NGF increases Connexin-43 expression and function in pulmonary arterial smooth muscle cells to induce pulmonary artery hyperreactivity.

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.

Pathophysiological Roles of the TRPV4 Channel in the Heart.

The transient receptor potential vanilloid 4 (TRPV4) channel is a non-selective cation channel that is mostly permeable to calcium (Ca2+), which participates in intracellular Ca2+ handling in cardiac cells. It is widely expressed through the body and is activated by a large spectrum of physicochemical stimuli, conferring it a role in a variety of sensorial and physiological functions. Within the cardiovascular system, TRPV4 expression is reported in cardiomyocytes, endothelial cells (ECs) and smooth muscle cells (SMCs), where it modulates mitochondrial activity, Ca2+ homeostasis, cardiomyocytes electrical activity and contractility, cardiac embryonic development and fibroblast proliferation, as well as vascular permeability, dilatation and constriction. On the other hand, TRPV4 channels participate in several cardiac pathological processes such as the development of cardiac fibrosis, hypertrophy, ischemia-reperfusion injuries, heart failure, myocardial infarction and arrhythmia. In this manuscript, we provide an overview of TRPV4 channel implications in cardiac physiology and discuss the potential of the TRPV4 channel as a therapeutic target against cardiovascular diseases.

OP2113, a new drug for chronic hypoxia-induced pulmonary hypertension treatment in rat.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a cardiovascular disease characterised by an increase in pulmonary arterial (PA) resistance leading to right ventricular (RV) failure. Reactive oxygen species (ROS) play a major role in PH. OP2113 is a drug with beneficial effects on cardiac injuries that targets mitochondrial ROS. The aim of the study was to address the in vivo therapeutic effect of OP2113 in PH. EXPERIMENTAL APPROACH: PH was induced by 3 weeks of chronic hypoxia (CH-PH) in rats treated with OP2113 or its vehicle via subcutaneous osmotic mini-pumps. Haemodynamic parameters and both PA and heart remodelling were assessed. Reactivity was quantified in PA rings and in RV or left ventricular (LV) cardiomyocytes. Oxidative stress was detected by electron paramagnetic resonance and western blotting. Mitochondrial mass and respiration were measured by western blotting and oxygraphy, respectively. KEY RESULTS: In CH-PH rats, OP2113 reduced the mean PA pressure, PA remodelling, PA hyperreactivity in response to 5-HT, the contraction slowdown in RV and LV and increased the mitochondrial mass in RV. Interestingly, OP2113 had no effect on haemodynamic parameters, both PA and RV wall thickness and PA reactivity, in control rats. Whereas oxidative stress was evidenced by an increase in protein carbonylation in CH-PH, this was not affected by OP2113. CONCLUSION AND IMPLICATIONS: Our study provides evidence for a selective protective effect of OP2113 in vivo on alterations in both PA and RV from CH-PH rats without side effects in control rats.

Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

Genetically-encoded BRET probes shed light on ligand bias-induced variable ion selectivity in TRPV1 and P2X5/7.

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-ß1-Dependent Mechanism.

Expression of the nerve growth factor NGF is increased in pulmonary hypertension (PH). We have here studied whether oxidative stress and inflammation, two pathological conditions associated with transforming growth factor-ß1 (TGF-ß1) in PH, may trigger NGF secretion by pulmonary arterial (PA) cells. Effects of hydrogen peroxide (H2O2) and interleukin-1ß (IL-1ß) were investigated ex vivo on rat pulmonary arteries, as well as in vitro on human PA smooth muscle (hPASMC) or endothelial cells (hPAEC). TßRI expression was assessed by Western blotting. NGF PA secretion was assessed by ELISA after TGF-ß1 blockade (anti-TGF-ß1 siRNA, TGF-ß1 blocking antibodies, TßRI kinase, p38 or Smad3 inhibitors). TßRI PA expression was evidenced by Western blotting both ex vivo and in vitro. H2O2 or IL-1ß significantly increased NGF secretion by hPASMC and hPAEC, and this effect was significantly reduced when blocking TGF-ß1 expression, binding to TßRI, TßRI activity, or signaling pathways. In conclusion, oxidative stress and inflammation may trigger TGF-ß1 secretion by hPASMC and hPAEC. TGF-ß1 may then act as an autocrine factor on these cells, increasing NGF secretion via TßRI activation. Since NGF and TGF-ß1 are relevant growth factors involved in PA remodeling, such mechanisms may therefore be relevant to PH pathophysiology.

Piezo1 Channel Activation Reverses Pulmonary Artery Vasoconstriction in an Early Rat Model of Pulmonary Hypertension: The Role of Ca2+ Influx and Akt-eNOS Pathway.

In intrapulmonary arteries (IPAs), mechanical forces due to blood flow control vessel tone, and these forces change during pulmonary hypertension (PH). Piezo1, a stretch-activated calcium channel, is a sensor of mechanical stress present in both endothelial cells (ECs) and smooth muscle cells (SMCs). The present study investigated the role of Piezo1 on IPA in the chronic hypoxia model of PH. Rats were raised in chronically hypoxic conditions for 1 (1W-CH, early stage) or 3 weeks (3W-CH, late-stage) of PH or in normoxic conditions (Nx). Immunofluorescence labeling and patch-clamping revealed the presence of Piezo1 in both ECs and SMCs. The Piezo1 agonist, Yoda1, induced an IPA contraction in Nx and 3W-CH. Conversely, Yoda1 induced an endothelial nitric oxide (eNOS) dependent relaxation in 1W-CH. In ECs, the Yoda1-mediated intracellular calcium concentration ([Ca2+]i) increase was greater in 1W-CH as compared to Nx. Yoda1 induced an EC hyperpolarization in 1W-CH. The eNOS levels were increased in 1W-CH IPA compared to Nx or 3W-CH PH and Yoda1 activated phosphorylation of Akt (Ser473) and eNOS (Ser1177). Thus, we demonstrated that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i, endothelial-dependent hyperpolarization, and Akt-eNOS pathway activation in the early stage of PH.

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

NiONP-Induced Oxidative Stress and Mitochondrial Impairment in an In Vitro Pulmonary Vascular Cell Model Mimicking Endothelial Dysfunction.

The development and use of nanomaterials, especially of nickel oxide nanoparticles (NiONPs), is expected to provide many benefits but also has raised concerns about the potential human health risks. Inhaled NPs are known to exert deleterious cardiovascular side effects, including pulmonary hypertension. Consequently, patients with pulmonary hypertension (PH) could be at increased risk for morbidity. The objective of this study was to compare the toxic effects of NiONPs on human pulmonary artery endothelial cells (HPAEC) under physiological and pathological conditions. The study was conducted with an in vitro model mimicking the endothelial dysfunction observed in PH. HPAEC were cultured under physiological (static and normoxic) or pathological (20% cycle stretch and hypoxia) conditions and exposed to NiONPs (0.5-5 µg/cm2) for 4 or 24 h. The following endpoints were studied: (i) ROS production using CM-H2DCF-DA and MitoSOX probes, (ii) nitrite production by the Griess reaction, (iii) IL-6 secretion by ELISA, (iv) calcium signaling with a Fluo-4 AM probe, and (v) mitochondrial dysfunction with TMRM and MitoTracker probes. Our results evidenced that under pathological conditions, ROS and nitrite production, IL-6 secretions, calcium signaling, and mitochondria alterations increased compared to physiological conditions. Human exposure to NiONPs may be associated with adverse effects in vulnerable populations with cardiovascular risks.

NiONPs-induced alteration in calcium signaling and mitochondrial function in pulmonary artery endothelial cells involves oxidative stress and TRPV4 channels disruption.

In New Caledonia, anthropic activities, such as mining, increase the natural erosion of soils in nickel mines, which in turn, releases nickel oxide nanoparticles (NiONPs) into the atmosphere. Pulmonary vascular endothelial cells represent one of the primary targets for inhaled nanoparticles. The objective of this in vitro study was to assess the cytotoxic effects of NiONPs on human pulmonary artery endothelial cells (HPAEC). Special attention will be given to the level of oxidative stress and calcium signaling, which are involved in the physiopathology of cardiovascular diseases. HPAEC were exposed to NiONPs (0.5-150 µg/cm2) for 4 or 24 h. The following different endpoints were studied: (i) ROS production using CM-H2DCF-DA probe, electron spin resonance, and MitoSOX probe; the SOD activity was also measured (ii) calcium signaling with Fluo4-AM, Rhod-2, and Fluo4-FF probes; (iii) inflammation by IL-6 production and secretion and, (iv) mitochondrial dysfunction and apoptosis with TMRM and MitoTracker probes, and AnnexinV/PI. Our results have evidenced that NiONPs induced oxidative stress in HPAEC. This was demonstrated by an increase in ROS production and a decrease in SOD activity, the two mechanisms seem to trigger a pro-inflammatory response with IL-6 secretion. In addition, NiONPs exposure altered calcium homeostasis inducing an increased cytosolic calcium concentration ([Ca2+]i) that was significantly reduced by the extracellular calcium chelator EGTA and the TRPV4 inhibitor HC-067047. Interestingly, exposure to NiONPs also altered TRPV4 activity. Finally, HPAEC exposure to NiONPs increased intracellular levels of both ROS and calcium ([Ca2+]m) in mitochondria, leading to mitochondrial dysfunction and HPAEC apoptosis.

Mechanosensitivity in Pulmonary Circulation: Pathophysiological Relevance of Stretch-Activated Channels in Pulmonary Hypertension.

A variety of cell types in pulmonary arteries (endothelial cells, fibroblasts, and smooth muscle cells) are continuously exposed to mechanical stimulations such as shear stress and pulsatile blood pressure, which are altered under conditions of pulmonary hypertension (PH). Most functions of such vascular cells (e.g., contraction, migration, proliferation, production of extracellular matrix proteins, etc.) depend on a key event, i.e., the increase in intracellular calcium concentration ([Ca2+]i) which results from an influx of extracellular Ca2+ and/or a release of intracellular stored Ca2+. Calcium entry from the extracellular space is a major step in the elevation of [Ca2+]i, involving a variety of plasmalemmal Ca2+ channels including the superfamily of stretch-activated channels (SAC). A common characteristic of SAC is that their gating depends on membrane stretch. In general, SAC are non-selective Ca2+-permeable cation channels, including proteins of the TRP (Transient Receptor Potential) and Piezo channel superfamily. As membrane mechano-transducers, SAC convert physical forces into biological signals and hence into a cell response. Consequently, SAC play a major role in pulmonary arterial calcium homeostasis and, thus, appear as potential novel drug targets for a better management of PH.

High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique.

Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.

Transient receptor potential vanilloid 4 channel participates in mouse ventricular electrical activity.

The TRPV4 channel is a calcium-permeable channel (PCa/PNa ∼ 10). Its expression has been reported in ventricular myocytes, where it is involved in several cardiac pathological mechanisms. In this study, we investigated the implication of TRPV4 in ventricular electrical activity. Left ventricular myocytes were isolated from trpv4+/+ and trpv4-/- mice. TRPV4 membrane expression and its colocalization with L-type calcium channels (Cav1.2) was confirmed using Western blot biotinylation, immunoprecipitation, and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4-/- compared with trpv4+/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD90) in trpv4+/+ but not in trpv4-/- myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased calcium transient (CaT) amplitude in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 carries an inward Ca2+ current in myocytes. Conversely, TRPV4 inhibitor GSK2193874 (100 nM) alone reduced APD90 in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 prolongs AP duration in basal condition. Finally, introducing TRPV4 parameters in a mathematical model predicted the development of an inward TRPV4 current during repolarization that increases AP duration and CaT amplitude, in accord with what was found experimentally. This study shows for the first time that TRPV4 modulates AP and QTc durations. It would be interesting to evaluate whether TRPV4 could be involved in long QT-mediated ventricular arrhythmias.NEW & NOTEWORTHY Transient receptor potential vanilloid 4 (TRPV4) is expressed at the membrane of mouse ventricular myocytes and colocalizes with non-T-tubular L-type calcium channels. Deletion of trpv4 gene in mice results in shortened QT interval on electrocardiogram and reduced action potential duration of ventricular myocytes. Pharmacological activation of TRPV4 channel leads to increased action potential duration and increased calcium transient amplitude in trpv4-/- but not in trpv4-/- ventricular myocytes. To the contrary, TRPV4 channel pharmacological inhibition reduces action potential duration in trpv4+/+ but not in trpv4-/- myocytes. Integration of TRPV4 channel in a computational model of mouse action potential shows that the channel carries an inward current contributing to slowing down action potential repolarization and to increase calcium transient amplitude, similarly to what is observed experimentally. This study highlights for the first time the involvement of TRPV4 channel in ventricular electrical activity.

CD95/Fas and metastatic disease: What does not kill you makes you stronger.

CD95 (also known as Fas) is the prototype of death receptors; however, evidence suggests that this receptor mainly implements non-apoptotic signaling pathways such as NF-κB, MAPK, and PI3K that are involved in cell migration, differentiation, survival, and cytokine secretion. At least two different forms of CD95 L exist. The multi-aggregated transmembrane ligand (m-CD95 L) is cleaved by metalloproteases to release a homotrimeric soluble ligand (s-CD95 L). Unlike m-CD95 L, the interaction between s-CD95 L and its receptor CD95 fails to trigger apoptosis, but instead promotes calcium-dependent cell migration, which contributes to the accumulation of inflammatory Th17 cells in damaged organs of lupus patients and favors cancer cell invasiveness. Novel inhibitors targeting the pro-inflammatory roles of CD95/CD95 L may provide attractive therapeutic options for patients with chronic inflammatory disorders or cancer. This review discusses the roles of the CD95/CD95 L pair in cell migration and metastasis.

TRPV4 channel mediates adventitial fibroblast activation and adventitial remodeling in pulmonary hypertension.

Pulmonary arterial adventitial fibroblasts (PAF), the most abundant cellular constituent of adventitia, act as a key regulator of pulmonary vascular wall structure and function from the outside-in. Previous studies indicate that transient receptor potential vanilloid 4 (TRPV4) channel plays an important role in the development of pulmonary hypertension (PH), but no attention has been given so far to its role in adventitial remodeling. In this study, we thus investigated TRPV4 implication in PAF activation occurring in PH. First, we isolated and cultured PAF from rat adventitial intrapulmonary artery. RT-PCR, Western blot, immunostaining, and calcium imaging (fluo-4/AM) showed that PAF express functional TRPV4 channels. In extension of these results, using pharmacological and siRNA approaches, we demonstrated TRPV4 involvement in PAF proliferation (BrdU incorporation) and migration (wound-healing assay). Then, Western blot experiments revealed that TRPV4 activation upregulates the expression of extracellular matrix protein synthesis (collagen type I and fibronectin). Finally, we explored the role of TRPV4 in the adventitial remodeling occurring in PH. By means of Western blot, we determined that TRPV4 protein expression was upregulated in adventitia from chronically hypoxic and monocrotaline rats, two animal models of PH. Furthermore, morphometric analysis indicated that adventitial remodeling is attenuated in PH-induced trpv4-/- mice. These data support the concept that PAF play an essential role in hypertensive pulmonary vascular remodeling and point out the participation of TRPV4 channel activity in PAF activation leading to excessive adventitial remodeling.

Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

Stretch-activated Piezo1 Channel in Endothelial Cells Relaxes Mouse Intrapulmonary Arteries.

In intrapulmonary arteries (IPA), endothelial cells (EC) respond to mechanical stimuli by releasing vasoactive factors to set the vascular tone. Piezo1, a stretch-activated, calcium-permeable channel, is a sensor of mechanical stress in EC. The present study was undertaken to investigate the implication of Piezo1 in the endothelium-dependent regulation of IPA tone and potential involvement of Piezo1 in pulmonary hypertension, the main disease of this circulation. IPA tone was quantified by means of a myograph in control Piezo1+/+ mice and in mice lacking endothelial Piezo1 (EC-Piezo1-/-). Endothelial intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) production were measured, in mouse or human EC, with Fluo-4 or DAF-FM probe, respectively. Immunofluorescent labeling and patch-clamp experiments revealed the presence of Piezo1 channels in EC. Yoda1, a Piezo1 agonist, induced an endothelium-dependent relaxation that was significantly reduced in pulmonary arteries in EC-Piezo1-/- compared with Piezo1+/+ mice. Yoda1 as well as mechanical stimulation (by osmotic stress) increased [Ca2+]i in mouse or human EC. Consequently, both stimuli increased the production of NO. NO and [Ca2+]i increases were reduced in EC from Piezo1-/- mice or in the presence of Piezo1 inhibitors. Furthermore, deletion of Piezo1 increased α-adrenergic agonist-mediated contraction. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, Piezo1 still mediated arterial relaxation, and deletion of this channel did not impair the development of the disease. The present study thus demonstrates that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i and NO production and that this effect is still present in pulmonary hypertension.

The Role of the Anti-Aging Protein Klotho in IGF-1 Signaling and Reticular Calcium Leak: Impact on the Chemosensitivity of Dedifferentiated Liposarcomas.

By inhibiting Insulin-Like Growth Factor-1-Receptor (IGF-1R) signaling, Klotho (KL) acts like an aging- and tumor-suppressor. We investigated whether KL impacts the aggressiveness of liposarcomas, in which IGF-1R signaling is frequently upregulated. Indeed, we observed that a higher KL expression in liposarcomas is associated with a better outcome for patients. Moreover, KL is downregulated in dedifferentiated liposarcomas (DDLPS) compared to well-differentiated tumors and adipose tissue. Because DDLPS are high-grade tumors associated with poor prognosis, we examined the potential of KL as a tool for overcoming therapy resistance. First, we confirmed the attenuation of IGF-1-induced calcium (Ca2+)-response and Extracellular signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation in KL-overexpressing human DDLPS cells. KL overexpression also reduced cell proliferation, clonogenicity, and increased apoptosis induced by gemcitabine, thapsigargin, and ABT-737, all of which are counteracted by IGF-1R-dependent signaling and activate Ca2+-dependent endoplasmic reticulum (ER) stress. Then, we monitored cell death and cytosolic Ca2+-responses and demonstrated that KL increases the reticular Ca2+-leakage by maintaining TRPC6 at the ER and opening the translocon. Only the latter is necessary for sensitizing DDLPS cells to reticular stressors. This was associated with ERK1/2 inhibition and could be mimicked with IGF-1R or MEK inhibitors. These observations provide a new therapeutic strategy in the management of DDLPS.

Disrupting the CD95-PLCγ1 interaction prevents Th17-driven inflammation.

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.