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Department of Physiology (X.-C.H., C.S.) and Department of Biochemistry and Molecular Biology (T.D.), College of Medicine, University of Florida, Gainesville, Florida 32610
Address all correspondence and requests for reprints to: Colin Sumners, Department of Physiology, Box 100274, 1600 Southwest Archer Road University of Florida, Gainesville, Florida 32610. E-mail: csumners{at}phys.med.ufl.edu
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Introduction |
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protein. In addition, in the same cells
Ang II (via AT1 receptors) also increases mitogen-activated
protein (MAP) kinase activities via a Gß
/Ras/Raf-1
pathway (10, 11). It is clear that the above physiological actions of Ang II involve modulation of brain catecholaminergic neurons (8, 12, 13, 14), and are associated with increased expression of the proto-oncogenes c-fos and c-jun (and their protein products c-Fos and c-Jun) in the same brain regions (15, 16, 17, 18). Evidence for the involvement of catecholamines and c-Fos/c-Jun in the AT1 receptor-mediated actions of Ang II in the brain has also come from numerous in vitro studies using primary neuronal cultures of newborn rat hypothalamus and brain stem (8, 11, 19, 20, 21). A population of catecholaminergic neurons within these cultures contain AT1 receptors (22). Studies using these cultures have shown that Ang II increases neuronal norepinephrine (NE) synthesis and re-uptake (11, 23) through its actions on AT1 receptors. Mechanisms for the longer term (hours) effects of Ang II on these processes include increased expression of tyrosine hydroxylase (TH) and NE transporter messenger RNAs (mRNAs) (11). Furthermore, studies in neuronal cultures have demonstrated that Ang II, via AT1 receptors, increases the levels of c-fos mRNA (8, 11, 19, 20), and indicate that one factor involved in the increased expression of TH and NE transporter mRNAs is c-Fos (11). This is reasonable considering that the TH gene contains an activating protein-1 (AP-1) binding site within its promotor region (24), to which c-Fos/c-Jun dimers (AP-1 transcription factor) can bind and initiate transcription (25, 26, 27). The transcriptional activity of c-Fos and c-Jun is augmented by phosphorylation (28, 29, 30, 31). Earlier studies have shown that several kinases are responsible for phosphorylating and regulating c-Fos and c-Jun. For example, c-Fos protein can be phosphorylated at Ser 374 by extracellular signal-regulated kinases (Erk1 and Erk2) (32), which are a subclass of the MAP kinases. In addition, c-Fos can be phosphorylated by Fos-regulating kinase (FRK) at Ser 133 and Thr 232 (33). c-Fos protein can also be phosphorylated at Ser 362 by ribosomal S6 kinase and protein kinase A in vitro (34, 35), and by a novel 37-kDa Fos kinase (36). In contrast, c-Jun is phosphorylated by other members of the MAP kinase family, the c-Jun NH2-terminal kinases (JNKs). JNKs phosphorylate c-Jun at Ser 63 and Ser 73 (37). It has been demonstrated that Ang II, via AT1 receptors, increases Erk1 and Erk2 activities in neuronal cultures (10, 11), and this led to the suggestion that Ang II stimulates phosphorylation of neuronal c-Fos via MAP kinases (11). However, the effects of Ang II on neuronal FRK and JNK activities have not been studied. This is important because activation of only these enzymes has been shown to augment c-Fos and c-Jun transcriptional activities (33, 37). Thus, actions of Ang II on c-Fos and c-Jun via stimulatory effects on FRK and JNK may provide an alternative pathway (distinct from MAP kinases) for the ultimate regulation of NE transporter and TH gene transcription.
The studies presented here indicate that Ang II, via AT1 receptors, causes time-dependent increases in both FRK and JNK activities in neuronal cultures. Furthermore, whereas the Ang II-stimulated increase in FRK activity occurs via a calcium-dependent PKC, the stimulation of JNK activity does not.
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Materials and Methods |
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-nicotinoyl-Tyr-Lys (N-CBZ-Arg)-His-Pro-Ile-OH (CGP42112A) was
purchased from Bachem (Torrance, CA). [-32P]ATP (3,000
Ci/mmol) was from Dupont-NEN (Boston, MA). All other peptides and
chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Neuronal cultures
Primary neuronal cocultures of newborn rat hypothalamus and
brain stem were prepared as detailed previously (8, 9). In brief,
trypsin-dissociated cells were resuspended in DMEM/10% PDHS and plated
in 35-mm Nunc (Boca Raton, FL) plastic tissue culture dishes (3.0
x 106 cells/dish). Cells were incubated in a humidified
incubator in 5% CO2/95% room air for 3 days at 37 C.
Next, cultures were incubated with fresh media containing 1
µM cytosine arabinoside to produce neuronally enriched
cultures. After 2 days, this was replaced with fresh DMEM/10% PDHS,
and cultures were allowed to grow for a further 6–9 days before use in
experiments. At this time, they contain approximately 90% neurons and
a 10% astroglia/microglia, as evidenced by immunohistochemical
staining (8). Animal use for these studies was approved by the
University of Florida Institutional Animal Care and Use Committee
(Approval no. 1751) and meets with NIH guidelines.
Preparation of glutathione-S-transferase (GST)-c-Fos and
GST-c-Jun fusion proteins
FRK and JNK activities were assayed by the ability of neuronal
extracts to phosphorylate GST-c-Fos and GST-c-Jun fusion proteins.
These fusion proteins were prepared as follows. The GST-protein
expression vectors TGEX-c-Fos (containing amino acids 1–152 of c-Fos)
and pGEX-3X-c-Jun (containing amino acids 1–93 of c-Jun) were
constructed as described previously (37). The GST-c-Fos fusion protein
contains a phosphorylation site at Ser 133 for FRK, and GST-c-Jun
fusion protein contains phosphorylation sites at Ser 63 and Ser 73 for
JNKs. Phosphorylation of these residues is sufficient for activation of
c-Fos and c-Jun. The GST-protein expression vectors were transformed
into Escherichia coli, and the GST-c-Fos and GST-c-Jun
fusion proteins were purified as follows. The bacteria containing
protein of either expression vectors were grown in 2x TD medium (5
g/liter NaCl, 7.5 g/liter yeast extract, 15 g/liter tryptone) at 37 C
until the A600 reading reached approximately 1.0. Next,
isopropyl ß-D-thiogalactopyranoside was added to the medium at a
final concentration of 1 mM, and bacteria were incubated
for 3 h at 37 C to induce GST fusion protein synthesis. The
bacteria were then ruptured by sonication, and GST fusion proteins were
purified by binding to and precipitation with glutathione agarose
beads. The concentrations of purified proteins were estimated by SDS
PAGE followed by staining with Coomassie blue.
Preparation of whole cell extracts. Neuronal cultures were washed three times with ice-cold PBS (pH 7.4), scraped from dishes, and suspended in buffer containing 25 mM HEPES (pH 7.7), 30 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DL-dithiothreitol (DTT), 20 mM ß-glycerophosphate, 0.1 mM Na3VO4, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonylfluoride. The cell suspension was rotated for 30 min at 4 C followed by centrifugation at 10,000 x g and 4 C for 10 min. The protein concentration in the supernatant was measured by a protein assay (Bio-Rad Labs., Melville, NY).
Solid-phase FRK and JNK assays
In vitro FRK and JNK activities in control, Ang II-,
and drug-treated neuronal cultures were assayed as described previously
(37). Whole cell extracts prepared as above were diluted in buffer
containing (final concentrations) 20 mM HEPES (pH 7.7), 75
mM NaCl, 2.5 mM MgCl2, 0.1
mM EDTA, 0.05% Triton X-100, 0.5 mM DL-DTT, 20
mM ß-glycerophosphate, 0.1 mM
Na3VO4, 2 µg/ml leupeptin, and 100 µg/ml
phenylmethylsulfonylfluoride. The diluted cell extracts (10 µg in 900
µl) were mixed by rotation at 4 C for 2 h with 10 µl GST
fusion protein (GST-c-Fos or GST-c-Jun)-bound glutathione agarose,
which contained approximately 20 µg GST fusion protein. The mixtures
were centrifuged at 10,000 x g for 20 secs, and the
pellet beads were washed four times with ice-cold HEPES binding buffer
(20 mM HEPES [pH 7.7), 50 mM NaCl, 2.5
mM MgCl2, 0.1 mM EDTA and 0.05%
Triton X-100). The kinase activity assays were carried out by
incubating the beads for 20 min at 30 C in 30 µl kinase buffer (20
mM HEPES [pH 7.6), 20 mM MgCl2, 20
mM ß-glycerophosphate, 20 mM
p-nitrophenylphosphate, 0.1 mM
Na3VO4, 2 mM DL-DTT) containing 20
µM ATP and 5 µCi [-32P]ATP. Control
incubations were made using GST without fused c-Fos or c-Jun proteins.
Reactions were terminated by the addition of 30 µl 3x Laemmli (38)
sample buffer and placing the tubes on ice. The reaction mixtures were
then boiled for 5 min, and the phosphorylated proteins were separated
on a 10% SDS-PAGE using the buffer system of Laemmli (38). Gels were
dried and bands corresponding to phosphorylated GST-c-Fos or GST-c-Jun
were visualized by exposure to Kodak XAR-5 films (Eastman Kodak,
Rochester, NY). Control incubations (no fusion protein present)
produced no bands. Kinase activity was measured by the incorporation of
32P from [-32P]ATP into GST-c-Fos (Ser
133) or GST-c-Jun (Ser 63 and/or Ser 73) and was quantified using a
series 400 PhosphorImager with Image Quant (Molecular Dynamics,
Sunnyvale, CA) and Microsoft Excel software (Redmond, WA).
Experimental groups and data analysis
All results are expressed as means ± SEM and
were obtained by combining data from individual experiments.
Comparisons of multiple means were made by ANOVA followed by a
Newman-Keuls test to assess statistical significance. Analyses were
preformed using Sigma Stat Software (Jandel, San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In contrast, incubation of neuronal
cultures with the selective Ang II type 2 (AT2) receptor
ligand CGP42112A (10 nM; 1–60 min), which is an agonist at
this concentration (39), elicited no significant effects on FRK
activity (n = 3 experiments; data not shown). This data implies
that the stimulatory effects of Ang II are via AT1
receptors. This was confirmed by experiments that demonstrated that the
stimulation of FRK activity by Ang II (100 nM, 15 min) was
abolished by the AT1 receptor blocker losartan (Los; 1
µM), but not by the AT2 receptor blocker
PD123,319 (PD; 1 µM) (Fig. 2).
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). This stimulatory effect was maximal
at 30 min, but declined toward control values at 45 and 60 min (Fig. 3). The AT2 receptor selective ligand CGP42112A (10
nM; 1–60 min) had no significant effects on JNK activity
(n = 3 experiments; data not shown). The stimulatory effects of
Ang II on JNK activity were completely inhibited by Los (1
µM) but not by PD (1 µM), indicating
that they are mediated via AT1 receptors (Fig. 4).
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). By contrast,
the PMA analog 4-phorbol (100 nM, 15 min), which does
not activate PKC, did not stimulate neuronal FRK activity (Fig. 5).
Furthermore, the effects of Ang II on FRK activity were abolished by
pretreatment of cultures for 30 min with GF109203X (10 nM)
(Fig. 5), which is a specific PKC inhibitor (40). These data suggest
the involvement of PKC in the stimulation of neuronal FRK by Ang II,
and it appears that a calcium-dependent PKC may be involved, because
the effects of Ang II were also blocked by chelation of intracellular
calcium with 10 mM BAPTA/AM (Figs. 5 and 6). Ang II also stimulates, via
AT1 receptors, increases in intracellular free calcium in
these cultured neurons (C. H. Gelband, P. Posner, and C. Sumners,
unpublished data). This can lead to stimulation of the
calcium/calmodulin pathway and subsequent activation of
calcium/calmodulin-dependent protein kinase II (CAM kinase II), which
is the case in vascular smooth muscle cells (41). Even though the
stimulatory action of Ang II on FRK is abolished by BAPTA/AM (Figs. 5 and 6), there was no significant effect of the calmodulin antagonist
W-7 (50 µM, 30 min) or the CAM kinase II inhibitor KN-93
(10 µM, 30 min) on this Ang II-induced response (Fig. 6).
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). Furthermore, GF109203X and KN-93 did
not alter the stimulation of JNK by Ang II, suggesting that PKC and CAM
kinase II are not involved in the activation of JNK by Ang II
in neurons (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our study FRK activity was measured by the ability of neuronal cell extracts to phosphorylate a GST-c-Fos fusion protein. It might be argued that the observed increase in phosphorylation of the GST-c-Fos fusion protein is simply due to an increase in the activity of the MAP kinases Erk1 and Erk2. These kinases are activated in cultured neurons by Ang II via AT1 receptors (10, 11), and are known to phosphorylate c-Fos (32). However, Erk1 and Erk2 phosphorylate c-Fos at Ser 374 (32). The GST-c-Fos fusion protein used here contained amino acid residues 1–152 of c-Fos and so does not include Ser 374. Therefore, the increase in phosphorylation of the GST-c-Fos fusion protein induced by Ang II is unlikely to be due to Erk1 and Erk2. Rather, the phosphorylation site in this GST fusion protein is at Ser 133, which is one of the sites phosphorylated by FRK (33). JNK activity was measured by the ability of cell extracts to phosphorylate a GST-c-Jun fusion protein that contains residues 1–93 of c-Jun. This contains phosphorylation sites at Ser 63 and Ser 73, which are known to be activated by JNK 1 and JNK 2 (37).
The overall scheme by which Ang II stimulates via AT1
receptors the formation of active c-Fos/c-Jun dimers, which
subsequently alter gene transcription, is multifaceted. The rapidity of
stimulation of FRK and JNK activities by Ang II suggests a
posttranslational activation of these enzymes. The pathways leading to
such activation are complex. For example, other studies have shown that
Ang II stimulates (possibly via Gßy) a Ras/Raf-1/MAP
kinase pathway to modulate transcription of the c-fos gene
and increase production of c-fos mRNA (11). This involves
translocation of MAP kinase to the nucleus and its phosphorylation of a
transcription factor ElkC bound at the SRE site in the c-fos
gene promotor (33). However, according to the data presented here, the
c-Fos protein that is produced following the increase in c-Fos mRNA may
be phosphorylated (and activated) by FRK, rather than MAP kinases.
Further, our data suggest that the activation of FRK by Ang II involves
a calcium-dependent PKC. This implies the involvement of
Gq-activated phospholipase C. The involvement of PKC in
the activation of FRK is consistent with our previous observation that
Ang II increases the activation of PKC in neuronal cultures (8). Based
on our data, the effects of Ang II on neuronal JNK activity probably do
not involve PKC or another calcium-dependent mechanism. This is in
contrast to findings from GN4 liver cells, in which Ang
II-stimulated JNK activity was found to occur via a calcium-dependent
mechanism (42). These data may suggest that the regulatory effects of
Ang II on JNK activity occur in a cell type-specific manner. The actual
mechanism by which Ang II stimulates activation of JNK on cultured
neurons is therefore not established. However, other studies indicate
that activation of JNK is dependent on MAP kinase kinase kinase (MEKK
or MEK kinase) (43). Furthermore, a kinase that directly activates JNK,
termed Jun kinase kinase (JNKK), was identified that functions between
MEKK and JNK (43). Therefore, MEKK and JNKK could conceivably be
involved in the activation of neuronal JNK by Ang II.
Taken together, data from the present study and from other studies are
consistent with the idea that Ang II acts at neuronal AT1
receptors to elicit a parallel set of events that lead to the
production of active (phosphorylated) c-Fos/c-Jun dimers (AP-1).
Certainly, the time courses for activation of FRK (Fig. 1
) and
increased c-fos mRNA induced by Ang II (20) are consistent.
The effects of Ang II on neuronal c-junmRNA and c-Jun
protein have not been evaluated. However, the time optimum observed for
Ang II-stimulated JNK activity (Fig. 4) is in the range to be
consistent with induction of proto-oncogenes. The phosphorylated
c-Fos/c-Jun dimers (AP-1) may then mediate modulatory actions of Ang II
on target genes such as the TH gene. In support of this idea, Ang II
has been shown to stimulate AP-1 binding activity in these cultured
neurons (19).
The fact that Ang II stimulates JNK activity in cultured neurons probably has more far-reaching consequences than neuromodulation alone. For example, the activation of JNK, along with concurrent inhibition of Erk1 and Erk2, are critical events for the induction of apoptosis (44). Thus, in combination with a decrease in Erk activities, a stimulation of JNK activity by Ang II via AT1 receptors may elicit neuronal apoptosis. We have determined that stimulation of neuronal AT2 receptors decreases Erk activities (10), and that a small population of neurons in these cultures contain both AT1 and AT2 receptors (22). Thus, costimulation of AT1 and AT2 receptors on the same neurons might lead to apoptosis of these cells. This suggestion is reasonable because costimulation of PC-12W pheochromocytoma cells with Ang II (which inhibits Erk via AT2 receptors) and NGF (which stimulates JNK) causes apoptosis (45).
Besides Erk (10, 11) and Janus Kinase/signal transducer and activator of transcription (Jak/STAT) (46) activation, our demonstration that Ang II stimulates activation of FRK and JNK illustrates the intricacies of AT1 receptor signaling pathways. It is possible that stimulation of AT1 receptors in a given neuron may activate either FRK or JNK or both. Different combinations of activation/inactivation of these kinases as well as Jak/STAT and Erk may explain the wide range of physiological and pathophysiological functions of AT1 receptors from the brain to peripheral tissues. Furthermore, AT1 receptor-mediated activation of FRK and/or JNK may also play a role in growth promotion, because this process has been shown to require the involvement of proto-oncogenes such as c-fos (47, 48, 49).
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Acknowledgments |
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Footnotes |
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Received July 16, 1997.
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