PMC

SphK1 silencing downregulates Akt/mTOR signaling pathway in VHL-defective A498 and 786-O ccRCC cells. A498 and 786-O cells were untransfected or transfected with 20 nmol/l of siSphK1 or siScr for 72 h before the experiments followed by 4 h under normoxic or hypoxic condition. Cell lysates were assayed for Ser2448 phosphorylated mTOR (p-mTOR Ser2448) and mTOR expression (a); Thr389 phosphorylated p70S6K (p-p70S6K Thr389), Thr421/Ser424 phosphorylated p70S6K (p-p70S6K Thr421/Ser424) and p70S6K expression (b); Ser65 phosphorylated 4E-BP1 (p-4E-BP1 Ser65) and 4E-BP1 expression (c); Ser473 phosphorylated Akt (P-Akt Ser473) and Akt expression (d) were analyzed by immunoblotting. Similar results were obtained in three independent experiments.

SphK1 silencing prevents HIF-2α accumulation in multiple human cancer cell lines under hypoxia. (a), relative mRNA expression of SphK1 expression and SphK1 activity cells were measured in prostate (PC-3), lung (A549), brain (U87) and renal cancer (CAKI-1 and A498) cells after 72 h of treatment with 20 nmol/l of siSphK1 or siScr. Columns, mean of at least three independent experiments; bars, s.e.m. **P<0.01; ***P<0.001. (b–f) PC-3 (b), A549 (c), U87 (d), CAKI-1 (e) and A498 (f) cells were untreated or treated with 20 nmol/L of siSphK1 or siScr then incubated under normoxia or hypoxia for an additional 6 h. HIF-2α expression was analyzed by immunoblotting. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin.

Exogenous S1P regulates HIF-2α accumulation in CAKI-1, A498 and 786-O ccRCC cells. (a) CAKI-1, A498 and 786-O cells were treated with the indicated concentrations of anti-S1P mAb for 2 h, then incubated under normoxia or hypoxia for an additional 6 h and HIF-2α expression was analyzed by immunoblotting. (b) Relative mRNA expression of Spns2 expression in CAKI-1 and A498 cells was measured after 72 h of treatment with 90 nmol/l of two different siSpns2 (siSpns2a and siSpns2b) or siScr followed by 6 h under normoxic or hypoxic condition. Columns, mean of at least four independent experiments; bars, s.e.m. *P<0.05; **P<0.01. (c) CAKI-1 and A498 cells were transfected with 90 nmol/l of two different siSpns2 (siSpns2a and siSpns2b) or siScr for 72 h, then incubated under normoxia or hypoxia for an additional 6 h. Cell lysates were assayed for Spns2 and HIF-2α expression by immunoblotting. For all experiments, similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin.

PLD regulates SphK1-dependent HIF-2α expression in CAKI-1 and A498 ccRCC cells. (a), CAKI-1 (left) and A498 (right) cells were incubated under hypoxia for the indicated times and then tested for PLD and SphK1 enzymatic activities. Points, mean of at least three experiments; bars, s.e.m. **P<0.01; ***P<0.001. Inset, HIF-2α expression in CAKI-1 cells exposed to hypoxia for the indicated times. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to α-tubulin. (b, c), CAKI-1 (left) and A498 (right) cells were untreated or treated with 1-butanol (1-ButOH) or tert-butanol (t-ButOH) as control (0.8%). SphK1 activity (b) and HIF-2α expression (c) were determined in normoxia or after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001. (d, e) CAKI-1 (left) and A498 (right) cells were transfected with siPLD1 (50 nmol/L), siPLD2 (50 nmol/L) or siPLD1 (50 nmol/L) and siPLD2 (50 nmol/L) or siScr (50 nmol/L) for 72 h then incubated under normoxia or hypoxia. SphK1 activity (d) and HIF-2α expression (e) were determined after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001.

S1P₁ mediates the effect of S1P on HIF-1α and HIF-2α protein content in CAKI-1 and A498 ccRCC cells. (a) The relative mRNA expression of S1P_1–5 in A498 and CAKI-1 was measured after 1 h of incubation under normoxic (black) or hypoxic (white) conditions. Columns, mean of at least five independent experiments; bars, s.e.m. **P<0.01. (b) A498 and CAKI-1 cells were treated with W146 (5 μm) or ethanol (control), then incubated under normoxia (Nx) or hypoxia for 6 h. HIF-1α and HIF-2α expression was analyzed by immunoblotting. (c) A498 and CAKI-1 cells were transfected with 50 nmol/l of siS1P₁ or siScr for 72 h, then incubated under normoxia (Nx) or hypoxia for an additional 6 h. Cell lysates were assayed for HIF-1α and HIF-2α expression by immunoblotting. (d) CAKI-1 shS1P₁ and CAKI-1 shCtrl cell lines were incubated under normoxia (Nx) or hypoxia (Hx) for 6 h, and HIF-1α and HIF-2α expression was analyzed by immunoblotting. For all experiments, similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin.

SphK1 silencing leads to a decrease in HIF-2 transcriptional activity in A498 and 786-O VHL-defective ccRCC cells. A498 and 786-O cells were treated with 20 nmol/l of siSphK1 or siScr for 72 h then incubated for an additional 16 h under normoxia or hypoxia. (a) HRE reporter gene assay (left) and protein HIF-2α expression (right) in transiently transfected A498 (upper) and 786-O (lower) cells. The y axis shows normalized Firefly luciferase over Renilla luciferase activity relative to the wild-type normoxic response. HIF-2α expression was analyzed by immunoblotting. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of at least four independent experiments; bars, s.e.m. ***P<0.001. Cell lysates were assayed for GLUT-1 (b) and cyclin D1 (c) expression by western blot analysis. Similar results were obtained in three independent experiments, and equal loading was monitored using antibody to tubulin. (d) Cell proliferation and viability was respectively assessed using [³H]-thymidine incorporation assay and MTT assay. Columns, mean of at least four independent experiments; bars, s.e.m. **P<0.01; ***P<0.001.

SphK1 signaling does not impact on protein synthesis and stability of HIF-2α in A498 ccRCC cells. (a) Relative mRNA expression of HIF-2α and SphK1 expression in A498 cells was measured after 72 h of treatment with 20 nmol/l of siSphK1 or siScr followed by 6 h under normoxic or hypoxic condition. Columns, mean of at least four independent experiments; bars, s.e.m. ***P<0.001. (b) A498 cells were untransfected or transfected with 20 nmol/l of siSphK1 or siScr for 72 h before the experiments. Cells were then incubated for 6 h under normoxic or hypoxic condition in presence or absence of the proteasome inhibitor MG132 (10 μm). Cell lysates were assayed for HIF-2α expression by immunoblotting. Similar results were obtained in three independent experiments, and equal loading was monitored using antibody to tubulin. (c) A498 cells were transfected with siSphK1 or siScr as described in . 48 h later, cells were treated for 10 min with 10 μg/ml of puromycin, before cells collection. Whole-cell extracts were analyzed by western blot. Puromycin incorporation was measured as readout of protein synthesis using anti-puromycin antibody. (d) A498 cells were treated as in (c) and subjected to hypotonic lysis. Extracts were separated on 10–45% sucrose gradient and subjected to polysomal profile analysis. 40S ribosomal subunits, 80S ribosomes and heavy polysomes (actively translated mRNA) are indicated. (e) RT-qPCR experiments were performed for the indicated mRNAs on RNA extracted from samples in (d). Results are presented as fold change normalized to β-actin mRNA and indicate relative mRNA expression. (f) RNA was extracted from light fractions (fractions 1–4) and heavy polysomes fractions (fractions 5–7) of the sucrose gradient from (d). Relative abundance of the indicated mRNAs in heavy polysomes was quantified by RT-qPCR and presented as translation efficiency for each mRNA. TATA binding protein and β-actin mRNAs were used as controls.