Classical transient receptor potential 6 (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis

Abstract

Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3-5years of diagnosis. The pathological process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor β (TGF-β1). Classical transient receptor potential 6 (TRPC6), a Na- and Ca-permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6-/-) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-β1 induced TRPC6 up-regulation, increased Ca influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-β1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF.

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

“…To measure receptor-operated Ca 2+ entry (ROCE) endothelin-1 (4 µM, Merck, Darmstadt, Germany) was applied in HBSS buffer with (2 mM) Ca 2+ or in nominal Ca 2+ free (0.5 mM EGTA) buffer after adding Ca 2+ (2 mM). Store-operated Ca 2+ entry (SOCE) was analyzed after depletion of internal Ca 2+ stores by 1 µM thapsigargin (Sigma, Taufkirchen, Germany) in Ca 2+ free HBSS solution containing 0.5 mM EGTA by adding extracellular Ca 2+ (2 mM) 30 . An increase in intracellular Ca 2+ ([Ca 2+ ] i ) was recorded using a Polychrome V monochromator (Till Photonics, Martinsried, Germany) and a 14-bit EMCCD camera (iXON3 885, Andor, Belfast, UK) coupled to an inverted microscope (IX71with an UPlanSApo 20×/0.85 oil immersion objective, Olympus, Hamburg, Germany) at 340 and 380 nm as described 30 .…”

Transient receptor vanilloid 4 (TRPV4) channels are essential for alveolar epithelial cell function

“…Western Blotting was done as previously described ( Hofmann et al, 2017 ). Chemiluminescence was detected in an Odyssey®Fc unit (Licor, Lincoln, NE, USA).…”

“…Isolation of nuclear protein extracts from ATI-like cells after 6 days of culture was performed with a Nuclear Extract Kit according to the manufacturer’s instructions (Active Motif, 40010, La Hulpe, Belgium) as described (Hofmann et al, 2017). In brief, cells were first washed with PBS containing phosphatase inhibitors.…”

Ca2+ signalling in fibroblasts and the therapeutic potential of KCa3.1 channel blockers in fibrotic diseases

“…Human and mouse lung fibroblasts (Hofmann et al, 2017; Rahaman et al, 2014; Wei et al, 2009; Yonezawa et al, 2016)…”

“…TRPC6 has been implicated in cardiac wound healing through myofibroblast transdifferentiation (Davis et al, ). In model lung fibrosis, TRPM2 contributed to bleomycin‐induced lung inflammation (Yonezawa et al, ), TRPC6 contributed to TGF‐β1‐induced myofibroblast differentiation during fibrosis (Hofmann et al, ) and TRPV4 activity mediated pulmonary fibrosis through the regulation of myofibroblast differentiation (Rahaman et al, ). TRPC3 channels were central in renal fibroblast proliferation, differentiation and activation through Ca 2+ ‐mediated ERK signalling, suggesting that TRPC3 inhibitors could significantly improve renal remodelling in kidney disease (Saliba et al, ).…”