An autocrine sphingosine-1-phosphate signaling loop enhances NF-kappaB-activation and survival

Peer reviewe

Endocrine modulating actions of a phytosterol mixture and its oxidation products in zebrafish (Danio rerio)

V2007okL-talon kirjasto (Yha

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

Hypoxia up-regulates S1P₁ but does not affect ML-1 migration.

<p>(<b>A</b>) Hypoxia increases S1P₁ protein expression. Cells were incubated in hypoxia (1% O₂) for 24 h. (<b>B</b>) Hypoxia does not affect basal or S1P-induced migration or haptotaxis. Cells were allowed to migrate in normoxia or hypoxia (1% O₂) towards serum and S1P in the migration experiments or towards collagen and S1P in the haptotaxis experiments for 8 h. (<b>C</b>) Hypoxia does not affect proliferation. Cells were incubated in normoxia or hypoxia (1% O₂) for the indicated times. Results are mean ± SEM, n ≥ 3. **P < 0.01 and ***P < 0.001 indicate statistically significant difference between S1P treatment and respective control.</p

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

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

Schematic representation of the putative signaling involved in S1P-induced HIF-1α expression in ML-1 cells.

<p>S1P stimulation up-regulates the HIF-1α protein in normoxia. This effect is dependent on activity of S1P₃ and G_i as well as their downstream effectors PKCβI, PI3K and MEK (S1P receptor signaling reviewed in 3 and 4). We suggest S1P to regulate both stability and translation of HIF-1α. S1P stimulation increases phosphorylation of mTOR via MEK and PI3K and phoshorylation of p70S6K, eIF-4E and 4E-BP1 via MEK and/or PI3K/mTOR and inhibition of p70S6K prevents S1P-induced up-regulation of HIF-1α. HIF-1 is involved in both basal and S1P-induced ML-1 migration.</p