C-Type Natriuretic Peptide Down-Regulates Expression of Its Cognate Receptor in Rat Aortic Smooth Muscle Cells
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《内分泌学杂志》
Department of Medicine and Diabetes Center, University of California at San Francisco, San Francisco, California 94143-0540
Abstract
The C-type natriuretic (CNP) peptide signals through the type B natriuretic peptide receptor (NPR-B) in vascular smooth muscle cells to activate the particulate guanylyl cyclase activity intrinsic to that receptor and raise cellular cyclic GMP levels. In the present study, we demonstrate that CNP down-regulates the expression of this receptor leading to a reduction in NPR-B activity. Pretreatment of rat aortic smooth muscle cells with CNP reduces NPR-B activity, NPR-B protein levels, NPR2 (NPR-B gene) mRNA levels, and NPR2 promoter activity. The decrease in NPR2 promoter activity is dependent on DNA sequence present between –441 and –134 relative to the transcription start site. The reduction in NPR2 gene expression appears to operate through generation of cyclic GMP. 8-Bromo cyclic GMP, a membrane-permeable cyclic GMP analog, reduced NPR2 mRNA levels and NPR2 promoter activity. Atrial natriuretic peptide, which signals through the type A natriuretic peptide receptor (NPR-A) to increase cyclic GMP levels in these cells, also reduced NPR-B mRNA levels and inhibited NPR-B promoter activity; however, this inhibition was not additive with that produced by CNP, implying that the two ligands traffic over a common signal transduction pathway. This report provides the first documentation that CNP is capable of autoregulating the expression of its cognate receptor.
Introduction
THE NATRIURETIC PEPTIDES constitute a family of peptide hormones with important effects on cardiovascular, renal, and endocrine physiology (1). Atrial natriuretic peptide (ANP) and brain, or B-type, natriuretic peptide are produced in the atria and ventricles of the heart. They promote reductions in blood pressure, increases in urinary volume and urinary sodium excretion, and suppression of renin and aldosterone secretion (1). C-type natriuretic peptide (CNP) is produced only to a limited degree in the heart, and its role in the regulation of blood pressure or renal sodium handling appears to be limited. CNP is produced in the endothelial cells of the vasculature (2), cardiac fibroblasts (3), and the endochondral growth plate (4) in which it is thought to play important roles in the control of vascular remodeling (5, 6, 7), fibroblast proliferation (3), and linear growth of the long bones (8), respectively.
ANP and BNP signal their activity through a single receptor called the type A natriuretic peptide receptor (NPR-A), whereas CNP signals through its own receptor, the type B receptor (NPR-B). Both NPR-A and NPR-B share a similar overall structure. They each possess a large extracellular ligand binding domain linked to an intracellular particulate guanylyl cyclase domain through a single transmembrane segment. NPR-A activity is regulated at the posttranslational level (9, 10) as well as the level of gene expression (11, 12, 13). Like other peptide hormone receptors, NPR1 (NPR-A) gene expression is down-regulated by its cognate ligand, ANP, in part through inhibition of NPR1 gene transcription (11, 12). Regulation of NPR2 gene expression is less well understood. Whereas selected studies suggest that it is subject to similar posttranslational regulation (vs. that seen with NPR-A) (14, 15), relatively few studies of NPR2 gene regulation have been published (16, 17) and nothing has been shown to regulate NPR2 gene transcription or promoter activity.
In the present study, we demonstrate that CNP, the primary ligand for NPR-B in rat aortic smooth muscle (RASM) cells reduces NPR-B activity through inhibition of NPR2 mRNA levels. It does so through suppression of NPR2 promoter activity, requiring promoter elements positioned between –441 and –134 relative to the transcription start site. This down-regulation of promoter activity appears to operate through a cyclic GMP-dependent mechanism.
Materials and Methods
Materials
ANP (human, 28 amino acids) and CNP (human, 22 amino acids) were purchased from American Peptide, Inc. (Sunnyvale, CA). 3-Isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor and 8-bromo-cGMP were purchased from Sigma (St. Louis, MO). Cyclic GMP RIA kit was purchased from PerkinElmer/Life Sciences (Norwalk, CT). RNeasy minikit was obtained from QIAGEN Inc. (Valencia, CA). The luciferase assay kit was purchased from Promega (Madison, WI). Restriction enzymes were obtained from New England Biotech, Inc. (Beverly, MA). All oligonucleotides were synthesized by Invitrogen (Carlsbad, CA). Other reagents were obtained from standard commercial suppliers.
Cell culture
Neonatal RASM cells were obtained from H. Ives (University of California at San Francisco, San Francisco, CA) (18). Cells were cultured at 37 C in a 5% CO2 humidified incubator in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2% (vol/vol) broth, tryptose phosphate for 24 h. Cells were then changed to 0.5% FBS/DMEM and cultured for an additional 24 h. At that point, media were changed (0.5% FBS/DMEM) and indicated agents were added to the culture media to begin the experiment.
Measurement of CNP-stimulated cGMP levels
Confluent RASM cells were preincubated with 10–11 to 10–7 M CNP for 12 h in presence of IBMX (10–4 M). Cells (105/well) were preincubated for 10 min in media with IBMX (5 x10–4 M) as indicated. Medium was then changed to include CNP 10–8 M in the presence of the phosphodiesterase inhibitor. After 10 min the reaction was stopped by removing the medium and adding 0.3 ml of 12% trichloroacetic acid. The extraction was continued for 30 min at 4 C. The contents of the plate were collected and centrifuged to pellet particulate material. The supernatant fraction was extracted four times with 0.5 ml water-saturated ether. cGMP levels were determined by RIA after acetylation of the sample and standard using a commercial 125I-cGMP RIA kit (PerkinElmer Life Sciences). Results are reported as picomoles per well.
Preparation of crude membranes
To prepare crude RASM cell membranes, 25-cm plates at approximately 85% confluence were washed and incubated overnight with DMEM in the absence of serum. After incubation with DMEM containing 10–8 M CNP for varying periods of time, cells were washed twice with PBS, scraped into 0.5 ml of buffer containing protease inhibitors [25 mM HEPES (pH 7.4), 20% glycerol, 50 mM NaCl, 50 mM -glycerol phosphate, 2 mM EDTA, 1 μM microcystin, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin], sonicated for 6 sec with a Misonix Sonicator (Branson 2510; Farmingdale, NY), and centrifuged at 20,000 x g for 20 min at 2 C. Pellets were resuspended in buffer and centrifuged at 1000 x g for 2 min at 4 C. Pellets were then resuspended in the same buffer and stored at –80 C. Protein concentrations were estimated using a bicinchoninic acid assay or Coomassie dye binding protein assay reagent (Pierce Chemical Co., Rockford, IL).
Immunoblot analysis
Sixty micrograms of protein from the resuspended pellet was electrophoresed on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred electrophoretically onto polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences). Membranes were incubated with goat polyclonal anti-NPR-B antibody (NPR-B c-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1:80 dilution) or rabbit polyclonal anti- tubulin antibody (-tubulin sc-9104, Santa Cruz Biotechnology) (1:400 dilution) in Tris-buffered saline/Tween buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20, and 6% nonfat milk] overnight at 4 C and then washed with Tris-buffered saline/Tween buffer. Membranes were then incubated with horseradish peroxidase-conjugated bovine antigoat antibody (SC-2350; Santa Cruz Biotechnology) or goat antirabbit antibody (SC-2004; Santa Cruz Biotechnology) for 1 h at room temperature and washed with Tris-buffered saline/Tween. Blots were immersed for 1 min in enhanced chemiluminescence detection reagent (Amersham Biosciences, Piscataway, NJ) and then exposed to film. Signals were quantified using the Image Station (440CF; Kodak, Rochester, NY).
Primer and probe design
Using the Primer Express (PE Applied Biosystems, Inc., Foster City, CA) software program, primers and probe were designed to recognize rat NPR2 (amplicon is 101 bp) and glyceraldehyde phosphate dehydrogenase (GAPDH) coding sequence. Primers for this gene target were selected to contain minimal internal structure (i.e. hairpins and potential for primer-dimer formation) and compatible melting temperature (i.e. each within 1 C of the other). Probes were selected with a melting temperature that was approximately 5–10 C higher than the matching primer pair. Probes were all less than 40 bp in total length. GAPDH primers and probe were designed and optimized at ABI. Table 1 depicts the primer and probe sequences used for measurement of NPR2 and GAPDH mRNA transcript levels.
Real-time PCR analysis
Total RNA was prepared from RASM cells using the QIAGEN RNeasy kit. The cells were harvested per the manufacturer’s instructions. RNA was treated with DNase I to reduce genomic DNA contamination. Then 1.2 μg total RNA was used to reverse transcribe cDNA using the reverse transcription kit (Clontech, Palo Alto, CA). Products were examined by agarose gel electrophoresis to assure near equivalent levels of cDNA synthesis.
Rat NPR2 and GAPDH primer pairs and probes (Table 1) were synthesized by PE Applied Biosystems, Inc. Real-time PCR was performed using Taqman master mix (PE Applied Biosystems, Inc.) with an ABI 7700 (PE Applied Biosystems, Inc.). Negative controls without input cDNA were used to assess signal specificity. NPR2 transcript levels were quantified and normalized for GAPDH transcript levels in each sample. Equivalent amounts (0.5 μl) of cDNA were used for measurement of NPR2 and GAPDH gene transcripts. Thermal cycling conditions involved 2 min at 50 C and 10 min of initial denaturation at 95 C, followed by 40 cycles of two-step PCR consisting of 15 sec at 95 C and 1 min at 60 C. All samples were amplified in triplicate. Relative quantification was achieved by the comparative: 2– Ct method (19). The relative increase/decrease (fold-induction/repression) of mRNA of target x in the experimental group (NPR2) was calculated using the control group as the calibrator: 2–Ct, where cycle threshold (Ct) is: {Ct.x[treatment] – Ct.GAPDH[treatment]} – {Ct.x[control] – Ct.GAPDH[control]}.
Construction of human natriuretic peptide receptor type 2 (hNPR)-B-luciferase (Luc) expression vectors
To obtain deletion constructs, PCR was performed using LA-Taq DNA polymerase (TaKaRa La Taq), a subcloned 9-kb human genomic clone as a template, and the following sense primers: –34 hNPR2 LUC, 5'-GGGGTACCCCAGGCGACCTGACCCGGAC-3'; –84 hNPR2 LUC, 5'-GGGGTACCCCGGGAGTCGCACTCG-3'; –134 hNPR2 LUC, 5'-GGGGTACCCCAGGGAGGAGGAGAGGCAG-3'; –219 hNPR2 LUC, 5'-GGGGTACCCCACCGGGGTGGGTTTGGGAACTG-3'; –441 hNPR2 LUC, 5'-GGGGTACCCCTGAGGGACTCAGGGAGTTC-3', together with antisense primer NPR2 R1 5'-GAAGATCTTCTGTGAAGGATTCTCCCG, positioned between +148 and +166, relative to the transcription start site. We incorporated restriction sites (KpnI and BglII, respectively) on sense and antisense primers. PCR products were digested with KpnI and BglII and inserted between the KpnI/BglII sites of pGL3 (Promega), a luciferase reporter that lacks eukaryotic promoter and enhancer sequences. The structure of all constructs was confirmed by DNA sequencing.
Transfection, luciferase, and -galactosidase assays RASM cells were transiently transfected with 0.5 μg of each hNPR2 5' deletion mutant and 0.5 μg Rous sarcoma virus--galactosidase (RSV- gal) by electroporation (Gene-Pulser; Bio-Rad Laboratories, Inc., Hercules, CA) at 250 mV and 960 μF. After transfection, cells were plated in six-well plastic plates and cultured for 48 h. Cells were harvested and lysed in 100 μl cell culture lysis reagent (Promega). The protein concentration of each cell extract was measured using Coomassie protein reagent (Pierce). Cell lysates were processed (30 μg protein/sample) and assayed for luciferase as described previously (12). Measurements of -galactosidase activity were made using the Galacto-Light Plus kit (Tropix, Inc., Bedford, MA).
Statistical analysis
Data were evaluated by one-way ANOVA and the Newman-Keuls test for significance.
Results
Chronic (i.e. 12 h) treatment of cultured RASM cells with CNP, in the presence of the nonselective phosphodiesterase inhibitor IBMX (10–4 M), led to a dose-dependent reduction in the subsequent ability of CNP to stimulate cyclic GMP accumulation in these cultures. Pretreatment with 10–8 M CNP resulted in an 80% reduction in the CNP-dependent cGMP accumulation (Table 2), suggesting that CNP promotes a ligand-dependent desensitization and/or down-regulation of the NPR-B receptor.
Ligand-dependent desensitization of NPR-B has been documented previously (14); however, down-regulation of receptor expression (e.g. NPR2 mRNA and protein levels) has not been reported. In our hands, treatment with CNP led to a time-dependent reduction in NPR2 mRNA levels in cultured RASM cells (Fig. 1). This was maximal (70% inhibition) at 12 h and had partially recovered after 24 h of incubation. The reduction in NPR2 transcript levels was also dose dependent with maximal inhibition seen at 10–8 M CNP after a 12-h incubation. The nonselective phosphodiesterase inhibitor IBMX (10–4 M) reduced basal NPR2 mRNA levels and increased the magnitude of inhibition at each concentration of CNP studied (Fig. 2), suggesting that the generation of cyclic GMP by CNP is linked to the down-regulation of NPR2 gene expression.
The reduction in NPR2 mRNA levels was accompanied by a reduction in NPR-B protein. As shown in Fig. 3, treatment with CNP (10–8 M) led to a time-dependent reduction in NPR-B levels, based on immunoblot analysis, with maximal inhibition (35%) seen at 12 h into the incubation.
Transient transfection of a series of 5' deletion mutants of the NPR2 promoter revealed a robust level of reporter activity in RASM cells. As shown in Fig. 4, a construct harboring 443 bp of 5'-flanking sequence generated the highest levels of expression, whereas progressive deletion of 5'-flanking sequence led to a stepwise reduction in promoter activity. Of note, CNP inhibited –441 hNPR2 LUC reporter activity, suggesting that the reduction in NPR2 mRNA levels described above derives, in part, from a reduction in transcription of the NPR2 gene. This inhibitory effect was partially preserved as the deletion was moved from –441 to –219 but lost completely when the deletion was extended to –134 relative to the transcription start site. This implies that the CNP-sensitive elements are positioned between –441 and –134 within the NPR2 promoter.
CNP-dependent suppression of the NPR2 promoter was both time and dose dependent. As shown in Fig. 5A, treatment with CNP led to a time-dependent reduction in reporter activity, which was first significant at 6 h and peaked 12 h into the incubation. Again, as seen with the mRNA studies above, there was partial recovery of the inhibition after 24 h. In addition, 10–8 M CNP proved to be the most effective dose of ligand in driving the inhibition of –441 hNPR2 promoter activity (Fig. 5B), and, once again, mirroring the RNA studies described above, inclusion of IBMX in the incubations led to a reduction in basal promoter activity and amplified the CNP-dependent inhibition.
The amplification of CNP’s inhibitory properties by inhibition of phosphodiesterase activity in these cultures implies that CNP-generated cyclic GMP is responsible for signaling the reduction in both NPR2 mRNA levels and promoter activity. To address this question more directly, we treated RASM cells with different doses of the membrane-permeable cyclic GMP analog 8-bromo-cGMP for different periods of time. As shown in Fig. 6, 10–5 M 8-bromo-cGMP led to a reduction in NPR2 mRNA levels. The inhibition appeared at 3 h, was maximal (50% inhibition) at 6 h and persisted for 12 h into the incubation. 8-Bromo-cGMP also reduced NPR2 promoter activity. Examination of the dose-response relationship (Fig. 7A) suggested a downward trend in NPR2 promoter activity between 10–8 and 10–6 M, but only 10–5 M 8-bromo-cGMP proved capable of generating a statistically significant reduction (25% in these studies). In the time-course analysis (Fig. 7B), 8-bromo-cGMP (10–5 M) treatment led to a reduction in promoter activity (maximal 35%) that was noticeable after 3 h and remained stable for the remainder of the time course (24 h).
Finally, if cyclic GMP is the effector molecule responsible for the reduction in NPR2 promoter activity, one would predict that other maneuvers that raise cyclic GMP levels in these cells (aside from phosphodiesterase inhibition and the use of membrane permeable analogs) should lead to similar reductions in gene expression and promoter activity. ANP is known to increase cyclic GMP levels in RASM cells (11) presumably through activation of the NPR-A receptor present on the cell surface (1). As shown in Fig. 8, both NPR2 and NPR1 were subject to down-regulation by a heterologous cyclic GMP-generating ligand. Treatment of RASM with ANP (10–8 M) for 24 h resulted in a modest (20%) but significant reduction in NPR2 mRNA levels (Fig. 7A), whereas treatment with CNP (10–8 M) for the same time interval resulted in approximately 35% reduction in NPR1 transcript levels (Fig. 7B). In these experiments, the ligands were used at concentrations that would not be predicted to promote occupancy and/or activation of the heterologous receptors (e.g. CNP for NPR-A and ANP for NPR-B) (20). Furthermore, as shown in Fig. 9, treatment with ANP (10–7 M) led to a statistically significant reduction in NPR2 promoter activity. This approached but did not quite equal that seen with CNP alone. When the combination of ANP and CNP was used together at 10–7 M (total natriuretic peptide concentration of 2 x 10–7 M), they generated a level of inhibition (35%) that was less than additive (compared with the combined effects of 10–7 M ANP and CNP used alone) and not different from that seen with 2 x 10–7 M CNP. These findings suggest that ANP and CNP operate over a common signaling network (e.g. cyclic GMP-dependent network) to down-regulate NPR2 promoter activity.
Discussion
This study documents for the first time that CNP, the primary ligand for NPR-B, is capable of down-regulating the expression of this receptor. This down-regulation provides a mechanism for dampening the activity of the CNP/NPR-B signal transduction system in situations associated with prolonged elevations in ligand levels, thereby allowing the system to move back toward homeostatic equilibrium. Thus, high levels of local CNP generation in the damaged endothelium or at the endochondral growth plate could result in dampening of NPR-B’s antiproliferative activity in the former (5, 6, 7) or developmental activity in the latter (8). CNP reduces NPR-B activity, NPR-B protein, NPR2 mRNA levels, and NPR2 promoter activity. These reductions were not quantitatively comparable. This may reflect simply differential sensitivity of the different assays used here but might also suggest that CNP selectively targets individual loci in the NPR-B synthetic pathway. The reduction in promoter activity requires an element or elements present between –441 and –134 relative to the transcription start site. The reduction in NPR2 gene expression appears to operate through generation of cyclic GMP, the second messenger of liganded NPR-B in these cells. Phosphodiesterase inhibition with IBMX increased the magnitude of the CNP-dependent inhibition. 8-bromo cyclic GMP, a membrane-permeable cyclic GMP analog, reproduced the inhibition of NPR2 mRNA levels and NPR2 promoter activity. In addition, ANP, which signals through NPR-A to increase cyclic GMP levels in these cells (11), also reduced NPR2 mRNA levels and suppressed NPR2 promoter activity; however, this latter activity was not additive with that produced by CNP, implying that the two ligands traffic over a common signal transduction pathway (i.e. cyclic GMP generation).
The studies presented here, along with previous studies from our own laboratory (11, 12) and those of others (9, 14), indicate that the natriuretic peptides are effective regulators of their individual signal transduction systems. ANP desensitizes NPR1 through dephosphorylation of the receptor protein (9) and, as mentioned above, decreases expression of the receptor through a transcriptional mechanism (11, 12). CNP has been shown to desensitize NPR2 through a similar dephosphorylation mechanism (14), and the present work demonstrates that it also down-regulates expression of NPR2 through a transcriptional mechanism. Both NPR1 and NPR2 are expressed in cultured RASM cells, and both signal vasorelaxant (21, 22) and antimitogenic (23, 24) activity, presumably through their intrinsic particulate guanylyl cyclase activities. Therefore, cyclic GMP-dependent feedback control of both signal transduction pathways may provide a convenient mechanism for regulating these shared activities. NPR-A expression has been suggested to be more prominent in quiescent aortic smooth muscle cells in situ, whereas NPR-B is more avidly expressed in dividing cells in culture (25) (similar to the system employed here) and in the damaged arterial wall in vivo (6), paradigms that are more compatible with the proliferative vs. contractile phenotype (26, 27). Differential expression of these two receptors in different vascular beds or damaged vs. intact vessels in vivo would limit ligand responsiveness and, inferentially, ligand-dependent receptor down-regulation to those ligands capable of increasing cyclic GMP in the target cell.
We have reported previously that ANP down-regulates expression of NPR-A in RASM cells. This down-regulation was seen at the level of the NPR1 mRNA and NPR1 promoter activity. The ability to promote this down-regulation was shared by BNP; 8-bromo cyclic GMP (11); and, interestingly, CNP (see Fig. 8A). Down-regulation of NPR2 gene regulation was shared by CNP, cyclic GMP, and ANP (Fig. 9B). Thus, it appears that down-regulation of both NPR-A and NPR-B are linked more closely to cyclic GMP generation rather than occupancy of a specific receptor subtype by ligand. In the case of NPR1, the down-regulation appears to involve elements positioned between –1575 and –1290 relative to the transcription start site (11). We have scanned the NPR1 genomic sequence between –1575 and –1290, which includes the putative cyclic GMP regulatory element, for short segments of DNA sequence homology with the region spanning –441 and –135 in the NPR2 gene and found no regions of highly conserved sequence. We conclude either that the NPR1 and NPR2 genes employ different elements for sensing cyclic GMP regulatory activity or that there is sufficient flexibility in the sequence requirements of the cyclic GMP regulatory element to preclude detection of sequence homology using conventional algorithms. Alternatively, cyclic GMP may function indirectly through interference with one or more transcription factors that are not shared between these two promoters.
This study adds to a growing body of information suggesting that control of natriuretic peptide receptor gene expression may represent an important locus for the regulation of cardiovascular and renal function (1). By inference, it also implies the presence of a key cyclic GMP-dependent regulatory element or elements in the promoters of these receptor genes, analogous to the cAMP-dependent regulatory element described in other systems (28), that play an important role in controlling their transcriptional activity.
Acknowledgments
The authors are grateful for the advice of Dr. Songcang Chen in carrying out these studies.
Footnotes
This work was supported by National Institutes of Health Grant HL45637. D.R. was supported by a postdoctoral fellowship from the American Heart Association (Western States Affiliate).
Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; Ct, cycle threshold; FBS, fetal bovine serum; GAPDH, glyceraldehyde phosphate dehydrogenase; hNPR, human natriuretic peptide receptor; IBMX, 3-isobutyl-1-methylxanthine; Luc, luciferase; NPR-A, type A natriuretic peptide receptor; NPR-B, type B natriuretic peptide receptor; RASM, rat aortic smooth muscle; RSV- gal, Rous sarcoma virus--galactosidase.
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Abstract
The C-type natriuretic (CNP) peptide signals through the type B natriuretic peptide receptor (NPR-B) in vascular smooth muscle cells to activate the particulate guanylyl cyclase activity intrinsic to that receptor and raise cellular cyclic GMP levels. In the present study, we demonstrate that CNP down-regulates the expression of this receptor leading to a reduction in NPR-B activity. Pretreatment of rat aortic smooth muscle cells with CNP reduces NPR-B activity, NPR-B protein levels, NPR2 (NPR-B gene) mRNA levels, and NPR2 promoter activity. The decrease in NPR2 promoter activity is dependent on DNA sequence present between –441 and –134 relative to the transcription start site. The reduction in NPR2 gene expression appears to operate through generation of cyclic GMP. 8-Bromo cyclic GMP, a membrane-permeable cyclic GMP analog, reduced NPR2 mRNA levels and NPR2 promoter activity. Atrial natriuretic peptide, which signals through the type A natriuretic peptide receptor (NPR-A) to increase cyclic GMP levels in these cells, also reduced NPR-B mRNA levels and inhibited NPR-B promoter activity; however, this inhibition was not additive with that produced by CNP, implying that the two ligands traffic over a common signal transduction pathway. This report provides the first documentation that CNP is capable of autoregulating the expression of its cognate receptor.
Introduction
THE NATRIURETIC PEPTIDES constitute a family of peptide hormones with important effects on cardiovascular, renal, and endocrine physiology (1). Atrial natriuretic peptide (ANP) and brain, or B-type, natriuretic peptide are produced in the atria and ventricles of the heart. They promote reductions in blood pressure, increases in urinary volume and urinary sodium excretion, and suppression of renin and aldosterone secretion (1). C-type natriuretic peptide (CNP) is produced only to a limited degree in the heart, and its role in the regulation of blood pressure or renal sodium handling appears to be limited. CNP is produced in the endothelial cells of the vasculature (2), cardiac fibroblasts (3), and the endochondral growth plate (4) in which it is thought to play important roles in the control of vascular remodeling (5, 6, 7), fibroblast proliferation (3), and linear growth of the long bones (8), respectively.
ANP and BNP signal their activity through a single receptor called the type A natriuretic peptide receptor (NPR-A), whereas CNP signals through its own receptor, the type B receptor (NPR-B). Both NPR-A and NPR-B share a similar overall structure. They each possess a large extracellular ligand binding domain linked to an intracellular particulate guanylyl cyclase domain through a single transmembrane segment. NPR-A activity is regulated at the posttranslational level (9, 10) as well as the level of gene expression (11, 12, 13). Like other peptide hormone receptors, NPR1 (NPR-A) gene expression is down-regulated by its cognate ligand, ANP, in part through inhibition of NPR1 gene transcription (11, 12). Regulation of NPR2 gene expression is less well understood. Whereas selected studies suggest that it is subject to similar posttranslational regulation (vs. that seen with NPR-A) (14, 15), relatively few studies of NPR2 gene regulation have been published (16, 17) and nothing has been shown to regulate NPR2 gene transcription or promoter activity.
In the present study, we demonstrate that CNP, the primary ligand for NPR-B in rat aortic smooth muscle (RASM) cells reduces NPR-B activity through inhibition of NPR2 mRNA levels. It does so through suppression of NPR2 promoter activity, requiring promoter elements positioned between –441 and –134 relative to the transcription start site. This down-regulation of promoter activity appears to operate through a cyclic GMP-dependent mechanism.
Materials and Methods
Materials
ANP (human, 28 amino acids) and CNP (human, 22 amino acids) were purchased from American Peptide, Inc. (Sunnyvale, CA). 3-Isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor and 8-bromo-cGMP were purchased from Sigma (St. Louis, MO). Cyclic GMP RIA kit was purchased from PerkinElmer/Life Sciences (Norwalk, CT). RNeasy minikit was obtained from QIAGEN Inc. (Valencia, CA). The luciferase assay kit was purchased from Promega (Madison, WI). Restriction enzymes were obtained from New England Biotech, Inc. (Beverly, MA). All oligonucleotides were synthesized by Invitrogen (Carlsbad, CA). Other reagents were obtained from standard commercial suppliers.
Cell culture
Neonatal RASM cells were obtained from H. Ives (University of California at San Francisco, San Francisco, CA) (18). Cells were cultured at 37 C in a 5% CO2 humidified incubator in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2% (vol/vol) broth, tryptose phosphate for 24 h. Cells were then changed to 0.5% FBS/DMEM and cultured for an additional 24 h. At that point, media were changed (0.5% FBS/DMEM) and indicated agents were added to the culture media to begin the experiment.
Measurement of CNP-stimulated cGMP levels
Confluent RASM cells were preincubated with 10–11 to 10–7 M CNP for 12 h in presence of IBMX (10–4 M). Cells (105/well) were preincubated for 10 min in media with IBMX (5 x10–4 M) as indicated. Medium was then changed to include CNP 10–8 M in the presence of the phosphodiesterase inhibitor. After 10 min the reaction was stopped by removing the medium and adding 0.3 ml of 12% trichloroacetic acid. The extraction was continued for 30 min at 4 C. The contents of the plate were collected and centrifuged to pellet particulate material. The supernatant fraction was extracted four times with 0.5 ml water-saturated ether. cGMP levels were determined by RIA after acetylation of the sample and standard using a commercial 125I-cGMP RIA kit (PerkinElmer Life Sciences). Results are reported as picomoles per well.
Preparation of crude membranes
To prepare crude RASM cell membranes, 25-cm plates at approximately 85% confluence were washed and incubated overnight with DMEM in the absence of serum. After incubation with DMEM containing 10–8 M CNP for varying periods of time, cells were washed twice with PBS, scraped into 0.5 ml of buffer containing protease inhibitors [25 mM HEPES (pH 7.4), 20% glycerol, 50 mM NaCl, 50 mM -glycerol phosphate, 2 mM EDTA, 1 μM microcystin, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin], sonicated for 6 sec with a Misonix Sonicator (Branson 2510; Farmingdale, NY), and centrifuged at 20,000 x g for 20 min at 2 C. Pellets were resuspended in buffer and centrifuged at 1000 x g for 2 min at 4 C. Pellets were then resuspended in the same buffer and stored at –80 C. Protein concentrations were estimated using a bicinchoninic acid assay or Coomassie dye binding protein assay reagent (Pierce Chemical Co., Rockford, IL).
Immunoblot analysis
Sixty micrograms of protein from the resuspended pellet was electrophoresed on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred electrophoretically onto polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences). Membranes were incubated with goat polyclonal anti-NPR-B antibody (NPR-B c-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1:80 dilution) or rabbit polyclonal anti- tubulin antibody (-tubulin sc-9104, Santa Cruz Biotechnology) (1:400 dilution) in Tris-buffered saline/Tween buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20, and 6% nonfat milk] overnight at 4 C and then washed with Tris-buffered saline/Tween buffer. Membranes were then incubated with horseradish peroxidase-conjugated bovine antigoat antibody (SC-2350; Santa Cruz Biotechnology) or goat antirabbit antibody (SC-2004; Santa Cruz Biotechnology) for 1 h at room temperature and washed with Tris-buffered saline/Tween. Blots were immersed for 1 min in enhanced chemiluminescence detection reagent (Amersham Biosciences, Piscataway, NJ) and then exposed to film. Signals were quantified using the Image Station (440CF; Kodak, Rochester, NY).
Primer and probe design
Using the Primer Express (PE Applied Biosystems, Inc., Foster City, CA) software program, primers and probe were designed to recognize rat NPR2 (amplicon is 101 bp) and glyceraldehyde phosphate dehydrogenase (GAPDH) coding sequence. Primers for this gene target were selected to contain minimal internal structure (i.e. hairpins and potential for primer-dimer formation) and compatible melting temperature (i.e. each within 1 C of the other). Probes were selected with a melting temperature that was approximately 5–10 C higher than the matching primer pair. Probes were all less than 40 bp in total length. GAPDH primers and probe were designed and optimized at ABI. Table 1 depicts the primer and probe sequences used for measurement of NPR2 and GAPDH mRNA transcript levels.
Real-time PCR analysis
Total RNA was prepared from RASM cells using the QIAGEN RNeasy kit. The cells were harvested per the manufacturer’s instructions. RNA was treated with DNase I to reduce genomic DNA contamination. Then 1.2 μg total RNA was used to reverse transcribe cDNA using the reverse transcription kit (Clontech, Palo Alto, CA). Products were examined by agarose gel electrophoresis to assure near equivalent levels of cDNA synthesis.
Rat NPR2 and GAPDH primer pairs and probes (Table 1) were synthesized by PE Applied Biosystems, Inc. Real-time PCR was performed using Taqman master mix (PE Applied Biosystems, Inc.) with an ABI 7700 (PE Applied Biosystems, Inc.). Negative controls without input cDNA were used to assess signal specificity. NPR2 transcript levels were quantified and normalized for GAPDH transcript levels in each sample. Equivalent amounts (0.5 μl) of cDNA were used for measurement of NPR2 and GAPDH gene transcripts. Thermal cycling conditions involved 2 min at 50 C and 10 min of initial denaturation at 95 C, followed by 40 cycles of two-step PCR consisting of 15 sec at 95 C and 1 min at 60 C. All samples were amplified in triplicate. Relative quantification was achieved by the comparative: 2– Ct method (19). The relative increase/decrease (fold-induction/repression) of mRNA of target x in the experimental group (NPR2) was calculated using the control group as the calibrator: 2–Ct, where cycle threshold (Ct) is: {Ct.x[treatment] – Ct.GAPDH[treatment]} – {Ct.x[control] – Ct.GAPDH[control]}.
Construction of human natriuretic peptide receptor type 2 (hNPR)-B-luciferase (Luc) expression vectors
To obtain deletion constructs, PCR was performed using LA-Taq DNA polymerase (TaKaRa La Taq), a subcloned 9-kb human genomic clone as a template, and the following sense primers: –34 hNPR2 LUC, 5'-GGGGTACCCCAGGCGACCTGACCCGGAC-3'; –84 hNPR2 LUC, 5'-GGGGTACCCCGGGAGTCGCACTCG-3'; –134 hNPR2 LUC, 5'-GGGGTACCCCAGGGAGGAGGAGAGGCAG-3'; –219 hNPR2 LUC, 5'-GGGGTACCCCACCGGGGTGGGTTTGGGAACTG-3'; –441 hNPR2 LUC, 5'-GGGGTACCCCTGAGGGACTCAGGGAGTTC-3', together with antisense primer NPR2 R1 5'-GAAGATCTTCTGTGAAGGATTCTCCCG, positioned between +148 and +166, relative to the transcription start site. We incorporated restriction sites (KpnI and BglII, respectively) on sense and antisense primers. PCR products were digested with KpnI and BglII and inserted between the KpnI/BglII sites of pGL3 (Promega), a luciferase reporter that lacks eukaryotic promoter and enhancer sequences. The structure of all constructs was confirmed by DNA sequencing.
Transfection, luciferase, and -galactosidase assays RASM cells were transiently transfected with 0.5 μg of each hNPR2 5' deletion mutant and 0.5 μg Rous sarcoma virus--galactosidase (RSV- gal) by electroporation (Gene-Pulser; Bio-Rad Laboratories, Inc., Hercules, CA) at 250 mV and 960 μF. After transfection, cells were plated in six-well plastic plates and cultured for 48 h. Cells were harvested and lysed in 100 μl cell culture lysis reagent (Promega). The protein concentration of each cell extract was measured using Coomassie protein reagent (Pierce). Cell lysates were processed (30 μg protein/sample) and assayed for luciferase as described previously (12). Measurements of -galactosidase activity were made using the Galacto-Light Plus kit (Tropix, Inc., Bedford, MA).
Statistical analysis
Data were evaluated by one-way ANOVA and the Newman-Keuls test for significance.
Results
Chronic (i.e. 12 h) treatment of cultured RASM cells with CNP, in the presence of the nonselective phosphodiesterase inhibitor IBMX (10–4 M), led to a dose-dependent reduction in the subsequent ability of CNP to stimulate cyclic GMP accumulation in these cultures. Pretreatment with 10–8 M CNP resulted in an 80% reduction in the CNP-dependent cGMP accumulation (Table 2), suggesting that CNP promotes a ligand-dependent desensitization and/or down-regulation of the NPR-B receptor.
Ligand-dependent desensitization of NPR-B has been documented previously (14); however, down-regulation of receptor expression (e.g. NPR2 mRNA and protein levels) has not been reported. In our hands, treatment with CNP led to a time-dependent reduction in NPR2 mRNA levels in cultured RASM cells (Fig. 1). This was maximal (70% inhibition) at 12 h and had partially recovered after 24 h of incubation. The reduction in NPR2 transcript levels was also dose dependent with maximal inhibition seen at 10–8 M CNP after a 12-h incubation. The nonselective phosphodiesterase inhibitor IBMX (10–4 M) reduced basal NPR2 mRNA levels and increased the magnitude of inhibition at each concentration of CNP studied (Fig. 2), suggesting that the generation of cyclic GMP by CNP is linked to the down-regulation of NPR2 gene expression.
The reduction in NPR2 mRNA levels was accompanied by a reduction in NPR-B protein. As shown in Fig. 3, treatment with CNP (10–8 M) led to a time-dependent reduction in NPR-B levels, based on immunoblot analysis, with maximal inhibition (35%) seen at 12 h into the incubation.
Transient transfection of a series of 5' deletion mutants of the NPR2 promoter revealed a robust level of reporter activity in RASM cells. As shown in Fig. 4, a construct harboring 443 bp of 5'-flanking sequence generated the highest levels of expression, whereas progressive deletion of 5'-flanking sequence led to a stepwise reduction in promoter activity. Of note, CNP inhibited –441 hNPR2 LUC reporter activity, suggesting that the reduction in NPR2 mRNA levels described above derives, in part, from a reduction in transcription of the NPR2 gene. This inhibitory effect was partially preserved as the deletion was moved from –441 to –219 but lost completely when the deletion was extended to –134 relative to the transcription start site. This implies that the CNP-sensitive elements are positioned between –441 and –134 within the NPR2 promoter.
CNP-dependent suppression of the NPR2 promoter was both time and dose dependent. As shown in Fig. 5A, treatment with CNP led to a time-dependent reduction in reporter activity, which was first significant at 6 h and peaked 12 h into the incubation. Again, as seen with the mRNA studies above, there was partial recovery of the inhibition after 24 h. In addition, 10–8 M CNP proved to be the most effective dose of ligand in driving the inhibition of –441 hNPR2 promoter activity (Fig. 5B), and, once again, mirroring the RNA studies described above, inclusion of IBMX in the incubations led to a reduction in basal promoter activity and amplified the CNP-dependent inhibition.
The amplification of CNP’s inhibitory properties by inhibition of phosphodiesterase activity in these cultures implies that CNP-generated cyclic GMP is responsible for signaling the reduction in both NPR2 mRNA levels and promoter activity. To address this question more directly, we treated RASM cells with different doses of the membrane-permeable cyclic GMP analog 8-bromo-cGMP for different periods of time. As shown in Fig. 6, 10–5 M 8-bromo-cGMP led to a reduction in NPR2 mRNA levels. The inhibition appeared at 3 h, was maximal (50% inhibition) at 6 h and persisted for 12 h into the incubation. 8-Bromo-cGMP also reduced NPR2 promoter activity. Examination of the dose-response relationship (Fig. 7A) suggested a downward trend in NPR2 promoter activity between 10–8 and 10–6 M, but only 10–5 M 8-bromo-cGMP proved capable of generating a statistically significant reduction (25% in these studies). In the time-course analysis (Fig. 7B), 8-bromo-cGMP (10–5 M) treatment led to a reduction in promoter activity (maximal 35%) that was noticeable after 3 h and remained stable for the remainder of the time course (24 h).
Finally, if cyclic GMP is the effector molecule responsible for the reduction in NPR2 promoter activity, one would predict that other maneuvers that raise cyclic GMP levels in these cells (aside from phosphodiesterase inhibition and the use of membrane permeable analogs) should lead to similar reductions in gene expression and promoter activity. ANP is known to increase cyclic GMP levels in RASM cells (11) presumably through activation of the NPR-A receptor present on the cell surface (1). As shown in Fig. 8, both NPR2 and NPR1 were subject to down-regulation by a heterologous cyclic GMP-generating ligand. Treatment of RASM with ANP (10–8 M) for 24 h resulted in a modest (20%) but significant reduction in NPR2 mRNA levels (Fig. 7A), whereas treatment with CNP (10–8 M) for the same time interval resulted in approximately 35% reduction in NPR1 transcript levels (Fig. 7B). In these experiments, the ligands were used at concentrations that would not be predicted to promote occupancy and/or activation of the heterologous receptors (e.g. CNP for NPR-A and ANP for NPR-B) (20). Furthermore, as shown in Fig. 9, treatment with ANP (10–7 M) led to a statistically significant reduction in NPR2 promoter activity. This approached but did not quite equal that seen with CNP alone. When the combination of ANP and CNP was used together at 10–7 M (total natriuretic peptide concentration of 2 x 10–7 M), they generated a level of inhibition (35%) that was less than additive (compared with the combined effects of 10–7 M ANP and CNP used alone) and not different from that seen with 2 x 10–7 M CNP. These findings suggest that ANP and CNP operate over a common signaling network (e.g. cyclic GMP-dependent network) to down-regulate NPR2 promoter activity.
Discussion
This study documents for the first time that CNP, the primary ligand for NPR-B, is capable of down-regulating the expression of this receptor. This down-regulation provides a mechanism for dampening the activity of the CNP/NPR-B signal transduction system in situations associated with prolonged elevations in ligand levels, thereby allowing the system to move back toward homeostatic equilibrium. Thus, high levels of local CNP generation in the damaged endothelium or at the endochondral growth plate could result in dampening of NPR-B’s antiproliferative activity in the former (5, 6, 7) or developmental activity in the latter (8). CNP reduces NPR-B activity, NPR-B protein, NPR2 mRNA levels, and NPR2 promoter activity. These reductions were not quantitatively comparable. This may reflect simply differential sensitivity of the different assays used here but might also suggest that CNP selectively targets individual loci in the NPR-B synthetic pathway. The reduction in promoter activity requires an element or elements present between –441 and –134 relative to the transcription start site. The reduction in NPR2 gene expression appears to operate through generation of cyclic GMP, the second messenger of liganded NPR-B in these cells. Phosphodiesterase inhibition with IBMX increased the magnitude of the CNP-dependent inhibition. 8-bromo cyclic GMP, a membrane-permeable cyclic GMP analog, reproduced the inhibition of NPR2 mRNA levels and NPR2 promoter activity. In addition, ANP, which signals through NPR-A to increase cyclic GMP levels in these cells (11), also reduced NPR2 mRNA levels and suppressed NPR2 promoter activity; however, this latter activity was not additive with that produced by CNP, implying that the two ligands traffic over a common signal transduction pathway (i.e. cyclic GMP generation).
The studies presented here, along with previous studies from our own laboratory (11, 12) and those of others (9, 14), indicate that the natriuretic peptides are effective regulators of their individual signal transduction systems. ANP desensitizes NPR1 through dephosphorylation of the receptor protein (9) and, as mentioned above, decreases expression of the receptor through a transcriptional mechanism (11, 12). CNP has been shown to desensitize NPR2 through a similar dephosphorylation mechanism (14), and the present work demonstrates that it also down-regulates expression of NPR2 through a transcriptional mechanism. Both NPR1 and NPR2 are expressed in cultured RASM cells, and both signal vasorelaxant (21, 22) and antimitogenic (23, 24) activity, presumably through their intrinsic particulate guanylyl cyclase activities. Therefore, cyclic GMP-dependent feedback control of both signal transduction pathways may provide a convenient mechanism for regulating these shared activities. NPR-A expression has been suggested to be more prominent in quiescent aortic smooth muscle cells in situ, whereas NPR-B is more avidly expressed in dividing cells in culture (25) (similar to the system employed here) and in the damaged arterial wall in vivo (6), paradigms that are more compatible with the proliferative vs. contractile phenotype (26, 27). Differential expression of these two receptors in different vascular beds or damaged vs. intact vessels in vivo would limit ligand responsiveness and, inferentially, ligand-dependent receptor down-regulation to those ligands capable of increasing cyclic GMP in the target cell.
We have reported previously that ANP down-regulates expression of NPR-A in RASM cells. This down-regulation was seen at the level of the NPR1 mRNA and NPR1 promoter activity. The ability to promote this down-regulation was shared by BNP; 8-bromo cyclic GMP (11); and, interestingly, CNP (see Fig. 8A). Down-regulation of NPR2 gene regulation was shared by CNP, cyclic GMP, and ANP (Fig. 9B). Thus, it appears that down-regulation of both NPR-A and NPR-B are linked more closely to cyclic GMP generation rather than occupancy of a specific receptor subtype by ligand. In the case of NPR1, the down-regulation appears to involve elements positioned between –1575 and –1290 relative to the transcription start site (11). We have scanned the NPR1 genomic sequence between –1575 and –1290, which includes the putative cyclic GMP regulatory element, for short segments of DNA sequence homology with the region spanning –441 and –135 in the NPR2 gene and found no regions of highly conserved sequence. We conclude either that the NPR1 and NPR2 genes employ different elements for sensing cyclic GMP regulatory activity or that there is sufficient flexibility in the sequence requirements of the cyclic GMP regulatory element to preclude detection of sequence homology using conventional algorithms. Alternatively, cyclic GMP may function indirectly through interference with one or more transcription factors that are not shared between these two promoters.
This study adds to a growing body of information suggesting that control of natriuretic peptide receptor gene expression may represent an important locus for the regulation of cardiovascular and renal function (1). By inference, it also implies the presence of a key cyclic GMP-dependent regulatory element or elements in the promoters of these receptor genes, analogous to the cAMP-dependent regulatory element described in other systems (28), that play an important role in controlling their transcriptional activity.
Acknowledgments
The authors are grateful for the advice of Dr. Songcang Chen in carrying out these studies.
Footnotes
This work was supported by National Institutes of Health Grant HL45637. D.R. was supported by a postdoctoral fellowship from the American Heart Association (Western States Affiliate).
Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; Ct, cycle threshold; FBS, fetal bovine serum; GAPDH, glyceraldehyde phosphate dehydrogenase; hNPR, human natriuretic peptide receptor; IBMX, 3-isobutyl-1-methylxanthine; Luc, luciferase; NPR-A, type A natriuretic peptide receptor; NPR-B, type B natriuretic peptide receptor; RASM, rat aortic smooth muscle; RSV- gal, Rous sarcoma virus--galactosidase.
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