Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression.

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation.

BACKGROUND: Agonist stimulation of airway smooth muscle (ASM) results in IP3 mediated Ca2+ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells. METHODS: The expression profile of NCX (which encodes the Na+/Ca2+ exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function. RESULTS: NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 µM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 µM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine peripheral airways. CONCLUSIONS: Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca2+ homeostasis and link store depletion via STIM1 directly with NCX activation.

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells.

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca(2+)-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (-logEC(50)=4.67+/-0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20muM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca(2+)-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G(q)-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, alpha(1)(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca(2+) mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.

ORAI and store-operated calcium influx in human airway smooth muscle cells.

The initial bronchoconstrictor response of the asthmatic airway depends on airway smooth muscle (ASM) contraction. Intracellular calcium is a key signaling molecule, mediating a number of responses, including proliferation, gene expression, and contraction of ASM. Ca(2+) influx through receptor-operated calcium (ROC) or store-operated calcium (SOC) channels is believed to mediate longer term signals. The mechanisms of SOC activation in ASM remain to be elucidated. Recent literature has identified the STIM and ORAI proteins as key signaling players in the activation of the SOC subtype; calcium release-activated channel current (I(CRAC)) in a number of inflammatory cell types. However, the role for these proteins in activation of SOC in smooth muscle is unclear. We have previously demonstrated a role for STIM1 in SOC channel activation in human ASM. The aim of this study was to investigate the expression and define the potential roles of the ORAI proteins in SOC-associated Ca(2+) influx in human ASM cells. Here we show that knockdown of ORAI1 by siRNA resulted in reduced thapsigargin- or cyclopiazonic acid (CPA)-induced Ca(2+) influx, without affecting Ca(2+) release from stores or basal levels. CPA-induced inward currents were also reduced in the ORAI1 knockdown cells. We propose that ORAI1 together with STIM1 are important contributors to SOC entry in ASM cells. These data extend the major tissue types in which these proteins appear to be major determinants of SOC influx, and suggest that modulation of these pathways may prove useful in the treatment of bronchoconstriction.

A key role for STIM1 in store operated calcium channel activation in airway smooth muscle.

BACKGROUND: Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular Ca2+ stores can modulate contractile responses, modulate proliferation and regulate synthetic activity. Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC) or receptor operated channels (ROC). Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified. METHODS: To investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay. RESULTS: Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70%) of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ influx in response to store depletion by cyclopiazonic acid (60%) or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp. CONCLUSION: Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.