[Show abstract][Hide abstract] ABSTRACT: Involvement of reactive oxygen species derived from NADPH oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology.
Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and TUNEL staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors were implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene.
These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.
[Show abstract][Hide abstract] ABSTRACT: Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease. Among 12 reported Nphp gene products, Inv/Nphp2, Nphp3 and Nek8/Nphp9 are localized to the proximal segment in the primary cilium. However, the functional relationships are unknown. This study focused on phenotype analysis of nek8 knockdown embryos and the genetic relationship between nek8 and inv in zebrafish. Knockdown of nek8 produced both pronephric cysts and abnormal cardiac looping. Simultaneous knockdown of nek8 and inv synergistically increased the incidence of these defects. Interestingly, nek8 mRNA rescued inv morphant phenotypes, although inv mRNA could not rescue nek8 morphant phenotypes. These results suggest that Nek8 acts downstream of Inv function.
[Show abstract][Hide abstract] ABSTRACT: Primary cilia are organelles that extend from the cell surface. More than 600 proteins have been identified in cilia, but ciliary targeting mechanisms are poorly understood. Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease with 11 responsible genes (NPHP1-11) thus far being identified. The mouse Nphp3 gene product is localized in the cilia and contains coiled-coil (CC) domains and tetratricopeptide repeats, but the ciliary targeting sequences (CTSs) are unknown. In the present study, we generated a series of GFP-tagged deletion constructs of Nphp3 and tried to find the CTSs of Nphp3. We found that the N-terminal 201 amino acid fragment (Nphp3 [1-201]), which contains two CC domains, is necessary and sufficient for cilia localization. Further analysis revealed that an N-terminal glycine (G2), which is a conserved myristoylation site among vertebrates, is also essential for trafficking of Nphp3 to the ciliary shaft. Interestingly, the N-terminal fragments, Nphp3 (8-201), Nphp3 (52-201), and Nphp3 (96-201), that contain the CC domains, targeted the basal body, but could not enter into the ciliary shaft. Our results showed the importance of myristoylation in ciliary trafficking, and suggest that Nphp3 trafficking to the ciliary shaft occurs in a two-step process.
[Show abstract][Hide abstract] ABSTRACT: Loss of cilia and ciliary protein causes various abnormalities (called ciliopathy), including situs inversus, renal cystic diseases, polydactyly and dysgenesis of the nervous system. Renal cystic diseases are the most frequently observed symptoms in ciliopathies. Cilia are microtubule-based organelles with the following regions: a ciliary tip, shaft, transitional zone and basal body/mother centriole. Joubert syndrome (JBTS), Meckel Gruber syndrome (MKS) and Nephronophthisis (NPHP) are overlapping syndromes. Recent studies show that JBST and MKS responsible gene products are localized in the transitional zone of the cilia, where they function as a diffusion barrier, and control protein sorting and ciliary membrane composition. Nephrocystins are gene products of NPHP responsible genes, and at least 11 genes have been identified. Although some nephrocystins interact with JBST and MKS proteins, proteomic analysis suggests that they do not form a single complex. Localization analysis reveals that nephrocystins can be divided into two groups. Group I nephrocystins are localized in the transitional zone, whereas group II nephrocystins are localized in the Inv compartment. Homologs of group I nephrocystins, but not group II nephrocystins, have been reported in C. reinhardtii and C. elegans. In this review, we summarize the structure of the ciliary base of C. reinhardtii, C. elegans and mammalian primary cilia, and discuss function of nephrocystins. We also propose a new classification of nephrocystins.
[Show abstract][Hide abstract] ABSTRACT: A primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. It is structurally divided along its vertical axis into sub-compartments that include the ciliary tip, the shaft, the ciliary necklace segment, the transitional zone and the basal body. We recently discovered that the shaft of the primary cilia has a distinct molecular compartment, termed the "Inv compartment", which is characterized by the accumulation of Inv at the base of primary cilia. Inv was discovered as a causative gene in inv mutant mice. It was later found to be responsible for the infantile type of nephronophthisis (NPHP2). Nephronophthisis (NPHP) is an autosomal recessive kidney disease. Nine causative genes have been identified, with all examined products thought to function in cilia, basal body and/or centrioles. However, their exact intra-ciliary localization and relationship have not been clear. Here, we report that products of Nphp3 and Nek8 (the mouse orthologs of the causative genes for NPHP3 and NPHP9, respectively) localize to the Inv compartment. We also show that Inv is essential for the compartmental localization of Nphp3 and Nek8, whereas localization of Inv does not require Nphp3 or Nek8. Nphp1 and Nphp4 also localize at the proximal region of the cilium, but not in Inv compartment. Our results indicate that Inv acts as an anchor for Nphp3 and Nek8 in the Inv compartment, and suggest that Inv compartment is a candidate site for intra-ciliary interaction of Inv, Nphp3 and Nek8.
[Show abstract][Hide abstract] ABSTRACT: Glis3 is a member of the Gli-similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3-deficient mice by gene-targeting. The Glis3(-/-) mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulin-producing cell formation. The pancreatic phenotypes indicate that the Glis3-deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.
[Show abstract][Hide abstract] ABSTRACT: Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.
PLoS ONE 02/2009; 4(7):e6299. DOI:10.1371/journal.pone.0006299 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. Loss or mutation of ciliary proteins can lead to polycystic kidney disease and other developmental abnormalities. inv mutant mice develop multiple renal cysts and are a model for human nephronophthisis type 2. The mouse Inv gene encodes a 1062-amino-acid protein that is localized in primary cilia. In this study, we show that the Inv protein (also known as inversin) is localized at a distinctive proximal segment of the primary cilium, using GFP-tagged Inv constructs and anti-Inv antibody. We named this segment the Inv compartment of the cilium. Further investigation of the Inv protein showed that 60 amino acids at its C-terminal, which contains ninein homologous sequences, are crucial for its localization to the Inv compartment. Fluorescence recovery after photobleaching analysis revealed that the Inv protein was dynamic within this compartment. These results suggest that localization of the Inv protein to the Inv compartment is actively regulated. The present study revealed that the primary cilium has a distinct molecular compartment in the body of the primary cilium with a specific confining and trafficking machinery that has not been detected previously by morphological examination.
[Show abstract][Hide abstract] ABSTRACT: The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxide-generating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1(-/Y)), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed. In dorsal root ganglia (DRG) neurons of Nox1(+/Y), pretreatment with chemical mediators bradykinin, serotonin, or phorbol 12-myristate 13-acetate (PMA) augmented the capsaicin-induced calcium increase, whereas this increase was significantly attenuated in DRG neurons of Nox1(-/Y). Concomitantly, PMA-induced translocation of PKCepsilon was markedly perturbed in Nox1(-/Y) or Nox1(+/Y) DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCepsilon, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCepsilon but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCepsilon in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2008; 28(38):9486-94. DOI:10.1523/JNEUROSCI.1857-08.2008 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The involvement of the P2 receptor in the activation of ERK induced by a short transient fluid flow stimulation in MC3T3-E1 osteoblasts was examined in the current study. The ERK activation induced by this transient fluid flow stimulation was followed by an increase in c-fos mRNA expression. Suramin, a non-selective P2 receptor antagonist, and two different P2X7 receptor (P2X7R) antagonists, ATP analogue (oxidized ATP) and dye (Brilliant blue G), inhibited fluid flow-induced ERK activation. However, the P2Y receptor pathway inhibitor U73122 did not abolish this ERK activation. The P2X7R agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) significantly increased ERK activation and this activation could be completely inhibited by oxidized ATP and Brilliant blue G. Our results suggest that P2X7R is a highly sensitive P2 receptor for fluid flow-induced ERK activation in osteoblasts.
Biochemical and Biophysical Research Communications 09/2008; 372(3):486-90. DOI:10.1016/j.bbrc.2008.05.066 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary cilia are hypothesized to act as a mechanical sensor to detect renal tubular fluid flow. Anomalous structure of primary cilia and/or impairment of increases in intracellular Ca2+ concentration in response to fluid flow are thought to result in renal cyst formation in conditional kif3a knockout, Tg737 and pkd1/pkd2 mutant mice. The mutant inv/inv mouse develops multiple renal cysts like kif3a, Tg737 and pkd1/pkd2 mutants. Inv proteins have been shown to be localized in the renal primary cilia, but response of inv/inv cilia to fluid stress has not been examined. In the present study, we examined the mechanical response of primary cilia to physiological fluid flow using a video microscope, as well as intracellular Ca2+ increases in renal epithelial cells from normal and inv/inv mice in response to flow stress. Percentages of ciliated cells and the length of primary cilia were not significantly different between primary renal cell cultures from normal and inv/inv mutant mice. Localization of inv protein was restricted to the base of primary cilia even under flow stress. Inv/inv mutant cells had similar bending mechanics of primary cilia in response to physiological fluid flow compared to normal cells. Furthermore, no difference was found in intracellular Ca2+ increases in response to physiological fluid flow between normal and inv/inv mutant cells. Our present study suggests that the function of the inv protein is distinct from polaris (the Tg737 gene product), polycystins (pkd1 and pkd2 gene products).
Cell Structure and Function 02/2005; 30(2):93-100. DOI:10.1247/csf.30.93 · 1.68 Impact Factor