ETS-1-mediated transcriptional up-regulation of CD44 is required for sphingosine-1-phosphate receptor subtype 3-stimulated chemotaxis

Abstract

Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P3), thus providing a tractable in vitro system to characterize molecular mechanism(s) underlying the S1P3 receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P3/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P3 or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P3/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, i.e., ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P3-regulated chemotactic response.

Keywords: CD44; Chemotaxis; Ets Family Transcription Factor; Jun N-terminal Kinase (JNK); Lipids; Receptors; Signal Transduction.

FIGURE 1.

S1P stimulates expression and membrane localization of CD44. H1793 cells were stimulated with or without S1P (200 nm) for indicated times. A and B, levels of CD44 mRNAs (A) and proteins (B) were measured by qPCR and Western blot analysis, respectively. Note that S1P treatment significantly enhances CD44 expression. Lower panel in B, levels of CD44 proteins were quantitated by the National Institutes of Health ImageJ software. C, H1793 cells were treated with or without S1P (200 nm) for 4 h. CD44 proteins (green) were detected by immunofluorescence staining. Nuclei were stained with DAPI (lower panels). Note that S1P treatment markedly increases membrane localization of CD44 proteins. D, HBEC2-KT immortalized normal lung epithelial (2KT) and H1792, H23, and H1650 lung adenocarcinoma cells were treated with or without S1P (200 nm) for 4 h, followed by qPCR quantitation of CD44 expression. Note that CD44 expression was significantly elevated in lung adenocarcinoma cells after S1P stimulation, whereas S1P was unable to stimulate CD44 expression in HBEC2-KT cells. A and D, data represent means ± S.D. from three individual experiments performed in triplicate. *, p < 0.05; **, p < 0.01 (S1P versus control vehicle treatment), t test. Ctrl, control; IB, immunoblotting.

FIGURE 2.

S1P-induced CD44 expression is mediated by S1P₃/ROCK signaling pathway. A, S1P-increased CD44 expression was measured in H1793 cells transfected with sh-S1P₃ or sh-Ctrl (control) vector. B, expression of S1P receptor subtypes was measured in H1793 cells stably transfected with sh-S1P₃ or sh-Ctrl vector by real time PCR. C, immunofluorescence staining of CD44 (green, panels a, c, e, and g) in sh-S1P₃ (panels e–h) or sh-Ctrl (panels a–d) transfected H1793 cells, treated with or without S1P for 4 h. Cell nuclei were stained with DAPI (panels b, d, f, and h). D, H1793 were pretreated for 1 h with or without indicated pharmacological inhibitors, followed by stimulating in the presence or absence of S1P for 4 h. The expression of CD44 was quantitated by qPCR. LY, LY294002 for PI3K; BAY, Bay 11-7085 for NFκB; Y, Y-27632 for ROCK. Note that pretreatment with ROCK inhibitor completely diminished the S1P-induced CD44 expression. E, H1793 were transfected with nontargeting siRNA control or si-ROCK1 (Qiagen). Levels of ROCK1 were quantitated by qPCR. F, si-Ctrl or si-ROCK1 transfected H1793 cells were treated with or without S1P for 4 h. Levels of CD44 were measured by qPCR. G, H1793 cells were pretreated with control vehicle (panels a–d) or Y-27632 (Y) (panels e–h) for 1 h, followed by stimulating with or without S1P for 4 h. CD44 proteins were analyzed by immunofluorescence staining (panels a, c, e, and g), and cell nuclei were stained with DAPI (panels b, d, f, and h). Note that the S1P-increased CD44 at membrane regions was inhibited by pretreatment of ROCK inhibitor. The data represent means ± S.D. from three (A, B, and D) or two (E and F) individual experiments performed in triplicate. *, p < 0.01; **, p < 0.05; t test. Ctrl, control.

FIGURE 3.

CD44 mediates S1P/S1P₃-stimulated cell migration. A, H1793 cells were stably transfected with sh-CD44 or sh-Ctrl vector. Levels of CD44 mRNA were quantitated by qPCR. Note that levels of CD44 mRNA were profoundly knocked down in sh-CD44 stably transfected cells. B, H1793 cells stably transfected with sh-CD44 or sh-Ctrl vector were treated with or without S1P for 4 h. Levels of CD44 proteins were quantitated by Western blotting. Immunoblotting of β-actin was assessed for equal loading. Lower panel, levels of CD44 polypeptides were quantitated with a densitometer. C, migration was measured in H1793 cells stably transfected with sh-CD44 or sh-Ctrl control vector in the presence of indicated concentrations of S1P. Note that S1P-stimulated chemotaxis was completely inhibited in CD44 knocked down cells. D, migration was measured in si-ROCK1 or si-Ctrl transfected H1793 cells, stimulated with indicated concentrations of S1P. A, C, and D, the data are means ± S.D. from two experiments with triplicate determinations. IB, immunoblotting.

FIGURE 4.

S1P stimulates ETS-1 binding to CD44 promoter. A, CD44 promoter contains four candidate ETS-1 binding sites. P1, P2, and P3 primer pairs were used to differentially amplify these candidate sites. B, ETS-1 binds to sites 3 and/or 4. ChIP analysis was performed as described under “Experimental Procedures.” Note that a specific amplicon (expected size, 230 bp) was detected by using the P2 primer pair, whereas no amplicon was detected by using the P1 primer pair (top panel). Middle panel, PCR amplification of total input chromatin, used as a loading control. Bottom panel, ChIP analysis was performed with normal IgG (nIgG) or ETS-1 immunoprecipitates, followed by PCR amplification with P2 primer pair. C, cells were treated with or without S1P for the indicated times and analyzed for ETS-1 ChIP assay with P3 primer pair. PCR amplification of anti-ETS-1 immunoprecipitates and total input chromatin (expected size, 429 bp) are shown in the upper and lower panels, respectively. D, PCR amplification of anti-ETS-1 immunoprecipitates and total input DNA with P2 primer pair is shown in the upper and lower panels, respectively. Note that S1P treatment increased ETS-1 binding to sites 3 and/or 4 in the CD44 promoter region. B–D are images of a representative experiment that was repeated two times with similar results.

FIGURE 5.

S1P enhances ETS-1 expression via S1P₃/ROCK pathway. A, ETS-1 mRNAs were quantitated in H1793 cells treated with S1P for various times. B, S1P increased ETS-1 expression in H1792, H23, and H1650 lung adenocarcinoma cells and not in HBEC2-KT cells. C, Western blotting showed that S1P treatment increased ETS-1 proteins. D, H1793 cells were treated with or without S1P for 4 h. ETS-1 was detected by immunostaining with ETS-1 antibody (upper panels), and nuclei were stained with DAPI (lower panels). Note that S1P treatment stimulated the expression and nuclear localization of ETS-1 proteins (white arrows). E, knockdown of S1P₃ completely abrogated S1P-increased ETS-1 expression. H1793 cells transfected with sh-S1P₃ or sh-Ctrl vector were treated with or without S1P for 4 h. ETS-1 mRNAs were quantified by qPCR. F, inhibition of ROCK significantly diminished S1P-induced ETS-1 expression. H1793 were pretreated with or without pharmacological inhibitors, followed by stimulating with S1P for 4 h. ETS-1 mRNAs were measured by qPCR. G, levels of ETS-1 in si-Ctrl or si-ROCK1 transfected cells, in the presence or absence of S1P treatment (200 nm, 4 h). The data represent means ± S.D. from three (A, B, E, and F) or two (G) individual experiments performed in triplicate. *, p < 0.05; **, p < 0.01; t test. p values in A and B are S1P versus control. Ctrl, control; IB, immunoblotting; LY, LY294002; BAY, Bay 11-7085; Y, Y-27632.

FIGURE 6.

ETS-1 knockdown inhibits S1P-stimulated CD44 expression and chemotaxis. A and B, ETS-1 mRNAs and proteins were measured by qPCR (A) and Western blotting analysis (B) in H1793 cells transfected with sh-ETS-1 or sh-Ctrl vector. Western blotting was quantitated by a densitometer (lower panel in B). Cells transfected with sh-ETS-1 or sh-Ctrl were treated with or without S1P for 4 h. C and D, ETS-1 (C) and CD44 (D) proteins were assessed by Western blotting analysis. β-Actin blotting was used as a loading control. Note that knockdown of ETS-1 abrogated the S1P-stimulated expression of ETS-1 (C) and CD44 (D) proteins. E, levels of CD44 mRNA and proteins in sh-Ctrl or sh-ETS-1 transfected cells, treated with or without S1P (200 nm, 4 h). F, H1793 cells, transfected with sh-Ctrl or sh-ETS-1, were treated with S1P (200 nm) for indicated times. Levels of ETS-1 and CD44 were assessed by Western blotting. Lower panel, levels of ETS-1 and CD44 were normalized by actin proteins and shown as arbitrary units. G, ETS-1 knockdown inhibits S1P-stimulated cell migration. Chemotaxis was measured in cells transfected with sh-ETS-1 or sh-Ctrl vector following S1P stimulation. **, p < 0.01, analysis of variance (n = 6). A, *, p < 0.05; the data are means ± S.D. (n = 3) of a representative experiment that was repeated two times with similar result.

FIGURE 7.

The JNK/c-Jun signaling pathway mediates the S1P₃/ROCK1 up-regulated ETS-1 expression. A, H1793 cells were treated with S1P (200 nm) for various times. Protein lysates were immunoblotted with phospho-JNK antibody. Extracts of HEK293 cells treated without and with phorbol 12-myristate 13-acetate (“−”ve and “+”ve, respectively) were used as negative and positive control. B, sh-Ctrl or sh-S1P₃ stably transfected H1793 cells were treated with or without S1P (200 nm, 15 min), followed by Western blotting with anti-phospho-JNK. C, H1793 cells were pretreated in the presence or absence of a ROCK inhibitor (Y27632) for 30 min, followed by stimulating with or without S1P (200 nm, 15 min). Extracts were probed with anti-phospho-JNK. D, H1793 cells were treated with S1P (200 nm) for indicated times. AP-1 ChIP assays were performed by amplifying the anti-c-Jun precipitates with primer pairs specific for AP-1 site in the ETS-1 promoter region (SABiosciences; GPH1016833(−)01A). E, H1793 cells were pretreated with JNK inhibitor (SP600125, 10 μm) for 30 min, followed by stimulating with S1P for 4 h. Extracts were blotted with anti-ETS-1 or anti-CD44. F, levels of ETS-1 and CD44 were measured in H1793 cells, transfected with si-Ctrl or si-JNK1 (Ambion), following S1P stimulation. G, immunoblot was quantified by National Institutes of Health ImageJ software and normalized to actin. H and I, S1P-stimulated chemotaxis was measured in H1793 cells pretreated with or without JNK inhibitor (SP600125) (H) or transfected with si-Ctrl or si-JNK1 (I). Cell migration induced by FBS (10%) was used as a control. *, p < 0.05, t test (n = 6). M.W., molecular mass.