Yasato Komatsu

Dwarfism and early death in mice lacking C-type natriuretic peptide

Longitudinal bone growth is determined by endochondral ossification that occurs as chondrocytes in the cartilaginous growth plate undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. The natriuretic peptide family consists of three structurally related endogenous ligands, atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), and is thought to be involved in a variety of homeostatic processes. To investigate the physiological significance of CNP in vivo, we generated mice with targeted disruption of CNP (Nppc−/− mice). The Nppc−/− mice show severe dwarfism as a result of impaired endochondral ossification. They are all viable perinatally, but less than half can survive during postnatal development. The skeletal phenotypes are histologically similar to those seen in patients with achondroplasia, the most common genetic form of human dwarfism. Targeted expression of CNP in the growth plate chondrocytes can rescue the skeletal defect of Nppc−/− mice and allow their prolonged survival. This study demonstrates that CNP acts locally as a positive regulator of endochondral ossification in vivo and suggests its pathophysiological and therapeutic implication in some forms of skeletal dysplasia.

Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway.

Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP–GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia.

Phenotype-related alteration in expression of natriuretic peptide receptors in aortic smooth muscle cells.

To elucidate the physiological and pathophysiological roles of the natriuretic peptide family in vascular smooth muscle cells, in which the natriuretic peptide family is implicated in growth inhibition as well as vasorelaxation, we have examined the phenotype-related expression of three kinds of natriuretic peptide receptors in rat aortic smooth muscle cells. The expression of natriuretic peptide receptors at the mRNA level was studied by Northern blot hybridization, and the expression at the protein level was determined by the cGMP production method and receptor binding assay. In intact aortic media, atrial natriuretic peptide (ANP)-A receptor mRNA and ANP-B receptor mRNA were detected, and the potency of cGMP production by ANP was at least two orders of magnitude stronger than that by C-type natriuretic peptide. Clearance receptor mRNA was undetectable, and only a small amount of the clearance receptor was detected by the binding assay in intact aortic media. By contrast, in cultured aortic smooth muscle cells at the first, fifth, and 17th passages, the ANP-B receptor mRNA level markedly increased; meanwhile, the expression of the ANP-A receptor mRNA became undetectable. C-type natriuretic peptide was one order of magnitude more potent than ANP in cGMP production in cultured aortic smooth muscle cells. The clearance receptor density and its mRNA level increased tremendously in these cultured cells. These results demonstrate that the marked phenotype-related alteration occurs in the expression of natriuretic peptide receptors in rat aortic smooth muscle cells.

Regulation of Endothelial Production of C-Type Natriuretic Peptide in Coculture With Vascular Smooth Muscle Cells Role of the Vascular Natriuretic Peptide System in Vascular Growth Inhibition

Recently, we have demonstrated that C-type natriuretic peptide (CNP) is produced in vascular endothelial cells (ECs). In the present study, we investigated the interaction of ECs and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth, using the EC/SMC coculture system. The concentration of CNP-like immunoreactivity in the medium was increased 60-fold within 48 hours in the EC/SMC coculture with direct contact compared with that in EC alone. Northern blot analysis revealed the augmented expression of CNP mRNA in the EC/SMC coculture. The accumulation of intracellular cGMP in the coculture was concomitantly increased, and this response was blocked by anti-CNP monoclonal antibody and HS-142-1, a nonpeptide atrial natriuretic peptide receptor antagonist. The concentration of biologically active transforming growth factor-beta (TGF-beta) in the culture medium of the coculture with direct contact of ECs and SMCs was elevated to the level to stimulate endothelial production of CNP. Actually, the neutralizing antibody against TGF-beta abrogated the cGMP accumulation in the coculture. These results show that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-beta. Furthermore, the conditioned medium from ECs stimulated by TGF-beta was demonstrated to have a growth-inhibitory effect on SMCs, which was abolished by anti-CNP monoclonal antibody and HS-142-1. The treatment with anti-CNP monoclonal antibody and HS-142-1 also significantly increased the cell number of the EC/SMC coculture. The present study reveals the pathophysiological significance of endothelial CNP as a paracrine/autocrine vascular regulator for vascular growth in the interaction of ECs and SMCs.

Systemic Administration of C-Type Natriuretic Peptide as a Novel Therapeutic Strategy for Skeletal Dysplasias

Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Furthermore, we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in a mouse model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, to elucidate whether or not the systemic administration of CNP is a novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by iv administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.

Impaired bone resorption by lipopolysaccharide in vivo in mice deficient in the prostaglandin E receptor EP4 subtype

ABSTRACT In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions.

Human C-type natriuretic peptide. Characterization of the gene and peptide.

We isolated the human C-type natriuretic peptide gene and identified the peptide in the brain. The human C-type natriuretic peptide gene appeared to be composed of at least two exons and one intron. In the 5′-flanking region, there is an array of cis elements (an inverted CCA AT box, two GC boxes, and a cyclic AMP response element-like sequence) that is not present in upstream sequences of the atrial and brain natriuretic peptide genes. Analysis of the deduced amino acid sequence revealed that human prepro C-type natriuretic peptide comprises 126 amino acids and that the C-terminal 22-residue peptide (G-L-S-K-G-C-F-G-L-K-L-D-R-I-G-S-M-S-G-L-G-C) preceded by Lys-Lys is identical to the porcine counterpart. However, replacement of two amino acids took place in the C-terminal 53-residue sequence, corresponding to another endogenous form of the peptide. Reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay for C-type natriuretic peptide demonstrated that it occurs in the human brain. C-type natriuretic peptide–like immunoreactivity was detected in discrete regions of the human brain, and its level was 10-fold higher than the atrial and brain natriuretic peptide levels, raising the possibility that C-type natriuretic peptide is the major natriuretic peptide in the human brain.

Thyroid Hormones Promote Chondrocyte Differentiation in Mouse ATDC5 Cells and Stimulate Endochondral Ossification in Fetal Mouse Tibias Through Iodothyronine Deiodinases in the Growth Plate.

Thyroid hormones (THs), 3,3′,5‐triiodo‐L‐thyronine (T3) and L‐thyroxine (T4), are important for the normal development of the growth plate (GP); congenital TH deficiency leads to severe dwarfism. In mouse chondrogenic cell line, ATDC5, T3 enhanced differentiation and increased Alizarin red staining, but did not affect Alcian blue staining. In organ‐cultured mouse tibias, THs stimulated the cartilage growth, especially in the hypertrophic zone. Interestingly, T4 was as equally potent as T3 in organ‐cultured tibias, which suggests that T4 is metabolized locally to T3, because T4 is a prohormone and must be converted to T3 for its activity. Two enzymes catalyze the conversion; type I deiodinase (D1) and type II deiodinase (D2). D1 has a ubiquitous distribution and D2, with a high affinity for T4, is present where the maintenance of intracellular T3 concentration is critical. Messenger RNAs (mRNAs) for D1 and D2 were detected in neonatal mouse tibias and ATDC5 cells. The enzyme activity was unaffected by the D1 inhibitor 6‐propyl‐2‐thiouracil, suggesting that D2 mainly catalyzes the reaction. D2 mRNA was detected in differentiated ATDC5 cells. In organ‐cultured mouse tibias, D2 activity was greater at later stages. In contrast, thyroid hormone receptors (TRs) were expressed in neonatal mouse tibias and ATDC5 cells, but their expression levels in ATDC5 cells were stable throughout the culture periods. Therefore, increased T3 production at later stages by D2 is likely to contribute to the preferential effects of THs in the terminal differentiation of GP. This article is the first to show that T4 is activated locally in GP and enhances the understanding of TH effects in GP.

Significance of C-type natriuretic peptide (CNP) in endochondral ossification: analysis of CNP knockout mice.

CNP has a primary structure similar to that of ANP and BNP, consisting of 22 amino acids, and the ring portion, consisting of 17 amino acids, is highly homologous to ANP and BNP. Different from ANP and BNP, CNP lacks the C-terminal tail and has a Cys residue at the Cterminus. Another species of CNP is CNP-53, which has an N-terminal extension of 31 amino acids compared with CNP-22. Molecular cloning of the CNP precursor in the pig, rat, human, and mouse has revealed that the primary structure of CNP-22 is identical in these species [4,5]. However, two amino-acid substitutions are noted in CNP-53 between human and porcine/rat/mouse precursors. ProCNP consists of 103 amino acids, and a single arginine residue precedes CNP-53 and CNP-22. Using reverse-phase high-performance liquid chromatography (HPLC), we demonstrated that the major molecular forms in the human brain are CNP-22 and CNP-53 [3]. Using the specific radioimmunoassay (RIA) for CNP, it was elucidated that immunoreactive CNP was detected in porcine, rat, and human brains, but not in the peripheral organs, including the heart [3].

Vascular Endothelial Growth Factor Suppresses C-Type Natriuretic Peptide Secretion

Angiogenesis plays a pivotal role not only in wound healing and tumor progression but also in diabetic angiopathy, arteriosclerosis, and collateral formation of obstructive vascular diseases. Vascular endothelial growth factor (VEGF) is now thought to be an endothelium-specific and potent angiogenic factor. We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from porcine brain, is produced by endothelial cells and proposed that CNP can exert control over vascular tone and growth as a local vascular regulator. In the present study, we examined the effect of VEGF on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP we developed. VEGF (1 to 100 ng/mL) dose-dependently suppressed CNP secretion from cultured bovine endothelial cells, and 100 ng/mL VEGF suppressed endothelial CNP secretion to 28% of control levels (31.7 +/- 5.5 versus 8.9 +/- 0.8 fmol/mL, vehicle versus VEGF). VEGF also suppressed CNP mRNA expression in endothelial cells 9 hours after administration. In contrast, basic fibroblast growth factor (20 ng/mL), an endothelium-nonspecific angiogenic factor, significantly stimulated CNP secretion by 290%. These results indicate that VEGF can regulate vascular tone and growth in the process of angiogenesis through suppression of endothelial secretion of CNP, which is an endothelium-derived vasorelaxing and growth-inhibitory peptide.