-[
I]CYP
saturation experiments (see Table I) taking into account the percentage
of each affinity component (indicated in parentheses after the
B
values) obtained from competition experiments.
Results are expressed as mean ± S.E. of three independent
experiments.
-AR(s),
-adrenergic receptor(s);
BRL37344,
sodium-4-{2-[2-hydroxy-2-(3-chloro-phenyl)ethylamino]propyl}phenoxyacetate
sesquihydrate ( RR.SS distereoisomer); CGP12177,
(±)-4-(3- t-butylamino-2-hydroxypropoxy)-benzimidazole-2-one;
CGP20712A,
(±)-(2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino)-3-(4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol
methane sulfonate; (-)-[
I-CYP,
(-)-[
I]cyanopindolol; GTP
S,
guanosine 5`- O-(3-thiotriphosphate); ICI118551,
erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol;
ICI201651,
( R)-4-(2-hydroxy-3-phenoxypropylamino-ethoxy)- N-(2-methoxyethyl)phenoxyacetamide;
ISO, (-)-isoproterenol; kb, kilobase(s); MMLV RT, Moloney murine
leukemia virus reverse transcriptase; 4
-PDD, 4
-phorbol
12,13-didecanoate; PKA, cAMP-dependent protein kinase; PKC, protein
kinase C; PMA, 4
-phorbol 12-myristate 13-acetate; RT-PCR, reverse
transcriptase-polymerase chain reaction; Taq polymerase,
Thermus aquaticus polymerase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.