Figure 9.
TNF-α disrupts barrier function by mediating S1PR1 phosphorylation at Y143.(A) Pulldown of WT-S1PR1 from ECs without or with 50 ng/ml TNF-α stimulation at indicated times using anti-GFP antibody. Immunocomplexes were Western blotted with anti-phosphotyrosine (PY20/PY99) and anti-BiP antibodies. Western blotting with anti-S1PR1 antibody was used for total S1PR1 expression. (B) Mean ± SD of tyrosine phosphorylation. n = 3 experiments performed independently. ***, P < 0.0001 compared with unstimulated cells by one-way ANOVA with two-tailed paired t test. (C) Time-lapse images from ECs cotransducing indicated cDNAs following stimulation with TNF-α. Image taken at 63× magnification. Scale bar, 5 μm. Gray background indicates the area outside the image. (D) Mean ± SD of the surface expression of S1PR1 (5–7 cells/group) from experiments performed multiple times. ***, P < 0.0001 compared with unstimulated cells by one-way ANOVA with two-tailed paired t test. (E) TEER was recorded in control ECs after stimulation with vehicle or 50 ng/ml TNF-α. After 2 h, 1 μM S1P was added to assess the effect of TNF-α–mediated internalization of S1PR1 (observed in C and D) on barrier function. (F) Mean ± SD of the TEER from experiments performed three times. Basal TEER was calculated as mean between 10 and 20 min and between 3 and 4 h after S1P addition. ***, P < 0.0001 compared with unstimulated cells by one-way ANOVA with two-tailed paired t test. (G) TEER was recorded in BiP-depleted ECs after addition of a single dose of 50 ng/ml TNF-α. (H) Mean ± SD of the TEER from experiments performed three times. Basal TEER (−) in control and BiP-depleted ECs was analyzed as mean between 10 and 20 min. TNF-α (+)–induced loss of EC barrier function in control or BiP-depleted ECs was calculated as mean between 3.5 and 4 h. ***, P < 0.0001 compared with unstimulated siSc by one-way ANOVA with two-tailed paired t test. (I) Model shows the effect of tyrosine phosphorylation (Y143) of S1PR1 within ERY motif in regulating receptor function. S1P or TNF-α binds S1PR1 (i) and phosphorylates S1PR1 at Y143 (ii; represented as red circle). Dynamin pinches off the phosphorylated S1PR1 (iii). S1P also induces BiP ATPase activity and its translocation to cytosol (not shown). Phosphorylated S1PR1 (Y143D-S1PR1) interacts with BiP in the cytosol (iv). BiP imports the receptor to the ER (v). WT-S1PR1 induces Ca2+ signaling and returns to the cell surface after dephosphorylation (not shown). BiP traps the Y143D-S1PR1 mutant at the ER where it augments SOCE and induces barrier-disruptive signaling in a Gi-dependent manner. See also Fig. S5, C and D. A.U., arbitrary units; IB, immunoblot; IP, immunoprecipitation; p-Tyr, phosphorylated tyrosine; UD, undetectable. (A) Molecular weight marker in kD.

TNF-α disrupts barrier function by mediating S1PR1 phosphorylation at Y143. (A) Pulldown of WT-S1PR1 from ECs without or with 50 ng/ml TNF-α stimulation at indicated times using anti-GFP antibody. Immunocomplexes were Western blotted with anti-phosphotyrosine (PY20/PY99) and anti-BiP antibodies. Western blotting with anti-S1PR1 antibody was used for total S1PR1 expression. (B) Mean ± SD of tyrosine phosphorylation. n = 3 experiments performed independently. ***, P < 0.0001 compared with unstimulated cells by one-way ANOVA with two-tailed paired t test. (C) Time-lapse images from ECs cotransducing indicated cDNAs following stimulation with TNF-α. Image taken at 63× magnification. Scale bar, 5 μm. Gray background indicates the area outside the image. (D) Mean ± SD of the surface expression of S1PR1 (5–7 cells/group) from experiments performed multiple times. ***, P < 0.0001 compared with unstimulated cells by one-way ANOVA with two-tailed paired t test. (E) TEER was recorded in control ECs after stimulation with vehicle or 50 ng/ml TNF-α. After 2 h, 1 μM S1P was added to assess the effect of TNF-α–mediated internalization of S1PR1 (observed in C and D) on barrier function. (F) Mean ± SD of the TEER from experiments performed three times. Basal TEER was calculated as mean between 10 and 20 min and between 3 and 4 h after S1P addition. ***, P < 0.0001 compared with unstimulated cells by one-way ANOVA with two-tailed paired t test. (G) TEER was recorded in BiP-depleted ECs after addition of a single dose of 50 ng/ml TNF-α. (H) Mean ± SD of the TEER from experiments performed three times. Basal TEER (−) in control and BiP-depleted ECs was analyzed as mean between 10 and 20 min. TNF-α (+)–induced loss of EC barrier function in control or BiP-depleted ECs was calculated as mean between 3.5 and 4 h. ***, P < 0.0001 compared with unstimulated siSc by one-way ANOVA with two-tailed paired t test. (I) Model shows the effect of tyrosine phosphorylation (Y143) of S1PR1 within ERY motif in regulating receptor function. S1P or TNF-α binds S1PR1 (i) and phosphorylates S1PR1 at Y143 (ii; represented as red circle). Dynamin pinches off the phosphorylated S1PR1 (iii). S1P also induces BiP ATPase activity and its translocation to cytosol (not shown). Phosphorylated S1PR1 (Y143D-S1PR1) interacts with BiP in the cytosol (iv). BiP imports the receptor to the ER (v). WT-S1PR1 induces Ca2+ signaling and returns to the cell surface after dephosphorylation (not shown). BiP traps the Y143D-S1PR1 mutant at the ER where it augments SOCE and induces barrier-disruptive signaling in a Gi-dependent manner. See also Fig. S5, C and D. A.U., arbitrary units; IB, immunoblot; IP, immunoprecipitation; p-Tyr, phosphorylated tyrosine; UD, undetectable. (A) Molecular weight marker in kD.

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