Figure 5.
LPHN2 promotes EC TJ assembly and impairs vascular permeability and cancer cell extravasation.(A) Confocal microscopy analysis of TJs stained with ZO-1 (red) and VE-cadherin (green) reveals how in siCTL ECs seeded on 10 kPa substrates coated with increasing amounts of FN (1, 3, and 5 µg/ml) ZO-1 is progressively accumulating at VE-cadherin+ (VE-cad+) cell-to-cell junctions. Compared with siCTL ECs, LPHN2 silencing impairs ZO-1 but not VE-cadherin accumulation at intercellular contacts on increasing FN amounts. Scale bar, 25 µm. Results concerning the percentage of VE-cad+ intercellular area covered with ZO-1 are the mean ± SD of two independent experiments and a total of five confocal microscopy images for each condition. Statistical analysis: two-way ANOVA with Bonferroni’s post hoc analysis; *, P ≤ 0.05; ***, P ≤ 0.001. (B) Confocal microscopy analysis reveals how, compared with siCTL ECs, LPHN2 silencing impairs ZO-1 accumulation to VE-cad+ intercellular contacts of ECs plated on FN (5 µg/ml)-coated coverslips. Lentiviral delivery of WT Lphn2 restores ZO-1 localization to TJs, while the ΔOLF Lphn2 mutant does not rescue the phenotype. Scale bar, 25 µm. Results concerning the percentage of VE-cad+ intercellular area covered with ZO-1 are the mean ± SD of three independent experiments for a total of 14 confocal microscopy images for each condition. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; **, P ≤ 0.01; ***, P ≤ 0.001. (C) Top: Representative images of vascular permeability in lphn2a+/+ versus lphn2a−/− zebrafish embryos. 70 kD FITC-dextran was injected with or without 1 ng of VEGF-A (#V7259; Sigma). 70 kD Dextran is in green. Scale bars, 30 µm (left) and 3 µm (right). Bottom: Quantification of relative extravascular fluorescence. For each embryo, the fluorescence intensity of the dextran was measured in two intervascular areas between the intersegmental vessels (dashed box and shown in the zoom images on the right). lphn2a+/+ (n = 13), lphn2a+/+ with VEGF (n = 6), lphn2a−/− (n = 7), lphn2a−/− with VEGF (n = 5) embryos from two independent experiments. Results are the mean ± SD. Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparison test; *, P ≤ 0.05. (D) MemBright-560–labeled mouse B16F10 melanoma cells were microinjected into the duct of Cuvier of 48 hpf lphn2a+/+ or lphn2a−/−Tg(Kdrl:EGFP)s843 zebrafish embryos. After 36 h postinjection, extravasated metastatic melanoma cells were imaged by confocal analysis of the caudal plexus. Mouse B16F10 melanoma cell extravasation is enhanced in lphn2a−/− compared with lphn2a+/+ zebrafish embryos. Results are the mean ± SD of two independent assays, in which 17 animals were analyzed. Scale bars, 50 µm. Statistical analysis: Mann–Whitney test; *, P ≤ 0.05. (E) LPHN2 signaling controls endothelial FAs, TJs, and vascular permeability. Vascular ECs synthesize the FLRT2 ligand of LPHN2 that localizes at integrin-based ECM adhesion sites. FLRT2-activated LPHN2 triggers a canonical heterotrimeric G-protein α subunit (Gα)/adenylate cyclase (AC)/cAMP pathway that in turn, likely via the guanine nucleotide exchange factor EPAC, activates the small GTPase Rap1, which is a well-known regulator of cell-to-ECM adhesions (Coló et al., 2012; Lagarrigue et al., 2016). Furthermore, Rap1 promotes the formation of TJs (Sasaki et al., 2020), which in ECs are crucial for the control of vascular permeability. Hence, LPHN2 activation of Rap1 may act both to inhibit the formation of FAs and to promote the assembly of TJs, which increase EC barrier function. LPHN2 also binds the central PDZ domain of SHANK adaptor protein that in turn, through its N-terminal SPN domain, binds Rap1-GTP, suppressing talin-mediated integrin activation and FA development (Lilja et al., 2017) and promoting the assembly of TJs (Sasaki et al., 2020). Therefore, LPHN2 may favor the turnover of FAs and the formation of TJs by funneling Rap1-GTP toward SHANK. In addition, while TJs inhibit the nuclear translocation of YAP and TAZ through their Hippo pathway–dependent phosphorylation, FAs and the associated F-actin stress fibers exert exactly the opposite effect, promoting YAP/TAZ nuclear localization and transcriptional function (Karaman and Halder, 2018; Moya and Halder, 2019). Thus, the nuclear translocation and functional activation of YAP/TAZ caused by LPHN2 silencing or knock-down likely lie downstream of both the disassembly of TJs and the increased formation of FAs and stress fibers. In addition, the myosin II–mediated contraction of FA-linked stress fibers releases YAP/TAZ from their binding to the inhibitory switch/sucrose non-fermentable complex (not depicted) and transmits force from the ECM to the nucleus, changing nuclear pore conformation, finally promoting the translocation of YAP/TAZ into the nucleus and the transcription of target genes, such as CTGFA and CYR61. The lack of LPHN2 also results in an abnormal ECM-driven intercellular targeting of ZO-1 and assembly of TJs, which increases vascular permeability and favors cancer cell extravasation. KO, knock-out.

LPHN2 promotes EC TJ assembly and impairs vascular permeability and cancer cell extravasation. (A) Confocal microscopy analysis of TJs stained with ZO-1 (red) and VE-cadherin (green) reveals how in siCTL ECs seeded on 10 kPa substrates coated with increasing amounts of FN (1, 3, and 5 µg/ml) ZO-1 is progressively accumulating at VE-cadherin+ (VE-cad+) cell-to-cell junctions. Compared with siCTL ECs, LPHN2 silencing impairs ZO-1 but not VE-cadherin accumulation at intercellular contacts on increasing FN amounts. Scale bar, 25 µm. Results concerning the percentage of VE-cad+ intercellular area covered with ZO-1 are the mean ± SD of two independent experiments and a total of five confocal microscopy images for each condition. Statistical analysis: two-way ANOVA with Bonferroni’s post hoc analysis; *, P ≤ 0.05; ***, P ≤ 0.001. (B) Confocal microscopy analysis reveals how, compared with siCTL ECs, LPHN2 silencing impairs ZO-1 accumulation to VE-cad+ intercellular contacts of ECs plated on FN (5 µg/ml)-coated coverslips. Lentiviral delivery of WT Lphn2 restores ZO-1 localization to TJs, while the ΔOLF Lphn2 mutant does not rescue the phenotype. Scale bar, 25 µm. Results concerning the percentage of VE-cad+ intercellular area covered with ZO-1 are the mean ± SD of three independent experiments for a total of 14 confocal microscopy images for each condition. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; **, P ≤ 0.01; ***, P ≤ 0.001. (C) Top: Representative images of vascular permeability in lphn2a+/+ versus lphn2a−/− zebrafish embryos. 70 kD FITC-dextran was injected with or without 1 ng of VEGF-A (#V7259; Sigma). 70 kD Dextran is in green. Scale bars, 30 µm (left) and 3 µm (right). Bottom: Quantification of relative extravascular fluorescence. For each embryo, the fluorescence intensity of the dextran was measured in two intervascular areas between the intersegmental vessels (dashed box and shown in the zoom images on the right). lphn2a+/+ (n = 13), lphn2a+/+ with VEGF (n = 6), lphn2a−/− (n = 7), lphn2a−/− with VEGF (n = 5) embryos from two independent experiments. Results are the mean ± SD. Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparison test; *, P ≤ 0.05. (D) MemBright-560–labeled mouse B16F10 melanoma cells were microinjected into the duct of Cuvier of 48 hpf lphn2a+/+ or lphn2a−/−Tg(Kdrl:EGFP)s843 zebrafish embryos. After 36 h postinjection, extravasated metastatic melanoma cells were imaged by confocal analysis of the caudal plexus. Mouse B16F10 melanoma cell extravasation is enhanced in lphn2a−/− compared with lphn2a+/+ zebrafish embryos. Results are the mean ± SD of two independent assays, in which 17 animals were analyzed. Scale bars, 50 µm. Statistical analysis: Mann–Whitney test; *, P ≤ 0.05. (E) LPHN2 signaling controls endothelial FAs, TJs, and vascular permeability. Vascular ECs synthesize the FLRT2 ligand of LPHN2 that localizes at integrin-based ECM adhesion sites. FLRT2-activated LPHN2 triggers a canonical heterotrimeric G-protein α subunit (Gα)/adenylate cyclase (AC)/cAMP pathway that in turn, likely via the guanine nucleotide exchange factor EPAC, activates the small GTPase Rap1, which is a well-known regulator of cell-to-ECM adhesions (Coló et al., 2012; Lagarrigue et al., 2016). Furthermore, Rap1 promotes the formation of TJs (Sasaki et al., 2020), which in ECs are crucial for the control of vascular permeability. Hence, LPHN2 activation of Rap1 may act both to inhibit the formation of FAs and to promote the assembly of TJs, which increase EC barrier function. LPHN2 also binds the central PDZ domain of SHANK adaptor protein that in turn, through its N-terminal SPN domain, binds Rap1-GTP, suppressing talin-mediated integrin activation and FA development (Lilja et al., 2017) and promoting the assembly of TJs (Sasaki et al., 2020). Therefore, LPHN2 may favor the turnover of FAs and the formation of TJs by funneling Rap1-GTP toward SHANK. In addition, while TJs inhibit the nuclear translocation of YAP and TAZ through their Hippo pathway–dependent phosphorylation, FAs and the associated F-actin stress fibers exert exactly the opposite effect, promoting YAP/TAZ nuclear localization and transcriptional function (Karaman and Halder, 2018; Moya and Halder, 2019). Thus, the nuclear translocation and functional activation of YAP/TAZ caused by LPHN2 silencing or knock-down likely lie downstream of both the disassembly of TJs and the increased formation of FAs and stress fibers. In addition, the myosin II–mediated contraction of FA-linked stress fibers releases YAP/TAZ from their binding to the inhibitory switch/sucrose non-fermentable complex (not depicted) and transmits force from the ECM to the nucleus, changing nuclear pore conformation, finally promoting the translocation of YAP/TAZ into the nucleus and the transcription of target genes, such as CTGFA and CYR61. The lack of LPHN2 also results in an abnormal ECM-driven intercellular targeting of ZO-1 and assembly of TJs, which increases vascular permeability and favors cancer cell extravasation. KO, knock-out.

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