Store-operated Ca2+ entry in primary murine lung fibroblasts is independent of classical transient receptor potential (TRPC) channels and contributes to cell migration

Abstract

Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca 2+ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca 2+ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca 2+ entry (Roce). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca 2+ influx and cell function in TRPC1/6-deficient (TRPC1/6 −/− ) and STIM1/2-deficient (STIM1/2 ΔpmLf ) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2-deficient pmLF and ROCE was decreased in TRPC1/6 −/− pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6 −/− pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLf.Store-operated Ca 2+ entry (SOCE) also named capacitive Ca 2+ entry (CCE) was first described by J.W. Putney Jr. more than 30 years ago as depletion of intracellular Ca 2+ stores which induces the opening of plasma membrane (PM) Ca 2+ channels 1 . Since then, candidate proteins like classical transient receptor potential (TRPC) channels 2 and mechanisms, e.g. coupling of TRPC proteins to inositol 1-4-5 trisphosphate (IP3) receptor channels in the endoplasmic reticulum 3,4 for SOCE, were intensively discussed in the scientific community. In 2005 however, stromal interaction molecules (Stim in Drosophila and STIM1, STIM2 in humans) were identified as Ca 2+ sensors in the ER directly regulating SOCE in two different large-scale screening approaches 5,6 . One year later, Ca 2+ selective channels at the plasma membrane (Orai) were discovered 7-9 , which were responsible for Ca 2+ release activated Ca 2+ (CRAC) currents originally described in mast cells 10 . A molecular model was developed to support the concept that upon ER Ca 2+ depletion STIM proteins homo-multimerize and translocate to ER-PM junctions 11,12 , where they recruit and gate Orai channels via direct interaction 13 . Ca 2+ influx through Orai channels is important for cellular remodeling, e.g. in cardiovascular diseases 14 , and mutations in these channels are responsible for multiple channelopathies 15 . Irrespective of these events, TRP channels trigger Ca 2+ influx in response to extracellular stimuli or receptor activation (receptor-operated Ca 2+ influx, ROCE) independently of STIM and Orai 16 . Some labs, however, reported that TRPC channels also interact with STIM proteins 17 and/or Orai channels 18 . Along these lines, TRPC channels like TRPC1 were invoked in SOCE in certain cells of salivary glands 19 and pancreatic acini 20 , while in vascular smooth muscle cells TRPC1 channels work independently of SOCE 21 . The role of TRPC1 i...

Histamine activates an intracellular Ca2+ signal in normal human lung fibroblast WI-38 cells

“…WI-38 fibroblasts also express the transcripts encoding for TRPC1-TRPC6, but the pharmacological sensitivity of histamine-evoked Ca 2+ entry to 10 µM La 3+ and Gd 3+ argues against the involvement of TRPC isoforms. Moreover, a recent investigation demonstrated that TRPC channels do not support SOCE in primary murine lung fibroblasts (Bendiks et al, 2020). Conversely, TRPC channels, which present a single-channel conductance that is 1000-fold larger than Orai3, could be activated by transforming growth factor β and thereby lead to VOC activation via strong membrane depolarization (Mukherjee et al, 2015).…”

“…Extracellular Ca 2+ can permeate the plasma membrane through a wide variety of ion channels, including voltage-operated channels (VOC) (Janssen et al, 2015;Rahman et al, 2016) and agonist-operated channels, which comprise three types of channels: 1) receptor-operated channels (ROC) (Saliba et al, 2015), which are ionotropic receptors stimulated by direct ligand binding, 2) second messenger-operated channels (SMOC), which are activated by intracellularly generated mediators, such as cyclic nucleotides, diacylglycerol (Hofmann et al, 2017), and arachidonic acid, and 3) store-operated Ca 2+ channels (SOC), which are the main Ca 2+ entry pathway in non-excitable cells. In the ER, Stromal Interaction Molecules (STIM1/2) act as sensors of ER Ca 2+ concentration that, after a reduction in intraluminal Ca 2+ , multimerize and translocate towards peripheral ER cisternae to functionally interact with the Ca 2+ permeable Orai channels on the plasma membrane (Bendiks et al, 2020). The following influx of Ca 2+ has been termed store-operated Ca 2+ entry (SOCE) and mediates agonist-induced Ca 2+ influx in human fibroblasts isolated from several tissues, including lungs (Guzmán-Silva et al, 2015;Vazquez-de-Lara et al, 2018).…”

An Inhibitory Function of TRPA1 Channels in TGF-β1-driven Fibroblast to Myofibroblast Differentiation

“…The identification of new target proteins mediating this process is therefore an important step toward establishing new biomarkers and future therapeutic options. TRP-channels are expressed and essential for Ca 2+ homeostasis in many cells including fibroblasts (17, 34). We were able to demonstrate an essential role of the classical TRP channel 6 (TRPC6) in murine fibroblast to myofibroblast differentiation (17).…”

An ex vivo perfused ventilated murine lung model suggests lack of acute pulmonary toxicity of the potential novel anticancer agent (−)-englerin A

“…Coverslips were washed with HEPES buffered HBSS and placed under a microscope in a recording chamber with a 500 µl volume. An increase in intracellular Ca 2+ was recorded using a Leica DFC9000 GT camera coupled to an inverted microscope (DMi8, Leica, Wetzlar, Germany) with an 40x/0.85 oil immersions objective at 340 and 380 nm for quantification of [Ca 2+ ] i as described (Bendiks et al 2020 ).…”