(Received for publication, September 10, 1996, and in revised form, December 2, 1996)
From the § Cardiovascular Research Center and the Cardiac Unit of the General Medical Service at the Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Charlestown, Massachusetts 02129
Nitric oxide (NO) modulates neurotransmission in
the central and peripheral nervous systems. NO acts, in part, by
stimulating cGMP production by soluble guanylate cyclase (sGC), an
obligate heterodimer composed of and
subunits. To investigate
mechanisms that regulate responsiveness to NO in the nervous system,
sGC regulation was examined in a rat pheochromocytoma cell line (PC12) exposed to nerve growth factor (NGF). NGF decreased sGC
1 and
1
subunit mRNA and protein levels as well as NO-stimulated sGC enzyme
activity. The NGF-mediated decrease in sGC subunit mRNA levels was
blocked by 5
-deoxy-5
-methylthioadenosine (an inhibitor of NGF-induced
tyrosine phosphorylation). NGF did not decrease sGC subunit mRNA
levels in PC12 cells containing a mutant Ras protein that blocks
Ras-dependent intracellular signaling. Incubation of PC12
cells with a transcription inhibitor (actinomycin D) or protein
synthesis inhibitors (anisomycin or cycloheximide) attenuated the
ability of NGF to decrease sGC subunit mRNA levels. Moreover, sGC
subunit mRNA levels decreased more rapidly in NGF-treated cells
than in actinomycin D-treated cells, suggesting that NGF decreases sGC
subunit mRNA stability. Thus, NGF decreases sGC subunit mRNA
levels via mechanisms that are dependent on protein tyrosine
phosphorylation and Ras activation. The effect of NGF on sGC subunit
mRNA stability appears to be transcription- and translation-dependent. Modulation of sGC subunit levels and
enzyme activity in PC12 cells suggests that NO responsiveness may be regulated in the nervous system by NGF.
In central and peripheral nervous systems, nitric oxide (NO)1 has an important role as a physiologic messenger molecule (1, 2). The enzymes responsible for NO synthesis, NO synthases, are present in selected neuronal populations in the brain, retina, adrenal medulla, and intestine as well as nerve fibers in the posterior pituitary (3). Many of the effects of NO on neuronal functions are mediated by the intracellular second messenger, cGMP. cGMP regulates neurotransmitter release (4) and appears to have an important role in long term potentiation in hippocampal pyramidal neurons (5). In addition, cGMP has been reported to repress gonadotropin-releasing hormone gene expression in a hypothalamic cell line (6). Moreover, NO has been observed to increase viability of trophic factor-deprived PC12 cells and sympathetic neurons via a cGMP-dependent mechanism (7).
NO stimulates soluble guanylate cyclase (sGC) to synthesize cGMP. sGC
is an obligate heterodimer composed of and
subunits with two
isoforms of each subunit identified in the rat genome:
1,
2,
1, and
2 (8). cGMP interacts with several intracellular targets
including protein kinases, ion channels, and phosphodiesterases. cGMP
is metabolized to relatively inactive GMP by phosphodiesterases.
Although the regulation of NO production in the nervous system has been extensively investigated, the mechanisms regulating responsiveness to NO are less completely understood. We (9) and others (10) observed that agents which increase intracellular cAMP decrease sGC subunit mRNA levels and decrease the ability of cells to synthesize cGMP in response to NO-donor compounds. Ujiie et al. (11) reported that agents which increase intracellular cGMP concentrations also decrease sGC enzyme activity and subunit mRNA levels in rat medullary interstitial cells. Whether or not NO responsiveness is regulated in the nervous system has not been reported.
The rat pheochromocytoma cell line, PC12, is an extensively characterized model used for the study of cell differentiation and proliferation in response to receptor-mediated tyrosine kinase activation (12). Recently, Peunova and Enikolopov (13) reported that differentiation of PC12 cells in response to nerve growth factor (NGF) was associated with increased expression of NO synthases. In the present study, we investigated the effect of NGF on sGC function in PC12 cells. sGC subunit mRNA and protein levels as well as sGC enzyme activity decreased in PC12 cells exposed to NGF. Evidence is presented that NGF decreases sGC subunit mRNA levels via mechanisms that are tyrosine kinase- and Ras-dependent.
NGF (2.5 S, isolated from mouse submaxillary
glands) was purchased from Collaborative Biomedical Products (Bedford,
MA). Epidermal growth factor (EGF, isolated from mouse submaxillary
glands), basic fibroblast growth factor (bFGF, isolated from bovine
pituitary glands), sodium nitroprusside, actinomycin D, anisomycin,
cycloheximide, 5-deoxy-5
-methylthioadenosine (MeSAdo),
NG-nitro-L-arginine methyl
ester (L-NAME), 8-bromo-cGMP, dibutyryl cGMP, dibutyryl
cAMP, forskolin, isobutylmethylxanthine (IBMX), phorbol 12-myristate
13-acetate (PMA), and A23187 were purchased from Sigma.
PC12 rat pheochromocytoma cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 culture medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 112 units/ml penicillin, and 112 units/ml streptomycin. Adherent cells were passaged every 3-4 days into 100-mm tissue culture dishes at a density of 1 × 107 cells/plate, and cells were used 2 days following passage.
M-M17-26, a PC12 cell line expressing a dominant inhibitory mutant Ras (a point mutation in codon 17 resulting in the substitution of serine by asparagine) (14), was generously provided by Dr. G. M. Cooper (Dana Farber Cancer Institute, Boston, MA). M-M17-26 cells were maintained in the same medium as PC12 cells supplemented with geneticin (G418, 0.4 mg/ml).
RNA Blot HybridizationRNA was isolated from PC12 cells by
the guanidine isothiocyanate-cesium chloride method (15). Fifteen µg
of RNA were fractionated in 1.5% agarose-formaldehyde gel, transferred
to MAGNA CHARGE membranes (Micron Separations, Westborough, MA), and
cross-linked by exposure to UV light. Membranes were hybridized
overnight at 42 °C with either a 32P-radiolabeled
0.9-kilobase EcoRI/SacI restriction fragment of the rat sGC 1 subunit cDNA or a 32P-radiolabeled
1.4-kilobase KpnI/BglII restriction fragment of the rat sGC
1 subunit (both cDNAs generously provided by Dr. M. Nakane, Abbott) (16). Membranes were washed at high stringency in a
solution containing 3 mM sodium citrate, 30 mM
sodium chloride, and 0.1% sodium dodecyl sulfate at 65 °C and were
exposed to x-ray film. To quantitate the amount of RNA loaded on the
agarose-formaldehyde gels, the membranes were subsequently hybridized
with a 10-fold molar excess of a 32P-radiolabeled
oligonucleotide complementary to rat 18 S ribosomal RNA (17). In some
experiments, RNA blots were also hybridized with radiolabeled probes
derived from cDNAs that encoded rat c-jun or
c-fos (both kindly provided by Dr. T. Curran, Roche
Institute of Molecular Biology, Nutley, NJ) (18). Autoradiograms were scanned using a Color Image Scanner (Seiko Epson Corp., Japan). All RNA
blots shown are representative of at least three similar experiments.
PC12 cells were washed
twice with 10 ml of ice-cold phosphate-buffered saline and harvested by
scraping with a rubber policeman into buffer that contained 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 2 mM
phenylmethylsulfonyl fluoride (TED buffer). Cell membranes were
disrupted by passing through a 22-gauge needle 10 times. Cell extracts
were centrifuged at 100,000 × g for 30 min at 4 °C.
Cell supernatants containing 50 µg of protein were subjected to 8%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
transferred electrophoretically to nitrocellulose filters (Micron
Separations). Filters were blocked in phosphate-buffered saline
containing 5% nonfat milk at room temperature for 1 h and then
incubated with an antiserum directed against the rat 1 sGC subunit
(provided by Dr. M. Nakane) or with an immunoaffinity-purified polyclonal antiserum directed against the sGC
1
subunit2 for 1 h at room temperature.
Bound mouse and rabbit antibodies were detected by incubation of
filters with goat anti-mouse immunoglobulin-horseradish peroxidase
(Amersham Life Sciences, Inc.) and horseradish peroxidase-protein A
(Boehringer Mannheim), respectively, for 1 h at room temperature and were visualized using chemiluminescence (Enhanced Chemiluminescence Kit, Amersham Life Sciences, Inc.).
Protein concentrations in cell extracts were measured using the Bio-Rad dye concentration reagent (Bio-Rad) and bovine serum albumin as a standard.
Soluble Guanylate Cyclase Enzyme ActivitysGC activity was measured as described previously (20). Briefly, PC12 cells were extracted in TED buffer, and cell supernatants were prepared as described above. Cell extracts (10 µg) were incubated in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 4 mM MgCl2, 0.5 mM IBMX, 7.5 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 1 mM GTP with or without 1 mM sodium nitroprusside for 10 min at 37 °C. The reaction was terminated by addition of 0.9 ml of ice-cold 0.05 M HCl and boiling for 3 min. The concentration of cGMP in the reaction mixture was measured using a commercial radioimmunoassay kit (Biomedical Technologies Inc., Stoughton, MA). sGC enzyme activity is expressed as pmol of cGMP produced/min/mg of protein in the cell extract supernatant.
Characterization of Soluble Guanylate Cyclase Regulation by Nerve Growth Factor in a Rat Pheochromocytoma Cell Line
NGF Decreases sGC Subunit mRNA Levels in PC12 CellsTo investigate the effect of NGF on sGC subunit gene
expression, sGC subunit mRNA levels were measured in PC12 cells
incubated with and without 100 ng/ml NGF for 2-24 h. NGF decreased sGC
1 and
1 subunit mRNA levels (Fig. 1,
Panel A). Decreases in sGC subunit mRNA levels were
evident within 2 h after exposure to NGF, and minimum levels were
detected at 4 h. After 24 h of continuous exposure to NGF,
sGC subunit mRNA levels returned toward baseline. The NGF-mediated
decrease in sGC subunit mRNA levels was concentration-dependent (Fig. 1, Panel B). Decreased sGC subunit mRNA levels
were evident in PC12 cells exposed for 4 h to as low as 1 ng/ml
NGF, and 10 ng/ml produced a near-maximal effect.
NGF Decreases NO-activated sGC Enzyme Activity in PC12 Cells
To ascertain whether the NGF-mediated decrease in sGC
subunit mRNA levels was associated with changes in sGC enzyme
function, basal and NO-stimulated sGC enzyme activities were measured
in extracts of PC12 cells exposed to NGF. Basal sGC enzyme activity in
PC12 cells was low and was not altered in PC12 cells exposed to 100 ng/ml NGF for 2-24 h. In extracts from untreated PC12 cells, sodium
nitroprusside, a NO-donor compound, increased sGC enzyme activity
25-fold. NO-stimulated sGC enzyme activity decreased in PC12 cells
exposed to NGF for 24 h but not in cells exposed for 2, 4, and
8 h (data not shown). The effect of NGF on NO-stimulated sGC
enzyme activity was dose-dependent; exposure of PC12 cells to 10 and 100 ng/ml NGF decreased NO-stimulated sGC enzyme activity by
25 and 50%, respectively (*, p < 0.05 and **,
p < 0.01, respectively) (Fig. 2).
sGC subunit protein levels were measured in PC12 cells incubated in the
presence and absence of NGF. Consistent with the observation that
prolonged exposure to NGF was necessary to decrease NO-stimulated sGC
enzyme activity, decreased sGC subunit protein levels were evident in
PC12 cells incubated with NGF for 24 h but not in cells exposed
for 2, 4, and 8 h (data not shown). The effect of NGF on levels of
both sGC subunits was dose-dependent; decreased subunit protein levels were detected in cells exposed to NGF in concentrations of 10 ng/ml or greater (Fig. 3).
Signal Transduction Pathways Regulating sGC Subunit Gene Expression in PC12 Cells Exposed to NGF
Modulation of sGC Subunit Gene Expression Is Tyrosine Phosphorylation-dependentThe biological response to
NGF is initiated by autophosphorylation of its high affinity receptor
(p140) on tyrosine residues (21). To determine whether the mechanisms
involved in the regulation of sGC subunit gene expression are tyrosine
phosphorylation-dependent, sGC subunit mRNA levels were
measured in PC12 cells pretreated with 3 mM MeSAdo (a
methyltransferase inhibitor that inhibits tyrosine phosphorylation of
the NGF receptor as well as other proteins in PC12 cells exposed to
NGF) (21). At this concentration, MeSAdo effectively inhibited
NGF-induced tyrosine phosphorylation in PC12 cells (data not shown).
MeSAdo also blocked the NGF-induced decrease in sGC subunit mRNA
levels (Fig. 4). Incubation of PC12 cells with two other
growth factors that signal through receptor tyrosine phosphorylation,
EGF and bFGF, in concentrations sufficient to induce protein tyrosine
phosphorylation (22) and c-fos gene expression in PC12 cells
(14), failed to alter sGC subunit gene expression (Fig.
5).
Regulation of sGC Subunit Gene Expression by NGF Is cGMP-, cAMP-, Calcium-, NO-, and Protein Kinase C-independent
To further
characterize the intracellular signaling mechanisms participating in
the regulation of sGC subunit gene expression by NGF, sGC subunit
mRNA levels were measured in PC12 cells exposed to agents that
modulate several regulatory pathways. Incubation of PC12 cells with
membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, did
not decrease sGC subunit mRNA levels (Fig. 5). Moreover,
pretreatment of PC12 cells with 1 mM L-NAME, a
NO synthase inhibitor) did not attenuate the ability of NGF to
decrease sGC subunit mRNA levels, suggesting that NO synthase activity did not account for the effect of NGF (Fig. 6).
PC12 cells were also exposed to PMA (100 nM), an activator
of protein kinase C, and A23187 (5 µM), a calcium
ionophore. sGC subunit mRNA levels were not altered in PC12 cells
exposed to either PMA or A23187 (Figs. 5 and 6). In addition,
pretreatment of PC12 cells with 10 µM bisindolylmaleimide
I, a protein kinase C inhibitor, did not block the effect of NGF on sGC
subunit gene expression (data not shown).
To determine the effect of cAMP on sGC subunit mRNA levels in PC12 cells, cells were incubated for 4 h with 1 mM dibutyryl cAMP (a membrane-permeable cAMP analogue), 10 µM forskolin (an adenylate cyclase agonist), or 1 mM IBMX (a phosphodiesterase inhibitor). All three agents decreased sGC subunit mRNA levels (Fig. 5). However, incubation of PC12 cells with 100 ng/ml NGF for 1-30 min did not increase intracellular cAMP levels, whereas 10 µM forskolin increased intracellular cAMP levels 15-fold after 30 min (data not shown). These results suggest that although increased intracellular cAMP concentrations can decrease sGC subunit mRNA levels, they do not account for the effect of NGF on sGC subunit gene expression.
NGF Regulates sGC Subunit Gene Expression via Ras ActivationRas activation has a critical role in signaling many
of the effects of NGF binding to its receptor. To study the role of Ras in the regulation of sGC subunit gene expression by NGF, M-M17-26, a
PC12 cell line expressing a dominant inhibitory Ras mutant, was used.
Whereas NGF decreased sGC subunit mRNA levels in wild-type PC12
cells (Fig. 7, Panel A, lanes 3 and 4), sGC subunit mRNA levels did not differ in
M-M17-26 cells incubated in the presence or absence of NGF (lanes
5-8). In contrast, increased intracellular cAMP decreased sGC
subunit mRNA levels in M-M17-26 cells (Fig. 7, Panel B),
suggesting that regulation of sGC subunit gene expression by cAMP is
not mediated via Ras activation. The inability of NGF to decrease sGC
subunit mRNA levels in M-M17-26 cells was not due to absence of the
NGF receptor since tyrosine phosphorylation of cellular proteins,
including a 140-kDa protein likely to be the NGF receptor, was observed
in M-M17-26 cells exposed to NGF (data not shown). Furthermore,
exposure of M-M17-26 cells to NGF stimulated c-fos gene
expression in a time-dependent manner (Fig. 7, Panel
C), as described previously (14).
Molecular Mechanisms Involved in the Regulation of sGC Subunit Gene Expression in NGF-treated PC12 Cells
Destabilization of sGC Subunit mRNAs by NGF Is Dependent on Gene TranscriptionTo investigate the role of mRNA stability
on the effect of NGF on sGC subunit mRNA levels, we examined the
effect of actinomycin D, an RNA polymerase inhibitor, on sGC subunit
gene expression in PC12 cells. The levels of sGC 1 and
1 subunit
mRNAs did not change in PC12 cells exposed to 10 µM
actinomycin D for up to 6 h. In contrast, c-jun
mRNA levels decreased more than 50% within 1 h (Fig.
8, left panel). sGC subunit levels decreased
more rapidly in PC12 cells exposed to NGF than in cells exposed to
actinomycin D (see Fig. 1, Panel A), suggesting that NGF
decreases sGC subunit mRNA stability. Moreover, incubation of PC12
cells with actinomycin D blocked the ability of NGF to decrease sGC
subunit mRNA levels (Fig. 8, right panel). These results
suggest that NGF decreases the stability of sGC subunit mRNAs via a
transcription-dependent mechanism.
Destabilization of sGC Subunit mRNAs by NGF Is Dependent on Protein Synthesis
To further examine the mechanisms by which NGF
decreases sGC subunit mRNA stability, PC12 cells were pretreated
with cycloheximide or anisomycin, protein synthesis inhibitors, before
exposure to NGF. Inhibition of protein synthesis completely blocked the
ability of NGF to decrease sGC 1 subunit mRNA levels and
partially blocked the decrease in
1 subunit mRNA levels (Fig.
9). These data suggest that NGF decreases sGC subunit
mRNA levels through mechanisms that involve both RNA transcription
and de novo protein synthesis.
Soluble guanylate cyclase is a critical component in NO-mediated
signal transduction. In this study, regulation of sGC subunit gene
expression was investigated in a rat pheochromocytoma cell line, PC12
cells, exposed to the neurotrophic factor NGF. NGF decreased levels of
both sGC 1 and
1 subunit mRNAs in a dose- and
time-dependent manner. The half-maximal effect of NGF on
sGC subunit gene expression was observed between 1 and 10 ng/ml, which is consistent with the reported dissociation constants
(Kd) of the two classes of NGF receptors
(approximately 10
9 M, 25 ng/ml) (23, 24) and
the EC50 for the activation of mitogen-activated protein
kinase by NGF (3 × 10
10 M, 10 ng/ml)
(25) in PC12 cells. Decreased sGC subunit mRNA levels were observed
within 2 h after addition of NGF and reached lowest levels within
4 h. Decreased sGC subunit mRNA levels were associated with
decreased sGC subunit protein levels and NO-activated enzyme activity.
However, the decrease in subunit protein levels and enzyme activity was
detectable only after 24 h of continuous exposure to NGF. These
results suggest that both sGC subunits are relatively stable cellular
proteins in PC12 cells.
Although two forms of NGF receptor p75 and p140, are expressed in PC12 cells, one form, p140, appears to be required for NGF-induced receptor tyrosine kinase activity, Ras activation, and immediate early gene expression (including c-fos) (26). MeSAdo, a methyltransferase inhibitor, inhibits NGF-induced tyrosine kinase activation of the NGF receptor as well as other cellular proteins (21). MeSAdo blocked the ability of NGF to decrease sGC subunit mRNA levels. These observations suggest that receptor tyrosine kinase activation, the initiating event in NGF signal transduction, is involved in the NGF regulation of sGC subunit gene expression in PC12 cells. Moreover, the tyrosine kinase-mediated regulation of sGC subunit gene expression appeared to be NGF-selective, because EGF and bFGF, agonists for two other receptor tyrosine kinases in PC12 cells, failed to modulate sGC subunit mRNA levels.
NGF is known to generate cellular responses via multiple signal transduction pathways. It has been reported that NGF increases the half-life of GAP43 mRNA in PC12 cells through a protein kinase C-dependent mechanism (27). Protein kinase C activation alone was insufficient to account for the NGF-induced decrease in sGC subunit mRNA levels because incubation of PC12 cells with PMA, a protein kinase C agonist, did not alter sGC subunit gene expression. Moreover, the inability of bisindolylmaleimide I, a protein kinase C inhibitor, to block the effect of NGF on sGC subunit mRNA levels suggested that protein kinase C activation was not required for the regulation of sGC subunit gene expression by NGF.
Similar to observations in rat fetal lung fibroblasts (9) and rat aortic smooth muscle cells (10), agents that increase intracellular cAMP were found to decrease sGC subunit gene expression in PC12 cells. However, consistent with the observations of Hatanaka et al. (28) and Buskirk et al. (29), cAMP levels were not increased in PC12 cells exposed to NGF. These results suggested that regulation of sGC gene expression by NGF is not dependent on cAMP.
Several observations led us to consider the possibility that NO and cGMP may mediate the effect of NGF on sGC subunit gene expression. First, Peunova and Enikolopov (13) observed that NGF stimulated NO synthase expression in PC12 cells. Second, Ujiie et al. (11) reported that NO donor compounds and agents that increase intracellular cGMP levels decreased sGC subunit mRNA and enzyme levels in rat medullary interstitial cells. Finally, we recently observed that sGC subunit mRNA and protein levels and sGC enzyme activity were decreased in rat pulmonary artery smooth muscle cells exposed to NO-donor compounds.2 In the present study, incubation of PC12 cells with membrane-permeable cGMP analogues did not decrease sGC subunit mRNA levels. Moreover, pretreatment of PC12 cells with L-NAME did not block the effect of NGF on sGC subunit mRNA levels. These results suggested that the effect of NGF on sGC subunit gene expression was not mediated by NO or cGMP.
Ras appears to have an important role in the regulation of sGC subunit mRNA levels by NGF. Ras is located at the inner surface of the plasma membrane and transduces signals from tyrosine kinase receptors to intracellular target molecules (30). Intrinsic GTPase activity regulates the levels of active (GTP-bound) and inactive (GDP-bound) Ras. To investigate the role of Ras in the regulation of sGC subunit gene expression, the M-M17-26 cell line (14), a PC12 cell line stably transfected with the mutant p21(Asn-17)Ha-ras gene, was used. The encoded mutant Ras is inactive due to a high affinity for GDP and likely competes with normal Ras for guanine nucleotide exchange factors, sequestering them into nonfunctional complexes (19). Incubation of M-M17-26 cells with NGF did not decrease sGC subunit mRNA levels, suggesting that the NGF effect was Ras-dependent. The presence of a functional NGF receptor in M-M17-26 cells was confirmed by the observations that NGF induced tyrosine phosphorylation of cellular proteins and stimulated c-fos gene expression. Ras activates several signaling pathways, at least one of which, the Raf/MEK/ERK protein kinase cascade, participates in PC12 cell differentiation (12). It remains to be determined whether or not NGF regulation of sGC subunit gene expression is Raf/MEK/ERK-dependent. Of note, agents that increase cAMP concentrations were able to decrease sGC subunit mRNA levels in M-M17-26 cells, suggesting that the effect of cAMP on sGC subunit gene expression is Ras-independent.
To further investigate the mechanisms regulating sGC subunit gene
expression in PC12 cells, the effect of actinomycin D on NGF-mediated
regulation of sGC subunit mRNA levels was measured. sGC subunit
mRNA levels decreased more rapidly in PC12 cells exposed to NGF
than in PC12 cells exposed to actinomycin D. These results suggest that
NGF decreased sGC subunit mRNA levels, at least in part, by
decreasing sGC subunit mRNA stability. sGC subunit mRNA levels
did not differ in PC12 cells exposed to actinomycin D alone or in cells
exposed to actinomycin D with NGF. These results suggest that NGF
decreases sGC subunit mRNA stability via a mechanism that is
dependent on RNA transcription. Exposure of PC12 cells to agents that
inhibit protein synthesis blocked, at least partially, the ability of
NGF to decrease sGC subunit mRNA levels, suggesting that regulation
of mRNA stability was protein synthesis-dependent. Of
note, we have observed that exposure of rat pulmonary artery smooth
muscle cells to NO-donor compounds destabilizes sGC subunit mRNAs
via a similar transcription/translation-dependent
mechanism.2 These results suggest that regulation of sGC
subunit mRNA stability is an important mechanism involved in
modulating sGC function in multiple cell types. Similar factors may be
involved in the coordinate destabilization of sGC 1 and
1 subunit
mRNAs in PC12 cells exposed to NGF and rat pulmonary artery smooth
muscle cells exposed to NO.
Although other components of the NO/cGMP signal transduction system contribute to NO responsiveness, the NGF-induced decrease in sGC function may be expected to decrease responsiveness to NO by decreasing cGMP synthesis. Since cGMP modulates neurotransmission (1, 6), changes in sGC function are likely to permit regulation of the ability of NO to alter neuronal activity. Exposure of PC12 cells to NGF activates expression of a program of genes leading to neuronal differentiation with neurite outgrowth and cessation of cell proliferation. Recent evidence suggests that NGF induces expression of NO synthases (beginning after 24 h of exposure), which leads to cytostasis, thereby permitting differentiation (13). It is unknown whether or not the NO-mediated inhibition of PC12 cell proliferation is cGMP-dependent. Incubation of PC12 cells with NGF appears to decrease sGC function before induction of NO synthases. It is possible that the NGF-induced decrease in sGC activity prevents excess signaling via cGMP under conditions associated with high levels of NO production.
In summary, a neurotrophic factor, NGF, decreased sGC subunit mRNA
and protein levels and decreased NO-activated sGC subunit enzyme
activity. NGF-mediated regulation of sGC subunit gene expression appeared to require NGF receptor-stimulated tyrosine kinase activity and Ras activation. The effect of NGF on sGC subunit mRNA levels did not depend on NO, cGMP, or protein kinase C. Although agents that
increase cAMP levels decreased sGC subunit mRNA levels, NGF did not
increase cAMP levels, suggesting that NGF-mediated regulation of sGC
subunit gene expression is cAMP-independent. NGF decreased sGC 1 and
1 subunit mRNA levels coordinately, at least in part by
decreasing mRNA stability. Inhibitors of RNA transcription and
protein synthesis attenuated the ability of NGF to decrease sGC subunit
levels, suggesting that NGF induces synthesis of a factor that
selectively decreases subunit mRNA stability. The decrease in sGC
function in PC12 cells exposed to NGF appeared to precede the induction
of NO synthases. These results suggest that NGF-induced changes in NO
responsiveness as well as NO synthesis may contribute to the neuronal
differentiation of PC12 pheochromocytoma cells.
We thank Dr. M. Nakane for providing the sGC
subunit cDNAs and the monoclonal anti-1 subunit antiserum, Dr.
T. Curran for providing the c-fos and c-jun
cDNA probes, and Dr. Geoffrey M. Cooper for kindly providing
M-M17-26 cells. We thank Dr. Donald B. Bloch for assistance in the
preparation of the anti-
1 subunit antiserum and for critically
reviewing the manuscript. We thank Dr. Galina Filippov for providing
technical assistance.