Ludovic Gillet

Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells

Voltage-gated sodium channels (NaV) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of NaV1.5 and associated auxiliary β-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel®-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role (∼65%) in cancer invasiveness. Matrigel® invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on NaV function. Inhibiting NaV has no consequence for cathepsins at the transcription, translation, and secretion levels. However, NaV activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Nav enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.

Cardiac sodium channel NaV1.5 distribution in myocytes via interacting proteins: The multiple pool model

The cardiac sodium current (INa) is responsible for the rapid depolarization of cardiac cells, thus allowing for their contraction. It is also involved in regulating the duration of the cardiac action potential (AP) and propagation of the impulse throughout the myocardium. Cardiac INa is generated by the voltage-gated Na(+) channel, NaV1.5, a 2016-residue protein which forms the pore of the channel. Over the past years, hundreds of mutations in SCN5A, the human gene coding for NaV1.5, have been linked to many cardiac electrical disorders, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. Similar to many membrane proteins, NaV1.5 has been found to be regulated by several interacting proteins. In some cases, these different proteins, which reside in distinct membrane compartments (i.e. lateral membrane vs. intercalated disks), have been shown to interact with the same regulatory domain of NaV1.5, thus suggesting that several pools of NaV1.5 channels may co-exist in cardiac cells. The aim of this review article is to summarize the recent works that demonstrate its interaction with regulatory proteins and illustrate the model that the sodium channel NaV1.5 resides in distinct and different pools in cardiac cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

NaV1.5 enhances breast cancer cell invasiveness by increasing NHE1-dependent H+ efflux in caveolae

NaV1.5 sodium channels enhance the invasiveness of breast cancer cells through the acidic-dependent activation of cysteine cathepsins. Here, we showed that the Na+/H+ exchanger type 1 (NHE1) was an important regulator of H+ efflux in breast cancer cells MDA-MB-231 and that its activity was increased by NaV1.5. NaV1.5 and NHE1 were colocalized in membrane rafts containing caveolin-1. The inhibition of NaV1.5 or NHE1 induced a similar reduction in cell invasiveness and extracellular matrix degradation; no additive effect was observed when they were simultaneously inhibited. Our study suggests that NaV1.5 and NHE1 are functionally coupled and enhance the invasiveness of cancer cells by increasing H+ efflux.

PDZ Domain–Binding Motif Regulates Cardiomyocyte Compartment-Specific NaV1.5 Channel Expression and Function

Background— Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain–binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results— To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (&Dgr;SIV). &Dgr;SIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of &Dgr;SIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and &Dgr;SIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued &Dgr;SIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. Conclusions— Our results demonstrate the in vivo significance of the PDZ domain–binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

C-terminal Calmodulin-binding Motif Differentially Controls Human and Rat P2X7 Receptor Current Facilitation

P2X7 receptors (P2X7R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X7Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X7R resulted from a Ca2+-calmodulin-dependent process and a distinct calcium-independent process. However, P2X7Rs show striking species differences; thus, this study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X7R as well as rat/human, human/rat chimeric, and mutated P2X7Rs. Facilitation at the human P2X7R was 5-fold slower than at the rat P2X7R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X7R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X7R. Replacement of three critical residues of this binding domain from the rat into the human P2X7R (T541I, C552S, and G559V) reconstituted the Ca2+-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X7Rs. The absence of Ca2+-dependent facilitation at the human P2X7R may represent a protective adaptation of the innate immune response in which P2X7R plays significant roles.

Cysteine cathepsins S and L modulate anti-angiogenic activities of human endostatin.

Background: Cathepsins participate to the release of endostatin, a potent anti-angiogenic protein. Results: Both cathepsins L and S generate two peptides from human endostatin with increased angiostatic properties. Conclusion: Endostatin-derived peptides reduce tube formation of endothelial cells. Significance: Endostatin-derived peptides may represent novel molecular links between cysteine cathepsins and aminopeptidase N in the regulation of angiogenesis. Human endostatin, a potent anti-angiogenic protein, is generated by release of the C terminus of collagen XVIII. Here, we propose that cysteine cathepsins are involved in both the liberation and activation of bioactive endostatin fragments, thus regulating their anti-angiogenic properties. Cathepsins B, S, and L efficiently cleaved in vitro FRET peptides that encompass the hinge region corresponding to the N terminus of endostatin. However, in human umbilical vein endothelial cell-based assays, silencing of cathepsins S and L, but not cathepsin B, impaired the generation of the ∼22-kDa endostatin species. Moreover, cathepsins L and S released two peptides from endostatin with increased angiostatic properties and both encompassing the NGR sequence, a vasculature homing motif. The G10T peptide (residues 1455–1464: collagen XVIII numbering) displayed compelling anti-proliferative (EC50 = 0.23 nm) and proapoptotic properties. G10T inhibited aminopeptidase N (APN/CD13) and reduced tube formation of endothelial cells in a manner similar to bestatin. Combination of G10T with bestatin resulted in no further increase in anti-angiogenic activity. Taken together, these data suggest that endostatin-derived peptides may represent novel molecular links between cathepsins and APN/CD13 in the regulation of angiogenesis.

Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current

BACKGROUND Membrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization. OBJECTIVE The purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes. METHODS SAP97(fl/fl) mice were generated by inserting loxP sequences flanking exons 1-3 of the SAP97 gene. SAP97(fl/fl) mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97(fl/fl) mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase-polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice. RESULTS Action potential duration was greatly prolonged in αMHC-Cre/SAP97(fl/fl) cardiomyocytes compared to SAP97(fl/fl) controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97(fl/fl) mice. CONCLUSION These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.

Voltage-gated sodium channels and cancer: is excitability their primary role?

Voltage-gated sodium channels (NaV) are molecular characteristics of excitable cells. Their activation, triggered by membrane depolarization, generates transient sodium currents that initiate action potentials in neurons and muscle cells. Sodium currents were discovered by Hodgkin and Huxley using the voltage clamp technique and reported in their landmark series of papers in 1952. It was only in the 1980s that sodium channel proteins from excitable membranes were molecularly characterized by Catterall and his collaborators. Non-excitable cells can also express NaV channels in physiological conditions as well as in pathological conditions. These NaV channels can sustain biological roles that are not related to the generation of action potentials. Interestingly, it is likely that the abnormal expression of NaV in pathological tissues can reflect the re-expression of a fetal phenotype. This is especially true in epithelial cancer cells for which these channels have been identified and sodium currents recorded, while it was not the case for cells from the cognate normal tissues. In cancers, the functional activity of NaV appeared to be involved in regulating the proliferative, migrative, and invasive properties of cells. This review is aimed at addressing the non-excitable roles of NaV channels with a specific emphasis in the regulation of cancer cell biology.

Ultrastructural and quantitative analysis of the lipid droplet clustering induced by hepatitis C virus core protein

Hepatitis C virus (HCV) release is linked to the formation of lipid droplet (LD) clusters in the perinuclear area of infected cells, induced by the core protein. We used electron microscopy (EM) to monitor and compare the number and size of LD in cells producing the mature and immature forms of the HCV core protein, and 3D EM to reconstruct whole cells producing the mature core protein. Only the mature protein coated the LD and induced their clustering and emergence from endoplasmic reticulum membranes enriched in this protein. We found no particular association between LD clusters and the centrosome in reconstructed cells. The LD clustering induced by the mature core protein was associated with an increase in LD synthesis potentially due, at least in part, to the ability of this protein to coat the LD. These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis.

A-Kinase Anchoring Protein Lbc Coordinates a p38 Activating Signaling Complex Controlling Compensatory Cardiac Hypertrophy

ABSTRACT In response to stress, the heart undergoes a remodeling process associated with cardiac hypertrophy that eventually leads to heart failure. A-kinase anchoring proteins (AKAPs) have been shown to coordinate numerous prohypertrophic signaling pathways in cultured cardiomyocytes. However, it remains to be established whether AKAP-based signaling complexes control cardiac hypertrophy and remodeling in vivo. In the current study, we show that AKAP-Lbc assembles a signaling complex composed of the kinases PKN, MLTK, MKK3, and p38α that mediates the activation of p38 in cardiomyocytes in response to stress signals. To address the role of this complex in cardiac remodeling, we generated transgenic mice displaying cardiomyocyte-specific overexpression of a molecular inhibitor of the interaction between AKAP-Lbc and the p38-activating module. Our results indicate that disruption of the AKAP-Lbc/p38 signaling complex inhibits compensatory cardiomyocyte hypertrophy in response to aortic banding-induced pressure overload and promotes early cardiac dysfunction associated with increased myocardial apoptosis, stress gene activation, and ventricular dilation. Attenuation of hypertrophy results from a reduced protein synthesis capacity, as indicated by decreased phosphorylation of 4E-binding protein 1 and ribosomal protein S6. These results indicate that AKAP-Lbc enhances p38-mediated hypertrophic signaling in the heart in response to abrupt increases in the afterload.