S1P-evoked inhibition of MMP2 is mediated through the Rho-ROCK pathway.

<p>(A) Time-dependent effect of S1P on Rho activity in C643 cells. (B) Stimulatory effect of the Rho inhibitor C3-transferase or the ROCK inhibitor Y-27632 on C643 cell invasion. (C) C3-transferase or Y-27632 attenuated the S1P-evoked inhibition of MMP2 activity. (D and E) Lack of an effect of pretreatment with C3-transferase or Y-27632 on the expression of MMP2 but S1P-evoked-attenuation of MMP2 expression was abolished. The representative blots are shown. β-Actin was used as loading control, and the normalized results are presented in graphs. The data in the graphs are the mean ±S.E.M, n = 3. Asterisks (*) denote the statistically significant differences compared with respective control. (¤) indicates comparisons between S1P effects. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001; ¤ <i>P</i> < 0.05; ¤¤ <i>P</i> < 0.01).</p

S1P inhibits invasion and affects expression, secretion and activity of MMP2 and MMP9 in C643 cells.

<p>(A) Cells were treated with S1P (100 nM for 2 h, 4 h 6 h and 8 h). The representative western blots show the expression of MMP2 and MMP9. β-Actin was used as loading control and the normalized data are presented in the graphs. (B) Cells were treated with S1P (100 nM for 2 h, 4 h, 6 h and 8 h). MMP2 and MMP9 mRNA expression was measured with quantitative real-time PCR. (C) Cells were treated with S1P (100 nM for 2 h, 4 h, 6 h and 8 h). The representative western blots show the secretion of MMP2 and MMP9. (D) Cells were treated with S1P (100 nM for 2 h, 4 h, 6 h and 8 h). The representative zymography blot shows the activity of MMP2 and MMP9. The data were normalized to the protein content on the plates and are presented in respective graphs. (E) C643 cells were allowed to invade through collagen IV towards 5% lipid-stripped FBS in the presence or absence of S1P (100 nM for 2 h, 4 h, 6 h and 8 h). Representative images of invasion inserts are shown. The scale bar is 50 μm. The normalized data in the graphs are the mean ± S.E.M, n = 3–6. Asterisks (*) denote the statistically significant differences compared with respective control. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001).</p

MMP2 and MMP9 regulate C643 invasion.

<p>(A) Wound healing assay for C643 and FTC-133 cell migration. Images were taken immediately after wound scratch at 0 h and after 24 h. The cells were stimulated with vehicle or 100 nM S1P. The images are representative of three separate experiments. The data are presented as % wound in respective graphs. (B and C) C643 cells were transfected with negative control siRNA, MMP2 siRNA or with MMP9 siRNA. After 48 hours of transfection, the cells were allowed to invade through collagen IV towards 5% lipid-stripped FBS for 6 h. (D) C643 cells were pre-incubated with the MMP2/9 inhibitor SB (10 μM, 1 h) and were allowed to invade towards 5% lipid-stripped FBS for 6 hours in the presence or absence of 100 nM S1P. (E) C643 cells were transfected with negative control siRNA or with siRNA for MMP2 and -9. After 48 hours of transfection, the cells were allowed to invade towards 5% lipid-stripped FBS for 6 hours in the presence or absence of 100 nM S1P. (F) C643 cells were transfected with negative control siRNA or with siRNA for MMP2 and -9. After 48 hours of transfection, the cells were stimulated with S1P for 6 hours and the expression of MMP2 and -9 was measured. A representative western blot is shown. β-Actin was used as loading control, and the normalized results are presented in the graphs. (I) As in (F), but the medium was collected and the activity of MMP2 measured using the zymography assay. The data were normalized to the protein content on the plates. (G and H) C643 cells were transfected with negative control siRNA, with MMP2 siRNA or MMP9 siRNA. After 48 hours of transfection, the cells were stimulated with 100 nM S1P for 6 hours and the expression of MMP2 and -9 was measured. Representative western blots are shown. β-Actin was used as loading control, and the normalized results are presented in the graphs. The normalized results in the graphs are the mean ±S.E.M, n = 3. Asterisks (*) denote the statistically significant differences compared with respective control. (¤) indicates comparisons between S1P effects. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001; ¤ <i>P</i> < 0.05; ¤¤ <i>P</i> < 0.01; ¤¤¤ <i>P</i> <0.001).</p

Expression of S1P receptors in human thyroid cancer C643 and FTC-133 cells.

<p>(A) The mRNA expression of S1P_1-5. Representative agarose gels showing the PCR products stained with ethidium bromide. ML-1 cDNA was used as positive control for S1P_1-3 and S1P₅. Hela cDNA was used as positive control for S1P₄. HPRT was used as control reference gene. (B) Representative western blots showing the expression of S1P_1-5 on protein level. Hsc70 was used as loading control. Each gel or blot is a representative of at least three independent experiments.</p

S1P inhibits invasion and affects expression, secretion and activity of MMP2 and MMP9 in FTC-133 cells.

<p>(A) Cells were treated with S1P (100 nM for 6 h and 8 h). The representative western blots show the expression of MMP2 and MMP9. β-Actin was used as loading control and the normalized data are presented in the graphs. (B) Cells were treated with S1P (100 nM for 6 h and 8 h). The representative western blots show the secretion of MMP2 and MMP9 and the normalized data are presented in the graphs. (C) Cells were treated with S1P (100 nM for 6 h and 8 h). The representative zymography blot shows the activity of MMP2 and MMP9. The data were normalized to the protein content on the plates and are presented in respective graphs. (D) C643 cells were allowed to invade through collagen IV towards 5% lipid-stripped FBS in the presence or absence of S1P (100 nM for 6 h and 8 h). Representative images of invasion inserts are shown. The scale bar is 50 μm. (E) MMP2 and MMP9 mRNA expression was measured with quantitative real-time PCR and normalized to GAPDH and PBGD, respectively. The normalized data in the graphs are the mean ± S.E.M, n = 3–4. Asterisks (*) denote the statistically significant differences compared with the respective control, (¤) denotes the statistically significant differences between controls and (o) denotes the statistical difference between S1P treatments. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001; ¤ <i>P</i> < 0.05; o <i>P</i> < 0.05).</p

Calpains participate in the expression, activity and secretion of MMP2 and MMP9.

<p>(A) C643 cells were transfected with negative control or with S1P₂ siRNA for 48 h, and then stimulated with 100 nM S1P for 6 h, and the calpain activity was measured. (B) C643 cells were treated with the S1P₂ antagonist JTE-013 (10 μM for 1 h) and then stimulated with S1P (100 nM for 6 h), and the calpain activity was measured. (C) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and allowed to invade through collagen IV towards 5% lipid-stripped FBS in the presence or absence of 100 nM S1P for 6 h. The data are presented in the graph. (D) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and then stimulated with 100 nM S1P for 6 hours. The expression of MMP2 was then determined. Representative western blots are shown. β-Actin was used as loading control, and the normalized data are presented in respective graphs. (Ea,b) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and then stimulated with S1P for 6 h. The medium was collected and the zymography assay was performed. A representative zymography blot is shown. The data were normalized to the protein content on the plates and are presented as graphs. (F) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and then stimulated with S1P for 6 hours. The medium was collected and the secreted MMP2 was measured. A representative western blot is shown. The data were normalized to the protein content on the plates. The normalized results in the graphs are the mean ±S.E.M, n = 3–6. Asterisks (*) denote the statistically significant differences compared with respective control. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001).</p

HIF-1α mediates basal and S1P-induced migration of ML-1 cells.

<p>(<b>A</b>) Inhibition of HIF-1 attenuates S1P-induced migration. Cells were preincubated with HIF-1 inhibitor (HIFi, 10 µM, 30 min) and S1P (100 nM, 30 min) and allowed to migrate towards serum for 8 h. (<b>B</b>) Down-regulation of HIF-1α decreases basal migration. Cells were transfected with HIF-1α siRNA and allowed to migrate towards serum and S1P (100 nM) for 8 h. (<b>C</b>) Down-regulation of HIF-1α attenuates S1P-induced migration. Cells were transfected with HIF-1α siRNA and allowed to migrate towards S1P (100 nM) for 20 h. (<b>D</b>) Inhibition of p70S6K decreases basal migration and prevents S1P-induced migration. Cells were preincubated with p70S6K inhibitor (p70i, 10 µM, 30 min) and S1P (100 nM, 30 min) and allowed to migrate towards serum for 8 h. (<b>E</b>) Down-regulation of S1P₃ attenuates S1P-induced migration. Cells were transfected with S1P₃ siRNA and allowed to migrate towards serum and S1P (100 nM) for 8 h. Results are mean ± SEM, n ≥ 3. *P < 0.05 and ***P < 0.001 indicate statistically significant difference between S1P treatment and respective vehicle or siRNA control, ^oP < 0.05 and ^oooP < 0.001 indicate statistically significant difference between siRNA treatment and control siRNA, between siRNA+S1P treatment and control siRNA+S1P or between inhibitor treatment and vehicle control.</p

Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

<div><p>Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P₃), G_i proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.</p></div

S1P up-regulates HIF-1α via S1P₃ and G_i in ML-1 cells.

<p>Inhibition of (<b>A</b>) G_i proteins, (<b>B</b>) S1P₃ or (<b>C</b>) S1P₁ and S1P₃ and (<b>D</b>) knockdown of S1P₃ prevents S1P-induced HIF-1α expression. Cells were pretreated with Pertussis toxin (Ptx, 100 ng/ml, 24 h), CAY10444 (CAY, 10 µM, 1 h) or VPC-23019 (VPC, 10 µM, 30 min) or transfected with control siRNA (siC) or S1P receptor siRNA (si1-3) and stimulated with S1P (100 nM) for 6 h. Results are mean ± SEM, n ≥ 3. *P < 0.05 and ***P < 0.001 indicate statistically significant difference between S1P treatment and respective vehicle or siRNA control, ^oooP < 0.001 indicates statistically significant difference between inhibitor treatment and vehicle control.</p

S1P stabilizes HIF-1α independently of pVHL binding.

<p>(<b>A</b>) S1P prolongs HIF-1α half-life. Cells were either left untreated, treated with S1P (100 nM) for 6 h, incubated in hypoxia (1% O₂) for 6 h or treated with CoCl₂ (150 µM) for 3 h before the cycloheximide chase (Chx, 5 µg/ml). S1P, hypoxic conditions or CoCl₂ were present throughout the chase. Time points are mean ± SEM, n = 3–10. Curve fit was done with the one phase exponential decay equation. (<b>B</b>) S1P does not inhibit binding of pVHL to HIF-1α. Cells were treated with S1P (100 nM) for 6 h. The level of co-immunoprecipitated HIF-1α was compared with the level of immunoprecipitated pVHL and IgG bands were used as a loading control. **P < 0.01 indicates statistically significant difference between S1P treatment and vehicle control.</p