TRPV1-dependent NKCC1 activation in mouse lens involves integrin and the tubulin cytoskeleton

Abstract

Previously we showed hyperosmotic solution caused TRPV1-dependent NKCC1 activation in the lens by a mechanism that involved ERK1/2 signaling. In various tissues, integrins and the cytoskeletal network play a role in responses to osmotic stress. Here, we examined the association between integrins and TRPV1-dependent activation of NKCC1 in mouse lens epithelium. Wild-type (WT) lenses exposed to the integrin agonist leukadherin-1 (LA-1) for 10 min displayed a ~33% increase in the bumetanide-sensitive rate of Rb uptake indicating NKCC activation. Paclitaxel, a microtubule stabilizing agent, abolished the Rb uptake response. In primary cultured lens epithelium LA-1 caused a robust ERK1/2 activation response that was almost fully suppressed by paclitaxel. The TRPV1 agonist capsaicin caused a similar ERK1/2 activation response. Consistent with an association between integrins and TRPV1, the TRPV1 antagonist A889425 prevented the Rb uptake response to LA-1 as did the ERK inhibitor U0126. LA-1 did not increase Rb uptake by lenses from TRPV1 knockout mice. In cells exposed to a hyperosmotic stimulus, both the ERK1/2 activation and Rb uptake responses were prevented by paclitaxel. Taken together, the findings suggest TRPV1 activation is associated with integrins and the tubulin cytoskeleton. This aligned with the observation that LA-1 elicited a robust cytoplasmic calcium rise in cells from WT lenses but failed to increase calcium in cells from TRPV1 knockout lenses. The results are consistent with the notion that integrin activation by LA-1, or a hyperosmotic stimulus, causes TRPV1 channel opening and the consequent downstream activation of the ERK1/2 and NKCC1 responses.

Keywords: NKCC activity; TRPV1; calcium; integrin; mouse lens; tubulin.

Fig. 1.

The influence of bumetanide or paclitaxel on leukadherin-1 (LA-1)-induced Rb uptake response by intact wild-type mouse lenses. Freshly isolated lenses were incubated for 10 min in Rb-containing Krebs solution in the presence or absence (Control) of LA-1 (25 µM). Some lenses also were exposed to the NKCC inhibitor bumetanide (Bum) (1 µM, 20 min preincubation) or microtubule stabilizer paclitaxel (100 nM, 6h preincubation) and then exposed to LA-1 in the continued presence of either bumetanide (Panel A) or paclitaxel (Panel B). The values are the mean ± SEM of results from 7–14 lenses. *** (p<0.001) Indicates a significant difference from control.

Fig. 2.

Western blot showing the time-course of ERK1/2 activation in cultured wild-type mouse lens epithelium exposed to LA-1 (Panel A). Samples isolated from cultured lens epithelium, exposed to LA-1 (25 µM) for 2 −15 min, were separated by electrophoresis, then probed for phospho-ERK1/2 (pERK1/2, Upper bands) and total ERK1/2 (tERK1/2, Lower bands). pERK1/2 band density displayed a transient increase that reached a maximum at 5 min. Paclitaxel prevented the ERK1/2 activation response in cultured wild-type mouse lens epithelial cells exposed to LA-1 (Panel B). Cells were exposed to LA-1 (25 µM) for 5 min in the presence or absence (Control) of paclitaxel (100 nM, 6h preincubation) were separated by electrophoresis and then probed for phospho-ERK1/2 (pERK1/2, Upper bands) and total ERK1/2 (tERK1/2, Lower bands). In each panel a representative Western blot was shown on the left and the bar graph for the band density ratio of phospho-ERK/total ERK (pERK/tERK) was shown on the right. The results are the mean ± SEM of 3 independent experiments. * (p < 0.05) and ** (p<0.01) indicate significant differences from control.

Fig. 3.

The TRPV1 inhibitor A889425 prevented the Rb uptake response in wild-type mouse lenses exposed to LA-1 (Panel A). Rb uptake was measured in intact wild-type lenses exposed to LA-1 (25 µM) for 10 min in the presence or absence of A889425 (1 µM) that was added 20 min earlier. Control lenses received neither LA-1 nor A889425. The values are the mean ± SEM of results from 5–6 lenses. ** (p<0.01) Indicates a significant difference from control. LA-1 did not increase Rb uptake by intact lenses obtained fromTRPV1 knockout mice (Panel B). TRPV1 KO lenses were incubated for 10 min in Rb-containing Krebs solution in the presence or absence (Control) of LA-1 (25 µM). The values are the mean ± SEM of results from 7 lenses.

Fig. 4.

Western blot showing the time course of ERK1/2 activation in cultured wild-type mouse lens epithelium exposed to hyperosmotic solution. Samples isolated from cells exposed to hyperosmotic solution (350 mOsm) for 2 −15 min were separated by electrophoresis then probed for phospho-ERK1/2 (pERK1/2, Upper bands) and total ERK1/2 (tERK1/2, Lower bands). pERK1/2 band density displayed a transient increase reaching maximum at 5 min. The Left panel shows a representative Western blot. The bar graph in the Right panel shows mean ± SEM of 3 independent experiments. * (p < 0.05) and ** (p<0.01) indicate significant differences from control.

Fig. 5.

Paclitaxel prevented the ERK1/2 activation response in cultured wild-type mouse epithelial cells exposed to hyperosmotic solution (Panel A). Cells were exposed to hyperosmotic solution (350 mOsm) for 5 min in the presence or absence (Control) of paclitaxel (100 nM) that was added 6h earlier (preincubation time). A representative Western blot is shown on the Left. The bar chart on the Right shows the band density ratio of phospho-ERK/total ERK (pERK/tERK). The results are the mean ± SEM of 3 independent experiments. *** (p<0.001) Indicates a significant difference from control. Paclitaxel also prevented the increase of Rb uptake in intact wild-type lenses exposed to hyperosmotic solution (Panel B). Rb uptake was measured in lenses exposed to hyperosmotic solution for 10 min in the presence or absence (Control) of paclitaxel (100 nM) added 6h earlier (preincubation time). The results are the mean ± SEM of 6 – 7 independent experiments. *** (p<0.001) Indicates a significant difference from control.

Fig. 6.

Western blot showing the time course of ERK1/2 activation in cultured wild-type mouse lens epithelium exposed to the TRPV1 agonist capsaicin. Samples isolated from cells exposed to capsaicin (1 µM) for 2 −15 min were separated by electrophoresis then probed for phospho-ERK (Upper bands) and total ERK1/2 (Lower bands). pERK1/2 band density displayed a transient increase with a maximum at 5 min. The Left panel shows a representative Western blot. The bar chart in the Right panel shows the band density ratio of phospho-ERK/total ERK (pERK/tERK). The results are the mean ± SEM of 3 independent experiments. * (p<0.05) Indicates a significant difference from control.

Fig. 7.

The time course of ERK1/2 activation was similar in cultured wild-type mouse lens epithelial cells exposed to LA-1 or capsaicin. As described above (Figs 3, and 9), samples isolated from cells exposed to LA-1 (25 µM) or capsaicin (1 µM) for 2 −15 min were probed by Western blot for phospho-ERK1/2 and total ERK1/2. The line graph shows the band density ratios, pERK/total ERK, at the time points shown on the x-axis. The results are the mean ± SEM of 3 independent experiments. * (p<0.05 and ** (p<0.01) indicate a significant difference from control for each treatment. There is no statistical significance between capsaicin and LA-1 treatment at any of the time points.

Fig. 8.

A highly selective ERK1/2/ MEK inhibitor, U0126, prevented the Rb uptake response in wild-type mouse lenses exposed to LA-1. Rb uptake was measured in intact wild-type lenses exposed to LA-1 (25 µM) for 10 min in the presence or absence of U0126 (10 µM) that was added 20 min earlier (preincubation time). Control lenses received neither LA-1 nor U0126. The values are the mean ± SEM of results from 6 lenses. *** (p<0.001) Indicates a significant difference from control.

Fig. 9.

LA-1 caused an increase of cytoplasmic calcium in cultured lens epithelial cells from wild type but not TRPV1 knockout (KO) mice. The results were obtained in cells loaded with Fura-2 AM. LA-1 (25 µM) added as shown (↑) caused an immediate increase of cytoplasmic calcium concentration in wild-type cells (Left panel) that was absent in TRPV1 KO cells (Right panel). The calcium response of TRPV1 KO cells to ATP (100 µM) was recorded as a positive control. Data from 15–30 cells were averaged and considered as n=1. Results are means ± SEM of 5 independent experiments.

Fig. 10.

Schematic diagram showing the signaling cascade and its possible components involved in Rb uptake increase due to integrin activation by LA1 or by hyperosmotic solution. Note that signaling components following TRPV1 activation were published earlier (Shahidullah, et al 2018; 2020).