1Institute of Molecular Medicine, University of TexasHouston Health Science Center; and 2Department of Integrative Biology and Pharmacology, University of Texas Medical School at Houston, Houston, Texas
Submitted 10 February 2005 ; accepted in final form 4 May 2005
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ABSTRACT |
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As a NO receptor, sGC exists as an obligatory heme-containing heterodimeric protein composed of - and
-subunits. Although sGC
2- and
2-subunits do exist, the
1- and
1-subunits appear to be the predominantly expressed isoforms. Each sGC subunit is the product of an independent gene, but the genes for the
1- and
1-subunits are located adjacently on the same chromosome in mammals (26).
Regulation of the sGC1 and sGC
1 mRNA and protein subunits has been reported in several in vivo and cell culture models through various pathways, first in response to cyclic nucleotides or NO donors (20, 27). We also have demonstrated that the mRNA for the sGC
1- and
1-subunits rapidly decreased in the uterus of estradiol-treated rats (13). Others have shown that nerve growth factor (NGF) treatment of PC12 cells and TNF-
/IL-1
treatment of rat pulmonary artery smooth muscle cells decreased the expression of the sGC transcripts (17, 28). However, many of the signaling mechanisms and cell-type specificities involved in the regulation of sGC expression have yet to be explained.
Various disease states are thought to involve decreased expression of sGC. For example, models of hypertension, atherosclerosis, and Alzheimer's disease all correlate with decreased levels of sGC (2, 9, 19, 24). Downregulation of sGC also has been suggested in other processes, such as NGF-mediated neuronal differentiation and negative feedback regulation of NO-cGMP signaling (10, 17). The stimulation and cell type-specific effects on sGC regulation add to the already complex NO-cGMP signaling paradigm. Therefore, a compound that could affect sGC gene regulation would be useful as a tool to dissect the important cell signaling and gene regulatory pathways involved.
This article demonstrates that the c-Jun NH2-terminal kinase JNK II inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP-600125) can block inhibition of sGC1 mRNA expression by NGF in PC12 cells. As a result, sGC
1 protein levels and NO-stimulated sGC activity in NGF-treated cells were preserved. Additional experiments imply that JNK may affect sGC
1 mRNA regulation.
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EXPERIMENTAL PROCEDURES |
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Cell cultures.
Rat pheochromocytoma (PC12) and rat fetal lung (RFL-6) fibroblast cells were purchased from the American Type Culture Collection (Manassas, VA). PC12 cells were cultured in RPMI 1640 containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 10% heat-inactivated horse serum, and 5% fetal bovine serum. RFL-6 cells were cultured in Ham's F-12K medium containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, and 20% fetal bovine serum. Cells were maintained in a 37°C and 5% CO2 atmosphere. For experiments, PC12 cells were plated at a density of 8 x 105 cells/well in six-well dishes, incubated for 3 days in culture medium, and treated without further manipulation. RFL-6 cells were cultured in six-well plates until confluent and also were treated without further manipulation. In both cell lines, the inhibitors SP-600125, PD-98059, or SB-203580 were added to the culture medium 30 min before the addition of NGF (50 ng/ml), forskolin (10 µM), or the combination of TNF- (100 ng/ml) and IL-1
(20 ng/ml).
Real-time RT-PCR analysis of endogenous sGC1 mRNA.
sGC
1 mRNA levels were determined using a rat-specific real-time RT-PCR assay as previously described (13). Briefly, 100 ng of total RNA for each sample, extracted using UltraSpec RNA isolation solution (Biotecx Laboratories, Houston, TX), was reverse transcribed using gene-specific oligonucleotides. Real-time PCR reactions were performed using an Applied Biosystems Prism 7700 sequence detector (PerkinElmer, Boston, MA). The data were analyzed using Sequence Detector software. sGC
1 mRNA levels were normalized to the housekeeping gene 36b4 (14), which also was detected using a real-time RT-PCR assay (a generous gift from Dr. Greg Shipley, University of Texas at Houston, Houston, TX), and expressed as relative mRNA expression per 100 ng of total RNA in each sample. 36b4 expression levels were unchanged between samples and did not change in PC12 or RFL-6 cells treated with NGF, TNF-
/IL-1
, SP, PD, or SB or in their combination treatments in this study (data not shown). To detect sGC
1 mRNA levels in transiently transfected RFL-6 cells, the plates were washed twice in PBS, followed by RNA isolation. RNA was then used directly in the RT-PCR assay, and sGC
1 mRNA levels were normalized to 36b4.
Immunoblot analysis.
Total protein lysates were obtained by washing the cells twice in PBS and then immediately lysing them with 1x Laemmli loading buffer containing a protease inhibitor cocktail (RFL-6 cells) or hypotonic lysis buffer containing 20 mM Tris (pH 8.0), 500 µM sodium orthovanadate, 80 mM -glycerophosphate, and protease inhibitor cocktail (PC12 cells). For RFL-6 cells, equal volumes of lysate were fractioned using 10% SDS-PAGE and then electrophoretically transferred to immunoblotting polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). PC12 cell lysates were quantified using the Bradford method, and 100 µg/lane were loaded using the same technique. Membranes were blocked at room temperature for 30 min with 5% milk and incubated overnight with antibodies to c-Jun (0.4 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Ser63 and phospho-Ser73 c-Jun (1:1,000 dilution; Cell Signaling Technology, Beverly, MA), ERK2 (0.4 µg/ml, Santa Cruz Biotechnology), sGC
1 (1:10,000 dilution; Sigma), hemagglutinin (HA, 1:10,000 dilution; Roche, Indianapolis, IN), or GAPDH (1:10,000 dilution, clone 6C5; a generous gift from Dr. A. Katrukha, Hytest, Turku, Finland) in blocking buffer or 5% BSA in place of milk for the antiphosphorylated c-Jun antibodies. Membranes were then incubated with secondary antibodies to peroxidase-conjugated rabbit (for c-Jun, phospho-c-Jun, sGC
, and ERK2 antibodies, 1:5,000 dilution; Amersham, San Francisco, CA) or mouse (for HA and GAPDH antibodies, 1:5,000 dilution; Amersham) for 1 h at room temperature, followed by ECL detection (Amersham).
Assay of sGC activity. Frozen cell preparations were thawed on ice and sonicated in 50 mM Tris·HCl buffer, pH 7.8, containing 1 mM EDTA, 1 mM EGTA, 10% glycerol, and protease inhibitor cocktail (Roche). The homogenates were centrifuged at 100,000 g for 1 h at 4°C, and the supernatant (2080 µg protein/sample assayed using the Bio-Rad dye-binding method) was used to measure sGC activity. Incubation medium (final volume 50 µl) contained 50 mM triethanolamine-HCl buffer, pH 7.4, 5 mM MgCl2, 1 mM 3-isobutyl-1-methylxanthine, 1 mg/ml BSA, 1 mM GTP, 5 mM creatine phosphate, and 50 µg/ml creatine kinase. Reactions were started by addition of the cell extract, followed with or without the NO donor diethylamine (DEA)-NO (100 µM). Samples were incubated for 10 min at 37°C and boiled for 2 min, and the amount of cGMP formed was determined by performing a radioimmunoassay, with the results expressed as picomolar cGMP formed per minute per milligram of protein (7).
Transient transfection of RFL-6 cells.
RFL-6 cells were plated in six-well dishes such that their confluence reached 80% after 1824 h in maintenance medium. After this incubation, each well was transfected with 1 µg of DNA and 3 µl of Fugene-6 (Roche Diagnostics, Indianapolis, IN) per well according to the manufacturer's protocol. Four hours after addition of the transfection reagent and the DNA to each well, the medium was aspirated and new medium was added. Cells were collected for RNA isolation after 24 h. Using a GFP expression construct, we determined the transfection efficiency to be
3545%.
Statistical analyses. Data are presented as means ± SE or SD. ANOVA was used for all analyses, followed by the Scheffé's post hoc test for comparison between groups.
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RESULTS |
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SP-600125 blocks the NGF-mediated decrease of sGC1 protein and activity levels in PC12 cells.
Because SP-600125 blocked the decrease of sGC
1 mRNA expression by NGF, we were curious to determine whether this was also reflected in the sGC
1 protein expression and, importantly, in NO-stimulated sGC activity. PC12 cells were pretreated with SP-600125 and stimulated with NGF for 24 h, a duration within which sGC protein and activity levels were decreased (17). Figure 2A shows that after a 24-h incubation with NGF, sGC
1 protein expression levels were noticeably decreased, an effect that was blocked by SP-600125. Densitometry results from three independent Western blot experiments revealed that NGF treatment resulted in the significant decrease of sGC
1 protein to
60% of the controls, which were completely inhibited and even slightly increased in the presence of SP-600125 (Fig. 2B).
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SP-600125 does not block forskolin-mediated decrease of sGC1 mRNA levels in PC12 cells.
Forskolin treatment of various cell types, including PC12 cells, causes decreased sGC mRNA expression through a cAMP-dependent mechanism (17, 20). As shown in Fig. 4, SP-600125 had no effect on the decrease in sGC
1 mRNA by forskolin. This finding demonstrates that the effects of SP-600125 in blocking sGC
1 regulation are specific to a NGF-stimulated pathway in PC12 cells. Because there was no increase in sGC
1 mRNA, this also supports the hypothesis that the increased mRNA levels with SP-600125 described in Fig. 1 are not likely due to nonspecific induction of sGC
1 gene transcription.
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Expression of a constitutively active JNKK2-JNK1 fusion protein causes sGC1 mRNA levels to decrease in RFL-6 cells.
To investigate whether JNK signaling may lead to inhibition of sGC
1 mRNA expression in RFL-6 cells, we used a gene delivery approach with a plasmid containing a constitutively active JNKK2-JNK1 fusion protein, which specifically stimulates the JNK signaling pathway and not the ERK or p38 pathways (32). Therefore, we transiently transfected RFL-6 cells with the plasmid that expresses the JNKK2-JNK1 fusion protein or the parent plasmid, SR
, and collected samples for sGC
1 mRNA analysis after 24 h. Cultures transfected with the JNKK2-JNK1 fusion protein expressed 35% less endogenous sGC
1 mRNA transcripts compared with cells transfected with the parent control plasmid SR
(Fig. 6A). In a parallel experiment, cells were transiently transfected with a plasmid that expressed a constitutively active ERK2 protein (5). Compared with the parent control plasmid, pCMV5, the endogenous sGC
1 mRNA expression levels were not significantly changed by constitutively active ERK2 expression after the 24-h transfection period (Fig. 6A). Considering that the transfection efficiency in RFL-6 cells was
3545%, we did not expect more of a decrease in sGC
1 mRNA expression in the cell preparations transfected with the JNKK2-JNK1 plasmid. During the transfection incubation, the cell number did not change among the cells transfected with JNKK2-JNK1, ERK2, or control plasmids, and the total RNA collected from cells also was not significantly different. Therefore, we do not think that cell viability was a factor in the changed expression level of sGC
1 mRNA. Both the constitutively active JNKK2-JNK1 and ERK2 plasmids were expressed at abundant levels in a parallel experiment in which they were detected using anti-HA (for the JNKK2-JNK1 fusion protein) and anti-ERK2 (for the active ERK2 protein) antibodies (Fig. 6B).
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DISCUSSION |
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Previously, it was shown that NGF treatment of PC12 cells resulted in the inhibition of sGC expression, which did not occur in a stable PC12 cell line that expressed a dominant inhibitory Ras mutant protein (17). Ras-dependent cell signaling has been identified as having the ability to proceed through any of the downstream MAPK effectors, including ERK, JNK, or p38 (29). Herein we have shown that not only was sGC downregulation blocked by SP-600125 but also expression of a constitutively active JNK enzyme caused sGC downregulation. We also have collected data indicating that UV radiation, a common stress signal known to activate JNK signaling (23), caused sGC1 mRNA levels to decrease after 4 h (data not shown). It is interesting that many of the conditions that lead to sGC mRNA regulation also have been shown to stimulate the JNK signaling pathway, including NGF, estradiol, and the cytokines TNF-
and IL-1
(16, 22, 30). Our results suggest that JNK signaling could be important in the regulation of sGC, but more work needs to be done to expand on the data reported herein. The specificity of SP-600125 to the JNK signaling pathway has been challenged (1), and we have not ruled out the contribution of a nonspecific effect of this compound. Because so little is known about the pathways involved in sGC mRNA regulation, additional studies are required to determine whether a nonspecific effect independent of JNK could contribute to sGC regulation.
It is reasonable to predict that sGC gene regulation will prove to be complex, dependent not only on the stimulation but also on the cell or tissue type as well as the intracellular machinery. For instance, estradiol treatment of rats caused sGC regulation in the uterus, but not in several other tissues known to contain estrogen receptors (13). Each cell may also contain more than one signaling pathway that can facilitate sGC regulation, exemplified in this study by the ability of SP-600125 to block sGC regulation by NGF but not by forskolin. It is possible that distinct pathways that regulate sGC expression could converge on a common downstream mechanism. Of note, both cGMP- and cAMP-mediated sGC mRNA regulation was shown to involve the decreased expression of the RNA binding protein HuR (10, 11). However, cGMP does not necessarily cause sGC regulation in all cell types, such as PC12 cells (17). We analyzed HuR protein expression levels in RFL-6 cells treated with either forskolin or TNF-/IL-1
after 4 and 8 h of treatment. Our findings indicate that HuR protein levels did not decrease in RFL-6 cells in these conditions. This finding could imply that HuR may not have been involved in sGC regulation in this study. Importantly, these studies were performed in a different cell type that may use distinct signaling pathways.
The complexity and importance of sGC gene regulation will probably become apparent as more studies are conducted. For example, while our present study focused on the regulation of the sGC1-subunit, there are three additional sGC isoforms, including
2,
1, and
2. Whether these isoforms are regulated in a similar manner remains to be resolved. It would be interesting to determine whether preservation of sGC expression could be achieved using SP-600125 in the setting of disease states in which sGC downregulation is found, such as
-amyloid deposition in Alzheimer's disease (3). JNK pathway inhibitors are already being tested for their therapeutic potential in neurodegenerative and inflammatory conditions (4). Work by Fiscus et al. (6) suggests that preservation of cGMP levels in neuronal cell types may play a role in the survival of these cells and could be a mechanism that contributes to the beneficial effects of JNK pathway inhibitors.
We hope that the data described herein will shed some light on the signaling mechanisms of sGC1 gene regulation. In addition, the SP-600125 compound will provide a useful tool to study the intracellular components involved in sGC regulation in culture models, which may help to improve the understanding of the role of sGC in the various disease states mentioned above.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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