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Endocrinology Vol. 144, No. 6 2279-2284
Copyright © 2003 by The Endocrine Society

Gene Expression, Secretion, and Autocrine Action of C-Type Natriuretic Peptide in Cultured Adult Rat Cardiac Fibroblasts

Takeshi Horio, Takeshi Tokudome, Toshiyuki Maki, Fumiki Yoshihara, Shin-ichi Suga, Toshio Nishikimi, Masayasu Kojima, Yuhei Kawano and Kenji Kangawa

Department of Medicine (T.H., F.Y., Y.K.) and Research Institute (T.T., T.M., S.S., K.K.), National Cardiovascular Center, Suita, Osaka 565-8565, Japan; Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine (T.N.), Tochigi 321-0293, Japan; and Institute of Life Sciences, Kurume University (M.K.), Kurume, Fukuoka 839-0861, Japan

Address all correspondence and requests for reprints to: Takeshi Horio, M.D., Division of Hypertension and Nephrology, Department of Medicine, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: thorio{at}ri.ncvc.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is known to be synthesized in the central nervous system and vascular endothelial cells, in contrast to atrial natriuretic peptide and brain natriuretic peptide. However, there have been no studies concerning CNP production in cultured cardiac cells. Here, we examined the production and the local effect of CNP in cultured ventricular cells. Under serum-free conditions, adult rat cardiac fibroblasts secreted immunoreactive CNP time dependently. TGF-ß1, basic fibroblast growth factor, and endothelin-1 significantly stimulated CNP secretion. Northern blot analysis detected significant expressions of CNP and its specific receptor (guanylyl cyclase-B) mRNA in cardiac fibroblasts. CNP stimulated intracellular cGMP production in fibroblasts more intensely than atrial and brain natriuretic peptides. CNP inhibited both DNA and collagen syntheses of cardiac fibroblasts, and these inhibitory effects by CNP were stronger than by atrial and brain natriuretic peptides. The inhibition by CNP of DNA and collagen syntheses was reproduced by a cGMP analog, 8-bromo cGMP. The present findings demonstrate that CNP is synthesized in and secreted from cardiac fibroblasts and suggest that CNP has a suppressive effect on fibroblast proliferation and extracellular matrix production, probably via the guanylyl cyclase-B-mediated cGMP-dependent process. CNP produced by cardiac fibroblasts may play a role as an autocrine regulator against excessive cardiac fibrosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NATRIURETIC PEPTIDES are a family of cyclic molecules that consist of three members, designated atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP; Ref. 1). ANP and BNP are primarily cardiac hormones secreted mainly from atria and ventricles, respectively. Once in the circulation, ANP and BNP bind to their specific receptors in the vascular tissue, kidney, and adrenal gland and induce vasodilatation, natriuresis, and diuresis. CNP, which was originally isolated from porcine brain extracts, not only acts as a neuropeptide in the central nervous system, but also plays a role in the local regulation of vascular tone, because CNP is synthesized in vascular endothelial cells (2).

Subsequent studies have revealed the existence of two biological [guanylyl cyclase (GC)-containing] natriuretic peptide receptors, called GC-A and GC-B, in cardiac cells (3). In addition to acting as a circulating hormone, therefore, ANP and BNP may have some function as autocrine and/or paracrine factors. In fact, some studies, including our in vitro study, showed that endogenous ANP and BNP suppress the development of cardiac myocyte hypertrophy and interstitial fibrosis (4, 5, 6). With regard to CNP, however, its production in cardiac cells and the local effect on the heart itself have remained to be elucidated. Therefore, we conducted this study to investigate the production of CNP and the expression of GC-B, a specific receptor for CNP, in cultured ventricular cells and to examine the effects of CNP on DNA and collagen syntheses by fibroblast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Adult rat cardiac fibroblasts were prepared according to a previously described protocol (7) with minor modifications. Apical halves of cardiac ventricles from 7-wk-old male Wistar rats were separated, minced, and dispersed with 0.1% collagenase type II (Worthington Biochemical Corp., Freehold, NJ). The isolated cells were resuspended in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Life Technologies, Inc.), and the suspension was plated onto 10-cm culture dishes for 2 h, which allowed for the preferential attachment of fibroblasts to the bottom of the culture dish. Nonadherent and weakly attached cells were then removed, and attached fibroblast cells were allowed to grow to confluence, trypsinized, and passaged 1:3 or 1:4. Adult cardiac fibroblasts at the second or third passage were used in the present study. The purity of cardiac fibroblasts and the absence of myocytes were confirmed by a morphological examination (fibroblasts are thin and triangular cells with light cytoplasm). In addition, the contamination of endothelial cells in our fibroblast culture was excluded by the lack of positive immunostaining with an antibody against von Willebrand factor (DAKO Corp. A/S, Glostrup, Denmark).

Neonatal ventricular myocytes and fibroblasts were prepared as described previously (8). From cardiac ventricles of 1- to 2-d-old Wistar rats, myocytes and fibroblasts were separately collected by the discontinuous Percoll gradient method. Primary cultures of neonatal rat cardiac myocytes and neonatal cardiac fibroblasts at the second passage were used for the gene expression study.

Measurement of immunoreactive (ir-) ANP, BNP, and CNP
After adult cardiac fibroblasts were incubated for 12–72 h in 10-cm dishes with serum-free DMEM, the culture medium was collected. In the study examining the effect of various agents on ir-CNP secretion, cells were incubated for 24 h under treatment with IL-1ß (Genzyme Techne, Minneapolis, MN), TNF-{alpha} (Sigma, St. Louis, MO), TGF-ß1 (R Systems, Minneapolis, MN), basic fibroblast growth factor (bFGF; Sigma), IGF-1 (Sigma), insulin (Sigma), angiotensin II (Peptide Institute, Osaka, Japan), endothelin-1 (Peptide Institute), phenylephrine (Research Biochemicals International, Natick, MA), isoproterenol (Sigma), or norepinephrine (Research Biochemicals International). The collected medium (10 ml) was condensed with a Sep-Pak C18 cartridge (Waters, Milford, MA) and lyophilized. The RIA for rat ANP, BNP, and CNP was performed as previously reported (6, 9).

Characterization of ir-CNP
The conditioned medium (80 ml) from adult cardiac fibroblasts was condensed and separated by reverse-phase HPLC on a µ-Bondasphere C18 column (300 Å, 3.9 x 150 mm; Waters), as described previously (8). A linear gradient elution of acetonitrile for 10–60% in 0.1% trifluoroacetic acid was made at a flow rate of 1 ml/min, and each collected fraction (1 ml) was submitted for RIA for CNP.

Northern blot analysis
After incubation in DMEM with FCS, the cultured cells were rinsed with PBS and submitted for RNA extraction. Total RNA was extracted with TRIzol reagent (Life Technologies, Inc.) and poly(A)+ RNA was prepared using Oligotex-dT30 Super (Takara Biomedicals, Shiga, Japan). Poly(A)+ RNA (10 µg/lane) was electrophoresed on 1% agarose gel and then transferred to a nylon membrane. Hybridization and washing of the membrane were carried out with cDNA probes for rat CNP, ANP, GC-A, GC-B, and glyceraldehyde-3-phosphate dehydrogenase genes, according to the method previously reported (8, 9).

Measurement of cellular cGMP
After preincubation, adult cardiac fibroblasts grown in 24-well plates were treated for 10 min with various concentrations of synthetic ANP, BNP, or CNP (Peptide Institute) in the presence of 5 x 10-4 mol/liter 3-isobutyl-1-methylxanthine (Nacalai Tesque, Kyoto, Japan). The intracellular cGMP levels were determined by a RIA performed with a cGMP assay kit (Yamasa Shoyu, Chiba, Japan), as previously reported (6).

DNA and collagen syntheses
The effects of natriuretic peptides and a cGMP analog on DNA synthesis and collagen synthesis in adult rat cardiac fibroblasts were evaluated by the incorporation of [3H]thymidine and [3H]proline into cells, respectively, as described previously (10). Fibroblasts at the second or third passage were seeded at a density of 2.5 x 104 cells/well on 24-well plates. After incubation in DMEM with 10% FCS, subconfluent fibroblast cells (approximately 7.5 x 104 cells/well) were maintained in serum-free DMEM for 48 h. After the preconditioning period, the culture medium was replaced with fresh DMEM with 1% FCS. Then, ANP, BNP, CNP, or 8-bromo cGMP (Sigma) was added, and 0.5 µCi of [3H]thymidine or [3H]proline was also added. After the cells were incubated for 24 h, the radioactivity of aliquots of the trichloroacetic acid-insoluble material was determined using a liquid scintillation counter.

Statistical analysis
The significance of differences among groups was estimated by an unpaired ANOVA, and probability values were calculated by the Fisher method. A value of P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Secretion and characterization of ir-CNP in adult cardiac fibroblasts
The significant release of ir-CNP from adult cardiac fibroblasts into the serum-free medium was observed, and its level in fibroblasts increased in a time-dependent manner (Fig. 1AGo). Although the concentration of ir-ANP and ir-BNP in the same culture medium was also measured, the significant release of these peptides was not detected after 12–72 h of incubation.



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  		Figure 1. A, Basal secretion of ir-CNP ({bullet}) from cultured adult rat cardiac fibroblasts under serum-free conditions. Values are mean ± SE of five measurements. Neither ir-ANP ({circ}) nor ir-BNP ({blacktriangleup}) was detected in the same culture medium. B, Reverse-phase HPLC analysis of ir-CNP in the culture medium of adult rat cardiac fibroblasts. Arrows indicate the elution positions of 1) ANP, 2) CNP, and 3) BNP.

 
Ir-CNP secreted from cultured fibroblasts was characterized by reverse-phase HPLC. Ir-CNP in the culture medium of adult cardiac fibroblasts consisted of one major and some minor components, and the major component was eluted at a retention time identical with that of authentic CNP (Fig. 1BGo).

We further examined which humoral factor stimulates CNP release from adult cardiac fibroblasts. The secretion levels of ir-CNP were slightly but significantly increased by TGF-ß1 (10-9 mol/liter) and bFGF (10-8 mol/liter) among several cytokines and growth factors (Table 1Go). Among vasoactive peptides and catecholamines, only endothelin-1 (10-7 mol/liter) stimulated the ir-CNP release from cardiac fibroblasts.


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  		Table 1. Effects of various humoral factors on CNP secretion from adult cardiac fibroblasts

 
Expression of CNP and its receptor mRNA in adult and neonatal cardiac fibroblasts
By Northern blot analysis, a single band hybridizing to the rat CNP cDNA probe was found in adult rat cardiac fibroblasts at passages 2 and 3 (Fig. 2Go). The expression of ANP mRNA was not detected at all in the same membrane. We also investigated the CNP gene expression in cultured neonatal rat cardiac fibroblasts and myocytes. A weak transcript signal was observed in neonatal cardiac fibroblasts. On the other hand, no specific expression of CNP mRNA was found in neonatal cardiac myocytes, although strong gene expression of ANP was observed in those cells.



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  		Figure 2. The expression of rat CNP, ANP, GC-A, GC-B, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in cultured adult rat cardiac fibroblasts (left) and neonatal rat cardiac fibroblasts and myocytes (right). Adult rat cardiac fibroblasts at the second or third passage, neonatal fibroblasts at the second passage, and primary cultures of neonatal cardiac myocytes were evaluated. The radioimages are representative of two separate experiments.

 
As for the expression of natriuretic peptide receptors, both GC-A and GC-B transcripts were found in adult and neonatal cardiac fibroblasts. However, expression levels of the GC-B receptor were much more intense than those of the GC-A in adult cardiac fibroblasts.

Effects of natriuretic peptides on cellular cGMP production and on DNA and collagen syntheses in adult cardiac fibroblasts
CNP markedly increased the cellular level of cGMP in adult cardiac fibroblasts and its increase was concentration dependent (Fig. 3Go). Although ANP and BNP also increased the cGMP levels concentration dependently, the stimulation of cGMP formation by ANP or BNP was weaker than by CNP.



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  		Figure 3. Effects of ANP ({circ}), BNP ({blacktriangleup}), and CNP ({bullet}) on the production of cellular cGMP in cultured adult rat cardiac fibroblasts. Values are mean ± SE of four measurements. *, P < 0.001 vs. the same dose of ANP; {dagger}, P < 0.001 vs. the same dose of BNP.

 
ANP, BNP, and CNP decreased the incorporation of [3H]proline and [3H]thymidine into adult cardiac fibroblasts (Fig. 4Go, A and B). Above all, CNP clearly inhibited collagen synthesis at concentrations of 10-10 mol/liter or greater, and DNA synthesis at concentrations of 10-9 mol/liter or greater, and these inhibitory effects of CNP were stronger than those of ANP and BNP. 8-Bromo cGMP, a membrane-permeable analog of cGMP, reduced both [3H]proline and [3H]thymidine incorporation in a dose-dependent manner (Fig. 4Go, C and D). Therefore, inhibition by natriuretic peptides of collagen and DNA syntheses in adult cardiac fibroblasts could be mimicked by this cGMP analog.



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  		Figure 4. Effects of ANP ({circ}), BNP ({blacktriangleup}), and CNP ({bullet}) on collagen synthesis (A) and DNA synthesis (B), and effects of 8-bromo cGMP on collagen synthesis (C) and DNA synthesis (D) in cultured adult rat cardiac fibroblasts. Values are percentages of control (mean ± SE of six measurements). Control represents proline or thymidine incorporation in cells incubated in DMEM with 1% FCS. The mean radioactivities in the control wells were 9,001 ± 135 (A), 10,402 ± 250 (B), 7,911 ± 233 (C), and 7,503 ± 291 cpm/well (D), respectively. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has demonstrated for the first time that CNP is produced and secreted from cultured adult rat cardiac fibroblasts. Although CNP was initially thought to be distributed in the central nervous system and to act as a neuropeptide (1, 11), it has been shown that CNP is localized or synthesized in the kidney (12), bone (13), vascular cells (2, 14), and blood cells (15). As for the heart, discrepant findings have been reported. Some studies reported that ir-CNP or CNP mRNA was not detected in human or rat hearts (11, 16). On the other hand, Wei et al. (17) revealed the existence of CNP peptide in human ventricular myocardium by immunohistochemical staining. However, it does not necessarily indicate that CNP is synthesized in and secreted from cardiac myocytes. There is the possibility that the positive immunostaining of CNP by Wei et al. (17) might be attributable to the binding of CNP to its receptors, as they discussed. Vollmar et al. (18) and Minamino et al. (9) showed that CNP mRNA was detected in the rat heart using an RT-PCR technique. However, their studies have not clarified which type of cells synthesizes CNP in the heart. Moreover, the possibility that the CNP gene expression was derived from coronary endothelium could not be ruled out, because the detection was induced by the RT-PCR method. In the present study, we demonstrated that cardiac fibroblasts, in which no contamination of endothelial cells was confirmed, secreted ir-CNP in a time-dependent manner. Although ANP and BNP were recently reported to be synthesized by cardiac fibroblasts (or myofibroblasts; Refs. 19 and 20), the HPLC profiles in the present study proved that the ir-CNP observed in the culture medium was not due to the cross-reactivity of ANP or BNP. In fact, neither ir-ANP nor ir-BNP was detected in the medium of cultured fibroblasts. Furthermore, we clearly showed by the Northern blot analysis the existence of CNP mRNA in adult and neonatal cardiac fibroblasts. Significant expression of the CNP gene was not observed in neonatal cardiac myocytes, although we failed to examine its expression in adult cultured cardiac myocytes. These findings suggest that CNP is produced probably only by cardiac fibroblasts, differing from the production site of ANP and BNP.

In the present study, TGF-ß1, bFGF, and endothelin-1 among various humoral factors elicited a weak but significant stimulatory effect on CNP secretion. TGF-ß and bFGF have been shown to increase CNP secretion in cultured vascular endothelial cells and/or chondrocytes (2, 13). In addition, it has been shown that CNP production is enhanced by activation of protein kinase C (21), of which endothelin-1 is one of the activators. Therefore, the present findings concerning stimulation factors of CNP secretion are consistent with previous observations. Because TGF-ß1, bFGF, and endothelin-1 all increase collagen or DNA synthesis in cardiac fibroblasts, CNP induced by these factors may display a counter-action against cardiac fibrosis.

Previous studies showed that ANP and BNP inhibited collagen production and proliferation of cultured cardiac fibroblasts (22, 23, 24). However, little is known about the effect of CNP on those cells, except for one study reported that CNP as well as ANP and BNP decreased bFGF-induced DNA synthesis in neonatal cardiac fibroblasts (23). The present study has clearly demonstrated that CNP inhibits both DNA and collagen syntheses in adult cardiac fibroblasts, and that these suppressive effects by CNP are more potent than those by ANP or BNP. Furthermore, CNP increased the intracellular cGMP levels more markedly compared with ANP or BNP, and a cGMP analog reproduced the suppression of DNA and collagen syntheses. Three receptor subtypes for natriuretic peptides are presently known. Two of these receptors have GC activity (i.e. induce cGMP accumulation) and are called GC-A and GC-B. ANP and BNP bind specifically to GC-A rather than GC-B (25). In contrast, CNP binds to GC-B with a very high affinity but almost lacks the binding ability to GC-A (26). Because our study showed that cultured adult rat cardiac fibroblasts predominantly expressed the GC-B by Northern hybridization, the inhibitory effects of CNP on the proliferation and collagen production of cardiac fibroblasts are probably through a GC-B-mediated cGMP-dependent pathway. The present observations concerning the gene expression of the GC-B- and CNP-stimulated cGMP accumulation in fibroblasts were broadly consistent with the findings previously reported by Lin et al. (3). In their study, however, expression levels of the GC-A receptor in cultured cardiac fibroblasts were comparable to those of the GC-B, and ANP stimulated cGMP production as potently as CNP. The exact reason for such discrepant findings is unclear, but it may be partly due to the difference in the methods of preparation of cardiac fibroblasts and the conditions of the fibroblast cell culture used, including their purity and passages.

A previous study showed that GC-B mRNA levels were markedly increased in the hypertrophied left ventricle induced by volume overload (27). Furthermore, Doyle et al. (28) recently revealed that the distribution of the GC-B receptor in adult rat ventricles was confined to the nonmyocytes such as interstitial and vascular fibroblasts. These findings suggest that CNP released from cardiac fibroblasts may function effectively on themselves as an autocrine negative regulator against excessive cardiac fibrosis in pathological states. However, further investigations are necessary to clarify the physiological and pathophysiological roles of endogenous CNP in the heart.

In conclusion, the present study demonstrated that CNP was synthesized in and secreted from cultured adult cardiac fibroblasts and that CNP inhibited DNA and collagen syntheses in these cells more potently than ANP and BNP. Cardiac CNP produced by fibroblasts may function as a local modulator of cardiac fibrosis, defined as a proliferation of interstitial fibroblasts and the biosynthesis of extracellular matrix components.


   Acknowledgments
 
We thank Yoko Saito for her technical assistance.


   Footnotes
 
This work was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan, grants-in-aid for scientific research (14571044) from Japan Society for the Promotion of the Science, and U.S. Public Health Science Grants DK-52121 and HL-68607.

Abbreviations: ANP, Atrial natriuretic peptide; bFGF, basic fibroblast growth factor; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; FCS, fetal calf serum; GC, guanylyl cyclase; ir, immunoreactive.

Received January 27, 2003.

Accepted for publication March 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M. Wakeno, T. Minamino, O. Seguchi, H. Okazaki, O. Tsukamoto, K.-i. Okada, A. Hirata, M. Fujita, H. Asanuma, J. Kim, et al.
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M. D. Jarvis, M. T. Rademaker, L. J. Ellmers, M. J. Currie, J. L. McKenzie, B. R. Palmer, C. M. Frampton, A. M. Richards, and V. A. Cameron
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T. E. H. Christoffersen, M. Aplin, C. C. Strom, S. P. Sheikh, O. Skott, P. K. Busk, S. Haunso, and L. B. Nielsen
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T. Nishikimi, N. Maeda, and H. Matsuoka
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D. Rahmutula and D. G. Gardner
C-Type Natriuretic Peptide Down-Regulates Expression of Its Cognate Receptor in Rat Aortic Smooth Muscle Cells
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T. Tokudome, T. Horio, I. Kishimoto, T. Soeki, K. Mori, Y. Kawano, M. Kohno, D. L. Garbers, K. Nakao, and K. Kangawa
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T. Soeki, I. Kishimoto, H. Okumura, T. Tokudome, T. Horio, K. Mori, and K. Kangawa
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S. Murakami, N. Nagaya, T. Itoh, T. Fujii, T. Iwase, K. Hamada, H. Kimura, and K. Kangawa
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T. Tokudome, T. Horio, T. Soeki, K. Mori, I. Kishimoto, S.-i. Suga, F. Yoshihara, Y. Kawano, M. Kohno, and K. Kangawa
Inhibitory Effect of C-Type Natriuretic Peptide (CNP) on Cultured Cardiac Myocyte Hypertrophy: Interference between CNP and Endothelin-1 Signaling Pathways
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