GEMC1 is a critical regulator of multiciliated cell differentiation.

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1-deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.

Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells.

BACKGROUND: Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells. RESULTS: Using fluorometric measurements of intracellular Ca2+ concentration ([Ca2+]i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca2+]i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells. CONCLUSIONS: Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.

Mobilization of endothelial progenitor cells in acute cardiovascular events in the PROCELL study: time-course after acute myocardial infarction and stroke.

The mobilization pattern and functionality of endothelial progenitor cells after an acute ischemic event remain largely unknown. The aim of our study was to characterize and compare the short- and long-term mobilization of endothelial progenitor cells and circulating endothelial cells after acute myocardial infarction or atherothrombotic stroke, and to determine the relationship between these cell counts and plasma concentrations of vascular cell adhesion molecule (VCAM-1) and Von Willebrand factor (VWF) as surrogate markers of endothelial damage and inflammation. In addition, we assessed whether endothelial progenitor cells behave like functional endothelial cells. We included 150 patients with acute myocardial infarction or atherothrombotic stroke and 145 controls. Endothelial progenitor cells [CD45-, CD34+, KDR+, CD133+], circulating endothelial cells [CD45-, CD146+, CD31+], VWF, and VCAM-1 levels were measured in controls (baseline only) and in patients within 24h (baseline) and at 7, 30, and 180 days after the event. Myocardial infarction patients had higher counts of endothelial progenitor cells and circulating endothelial cells than the controls (201.0/mL vs. 57.0/mL; p<0.01 and 181.0/mL vs. 62.0/mL; p<0.01). Endothelial progenitor cells peaked at 30 days post-infarction (201.0/mL vs. 369.5/mL; p<0.01), as did VCAM-1 (573.7 ng/mL vs. 701.8 ng/mL; p<0.01). At 180 days post-infarction, circulating endothelial cells and VWF decreased, compared to baseline. In stroke patients, the number of endothelial progenitor cells - but not circulating endothelial cells - was higher than in controls (90.0/mL vs. 37.0/mL; p=0.01; 105.0/mL vs. 71.0/mL; p=0.11). At 30 days after stroke, however, VCAM-1 peaked (628.1/mL vs. 869.1/mL; p<0.01) but there was no significant change in endothelial progenitor cells (90/mL vs. 78/mL; p<0.34). At 180 days after stroke, circulating endothelial cells and VWF decreased, compared to baseline. Cultured endothelial progenitor cells from controls and myocardial infarction patients had endothelial phenotype characteristics and exhibited functional differences in adhesion and Ca(2+) influx, but not in proliferation and vasculogenesis. In myocardial infarction patients, VCAM-1 levels and mobilization of endothelial progenitor cells peaked at 30 days after the ischemic event. Although a similar VCAM-1 kinetic was observed in stroke patients, endothelial progenitor cells did not increase. Endothelial progenitor cells had mature endothelial capabilities in vitro.

The TRPV4 channel.

The widely distributed TRPV4 cationic channel participates in the transduction of both physical (osmotic, mechanical, and heat) and chemical (endogenous, plant-derived, and synthetic ligands) stimuli. In this chapter we will review TRPV4 expression, biophysics, structure, regulation, and interacting partners as well as physiological and pathological insights obtained in TRPV4 animal models and human genetic studies.

Ion channels in asthma.

Ion channels are specialized transmembrane proteins that permit the passive flow of ions following their electrochemical gradients. In the airways, ion channels participate in the production of epithelium-based hydroelectrolytic secretions and in the control of intracellular Ca(2+) levels that will ultimately activate almost all lung cells, either resident or circulating. Thus, ion channels have been the center of many studies aiming to understand asthma pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. In this minireview, we focus on molecular, genetic, and animal model studies associating ion channels with asthma.

The progesterone receptor regulates the expression of TRPV4 channel.

The transient receptor potential cationic channel TRPV4 contributes to different aspects of cell physiology via the generation of a Ca2+ signal and/or depolarization of the membrane potential. TRPV4 channel integrates distinct physical and chemical stimuli, including osmotic and mechanical stress, heat, acidic pH, endogenous ligands, and synthetic agonists such as 4alpha-phorbol 12,13-didecanoate (4alphaPDD). Although several regulatory sites controlling TRPV4 channel activity have been identified, very little is known about the regulation of TRPV4 expression, a situation common to other TRP channels. Here we show that TRPV4 expression is under the control of progesterone in both human airways and mammary gland epithelial cells, as well as in vascular smooth muscle cells. Exposure of human airways epithelial CFT1-LCFSN and mammary gland epithelial T47D cells to progesterone decreased TRPV4 mRNA and protein expression. Consequently, 4alphaPDD-induced cationic currents and Ca2+ signals were also diminished in progesterone-treated cells. The effect of progesterone was reverted by the progesterone receptor (PR) antagonist RU-486 or following transfection with small interference RNA (siRNA) against both PRA and PRB isoforms. Interestingly, TRPV4 expression and activity were increased in T47D mammary gland epithelial cells when PR was silenced with siRNA. Transcriptional regulation of -1.3 kB TRPV4 promoter-luciferase plasmids was also evaluated in vascular smooth muscle cells. TRPV4 promoter activity was reduced by coexpression with PR and further reduced in the presence of PG. Together, our data report the regulation of TRPV4 expression by progesterone, a process that requires a functional PR.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways.

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

Functional coupling of GABAA/B receptors and the channel TRPV4 mediates rapid progesterone signaling in the oviduct.

The molecular mechanism by which progesterone (P4) modulates the transport of ova and embryos along the oviduct is not fully resolved. We report a rapid response to P4 and agonists of γ-aminobutyric acid receptors A and B (GABAA/B) in the mouse oviduct that was characterized by oscillatory Ca2+ signals and increased ciliary beat frequency (CBF). Pharmacological manipulation, genetic ablation, and siRNA-mediated knockdown in oviductal cells, as well as overexpression experiments in HEK 293T cells, confirmed the participation of the cationic channel TRPV4, different subunits of GABAA (α1 to α3, ß2, and ß3), and GABAB1 in P4-induced responses. TRPV4-mediated Ca2+ entry in close proximity to the inositol trisphosphate receptor was required to initiate and maintain Ca2+ oscillations after P4 binding to GABAA and transactivation of Gi/o protein-coupled GABAB receptors. Coimmunoprecipitation experiments and imaging of native tissue and HEK 293T cells demonstrated the close association of GABAA and GABAB1 receptors and the activation of Gi/o proteins in response to P4 and GABA receptor agonists, confirming a molecular mechanism in which P4 and GABAergic agonists cooperatively stimulate cilial beating.

DYRK1A Kinase Positively Regulates Angiogenic Responses in Endothelial Cells.

Angiogenesis is a highly regulated process essential for organ development and maintenance, and its deregulation contributes to inflammation, cardiac disorders, and cancer. The Ca2+/nuclear factor of activated T cells (NFAT) signaling pathway is central to endothelial cell angiogenic responses, and it is activated by stimuli like vascular endothelial growth factor (VEGF) A. NFAT phosphorylation by dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) is thought to be an inactivating event. Contrary to expectations, we show that the DYRK family member DYRK1A positively regulates VEGF-dependent NFAT transcriptional responses in primary endothelial cells. DYRK1A silencing reduces intracellular Ca2+ influx in response to VEGF, which dampens NFAT activation. The effect is exerted at the level of VEGFR2 accumulation leading to impairment in PLCγ1 activation. Notably, Dyrk1a heterozygous mice show defects in developmental retinal vascularization. Our data establish a regulatory circuit, DYRK1A/ Ca2+/NFAT, to fine-tune endothelial cell proliferation and angiogenesis.

TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells.

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

A gain-of-function SNP in TRPC4 cation channel protects against myocardial infarction.

AIMS: The TRPC4 non-selective cation channel is widely expressed in the endothelium, where it generates Ca(2+) signals that participate in the endothelium-mediated vasodilatory response. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC4 gene that are associated with myocardial infarction (MI). METHODS AND RESULTS: Our candidate-gene association studies identified a missense SNP (TRPC4-I957V) associated with a reduced risk of MI in diabetic patients [odds ratio (OR) = 0.61; confidence interval (CI), 0.40-0.95, P= 0.02]. TRPC4 was also associated with MI in the Wellcome Trust Case-Control Consortium's genome-wide data: an intronic SNP (rs7319926) within the same linkage disequilibrium block as TRPC4-I957V showed an OR of 0.86 (CI, 0.81-0.94; P =10(-4)). Functional studies of the missense SNP were carried out in HEK293 and CHO cells expressing wild-type or mutant channels. Patch-clamp studies and measurement of intracellular [Ca(2+)] in response to muscarinic agonists and direct G-protein activation showed increased channel activity in TRPC4-I957V-transfected cells compared with TRPC4-WT. Site-directed mutagenesis and molecular modelling of TRPC4-I957V suggested that the gain of function was due to the presence of a less bulky Val-957. This permits a firmer interaction between the TRPC4 and the catalytic site of the tyrosine kinase that phosphorylates TRPC4 at Tyr-959 and facilitates channel insertion into the plasma membrane. CONCLUSION: We provide evidence for the association of a TRPC4 SNP with MI in population-based genetic studies. The higher Ca(2+) signals generated by TRPC4-I957V may ultimately facilitate the generation of endothelium- and nitric oxide-dependent vasorelaxation, thereby explaining its protective effect at the vasculature.

Altered sarcoplasmic reticulum calcium transport in the presence of the heavy metal chelator TPEN.

TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine) is a membrane-permeable heavy-metal ion chelator with a dissociation constant for Ca2+ comparable to the Ca2+ concentration ([Ca2+]) within the intracellular Ca2+ stores. It has been used as modulator of intracellular heavy metals and of free intraluminal [Ca2+], without influencing the cytosolic [Ca2+] that falls in the nanomolar range. In our previous studies, we gave evidence that TPEN modifies the Ca2+ homeostasis of striated muscle independent of this buffering ability. Here we describe the direct interaction of TPEN with the ryanodine receptor (RyR) Ca2+ release channel and the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA). In lipid bilayers, at negative potentials and low [Ca2+], TPEN increased the open probability of RyR, while at positive potentials it inhibited channel activity. On permeabilized skeletal muscle fibers of the frog, but not of the rat, 50 microM TPEN increased the number of spontaneous Ca2+ sparks and induced propagating events with a velocity of 273 +/- 7 microm/s. Determining the hydrolytic activity of the SR revealed that TPEN inhibits the SERCA pump, with an IC(50) = 692 +/- 62 microM and a Hill coefficient of 0.88 +/- 0.10. These findings provide experimental evidence that TPEN directly modifies both the release of Ca2+ from and its reuptake into the SR.