LRRC8/VRAC Channels and the Redox Balance: A Complex Relationship.

More than three decades after their first biophysical description, Volume Regulated Anion Channels (VRACs) still remain challenging to understand. Initially, VRACs were identified as the main pathway for the cell to extrude Cl- ions during the regulatory volume decrease (RVD) mechanism contributing in fine to the recovery of normal cell volume. For years, scientists have tried unsuccessfully to find their molecular identity, leading to controversy within the field that only ended in 2014 when two independent groups demonstrated that VRACs were formed by heteromers of LRRC8 proteins. This breakthrough gave a second breath to the research field and was followed by many publications regarding LRRC8/VRACs structure/ function, physiological roles and 3D structures. Nevertheless, far from simplifying the field, these discoveries have instead exponentially increased its complexity. Indeed, the channel's biophysical properties seem to be dependent on the LRRC8 subunits composition with each heteromer showing different ion/molecule permeabilities and regulatory mechanisms. One clear example of this complexity is the intricate relationship between LRRC8/VRACs and the redox system. On one hand, VRACs appear to be directly regulated by oxidation or reduction depending on their subunit composition. On the other hand, VRACs can also impact the redox balance within the cells, through their permeability to reduced glutathione or through other as yet uncharacterized pathways. Unravelling this issue is particularly crucial as LRRC8/VRACs play an important role in a wide variety of physiological processes involving oxidative stress signaling. In this regard, we have tried to systematically identify in the literature both preand post-LRRC8 discovery as well as the interplay between VRACs and the redox system to provide new insights into this complex relationship.

Inhibition of eIF5A hypusination pathway as a new pharmacological target for stroke therapy.

In eukaryotes, the polyamine pathway generates spermidine that activates the hypusination of the translation factor eukaryotic initiation factor 5A (eIF5A). Hypusinated-eIF5A modulates translation, elongation, termination and mitochondrial function. Evidence in model organisms like drosophila suggests that targeting polyamines synthesis might be of interest against ischemia. However, the potential of targeting eIF5A hypusination in stroke, the major therapeutic challenge specific to ischemia, is currently unknown. Using in vitro models of ischemic-related stress, we documented that GC7, a specific inhibitor of a key enzyme in the eIF5A activation pathway, affords neuronal protection. We identified the preservation of mitochondrial function and thereby the prevention of toxic ROS generation as major processes of GC7 protection. To represent a thoughtful opportunity of clinical translation, we explored whether GC7 administration reduces the infarct volume and functional deficits in an in vivo transient focal cerebral ischemia (tFCI) model in mice. A single GC7 pre- or post-treatment significantly reduces the infarct volume post-stroke. Moreover, GC7-post-treatment significantly improves mouse performance in the rotarod and Morris water-maze, highlighting beneficial effects on motor and cognitive post-stroke deficits. Our results identify the targeting of the polyamine-eIF5A-hypusine axis as a new therapeutic opportunity and new paradigm of research in stroke and ischemic diseases.

LRRC8/VRAC channels exhibit a noncanonical permeability to glutathione, which modulates epithelial-to-mesenchymal transition (EMT).

Volume-regulated anion channels (VRAC) are chloride channels activated in response to osmotic stress to regulate cellular volume and also participate in other cellular processes, including cell division and cell death. Recently, members of the LRRC8 family have been identified as the main contributors of VRAC conductance. LRRC8/VRAC is permeable to chloride ions but also exhibits significant permeability to various substrates that vary strongly in charge and size. In this study, we explored the intriguing ability of LRRC8/VRAC to transport glutathione (GSH), the major cellular reactive oxygen species (ROS) scavenger, and its involvement in epithelial-to-mesenchymal transition (EMT), a cellular process in which cellular oxidative status is a crucial step. First, in HEK293-WT cells, we showed that a hypotonic condition induced LRRC8/VRAC-dependent GSH conductance (PGSH/PCl of ~0.1) and a marked decrease in intracellular GSH content. GSH currents and GSH intracellular decrease were both inhibited by DCPIB, an inhibitor of LRRC8/VRAC, and were not observed in HEK293-LRRC8A KO cells. Then, we induced EMT by exposing renal proximal tubule epithelial cells to the pleiotropic growth factor TGFß1, and we measured the contribution of LRRC8/VRAC in this process by measuring (i) EMT marker expression (assessed both at the gene and protein levels), (ii) cell morphology and (iii) the increase in migration ability. Interestingly, pharmacologic targeting of LRRC8/VRAC (DCPIB) or RNA interference-mediated inhibition (LRRC8A siRNA) attenuated the TGFß1-induced EMT response by controlling GSH and ROS levels. Interestingly, TGFß1 exposure triggered DCPIB-sensitive chloride conductance. These results suggest that LRRC8/VRAC, due to its native permeability to GSH and thus its ability to modulate ROS levels, plays a critical role in EMT and might contribute to other physiological and pathophysiological processes associated with oxidative stress.

Matrix Stiffening and EGFR Cooperate to Promote the Collective Invasion of Cancer Cells.

In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg Cancer Res; 78(18); 5229-42. ©2018 AACR.

Selective EGF-Receptor Inhibition in CD4+ T Cells Induces Anergy and Limits Atherosclerosis.

BACKGROUND: Several epidermal growth factor receptor (EGFR) inhibitors have been successfully developed for the treatment of cancer, limiting tumor growth and metastasis. EGFR is also expressed by leukocytes, but little is known about its role in the modulation of the immune response. OBJECTIVES: The aim of this study was to determine whether EGFR expressed on CD4+ T cells is functional and to address the consequences of EGFR inhibition in atherosclerosis, a T cell-mediated vascular chronic inflammatory disease. METHODS: The authors used EGFR tyrosine kinase inhibitors (AG-1478, erlotinib) and chimeric Ldlr-/-Cd4-Cre/Egfrlox/lox mouse with a specific deletion of EGFR in CD4+ T cells. RESULTS: Mouse CD4+ T cells expressed EGFR, and the EGFR tyrosine kinase inhibitor AG-1478 blocked in vitro T cell proliferation and Th1/Th2 cytokine production. In vivo, treatment of Ldlr-/- mice with the EGFR inhibitor erlotinib induced T cell anergy, reduced T cell infiltration within atherosclerotic lesions, and protected against atherosclerosis development and progression. Selective deletion of EGFR in CD4+ T cells resulted in decreased T cell proliferation and activation both in vitro and in vivo, as well as reduced interferon-γ, interleukin-4, and interleukin-2 production. Atherosclerotic lesion size was reduced by 2-fold in irradiated Ldlr-/- mice reconstituted with bone marrow from Cd4-Cre/Egfrlox/lox mice, compared to Cd4-Cre/Egfr+/+ chimeric mice, after 4, 6, and 12 weeks of high-fat diet, associated with marked reduction in T cell infiltration in atherosclerotic plaques. Human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both in vitro and in vivo. CONCLUSIONS: EGFR blockade induced T cell anergy in vitro and in vivo and reduced atherosclerosis development. Targeting EGFR may be a novel strategy to combat atherosclerosis.

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance.

Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 µM and 55 ± 2 µM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 µM and 20 ± 1 µM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 µM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.