(Received for publication, February 27, 1996)
From the
The nuclear factor of activated T cells (NFAT) was discovered as
an inducible transcription factor activated by antigen stimulation of
the T cell receptor in lymphocytes. Stimulation of NFAT-mediated
transcription is now reported in both lymphoid and non-lymphoid cells
following activation of a neurotransmitter receptor. Carbachol induces
robust luciferase responses in Jurkat and pheochromocytoma PC12 cells
expressing an NFAT-luciferase reporter construct and a
G-coupled m3 muscarinic receptor. Cyclosporin blocks this
response in PC12 cells, as in Jurkat cells. In PC12 cells expressing a
G
-coupled m2 muscarinic receptor, carbachol induces
NFAT-mediated luciferase activity that is strictly dependent upon
co-expression of a chimeric G
/
subunit, which confers G
-effector coupling on
G
-linked receptors. These findings suggest that
neurotransmitters, autacoids, or hormones acting on
G
-protein-coupled receptors may serve as physiological
stimulators of NFAT in lymphoid and non-lymphoid cells.
The nuclear factor of activated T cells (NFAT), ()a
transcription factor activated by antigen stimulation of the T cell
receptor (TCR), is known mainly for its role in the co-ordinate
induction of interleukin and cytokine genes during immune cell
activation(1) . Stimulation of the TCR and subsequent
protein-tyrosine phosphorylation initiates several downstream events,
including the activation of
isoforms of phospholipase C
(PLC
). PLC
, like other PLC isoforms, catalyzes the breakdown
of phosphoinositides. The resultant co-activation of Ca
and protein kinase C (PKC) signaling pathways is required for
induction of NFAT-mediated
transcription(2, 3, 4) .
Stimulation of
receptors coupled to the G subfamily of heterotrimeric
G-proteins activates
isoforms of PLC (PLC
), and thus the
possibility exists that such receptors may participate in induction of
NFAT-dependent gene expression. Previous experiments have shown that
stimulation of an ectopically expressed muscarinic receptor in T cells
induces phosphoinositide hydrolysis(5) , interleukin 2 (IL-2)
production(6) , and lymphoid NFAT activity(7) .
G
-protein-coupled receptors are expressed in many cell
types, and NFAT or NFAT-like activities have recently been discovered
in a variety of lymphoid and non-lymphoid cells and
tissues(1, 8, 9, 10, 11, 12, 13, 14, 15) .
The present study demonstrates that NFAT can act as a downstream
effector of G
-protein-coupled receptors in the non-lymphoid
PC12 cell line, as well as in lymphoid cells.
Transcriptional responses to Ca and PKC
establish the salient features of NFAT-mediated gene expression in
lymphoid (Jurkat) and non-lymphoid (PC12) cells. These were transfected
with a well characterized NFAT-inducible luciferase reporter plasmid
containing a concatemer of the distal IL-2 gene NFAT-responsive element
on a minimal IL-2 gene promoter(16, 21) , as well as a
plasmid encoding the SV40 large T antigen (pRSV-Tag) to ensure plasmid
replication. In Jurkat cells (n = 4 experiments) and in
PC12 cells (n = 3 experiments), the respective basal
luciferase responses in the absence of added drugs (0.04 ± 0.01
and 0.06 ± 0.02 Turner light units; mean ± S.E.) are near
the lower limit of detectability. The PKC activator PMA does not
significantly stimulate luciferase activity in either cell line. In
Jurkat cells, the Ca
ionophore ionomycin induces a
modest increase in luciferase activity (5.5 ± 1.6-fold over
basal). Consistent with previous
reports(1, 7, 16, 22) , ionomycin
and PMA together elicit synergistic stimulation of luciferase activity
(550 ± 170-fold over basal) (Fig. 1a).
Similarly, in PC12 cells, modest luciferase responses elicited by
ionomycin alone (5.3 ± 1.4-fold) are potentiated by PMA (13
± 3-fold in PC12 cells) (Fig. 1b). NFAT-mediated
responses induced by ionomycin in PC12 cells exhibit dose-related
increases at concentrations up to 1 µM, which attenuate at
higher concentrations (Fig. 1c). Virtually identical
results have been obtained using ionomycin in Jurkat cells (data not
shown)(22) . These data suggest that PC12 cells, like Jurkat
cells, express a functional NFAT capable of mediating transcriptional
events in response to Ca
and PKC signaling.
Figure 1: Ionomycin and PMA synergistically increase NFAT-mediated responses in Jurkat and PC12 cells. a, in Jurkat cells transfected with NFAT-luciferase and pRSV-Tag plasmids, (n = 4 experiments, each performed in duplicate), ionomycin (iono; 0.25 µM), but not PMA (10 nM), induces significant (p < 0.01) luciferase activity compared with basal. Ionomycin and PMA together induce a luciferase response that is significantly greater than basal (p < 0.005) or than responses elicited by ionomycin (p < 0.005) or PMA (p < 0.005) alone. b, in similarly transfected PC12 cells (n = 3), ionomycin (0.5 µM), but not PMA (10 nM), induces a significant (p < 0.001) increase in luciferase activity compared with basal. The response elicited by ionomycin and PMA together is significantly greater than basal (p < 0.001) or responses elicited by ionomycin (p < 0.005) or PMA (p < 0.005). c, in transfected PC12 cells, ionomycin alone (open squares) induces a dose-dependent stimulation of luciferase activity, which is potentiated (closed squares) by 10 nM PMA. Student's t test was used for data analysis in these and all subsequent experiments.
TCR
activation of PLC defines the major known pathway for induction of
NFAT-mediated transcription in lymphocytes. Recent studies suggest that
lymphoid NFAT may also be induced through stimulation of receptors that
activate PLC
through coupling to heterotrimeric
G
-proteins(6, 7) . Carbachol (1
mM) elevates luciferase activity (410 ± 44-fold
increase; n = 5 experiments) in Jurkat cells expressing
a muscarinic m3 receptor but not in cells transfected with empty vector
as control (Fig. 2a). Similarly, carbachol elicits a
dose-dependent increase in luciferase activity that is 34 ±
7-fold over basal at the highest dose applied (1 mM carbachol)
in PC12 cells expressing the m3 receptor but not in control cells
transfected with empty vector (n = 5 experiments) (Fig. 2b). Carbachol-induced phosphoinositide
hydrolysis and luciferase responses in PC12 cells were compared in
side-by-side experiments. Both responses were elicited in cells
expressing the m3 receptor but not in those transfected with empty
vector instead (Table 1), although luciferase measurements
clearly serve as a more sensitive indicator of receptor stimulation
than inositol phosphate responses.
Figure 2:
Stimulation of G-coupled
receptors induces NFAT-mediated luciferase activity in Jurkat and PC12
cells. Cells were transfected with NFAT-luciferase and pRSV-Tag
plasmids, as well as either a plasmid encoding the m3 muscarinic
receptor or an empty vector as control. a, significantly
greater (p < 0.005) stimulation of NFAT-dependent
luciferase activity is elicited by 1 mM carbachol (+)
than by vehicle(-) in Jurkat cells transfected with the m3
receptor plasmid but not in cells transfected with empty vector instead (n = 5). b, carbachol elicits a dose-dependent
increase in luciferase activity in PC12 cells transfected with the m3
receptor (closed squares) but not in those transfected with
empty vector instead (open squares) (n = 5). c, PC12 cells were transfected with NFAT-luciferase and
pRSV-Tag plasmids, as well as either a plasmid coding for the m2
receptor or an empty vector as control. Transfections also included
either a plasmid coding for the chimeric G
/
subunit or wild type G
. Carbachol elicits a
dose-dependent increase in luciferase activity in PC12 cells
transfected with an m2 receptor plasmid (filled circles) or
empty vector (filled circles) in addition to a plasmid coding
for the chimeric G
/
subunit.
Carbachol does not increase luciferase activity in cells transfected
with an m2 receptor plasmid (filled squares) or empty vector (open squares) in addition to a plasmid coding for a wild type
G
subunit (n = 6). Student's t tests were used as statistical
analyses.
A chimeric
G/
subunit that switches the coupling
of G
-linked receptors from inhibition of adenylate cyclase
to stimulation of PLC (18) was used to establish that
heterotrimeric G-proteins are involved in muscarinic receptor-induced
NFAT responses. PC12 cells were transfected with reporter plasmids, and
either an empty vector, a plasmid coding for a wild type
G
, or a plasmid coding for
G
/
was added. A dose-related increase
in carbachol-stimulated luciferase activity is elicited in PC12 cells
expressing G
/
alone but not in
control cells expressing wild type G
(Fig. 2c) or transfected with empty vector (data
not shown). This response is likely mediated by a muscarinic m4
receptor endogenous to PC12 cells, which is known to couple negatively
to adenylate cyclase(23) . In parallel experiments, larger
dose-related increases in carbachol-stimulated luciferase activity are
elicited in cells expressing a G
-coupled m2 receptor, and
these responses are strictly dependent upon the presence of
G
/
(Fig. 2c).
Carbachol elicits both phosphoinositide hydrolysis and NFAT-luciferase
responses in side-by-side assays in PC12 cells expressing the m2
receptor in the presence, but not the absence, of the chimeric
G
/
subunit (Table 1). Since the
chimeric G-protein complements a response otherwise refractory in the
cells, the simplest interpretation of these data is that
G
-protein coupling is both necessary and sufficient for
induction of NFAT-mediated transcription by muscarinic receptors.
The immunosuppressant CsA forms a complex with a cyclophilin that
inhibits calcineurin, a Ca-dependent phosphatase
essential for the nuclear translocation of NFAT and subsequent
NFAT-dependent
transcription(22, 24, 25, 26) . As
in Jurkat cells (data not shown) (22) Ca
- and
PKC-dependent NFAT activation in PC12 cells is inhibited by CsA at
concentrations consistent with its effects on immune
cells(21, 22, 24, 26, 27, 28) .
CsA also inhibits carbachol-induced luciferase expression in cells
expressing the m3 receptor (Fig. 3). Co-dependence of NFAT
activation upon elevations of intracellular Ca
and
PKC activity and its sensitivity to CsA are principal characteristics
of NFAT activity (for reviews, see (2, 3, 4) ). Therefore, the finding that m3
receptor-stimulated luciferase responses are blocked by CsA in PC12
cells provides further evidence that G
-protein-coupled
receptors can mediate NFAT induction in non-immune cells.
Figure 3: CsA inhibits NFAT-mediated luciferase activity in PC12 cells. Luciferase responses elicited by co-application of 1 µM ionomycin and 10 nM PMA (filled squares) or by 1 mM carbachol (filled circles) or buffer (open squares) in PC12 cells transfected with NFAT-luciferase, pRSV-Tag, and m3 receptor plasmids are inhibited by increasing doses of CsA but not by vehicle containing 0.1% ethanol and 0.05% Tween 80 (no CsA). Data are presented as mean ± S.E. of 4 experiments, each performed in duplicate.
This study
establishes that NFAT transcription factors can serve as downstream
effectors for G-protein-coupled receptors that activate PLC
and that such receptors can induce NFAT activity in both lymphoid and
non-lymphoid cells. NFAT proteins or NFAT-like DNA binding and/or
transcriptional activities and NFAT isoform mRNA have been identified
in a variety of tissues (8, 14) and cell types,
including lymphoid(1, 9, 10, 12) ,
endothelial(13) , neuronal(11) , and mast
cells(15) . Receptors coupled to G
-proteins are
represented in numerous cell types and respond to a diverse array of
hormones, autacoids, and neurotransmitters. The present studies
indicate that these agents are capable of acting as physiological
activators of NFAT and that NFAT-mediated transcription represents a
heretofore unappreciated mechanism by which these receptor agonists can
modulate cellular physiology. Furthermore, immunosuppressant therapy
with CsA is limited by its toxicity to neural, hepatic, bone, and renal
systems. The present findings raise the possibility that some of these
side effects might arise in part through disruption of normal cellular
processes that are modulated by virtue of G
-protein-coupled
receptor regulation of NFAT-mediated transcriptional activities.