ARMC4 mutations cause primary ciliary dyskinesia with randomization of left/right body asymmetry.

The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.

Rho GAP myosin IXa is a regulator of kidney tubule function.

Mammalian class IX myosin Myo9a is a single-headed, actin-dependent motor protein with Rho GTPase-activating protein activity that negatively regulates Rho GTPase signaling. Myo9a is abundantly expressed in ciliated epithelial cells of several organs. In mice, genetic deletion of Myo9a leads to the formation of hydrocephalus. Whether Myo9a also has essential functions in the epithelia of other organs of the body has not been explored. In the present study, we report that Myo9a-deficient mice develop bilateral renal disease, characterized by dilation of proximal tubules, calyceal dilation, and thinning of the parenchyma and fibrosis. These structural changes are accompanied by polyuria (with normal vasopressin levels) and low-molecular-weight proteinuria. Immunohistochemistry revealed that Myo9a is localized to the circumferential F-actin belt of proximal tubule cells. In kidneys lacking Myo9a, the multiligand binding receptor megalin and its ligand albumin accumulated at the luminal surface of Myo9a-deficient proximal tubular cells, suggesting that endocytosis is dysregulated. In addition, we found, surprisingly, that levels of murine diaphanous-related formin-1, a Rho effector, were decreased in Myo9a-deficient kidneys as well as in Myo9a knockdown LLC-PK1 cells. In summary, deletion of the Rho GTPase-activating protein Myo9a in mice causes proximal tubular dilation and fibrosis, and we speculate that downregulation of murine diaphanous-related formin-1 and impaired protein reabsorption contribute to the pathophysiology.

The Myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization.

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.

Tropomyosin 4 expression is enhanced in dedifferentiating smooth muscle cells in vitro and during atherogenesis.

Dedifferentiation of smooth muscle cells (SMC) from the contractile to the synthetic phenotype is a key event in atherosclerosis. A comparable phenotypic change from the contractile to the synthetic state is rapidly incurred when SMC are grown in culture. To identify genes that characterize the contractile and synthetic phenotypes, we performed differential display reverse transcription polymerase chain reactions on RNA from porcine arterial contractile SMC obtained directly from medial tissues and from SMC made synthetic by cell culturing. One of the differentially expressed cDNAs we identified encoded tropomyosin 4 (TM4). Whereas basal levels of TM4 existed in contractile SMC, the amount of TM4 transcripts strongly increased in synthetic SMC (33% vs. 86-106%; p < 0.005). Induction of foam cell formation had no additional enhancing effect on the expression of TM4 in cultivated SMC. We also tested whether TM4 expression was correspondingly enhanced during atherogenesis. The number of TM4-expressing SMC increased with plaque development as demonstrated by simultaneous in situ hybridization and immunohistochemistry. We compared the localization patterns of myosin heavy chain isoforms in normal arteries and lesions of increasing severity and determined that TM4 expression was relegated mainly to SMC of the synthetic phenotype in the media and intima during atherogenesis. The present study demonstrates that upregulation of TM4 mRNA is a relevant marker of dedifferentiation in vascular SMC.

DYX1C1 is required for axonemal dynein assembly and ciliary motility.

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2-4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

Myosin IXa regulates epithelial differentiation and its deficiency results in hydrocephalus.

The ependymal multiciliated epithelium in the brain restricts the cerebrospinal fluid to the cerebral ventricles and regulates its flow. We report here that mice deficient for myosin IXa (Myo9a), an actin-dependent motor molecule with a Rho GTPase-activating (GAP) domain, develop severe hydrocephalus with stenosis and closure of the ventral caudal 3rd ventricle and the aqueduct. Myo9a is expressed in maturing ependymal epithelial cells, and its absence leads to impaired maturation of ependymal cells. The Myo9a deficiency further resulted in a distorted ependyma due to irregular epithelial cell morphology and altered organization of intercellular junctions. Ependymal cells occasionally delaminated, forming multilayered structures that bridged the CSF-filled ventricular space. Hydrocephalus formation could be significantly attenuated by the inhibition of the Rho-effector Rho-kinase (ROCK). Administration of ROCK-inhibitor restored maturation of ependymal cells, but not the morphological distortions of the ependyma. Similarly, down-regulation of Myo9a by siRNA in Caco-2 adenocarcinoma cells increased Rho-signaling and induced alterations in differentiation, cell morphology, junction assembly, junctional signaling, and gene expression. Our results demonstrate that Myo9a is a critical regulator of Rho-dependent and -independent signaling mechanisms that guide epithelial differentiation. Moreover, Rho-kinases may represent a new target for therapeutic intervention in some forms of hydrocephalus.

Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells.

Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 microM for 24 h) toxicity was significantly attenuated from 27.3+/-3.9% in ARF6-WT to 11.1+/-4.0% in ARF6-DN cells (n=6, P<0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0+/-4.6% versus 3.9+/-3.9% of ARF6-WT cells, n=3, P<0.01) and/or remained near the PM (89.3+/-5. 6% versus 45.2+/-14.3% of ARF6-WT cells, n=3, P<0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells.

Cadmium induces nuclear translocation of beta-catenin and increases expression of c-myc and Abcb1a in kidney proximal tubule cells.

Cadmium (Cd2+) induces renal proximal tubular (PT) damage, including disruption of the E-cadherin/beta-catenin complex of adherens junctions (AJs) and apoptosis. Yet, chronic Cd2+ exposure causes malignant transformation of renal cells. Previously, we have demonstrated that Cd(2+)-mediated up-regulation of the multidrug transporter Abcb1 causes apoptosis resistance in PT cells. We hypothesized that Cd2+ activates adaptive signaling mechanisms mediated by beta-catenin to evade apoptosis and increase proliferation. Here we show that 50 microM Cd2+, which induces cell death via apoptosis and necrosis, also causes a decrease of the trans-epithelial resistance of confluent WKPT-0293 Cl.2 cells, a rat renal PT cell model, within 45 min of Cd2+ exposure, as measured by electric cell-substrate impedance sensing. Immunofluorescence microscopy demonstrates Cd(2+)-induced decrease of E-cadherin at AJs and redistribution of beta-catenin from the E-cadherin/beta-catenin complex of AJs to cytosol and nuclei after 3 h. Immunoblotting confirms Cd(2+)-induced decrease of E-cadherin expression and translocation of beta-catenin to cytosol and nuclei of PT cells. RT-PCR shows Cd(2+)-induced increase of expression of c-myc and of the isoform Abcb1a at 3 h. The data prove for the first time that Cd2+ induces nuclear translocation of beta-catenin in PT cells. We speculate that Cd2+ activates beta-catenin/T-cell factor signaling to trans-activate proliferation and apoptosis resistance genes and promote carcinogenesis of PT cells.

Knockdown of endosomal/lysosomal divalent metal transporter 1 by RNA interference prevents cadmium-metallothionein-1 cytotoxicity in renal proximal tubule cells.

Chronic exposure to Cd2+ causes renal proximal tubular (PT) damage. Cd2+ reaches the PT mainly as cadmium-metallothionein 1 (CdMT-1) complexes that are filtered at the glomerulus and then internalized in part via endocytosis mediated by megalin and cubulin. Subsequently, Cd2+ is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports Cd2+ and is expressed exclusively in endosomes/lysosomes in rat PT cells. Using vector-based RNA interference with short-hairpin small-interfering RNAs (shRNAs) to downregulate DMT1 in the rat renal PT cell line WKPT-0293 Cl.2, we tested the hypothesis that endosomal/lysosomal DMT1 is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMT1 protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 +/- 9.6 and 36.9 +/- 14.4% (n = 5-6), respectively. Similarly, sh3 reduced perinuclear DMT1 immunostaining in transfected cells. Consistent with the assumed role of DMT1 in CdMT-1 cytotoxicity, sh3, but not the empty vector or sh5, significantly attenuated cell death induced by a 24-h exposure to 14.3 microM CdMT-1 by 35.6 +/- 4.2% (n = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free Cd2+ toxicity was altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and Cd2+-induced cell death signaling are not affected by DMT1 knockdown. Thus we conclude that endosomal/lysosomal DMT1 plays a role in renal PT CdMT-1 toxicity.

Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells.

The nephrotoxic metal cadmium at micromolar concentrations induces apoptosis of rat kidney proximal tubule (PT) cells within 3-6 h of exposure. The underlying cell death pathways remain poorly defined. Using Hoechst 33342/ethidium bromide nuclear staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell death assays, 10-50 microM cadmium induced apoptosis of immortalized rat kidney cells derived from the S1-segment of PT at 6 and 24 h, but necrosis at 24 h only. Cadmium (10-50 microM) also caused mitochondrial cytochrome c (cyt. c)- and apoptosis-inducing factor release at 24 h, but not at 6 h, as measured by immunofluorescence imaging and immunoblotting. Caspases-9 and -3 were activated only by 10 microM cadmium for 24 h, and accordingly apoptosis was significantly reduced by the respective inhibitors (z-LEHD-fmk, z-DEVD-fmk; 10 microg/ml) at 24 h, but not at 6 h, without affecting necrosis. At 6 h, 10 microM cadmium increased the activity of the calcium-activated protease calpain, but not at 24 h, and calpain inhibitors (ALLN, PD 150606; 10-30 microM) blocked apoptosis by 10 microM cadmium at 3-6 h. However, PD-150606 also attenuated caspase-3 activity and apoptosis at 24 h, suggesting calpain-dependent caspase activation. Thus cadmium-induced apoptosis of PT cells involves a complex and sensitive interplay of signaling cascades involving mitochondrial proapoptotic factors, calpains and caspases, whose activation is also determined by cadmium concentration and the duration of cadmium exposure.

Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells.

Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (molecular mass approximately 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 reabsorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concentration-dependent, saturating at approximately 15 microM. At 14.3 microM, metallothionein-1 uptake could be significantly attenuated by 30.9 +/- 6.6% (n = 4) by 1 muM of the receptor-associated protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 muM cadmium-metallothionein-1 was significantly reduced by 41.0 +/- 7.6%, 61.6 +/- 3.4%, and 26.2 +/- 1.8% (n = 4-5 each) by the presence of 1 microM RAP, 400 microg/ml anti-megalin antibody, or 5 microM of the cubilin-specific ligand, apo-transferrin, respectively. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.

Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes.

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.