Fig 2: Effects of peretinoin on SPHK1 promoter activity. A effects of peretinoin on the promoter activity of SPHK1 and SPHK2. (a) Plasmids encoding luciferase under the control of the promoter region of SPHK1, SPHK2, β-actin, and GAPDH were transfected into Huh-7 cells and, 24 h later, DMSO or peretinoin at 1, 5, 10, 20, or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. The luciferase activity in each condition was normalized to that of DMSO control. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by one-way ANOVA. (b) Possible binding sites of Sp1 in the SPHK1 promoter region and a representation of deletion mutants. This figure shows the possible binding sites of Sp1 in the promoter region of SPHK1 and the series of deletion mutants analyzed. (c) Enhancement of the promoter activity of SPHK1 by Sp1 overexpression. A plasmid encoding Sp1 or a control empty vector was co-transfected with a plasmid encoding luciferase under the control of the SPHK1 promoter region and, 48 h later, luciferase activity was determined. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by the Student’s t test. (d) Restoration of the suppressive effects of peretinoin on the promoter activity of SPHK1 by Sp1 overexpression. A plasmid encoding Sp1 or a control empty vector was transfected into Huh-7 cells. After 24 h, the cells were transfected with a plasmid encoding luciferase under the control of the promoter region of SPHK1 or a control vector without any promoter regions. After another 24 h, DMSO or peretinoin at 20 or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity from peretinoin-treated cells to that of DMSO-treated cells in cells transfected with the plasmid encoding Sp1 or the control empty vector. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by the Student’s t test. (e), attenuation of the suppressive effects of peretinoin on the promoter activity of SPHK1 by Sp1 knockdown. Three kinds of siRNA to Sp1, control siRNA (siCNT), or mock were transfected at 20 nM into Huh-7 cells and, 48 h later, the cells were transfected with the plasmid encoding luciferase under the control of the promoter region of SPHK1 or a control vector without any promoter regions. After another 24 h, DMSO or peretinoin at 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity of cells treated with 40 μM peretinoin to that of DMSO-treated cells for each siRNA or mock transfection. (f), effects of deletion of the Sp1 binding site on the promoter activity of SPHK1. The plasmids encoding luciferase under the control of various lengths of promoter regions of SPHK1 were transfected into Huh-7 cells and, 24 h later, DMSO or peretinoin at 20 or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity of peretinoin-treated cells to that of DMSO-treated cells for each plasmid. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.005.

Fig 3: mRNA level of SPHK1 in human liver. A. comparison of the mRNA level of SPHK1 between mild and severe fibrosis in the HCV-infected liver. (a) We performed liver biopsies on patients infected with HCV prior to antiviral treatment and quantified the mRNA levels of SPHK1 and β-actin in total RNA extracted from the liver by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each patient. The patients were categorized into two groups based on their liver fibrosis grade; mild fibrosis was defined as METAVIR Score F1 or F2 and severe fibrosis as F3 or F4. We compared the normalized mRNA level of SPHK1 between mild and severe fibrotic liver. Error bars show the standard deviation from 119 patients with mild liver fibrosis and 59 patients with severe fibrosis. The statistical significance of the difference in the average between the two groups was analyzed by the Student’s t test. (b) Comparison of the mRNA level of SPHK1 before and after HCV eradication. We performed liver biopsies on another 12 patients before they started antiviral treatment for HCV and after successful eradication of HCV. We quantitated the mRNA levels of SPHK1 and β-actin in total RNA extracted from the liver by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each patient. The relative mRNA level of SPHK1 after HCV eradication (post-SVR [sustained virologic response]) was normalized to that before antiviral treatment (prior to antiviral treatment) in the individual patients, with the mRNA level of SPHK1 before antiviral treatment set to 1. The statistical significance of the difference in the average between these two groups was analyzed by a paired t test. *p < 0.05, ***p < 0.005.

Fig 4: Effects of peretinoin on the mRNA level, protein expression, and enzymatic activity of SPHK1. (a) effects of peretinoin on the mRNA level of sphingolipid-related genes in Huh-7 cells. Huh-7 cells were treated with peretinoin at 10, 25, or 50 μM or with 0.5% DMSO for 72 h and total cellular RNA was extracted. The mRNA levels of SPHK1, SPHK2, S1P lyase, S1P receptor 1–3 (S1P1, S1P2, and S1P3), and β-actin were quantitated by qRT-PCR (SYBR green assay), and the mRNA levels of SPHK1, SPHK2, S1P lyase, S1P1, S1P2, and S1P3 were normalized to that of β-actin. The relative mRNA level of each gene under each condition was normalized to that of DMSO control. (b) Effects of peretinoin on the protein expression of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin 10, 20, or 40 μM. Total cell lysates were collected 12, 24, 48, and 72 h later and then probed by western blotting with anti-SPHK1 and β-actin antibodies. Full-length gels and blots before cropping are shown in supplemental Fig. S8. (c) Effects of peretinoin on the enzymatic activity of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin at 10, 20, and 40 μM and a plasmid encoding SPHK1 cDNA or an empty vector were transfected into Huh-7 cells. Total cell lysates were collected 72 h after initiation of peretinoin treatment and transfection and used in an in vivo SPHK activity assay. Synthesized S1P was visualized by using radioisotopes. The radioactive spots corresponding to S1P were scraped into vials and the radioactivity was measured in a scintillation counter and normalized to that of DMSO-treated cells or empty vector-transfected cells. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by one-way ANOVA. (d), effects of peretinoin on the enzymatic activity of SPHK1 in vitro. Human recombinant SPHK1 was incubated with DMSO or peretinoin at 5, 10, 25, or 50 μM or with SKI II at 5, 10, 25, or 50 μM as described in the Experimental procedures. Synthesized S1P was quantitated by a fluorescence-based method. The SPHK activity in each condition was normalized to that of DMSO control. *p < 0.05, **p < 0.01, ***p < 0.005.

Fig 5: Liver mRNA levels of SPHK1 in the PDGFC-Tg and Ath-HF diet mouse models. (a) mRNA level of SPHK1 in the liver of PDGF-C Tg mice determined by microarray analysis. We normalized the mRNA level of SPHK1 in the liver of PDGF-C Tg mice treated with or without 0.06% peretinoin to that of non-Tg mice. The mRNA levels were calculated from the microarray data published in one of our previous reports16. This figure shows the relative mRNA level of SPHK1 under each condition to that of non-Tg mice. Error bars indicate the standard deviation from three mice. The statistical significance of the difference in the average between the two groups was analyzed by the Student’s t test. (b) mRNA levels in the liver in the Ath-HF diet mouse model determined by microarray analysis. We normalized the mRNA level of SPHK1 in the liver of Ath-HF diet mice with or without 0.03% peretinoin to that of LF mice. The mRNA levels were calculated from the microarray data published in one of our previous reports17. This figure shows the relative mRNA level of SPHK1 under each condition to that in LF mice. Error bars indicate the standard deviation from three mice. The statistical significance was analyzed as above. (c) mRNA level of the liver in the Ath-HF diet mouse model determined by qRT-PCR. We quantitated the mRNA levels of SPHK1 and β-actin in the liver of Ath-HF diet mice with or without 0.03% peretinoin to that of LF mice by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each mouse. The normalized mRNA levels of SPHK1 in the liver of Ath-HF diet mice with or without peretinoin were further normalized to that of LF mice. This figure shows the relative mRNA level of SPHK1 of each condition to that of LF mice. Error bars indicate the standard deviation from at least 10 mice. Statistical significance was analyzed as above. *p < 0.05, ***p < 0.005.