The achondroplastic mouse is a spontaneous mutant characterized by disproportionate dwarfism with short limbs and tail due to disturbed chondrogenesis during endochondral ossification. These abnormal phenotypes are controlled by an autosomal recessive gene (cn). In this study, linkage analysis using 115 affected mice of F 2 progeny mapped the cn locus on an ϳ0.8-cM region of chromosome 4, and natriuretic peptide receptor 2 (Npr2) gene was identified as the most potent candidate for the cn mutant in this region. This gene encodes a receptor for C-type natriuretic peptide (CNP) that positively regulates longitudinal bone growth by producing cGMP in response to CNP binding to the extracellular domain. Sequence analyses of the Npr2 gene in cn/cn mice revealed a T to G transversion leading to the amino acid substitution of highly conserved Leu with Arg in the guanylyl cyclase domain. In cultured chondrocytes of cn/cn mice, stimulus with CNP did not significantly increase intracellular cGMP concentration, whereas it increased in ؉/؉ mice. Transfection of the mutant Npr2 gene into COS-7 cells also showed similar results, indicating that the missense mutation of the Npr2 gene in cn/cn mice resulted in disruption of the guanylyl cyclase activity of the receptor. We therefore concluded that the dwarf phenotype of cn/cn mouse is caused by a loss-offunction mutation of the Npr2 gene, and cn/cn mouse will be a useful model to further study the molecular mechanism regulating endochondral ossification by CNP/natriuretic peptide receptor B signal.The skeleton of vertebrates is formed by two different processes, namely intramembranous and endochondral ossifications. The latter process leads to the development of long bones that comprise the appendicular skeleton and vertebrae. During endochondral ossification, mesenchymal cells initially differentiate into chondrocytes, and progress through proliferating, maturating, and hypertrophic stages with strict columnar alignment. Distal hypertrophic chondrocytes then undergo apoptosis and are replaced by trabecular bone. A large number of genes have been implicated in the mechanisms regulating these processes (1), and mutations of these genes often cause skeletal dysplasias with shortened extremities (2, 3).The natriuretic peptide (NP) 1 family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (4), and three receptors for NPs, including natriuretic peptide receptor A (NPRA), natriuretic peptide receptor B (NPRB), and natriuretic peptide receptor C (NPRC), have been identified in mammals (5-7). NPRA and NPRB consist of extracellular ligand binding, transmembrane, protein kinase homology, and guanylyl cyclase catalytic domains. These receptors mediate ligand signals by producing an intracellular second messenger, cyclic GMP (cGMP) (2). Both ANP and BNP bind to NPRA with high affinity, and CNP prefers binding to NPRB (8). NPRC has a ligand-binding domain and a short cytoplasmic domain that lacks guanylyl cyclase activity and i...

Morphological changes of osteoclasts by a MEK1 inhibitor, PD98059,were investigated to clarify a role of ERK. PD98059 promoted apoptosis of osteoclasts and the loss of ruffled borders. This study supports the importance of ERK in survival and polarity of osteoclasts.Introduction: Extracellular signal-regulated kinase (ERK) is a mitogen activated protein kinase (MAPK) that has been reported to play a role in the survival and apoptosis of osteoclasts. However, the precise signal transduction mechanism is not fully understood. The aim of this study was to clarify the role of ERK in osteoclasts by histological analysis. Materials and Methods: Using a rat calvarial organ culture system, the inhibition of ERK phosphorylation by PD98059, a MAPK/ERK kinase 1 (MEK1) inhibitor, was assayed by immunoblotting. Morphological changes in osteoclasts induced by PD98059 were elucidated by light and electron microscopy. The cellular localization of ERK was also determined by immunoelectron microscopy. Results: PD98059 inhibited phosphorylated ERK after a 1-h incubation. Ultrastructural study demonstrated that PD98059 induced the accumulation of vesicles and vacuoles in osteoclasts and the loss of ruffled border at 1 h. At 3 h, some osteoclasts showed apoptosis with nuclear condensation, and at 6 h after PD98059 treatment, many osteoclasts were detached from the bone surface and had lost their cell polarity. Electron microscopic immunohistochemistry revealed that ERK was mainly localized in the cytoplasm of clear zones in control osteoclasts, but apoptotic osteoclasts also showed immunoreactivity in clear zone-like structures in contact with osteoblast-lineage cells. Conclusion: These findings indicate that ERK in osteoclasts is involved in their survival and may be involved in the formation of a ruffled border and the maintenance of cell polarity.

Oocyte meiosis is arrested at prophase I by factors secreted from surrounding somatic cells after oocytes acquire meiotic competence at an early antral stage, and meiosis resumes in preovulatory follicles as a result of the luteinizing hormone (LH) surge. Recently, signaling by C-type natriuretic peptide (CNP) through its receptor, natriuretic peptide receptor 2 (NPR2), was found to be essential for meiotic arrest at the late antral stage. Whether or not CNP/NPR2 signaling maintains oocyte meiotic arrest in earlier follicular stages and how it is associated with meiotic resumption induced by the LH surge is unclear. In this study, we examined the expression of Nppc and Npr2, respectively encoding CNP and NPR2, in the ovaries of immature mice. Nppc and Npr2 mRNA were specifically expressed in the outer and inner granulosa cell layers, respectively, in early antral follicles. Histological analysis of mice with a mutation in Npr2 revealed precocious resumption of oocyte meiosis in early antral follicles. Ovaries of mice treated with excess human chorionic gonadotropin (hCG) exhibited markedly decreased Nppc mRNA levels in granulosa cells of preovulatory follicles. Moreover, we found that amphiregulin, a mediator of LH/hCG activity through epidermal growth factor receptor (EGFR), suppressed Nppc mRNA levels in cultured granulosa cells. These results suggest that CNP/NPR2 signaling is essential for oocyte meiotic arrest in early antral follicles and that activated LH/amphiregulin/EGFR signaling pathway suppresses this signal by downregulating Nppc expression.

Summary Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome.