(Received for publication, November 28, 1995)
From the
While the functions of several G protein subunits such as
and
are relatively well understood,
the action of others such as
remain largely
undefined. Because of recent interest in regulation of nitric-oxide
synthase (NOS) by G protein-coupled signaling systems and findings that
receptors for two proinflammatory substances, thrombin and thromboxane
couple to
, we studied the effect of
on NOS activity in a renal epithelial cell line. We found that
stable overexpression of
or its GTPase-deficient
mutant,
, in a continuous renal epithelial cell
line (MCT) increased NOS activity. The increased NOS activity was due
to increased expression of the macrophage-inducible form of NOS (iNOS).
iNOS protein and activity were not increased in similar cells
expressing an activated
(
) or
were minimally increased in cells expressing activated
(
) and
(
), members of the three other G protein
chain families. Transient co-expression of
or
increased the activity of an iNOS promoter-CAT
construct demonstrating that
increases iNOS
expression through transcription. Consequently,
induces iNOS through a novel mechanism that is distinct from that
of other G protein
chains and that may mediate the actions of G
protein-dependent proinflammatory agents.
The G protein family of heterotrimeric signaling proteins
couples receptors on the outside of cells to effector molecules in cell
membranes or inside cells. The subunits have primary
responsibility for determining the specificity of receptor and effector
interactions. Individual
chains couple to multiple effectors
(enzymes and ion channels), and the effects of multiple G proteins
converge on single effector systems at multiple levels. These
characteristics create signaling networks and allow for integration and
modulation of signals in cells(1, 2) . The functions
of some
subunits such as
, that stimulates
adenylyl cyclase, and
, that regulates phospholipase
C
isoforms, are relatively well defined in biochemical and
physiologic terms (1, 2, 3) .
The
functions of other chains such as those of the
/
family remain largely undefined.
The
and
subunits are widely
expressed in mammalian tissues(4, 5) . We have
demonstrated that stable expression of
in epithelial
cells increases calcium influx(6) . Transient or stable
overexpression of
increases Na/H exchange (NHE-1)
activity, although the mechanism is not fully
defined(6, 7, 8) . Expression of an activated
form of
can transform cells in tissue culture and
increase expression of immediate early genes and activate
Jun/stress-activated
kinases(9, 10, 11, 12) .
Receptors for thrombin and thromboxane, both ligands that
participate in inflammatory reactions, stimulate guanine nucleotide
exchange by in platelet membranes(13) . This
finding suggests that
may be involved in signaling
by proinflammatory substances and could regulate cell processes that
are involved in inflammatory responses, including intracellular calcium
(Ca
)(
)(13) . Recently,
considerable interest has been expressed in regulation of NO production
by G protein-coupled signaling systems. To explore involvement of
in inflammation and provide direct evidence for
regulation of NO production by heterotrimeric G proteins, we studied
the effect of stable expression of
and members of
the three other G protein
chain families (
,
, and
) on NO production.
We obtained the iNOS promoter-CAT (p1 iNOS-CAT)
from Carl Nathan and Qiao-wen Xie (Cornell) and from Henry Bourne (University of California, San
Francisco)(8, 16) .
and
were expressed in Zem-228 and pCMV5 vectors (17, 18) . Truncation mutants of p1 iNOS-CAT were made
using convenient restriction sites, blunt-ending the overhangs with
polymerase, and religating the plasmids. cDNAs were expressed
transiently using a modification of the technique of Martin et
al.(19) . MCT cells were grown to near confluence,
suspended, and aliquots of approximately 10
cells incubated
with 4 µg of DNA (2 µg of p1 iNOS-CAT, 1 µg of
construct, and 1 µg of SV40
-galactosidase) in the
presence of DEAE-dextran (M
5
10
) and 5% serum. The control sample contained 1 µg of
pCMV5 in place of the
chain construct.
Our approach to studying regulation of NOS activity by
heterotrimeric G protein chains was to stably express members of
each of the four G protein
chain families,
,
,
, and
, in
MCT cells(23) . The expressed
chains
(
,
,
, and
) contained
mutations, substitution of leucine for glutamine at a conserved site in
the guanine nucleotide binding domain that blocks GTPase activity and
produces an activated protein(24) . The GTPase-deficient forms
of the
chains were studied because they provide the strongest
intracellular signals.
Figure 1:
NOS activity in cell
extracts from neomycin-resistant control cells and cells expressing
,
,
,
, and
. Open bars represent total citrulline conversion activity, and closed
bars represent NOS activity in the presence of 1 mMN
-nitro-L-arginine. Values represent the
means of multiple experiments expressed as multiples of the control
neomycin-resistant control cells value ± S.E.
(neomycin-resistant control cells (Neo), n =
9;
, n = 4;
, n = 4;
, n = 9;,
, n = 4;
, n =
4).
Figure 2:
Immunoblot of cell extracts from
neomycin-resistant control cells (Neo) and cells expressing
,
,
,
, and
using antibodies to
iNOS (A), bNOS (B), and eNOS (C). The first
lane contains 400 ng of cell extract enriched in iNOS (A),
bNOS (B), or eNOS protein. The blot shown is representative of
four separate blots.
The next question addressed was whether the increase in iNOS protein
was due to increased mRNA levels. We performed Northern blots with a
cDNA probe for iNOS. We found that expression of substantially increased iNOS mRNA abundance (approximately
10-fold) as shown in Fig. 3. For comparison, the increase in
iNOS mRNA in the cells expressing
, the cells
with the next highest increase in NOS activity and protein expression
was relatively small, approximately 2-fold.
Figure 3:
RNA blot using 8 µg of
poly(A) RNA from neomycin-resistant control cells (Neo) and cells expressing
and
. In the top panel, the blot was probed for
mouse iNOS, and in the bottom panel the blot was probed for
-actin to normalize for loading. The position of the ribosomes is
shown at the right.
Figure 4:
A, regulation of iNOS promoter activity by
and
in MCT cells. MCT cells
were transiently co-transfected with p1 iNOS-CAT and pCMV5 without
insert (control) or one of the
constructions
(
or
) in the vectors
indicated (Zem-228 or pCMV5). Cells were grown for 48 h, extracts
prepared, and equal amounts of cell extract (normalized for protein)
were assayed for CAT activity. The experiment shown is representative
of four separate experiments. B summarizes the effect of
protein kinase C inhibition on
and
-stimulated p1 iNOS-CAT activity (left
panel: open columns, control;
, filled
columns; shaded columns, calphostin C) and activation of
adenylyl cyclase on
and
-stimulated P1 iNOS-CAT activity (right
panel: open columns, control; filled columns, forskolin
+ isobutylmethylxanthine) relative to the
control. Cells were transiently transfected with the
chains
(in pCMV5 vectors) and p1 iNOS-CAT as indicated. After 24 h, the cells
were treated with phorbol 12-myristate 13-acetate (400 nM),
calphostin C (10 nM), or isobutylmethylxanthine and forskolin
(50 µM and 100 nM, respectively) for 24 h as
indicated. Cells were harvested and assayed for CAT activity as
described above. The experiments shown are representative of at least
three experiments.
G had a marked
stimulatory effect on iNOS expression, while
and
had smaller effects. In order to demonstrate
the specificity of the
effect and that the other
chains act by different mechanisms, we studied the effects of
chemical activators or inhibitors of several signaling pathways on
expression of p1 iNOS-CAT co-expressed with
,
, or
. Other investigators
have demonstrated that inhibition of protein kinase C blocks the
ability of
, but not
to stimulate
Na/H exchanger activity(7) . Consequently, in cells transiently
expressing p1 iNOS-CAT and
or
, we inhibited protein kinase C by
down-regulating it with a high dose of phorbol 12-myristate 13-acetate,
and by treatment with calphostin C. As shown in Fig. 4B (left panel), inhibition of protein kinase C reduced
stimulation of p1 iNOS-CAT by
, but had no effect
on its expression in response to
. In similar
experiments (Fig. 4B (right panel)),
isobutylmethylxanthine and forskolin prevented stimulation of p1
iNOS-CAT by
, but increased its expression
(rather than decreased it) to a small degree in the cells with
. These results demonstrate that
stimulates iNOS expression by a mechanism that is distinct from
those of
and
.
The iNOS
promoter-CAT reporter construct contains 161 bases of the
5`-untranslated region(16) , so in principle, could regulate expression of this construct through stabilization
of the mRNA. In order to determine what region of p1 iNOS-CAT responds
to
, we made a series of truncation mutations using
convenient restriction sites, -724 to +161 (P3 iNOS-CAT),
-333 to +161 (p4 iNOS-CAT), and -48 to +161 (p6
iNOS-CAT)(16) . Fig. 5shows that stimulation of
iNOS-CAT activity by
requires the 285-base
region between -333 and -48. Consequently, the principal
effect of
cannot be through stabilization of mRNA,
but must be through increased transcription. The exact sequence
responsible for the effect of
on iNOS expression is
currently under investigation.
Figure 5:
Effect of successive truncations of p1
iNOS-CAT on -stimulated iNOS-CAT activity. A shows a map of p1 iNOS-CAT and the truncations made. B shows CAT activity of p1 iNOS-CAT and the truncation mutants with
pCMV5 vector alone (top) and pCMV5-
(bottom). The cells were harvested and assayed for CAT
activity as described above.
The novel and central finding of these studies is that iNOS
is regulated at the transcriptional level by a specific G protein
chain, G
. Induction of iNOS is usually associated
with inflammatory reactions initiated by a variety of stimuli,
particularly bacterial products and/or cytokines, not by G
protein-dependent signaling systems(25) . Consequently,
induction of iNOS by
and the receptors that couple
to it represents a new mechanism for regulation of iNOS and possibly
other proteins that are induced during inflammatory reactions.
Regulation of iNOS expression by
is not limited to
epithelial cells. In experiments similar to those shown in Fig. 4,
and
increased
iNOS promoter activity in RAW 264.7 cells, a macrophage-like cell
line.
These studies indicate that substances that act
through receptors coupled to
such as thrombin and
thromboxane (13) could also initiate cellular responses similar
to those initiated by cytokines and bacterial products such as
lipopolysaccharide.
Both the wild type and mutant forms of
increase iNOS expression and activity. In the stable
pooled cells the level of iNOS expression and activity was similar in
response to
and
. One would
expect that expression of the mutant
chain might result in higher
levels of iNOS expression. However, higher levels of NOS could be toxic
to the cells and result in similar levels of expression in cells
containing mutant and wild-type
proteins. In the
transient system where the total number of cells and efficiency of
expression is lower than the stable system,
is
more effective than
.
induces
iNOS by a specific mechanism.
chains from other families either
do not induce iNOS (
) or do so to a much smaller
degree and by a different mechanism (
and
). We do not believe that
or
acts primarily through increased Ca
,
because the resting Ca
levels and calcium pool sizes are
similar in neomycin-resistant control cells and the cells expressing
the
chains(6, 26) . Cells expressing
have substantially elevated basal phospholipase
C activity and presumably protein kinase C activity.
probably induces iNOS through protein kinase C because inhibition
of protein kinase C blocks stimulation of iNOS by
. The small increase in iNOS activity and protein
in the cells that express
is due to inhibition
of adenylyl cyclase by overexpression of this
subunit, because
the low level of iNOS induction is blocked by activators of the
adenylyl cyclase system.
may signal by a
mechanism that has not been described previously for G protein
chains, because activated representatives of the other G protein
chain families either do not stimulate iNOS expression
(
) or cause only a minor increase in its
expression (
and
through
their respective second messengers and kinases). Regulation of iNOS
expression is relatively well characterized in macrophages and other
tissues where it is induced by cytokines and lipopolysaccharide via
interferon response elements and NF-
B(25, 27) .
Interferon-
signals through pathways involving JAK/STAT proteins,
and lipopolysaccharide activates a complex signaling pathway involving
NF-
B and mitogen-activated protein kinases.
appears not to require the upstream GAS or ISRE sequences in the
iNOS gene(19) , but may couple receptors for substances like
thrombin and thromboxane to regulation of iNOS expression through
activation of other sequences shared by cytokines or
lipopolysaccharide.