The cleaved FAS ligand activates the Na(+)/H(+) exchanger NHE1 through Akt/ROCK1 to stimulate cell motility

Abstract

Transmembrane CD95L (Fas ligand) can be cleaved to release a promigratory soluble ligand, cl-CD95L, which can contribute to chronic inflammation and cancer cell dissemination. The motility signaling pathway elicited by cl-CD95L remains poorly defined. Here, we show that in the presence of cl-CD95L, CD95 activates the Akt and RhoA signaling pathways, which together orchestrate an allosteric activation of the Na(+)/H(+) exchanger NHE1. Pharmacologic inhibition of Akt or ROCK1 independently blocks the cl-CD95L-induced migration. Confirming these pharmacologic data, disruption of the Akt and ROCK1 phosphorylation sites on NHE1 decreases cell migration in cells exposed to cl-CD95L. Together, these findings demonstrate that NHE1 is a novel molecular actor in the CD95 signaling pathway that drives the cl-CD95L-induced cell migration through both the Akt and RhoA signaling pathways.

Figure 1. cl-CD95L increases cell migration by activation of NHE1.

(a) PS120 cells, stably expressing different combinations of wild type or transport inactive mutant of NHE1 (D267V), and wild type or death domain mutant of CD95, were incubated for 16 h in Boyden chambers in the presence or absence of 100 ng/ml of cl-CD95 in the lower compartment. Migrating cells were then fixed with methanol and stained by Giemsa. For each experiment, five pictures of random fields were taken and a representative picture is shown (Bars = 70 μm).(b) For quantification, Giemsa-stained migrating cells were lysed, and absorbance was measured at 560 nm. Values represent means and SD of three independently performed experiments. (c) Cell migration of NHE1 deficient PS120-CD95 cells (▴), or PS120-CD95 cells stably expressing wild type NHE1 (◾) was tested using wound-healing assays in the presence (full symbols) or absence (empty symbols) of cl-CD95L (100 ng/ml). For this, straight scratches of identical size were executed on confluent cell monolayers preincubated in 0.5% Fetal Calf Serum. Medium containing 100 ng/ml of cl-CD95 ligand then was added and images were acquired for 24 hours using phase contrast microscopy. Quantification was performed using ImageJ (see methods). For each condition, images were acquired at five different positions along each scratch. The graphs are representative of 3 independent experiments. The corresponding images are displayed in Supplementary Figures. (d) NHE1 cooperative activation by intracellular protons was measured in PS120-NHE1-CD95 cells treated for 10 minutes with 100 ng/ml of cl-CD95L, CD95L or control medium. Intracellular pH was clamped at different pH values as described in Materials and Methods. Initial rates of NHE1 activity were measured using rapid kinetics of Li⁺ uptake and are represented as normalized values to the maximal uptake (V/Vmax). Each line represents the activity of NHE1 in one of the experimental conditions. ♦-Grey Line: CD95 ligand, ○-dotted line: untreated; ◾ dark line: cl-CD95 ligand. Data are representative for at least five independent experiments. Error bars are SEM.

Figure 2. CD95-mediate Akt stimulation and activates NHE1.

(a) After treatment with 100 ng/ml of cl-CD95L, cells were lysed and the levels of phosphorylated Akt (Ser 473) and total Akt were analyzed by immunoblotting using anti-Akt and anti-phosphoS473 Akt antibodies. Loading control: Total Akt. The analyzed cell lines are indicated on top of each Western Blot for p-Akt and total Akt. (b) Confluent cell monolayers (PS120-CD95-NHE1) were pre-incubated in presence or absence (noted DMSO) of the Triciribine (20 nM) for 1 hour and then treated as in Fig. 1c, in the presence or absence of cl-CD95L (100 ng/ml) for wound-healing assay. Histogram bars correspond to the surface covered after 24 hours. Error Bars are SEM, p < 0.05 (*), p < 0.01 (**). (c) The dose response of the PS120-CD95-NHE1 AKTA mutant (full lines, ▴) for intracellular protons was measured by Lithium uptake at clamped intracellular pH values in the presence (red line) or absence of cl-CD95L (100 ng/ml) (blue line) as described in Fig. 1d and compared to that the wild type of NHE1 response, (PS120-CD95-NHE1 WT dotted lines, ⦁) in the presence (red line) or absence of 100 ng/ml cl-CD95L (10 ng/ml) (blue line). Initial rates of NHE1 are represented normalized values to the maximal uptake (V/Vmax). Data are representative for at least three independent experiments. Error bars are SEM. (d) Cell migration of PS120-CD95 cells stably expressing wild type or AKTA or AKTD NHE1 mutants was assessed by wound healing assays. Histogram bars correspond to the surface covered after 24 hours. Error bars are SEMs. p < 0.05 (*), p < 0.01 (**). (e) The dose response of the PS120-CD95-NHE1 AKTD mutant (full lines, ◾) to intracellular protons was measured by lithium uptake in the presence (red line) or absence of 100 ng/ml cl-CD95L (blue line) as described in Fig. 1d and compared to that of wild type NHE1 response (PS120-CD95-NHE1 WT dotted red and blue lines, ⦁). Initial rates of NHE1 are represented normalized values to the maximal uptake (V/Vmax). Data are representative for at least three independent experiments. Error bars are SEM.

Figure 3. CD95-mediate RhoA stimulation and activates NHE1.

(a) cl-CD95L activates the RhoA signaling pathway: Cells treated with 100 ng/ml of cl-CD95L for the indicated times were lysed and active RhoA was pulled down using GST-agarose Rhotekin Rho-binding domain. Active and total RhoA were revealed by Western blotting. Data are representative of three independent experiments. (b) Cell migration of PS120-CD95-NHE1 wild type was assessed by wound healing in presence or absence of 100 ng/ml of cl-CD95L. Cells were pre-incubated in presence or absence (noted DMSO) of the ROCK1 inhibitor Y27632 (20 μM) for 1 hour. Histogram bars correspond to the surface covered after 24 hours. Error bars are SEMs. p < 0.05 (*), p < 0.01 (**). (c) The dose response of the PS120-CD95-NHE1 ROCKA mutant (full lines, ▴) for intracellular protons was measured by Lithium uptake at clamped intracellular pH values in the presence (red line) or absence of cl-CD95L (100 ng/ml) (blue line) as described in Fig. 1d and compared to that the wild type of NHE1 response, (PS120-CD95-NHE1 WT dotted lines, ⦁ red and blue lines). Initial rates of NHE1 are represented normalized values to the maximal uptake (V/Vmax). Data are representative for at least three independent experiments. Error bars are SEM.

Figure 4. Akt and RhoA pathways cooperate to activate NHE1 and stimulate migration in presence of cl-CD95L.

Cell migration of PS120-CD95 cells stably expressing wild type or ROCKA NHE1 mutants was assessed by wound healing assay in the presence or absence of cl-CD95 (100 ng/ml). Histogram bars correspond to the surface covered after 24 hours. Error bars are SEMs. p < 0.05 (*), p < 0.01 (**). (b) PS120-CD95 cells stably expressing the AKTD mutant were pre-treated with or without a non-cytotoxic dose of the ROCK1 inhibitor Y27632 (20 μM) for 1 h and then incubated for 24 h in the presence or absence of cl-CD95L (100 ng/ml). Migration was measured by wound healing assay. Histogram bars: surface area covered after 24 h. Error bars are the SEM. (c) Cell migration of PS120-CD95 cells stably expressing the AKTD-ROCKA NHE1 double mutants was measured by wound healing assay and compared to that of WT. Histogram bars correspond to the surface covered after 24 hours. Error bars are SEMs. p < 0.05 (*), p < 0.01 (**). (d) Summary of NHE1 activation by the non-conventional CD95 engagement.