S1P increased HIF-1 activity and expression of HIF-1 focus on genes also

S1P increased HIF-1 activity and expression of HIF-1 focus on genes also. FTC-133 cells. Cells had been treated with S1P (100 nM) for the indicated situations. Email address details are mean SEM, n 3. *P 0.05, **P 0.01 and ***P 0.001 indicate statistically significant difference between S1P treatment Angiotensin II and respective siRNA or automobile control, oooP 0.001 indicates significant difference between control siRNA+S1P and HIF-1 siRNA+S1P statistically.(TIF) pone.0066189.s001.tif (2.9M) GUID:?2142F368-85BD-453D-A03E-8665A0D32470 Figure S2: S1P1 activation will not increase HIF-1 expression in ML-1 cells. Cells had been treated with 10 M SEW-2871 (SEW) for 6 h. Result is normally mean SEM, n?=?6.(TIF) pone.0066189.s002.tif (258K) GUID:?A83D37B6-E2A4-4FF1-9DD3-DA0B6176F737 Figure S3: S1P up-regulates HIF-1 mRNA just after lengthy S1P incubation in ML-1 cells. (A) The original S1P-induced HIF-1 appearance isn’t mediated by elevated transcription. Cells had been treated with S1P (100 nM) for the indicated situations. (B) siRNA against HIF-1 triggered an around 90% knockdown of HIF-1 mRNA. Cells had been transfected with control siRNA (siC) or HIF-1 siRNA (siHIF) and treated with S1P (100 nM) or incubated in hypoxia (1% O2) for 9 h. Email address details are mean SEM, n 3. **P 0.01 and ***P 0.001 indicate significant difference between S1P treatment and automobile control statistically, oooP 0.001 indicates significant difference between HIF-1 control and siRNA siRNA.(TIF) pone.0066189.s003.tif (496K) GUID:?162515C3-28D6-4DB0-A0F5-4BE1383A31DF Amount S4: S1P may affect HIF-1 stability. (A) Inhibition of proteasomes highly elevates the basal HIF-1 proteins level and S1P struggles to boost it further. Cells had been pre-incubated with MG-132 (MG, 20 M, 1 Angiotensin II h) and activated with S1P (100 nM) for 6 h. (B-C) S1P inhibits hydroxylation of HIF-1 on Pro402 but will not inhibit hydroxylation of Pro564. Cells had been treated with S1P (100 nM) for 6 h. (D) Inhibition of Hsp90 lowers basal HIF-1 appearance and prevents S1P-induced up-regulation of HIF-1. Cells had been pre-incubated with 17-(allylamino)-17-desmethoxygeldanamycin (17-AAG, 2 M, 16 h) and activated with S1P (100 nM) for 6 h. (D) RACK1 and Hsp90 might not bind to HIF-1 in ML-1 cells. Cells had been treated with S1P (100 nM) for 6 h. Lysates had been immunoprecipitated using a RACK1 or Hsp90 antibody or an IgG control. A lysate of CoCl2-treated cells was utilized as positive control for HIF-1. Email address details are mean SEM, n 3. **P 0.01 indicates significant difference between S1P treatment and automobile control statistically, oP 0.05 and oooP 0.001 indicate significant difference between inhibitor treatment and automobile control statistically.(TIF) pone.0066189.s004.tif (2.5M) GUID:?D868BB63-28F3-400E-BC28-F42D64837C23 Figure S5: HIF-1 siRNA caused a knockdown of around 90% (in the qPCR experiments) and prevented S1P-induced HIF-1 expression. Cells had been transfected with control siRNA (siC) or HIF-1 siRNA (siHIF) and treated with S1P (100 nM) for 6 h.(TIF) pone.0066189.s005.tif (106K) GUID:?8C4FStomach62-99E5-4867-8EA3-C59563829271 Amount S6: qPCR results teaching expression of targeted mRNAs. siRNAs against Angiotensin II S1P1, S1P2, S1P3 and PCKI triggered a knockdown of 60C70% and siRNA against PKC triggered a knockdown of around 35%. Email address details are mean SEM, n 5. **P 0.01 and ***P 0.001 indicate significant difference between control siRNA and targeting siRNA statistically.(TIF) pone.0066189.s006.tif (809K) GUID:?B7FE9248-EEAB-4B86-A4DA-C450DE4100D5 Table S1: Primer information.(DOC) pone.0066189.s007.doc (36K) GUID:?B1E273D4-FA14-48DA-ADBF-B123B74CFFC2 Abstract 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 malignancy 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-1 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-1 attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1 expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCI. Half-life measurements with cycloheximide indicated that S1P.5D and 5G). significant difference between S1P treatment and respective vehicle or siRNA control, oooP 0.001 indicates statistically significant difference between control siRNA+S1P and HIF-1 siRNA+S1P.(TIF) pone.0066189.s001.tif (2.9M) GUID:?2142F368-85BD-453D-A03E-8665A0D32470 Figure S2: S1P1 activation does not increase HIF-1 expression in ML-1 cells. Cells were treated with 10 M SEW-2871 (SEW) for 6 h. Result is usually mean SEM, n?=?6.(TIF) pone.0066189.s002.tif (258K) GUID:?A83D37B6-E2A4-4FF1-9DD3-DA0B6176F737 Figure S3: S1P up-regulates HIF-1 mRNA only after long S1P incubation in ML-1 cells. (A) The initial S1P-induced HIF-1 expression is not mediated by increased transcription. Cells were treated with S1P (100 nM) for the indicated occasions. (B) siRNA against HIF-1 caused an approximately 90% knockdown of HIF-1 mRNA. Cells were transfected with control siRNA (siC) or HIF-1 siRNA (siHIF) and treated with S1P (100 nM) or incubated in hypoxia (1% O2) for 9 h. Results are mean SEM, n 3. **P 0.01 and ***P 0.001 indicate statistically significant difference between S1P treatment and vehicle control, oooP 0.001 indicates significant difference between HIF-1 siRNA and control siRNA.(TIF) pone.0066189.s003.tif (496K) GUID:?162515C3-28D6-4DB0-A0F5-4BE1383A31DF Physique S4: S1P may affect HIF-1 stability. (A) Inhibition of proteasomes strongly elevates the basal HIF-1 protein level and S1P is not able to increase it further. Cells were pre-incubated with MG-132 (MG, 20 M, 1 h) and stimulated with S1P (100 nM) for 6 h. (B-C) S1P inhibits hydroxylation of HIF-1 on Pro402 but does not inhibit hydroxylation of Pro564. Cells were treated with S1P (100 nM) for 6 h. (D) Inhibition of Hsp90 decreases basal HIF-1 expression and prevents S1P-induced up-regulation of HIF-1. Cells were pre-incubated with 17-(allylamino)-17-desmethoxygeldanamycin (17-AAG, 2 M, 16 h) and stimulated with S1P (100 nM) for 6 h. (D) RACK1 and Hsp90 may not bind to HIF-1 in ML-1 cells. Cells were treated with S1P (100 nM) for 6 h. Lysates were immunoprecipitated with a RACK1 or Hsp90 antibody or an IgG control. A lysate of CoCl2-treated cells was used as positive control for HIF-1. Results are mean SEM, n 3. **P 0.01 indicates statistically significant difference between S1P treatment and vehicle control, oP 0.05 and oooP 0.001 indicate statistically significant difference between inhibitor treatment and vehicle control.(TIF) pone.0066189.s004.tif (2.5M) GUID:?D868BB63-28F3-400E-BC28-F42D64837C23 Figure S5: HIF-1 siRNA caused a knockdown of approximately 90% (in the qPCR experiments) and prevented S1P-induced HIF-1 expression. Cells were transfected with control siRNA (siC) or HIF-1 siRNA (siHIF) and treated with S1P (100 nM) for 6 h.(TIF) pone.0066189.s005.tif (106K) GUID:?8C4FAB62-99E5-4867-8EA3-C59563829271 Physique S6: qPCR results showing expression of targeted mRNAs. siRNAs against S1P1, S1P2, S1P3 and PCKI caused a knockdown of 60C70% and siRNA against PKC caused a knockdown of approximately 35%. Results are mean SEM, n 5. **P 0.01 and ***P 0.001 indicate statistically significant difference between control siRNA and targeting siRNA.(TIF) pone.0066189.s006.tif (809K) GUID:?B7FE9248-EEAB-4B86-A4DA-C450DE4100D5 Table S1: Primer information.(DOC) pone.0066189.s007.doc (36K) GUID:?B1E273D4-FA14-48DA-ADBF-B123B74CFFC2 Abstract 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 malignancy 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 CSF2RB of tumor biology. We found S1P to increase expression of the regulatory HIF-1 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-1 attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1 expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1 protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1 synthesis. In conclusion, we have recognized S1P as a non-hypoxic regulator of HIF-1 activity in thyroid malignancy cells, studied.