Mechanotransduction via TRPV4 regulates inflammation and differentiation in fetal mouse distal lung epithelial cells

Abstract

Background: Mechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury.

Methods: Expression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively.

Results: TRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4.

Conclusion: These studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.

Fig. 1

TRPV4 expression increases with gestation. a Fetal lung tissue was collected at different times in gestation, as shown. RNA was isolated, reversed-transcribed, and the cDNA products for TRPV4 were analyzed by quantitative RT-PCR. N = 3. b Fetal lung tissue was collected at different times in gestation and proteins extracted to assess TRPV4 expression by Western blot. The upper panel is a representative blot. Results were normalized to GAPDH to control for protein loading. N = 3. c Fetal lung tissue from different times in gestation was fixed in formalin. Sections were process by immunohistochemistry using rabbit anti-TRPV4 polyclonal antibody, stained with diaminobenzidine and counterstained with hematoxylin. Immunohistochemistry pictures show distribution of TRPV4 during fetal lung development (arrows). At E16 (pseudoglandular stage), TRPV4 was minimally expressed in the respiratory bronchioles. Later in gestation, TRPV4 immunostaining was more apparent in the distal epithelium. Bar, 20 μm

Fig. 2

TRPV4 expression in isolated distal fetal epithelial cells and the effect of mechanical stretch. a E17-E19 fetal epithelial cells were isolated as described in methods and processed to analyze TRPV4 mRNA expression by qRT-PCR using the ∆∆C_T method for relative quantification (n = 4; *p < 0.02 vs E17 or E18, Tukey-Kramer Multiple Comparisons Test). b Fetal epithelial cells were isolated at E17-E19 of gestation and cultured on bioflex plates coated with fibronectin. Twenty four hours later, monolayers were exposed to 20 % cyclic stretch at 40 cycles/min for 24 h; unstretched samples were used as control. Samples were processed by qRT-PCR to assess TRPV4 mRNA expression (n = 4; *p < 0.05 vs E17 control, Tukey-Kramer Multiple Comparisons Test). c Fetal epithelial cells isolated from E17-E19 lungs were seeded on bioflex plates, as described above, and exposed to 20 % cyclic stretch for 24 h. Proteins were extracted and processed to determine TRPV4 protein abundance. The upper panel is a representative blot normalized to vinculin. Data in the lower panel are from 4 different experiments (*P < 0.01 vs E17 control, Tukey-Kramer Multiple Comparisons Test). C = control; S = stretch

Fig. 3

TRPV4 regulates stretch-induced release of IL-6. E17 epithelial cells were isolated and seeded on bioflex plates coated with fibronectin. 24 hours later, cells were exposed to 20 % cyclic stretch for 48 h in the presence or absence of the vehicle DMSO, the TRPV4 agonist GSK1016790A [100 nM] or TRPV4 antagonist HC-067047 [1 μM]. Unstretched cells served as controls. Supernatants were collected and processed to assess IL-6 concentrations by ELISA, as described in methods. Values are mean ± SEM from 5 different experiments. Results were normalized to the cell lysate protein concentrations. *p < 0.05 vs control vehicle; **p < 0.01 vs stretch vehicle. Tukey-Kramer Multiple Comparisons Test. Veh = vehicle, DMSO; ag = agonist GSK1016790A; ant = antagonist HC-067047

Fig. 4

Stretch-induced activation of TRPV4 is mediated via p38 and ERK pathways. E17 distal lung epithelial cells were isolated and seeded on plates coated with fibronectin. The following day, monolayers were exposed to 20 % cyclic stretch at 40 cycles/min for 15 min in the presence or absence of the vehicle DMSO, the TRPV4 agonist GSK1016790A [100 nM] or TRPV4 antagonist HC-067047 [1 μM]. The level of activation of the indicated proteins in the cell lysate was evaluated by Western blot using phospho-specific antibodies. Blots were then stripped and reprobed with total antibodies to control for protein loading. Upper panels are representative Western blots. Data in the lower panel are from 5 different experiments. *p < 0.05 vs negative control; **p < 0.01 vs negative stretch. Tukey-Kramer Multiple Comparisons Test

Fig. 5

Release of IL-6 by stretch or TRPV4 agonist is regulated via ERK and p38 pathways. a E17 epithelial cells seeded on bioflex plates coated with fibronectin were preincubated for 30 min with the ERK pathway inhibitor U0126 [20 μM] or the p38 inhibitor SB203580 [20 μM] and then exposed to 20 % cyclic stretch for 48 h. Supernatants were collected and the concentration of IL-6 was analyzed by ELISA, as described in methods. Data are normalized to the cell lysate concentrations. N = 4; *p < 0.01 vs vehicle control; **p < 0.01 vs vehicle stretch. Tukey-Kramer Multiple Comparisons Test. b E17 epithelial cells seeded on fibronectin-coated plates were incubated for 48 h with the ERK pathway inhibitor U0126 [20 μM], the p38 inhibitor SB203580 [20 μM] or the JNK inhibitor SP600125 [20 μM], in the presence of DMSO (vehicle) or the TRPV4 agonist GSK1016790A [100 nM]. Supernatants were collected and the concentration of IL-6 was analyzed by ELISA, as described above. N = 3; *p < 0.05 vs negative vehicle; **p < 0.01 vs TRPV4 agonist. Tukey-Kramer Multiple Comparisons Test

Fig. 6

Stretch-induced fetal epithelial cell differentiation is mediated via TRPV4. E17 epithelial cells were seeded on bioflex plates coated with laminin and then exposed to a physiologic 5 % cyclic stretch at 40 cycles/min for 24 h, in the presence or not of the TRPV4 agonist GSK1016790A [100 nM] or TRPV4 antagonist HC-067047 [1 μM]. Unstretched cells served as controls. RNA was extracted, as described in methods, and processed to assess SP-C mRNA abundance by qRT-PCR. Results are from 5 separate experiments. *P < 0.05 vs vehicle control; **P < 0.05 vs vehicle stretch; #P < 0.01 vs vehicle stretch. Tukey-Kramer Multiple Comparisons Test