SphK1 mRNA Levels Research Articles

Combined quercetin and simvastatin attenuate hepatic fibrosis in rats by modulating SphK1/NLRP3 pathways

Liver fibrosis involves several signalling pathways working in concert regulating the deposition of extracellular matrix. In this study, we evaluated the effect of quercetin and simvastatin alone and their combination on the treatment of experimentally induced hepatic fibrosis in rats. To decipher the potential mechanisms involved, liver fibrosis was induced in rats by administration of 40 % carbon tetrachloride (CCl4) (1 μl/g rat, i.p., twice weekly) for 6 weeks. Quercetin (50 mg/kg, orally), simvastatin (40 mg/kg, orally) either individually or combined were administered for another 4 weeks. The three treatment groups ameliorated hepatic dysfunction and altered parameters of sphingolipid and pyroptosis pathways. Yet, the combined group showed a more pronounced effect. Treatments lowered serum levels of GOT, GPT, ALP and elevated albumin and total protein levels. Histopathological and electron microscope examination of liver tissue revealed diminished fibrosis and inflammation. Protein expression levels of α-SMA, IL-1β, PPAR-γ, TGF-β1, caspase-1 and caspase-3 expression in liver tissues were reduced. Additionally, hepatic mRNA levels of SphK1 and NLRP3 decreased after treatment. Furthermore, the three groups lowered MDA levels and elevated total antioxidant capacity, GSH and Nrf2 expression levels. Treatments downregulated sphingolipid pathway and NLRP3-mediated pyroptosis and stimulated an anti-apoptotic, anti-proliferative and antioxidant activity. This suggests that targeting the SphK1/NLRP3 pathway could be a prospective therapeutic strategy against liver fibrosis.

Detrimental role of sphingosine kinase 1 in kidney damage in DOCA-salt hypertensive model: evidence from knockout mice

BackgroundSphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingolipids and produced by sphingosine kinases (SphK1 and SphK2). SphK1/S1P pathway is implicated in the progression of chronic kidney disease. However, the role of SphK1/S1P pathway in renal injury in hypertension has not been reported. This study tested the hypothesis that SphK1/S1P pathway mediates the kidney damage in DOCA-salt hypertensive mice.MethodsMale wild type (WT) C57BL6 and SphK1 knockout (KO) mice were subjected to unilateral nephrectomy, subcutaneous implant containing 50 mg of deoxycorticosterone acetate (DOCA) and 1% NaCl drinking water for 7 weeks. At the end of experiments, blood pressure data, 24 h urine and kidney samples were collected. Renal mRNA levels of SphK1 were measured by real-time RT-PCR. Markers for fibrogenesis and immune cell infiltration in kidneys were detected using Western blot and immunohistochemistray analysis, respectively. The glomerular morphological changes were examined in kidney tissue slides stained with Periodic-Acid Schiff. Four groups were studied: wild type control (WT-C), WT-DOCA, KO-C and KO-DOCA.ResultsThe renal SphK1 mRNA expression was significantly upregulated in WT-DOCA mice, whereas this upregulation of renal SphK1 mRNA was blocked in KO-DOCA mice. There was no difference in DOCA-salt-induced hypertension between WT and KO mice. The urinary albumin was increased in both DOCA-salt groups. However, the albuminuria was significantly lower in KO-DOCA than in WT-DOCA group. There were increases in glomerulosclerosis indices in both DOCA-salt groups, whereas the increases were also significantly lower in KO-DOCA than in WT-DOCA mice. Renal protein levels of α-smooth muscle actin were upregulated in both DOCA-salt groups, but the increase was significant lower in KO-DOCA than in WT-DOCA group. The increased staining areas of collagen detected by Sirius Red-staining in kidney tissue sections were also attenuated in KO-DOCA compared with WT-DOCA mice. In contrast, the increased infiltration of CD43+ (a T cell marker) or CD68+ (a macrophage marker) cells in DOCA-salt kidneys showed no significant difference between WT-DOCA and KO-DOCA mice.ConclusionsSphK1/S1P signaling pathway mediates kidney damage in DOCA-salt hypertensive mice independent of blood pressure and immune modulation.

Knockout of Sphingosine Kinase 1 Attenuates Renal Fibrosis in Unilateral Ureteral Obstruction Model

Background: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in various diseases. S1P also plays significant roles in the differentiation of fibroblasts into myofibroblasts, being implicated in fibrotic diseases. S1P is produced by the phosphorylation of sphingosine catalyzed by sphingosine kinases (SphK1 and SphK2). It remains unclear if the activation of endogenous SphK1 contributes to fibrogenesis in kidneys. The present study determined the effect of SphK1 gene knockout (KO) on fibrotic markers in kidneys. Methods: The renal fibrosis was produced using the unilateral ureteral obstruction (UUO) model in wild-type (WT) and SphK1 gene KO mice. Renal mRNA levels of SphK1 and S1P receptors (S1PR) were measured by real-time RT-PCR. Fibrotic and immune cell markers in kidneys were measured by Western blot analysis and immunostaining, respectively. Renal morphological damage was examined by Periodic-Acid Schiff staining. Results: The mRNA levels of SphK1 and S1PRs were dramatically increased in renal tissues of WT-UUO mice, whereas the increase in renal SphK1 mRNA was blocked in KO-UUO mice. Interestingly, the increased levels of fibrotic markers, collagen and α-smooth muscle actin, in kidneys were significantly attenuated in KO-UUO versus WT-UUO mice. Meanwhile, kidney damage indices were remarkably attenuated in KO-UUO mice compared with WT-UUO mice. However, increased numbers of CD43+ and CD48+ cells, markers for T cell and macrophage, respectively, showed no significant difference between ­WT-UUO and KO-UUO kidneys. Conclusion: The activation of the SphK1-S1P pathway may contribute to tubulointerstitial fibrosis in UUO kidneys by affecting fibrotic signaling within renal cells independent of immune modulation.

Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo

Sphingosine-1-phospate is a potent bioactive lipid metabolite that regulates cancer progression. Because sphingosine kinase 1 and sphingosine kinase 2 (SPHK 1/2) are both essential for sphingosine-1-phospate production, they could be a therapeutic target in various cancers. Peretinoin, an acyclic retinoid, inhibits post-therapeutic recurrence of hepatocellular carcinoma via unclear mechanisms. In this study, we assessed effects of peretinoin on SPHK expression and liver cancer development in vitro and in vivo. We examined effects of peretinoin on expression, enzymatic and promoter activity of SPHK1 in a human hepatoma cell line, Huh-7. We also investigated effects of SPHK1 on hepatocarcinogenesis induced by diethylnitrosamine using SPHK1 knockout mice. Peretinoin treatment of Huh-7 cells reduced mRNA levels, protein expression and enzymatic activity of SPHK1. Peretinoin reduced SPHK1 promoter activity; this effect of peretinoin was blocked by overexpression of Sp1, a transcription factor. Deletion of all Sp1 binding sites within the SPHK1 promoter region abolished SPHK1 promoter activity, suggesting that peretinoin reduced mRNA levels of SPHK1 via Sp1. Additionally, diethylnitrosamine-induced hepatoma was fewer and less frequent in SPHK1 knockout compared to wild-type mice. Our data showed crucial roles of SPHK1 in hepatocarcinogenesis and suggests that peretinoin prevents hepatocarcinogenesis by suppressing mRNA levels of SPHK1.

Gfi1 Transcription Factor Is a Pro-Survival Molecule in Multiple Myeloma Cells

Rational: Multiple myeloma (MM) is the second most common hematologic malignancy and is incurable for most patients. We reported that MM cells induce expression of the transcriptional repressor, Growth independent factor 1 (Gfi1), in bone marrow stromal cells (BMSC) repressing Runx2 gene transcription, and results in prolonged suppression of osteoblast differentiation. Since Gfi1 is an anti-apoptotic factor in other hematologic malignancies, we hypothesized that Gfi1 has an important pro-survival role in MM cells by blocking apoptosis and can attenuate the pro-apoptotic effects of bortezomib.Methods: CD138+ cells isolated from MM patients, healthy donors and human MM cell lines (HMCLs) (H929, JJN3, and MM1.S) were tested for Gfi1 expression levels and the effects of Gfi1 knock down (KD) on MM cell survival by transduction with pLKO.1-puro lentivirus vectors encoding Gfi1 or non-mammalian shRNAs. HMCLs were treated with IL-6, S1P or TNFa or co-cultured with a human BMSC line (SAKA-T) to assess their effects on Gfi1 expression. The anti-apoptotic effects of Gfi1 overexpression (o/e) in MM1.S and H929 were tested by transduction with a pUC2 lentivirus encoding Gfi1 or with the empty vector followed by bortezomib (2 - 5nM) or vehicle (DMSO) treatment for 24 and 48 hours. MTT assays and cleaved caspase 3 protein levels were used to assess cell viability and cell death. Since acetylation of Gfi1 and p53 affects their activity and ability to bind each other, we also characterized HDAC inhibitors (HDACi)-induced changes on p53 enrichment at the Noxa, PUMA and p21 gene promoters by ChIP assays and the effects of acetylation of Gfi1 on its p53 binding capacity in MM cells.Results:We found that Gfi1 is highly expressed, at the mRNA and protein level, in CD138+ cells from MM patients and cell lines than CD138+ cells from normal donors. Gfi1 expression was further increased in MM cells by exogenous IL-6 (5ng/ml) and sphingosine-1-phosphate (S1P) (0.1 µM), but not by TNFα (10 ng/ml). KD of Gfi1 inhibited the growth and induced apoptosis of MM cells, as measured by increased mRNA levels of Bax, PUMA, Noxa, increased cleaved caspase 3 protein levels and decreased protein levels of Mcl-1 and c-Myc. Gfi1 (o/e) in MM cells conferred a survival advantage over their respective empty vector transduced controls as assessed by cell counts and MTT assays. Further, Gfi1 o/e protected MM cells from apoptosis induced by treatment with bortezomib as measured by MTT and cleaved caspase 3 protein levels. Since SphK1 activitycan also prevent apoptosis of MM cells, we next determined if Gfi1 regulated SphK1 in MM cells.CD138+ cells from MM patients had increased SphK1 mRNA levels compared with normal donors, and SphK1 mRNA levels and protein activity were further increased in MM cells by exogenous IL-6 and S1P. Co-culture of MM cells with BMSC also enhanced Gfi1, IL6 (3 fold) and SphK1 (2.5 fold) mRNA levels in MM cells. Importantly, Gfi1 KD in MM cells profoundly downregulated SphK1 mRNA levels and reduced expression of phospho-SphK1, suggesting that Gfi1 enhances MM growth in part via increasing expression and activity of SphK1. Gfi1’s inhibition of apoptosis resulted in part from binding of Gfi1 to p53, which blocked p53’s access to its pro-apoptotic target gene promoters. HDACi treatment resulted in acetylation of Gfi1 and inhibited Gfi1’s suppression of apoptosis by preventing Gfi1-p53 binding and subsequent enrichment of p53 at the Noxa, PUMA and p21 promoters in MM cells.Conclusion: Taken together, our results suggest that Gfi1 may act as a key regulator of MM growth and survival through its regulation of p53 and SphK1 activity, and that targeting Gfi1 may be a novel therapeutic strategy for MM patients. DisclosuresGiuliani:Celgene: Research Funding; Janssen: Research Funding. Roodman:Amgen: Consultancy.

Role of Gfi1 Transcription Factor in Myeloma Cells Growth and Survival

RationalMultiple myeloma (MM) is a plasma cell malignancy that is the second most common hematologic malignancy and is currently incurable for the overwhelming majority of patients. We reported that MM cells induce expression of the transcriptional repressor, Growth independent factor 1 (Gfi1), in bone marrow stromal cells (BMSC) that results in prolonged suppression of osteoblast differentiation by repressing transcription of the Runx2 gene. Since Gfi1 is an anti‐apoptotic factor in other hematologic malignancies, we hypothesized that Gfi1 has an important pro‐survival role in MM cells by blocking apoptosis and regulating MM cell growth and survival.MethodsCD138+ cells isolated from MM patients and healthy donors and human MM cell lines (H929, JJN3, U266, RPMI‐1640 and MM1.S) were tested for Gfi1 expression levels and the effects of Gfi1 knock down (KD) on MM cell survival by transduction with pLKO.1‐puro lentivirus vectors encoding Gfi1 or non‐mammalian shRNAs. Since acetylation of Gfi1 and p53 can affect their activity and ability to bind each other, we also characterized HDAC inhibitor (HDACi)‐induced changes in p53 enrichment at the Noxa, PUMA and p21 gene promoters by ChIP assays and the effects on acetylation of Gfi1 on its p53 binding capacity in MM cells treated with trichostatin A (5 μM) and nicotinamide (10 mM). Gfi1 was immune‐precipitated followed by western blotting with anti‐acetylated lysine or anti‐p53.ResultsWe found that Gfi1 is highly expressed, at the mRNA and protein level, in CD138+ cells from MM patients and MM cell lines compared with CD138+ cells from normal donors. Gfi1 expression was further increased in MM cells by exogenous IL‐6 (5ng/ml) and sphingosine‐1‐phosphate (S1P) (0.1 μM), but not by TNFα (10 ng/ml). KD of Gfi1 inhibited the growth and induced apoptosis of MM cells, as measured by increased mRNA levels of Bax, PUMA, Noxa, increased cleaved caspase 3 protein levels and decreased protein levels of Mcl‐1 and c‐Myc. Gfi1's inhibition of apoptosis resulted in part from binding of Gfi1 to p53 which blocked p53's access to its pro‐apoptotic targets promoters. HDACi treatment inhibited Gfi1's suppression of apoptosis by acetylation of Gfi1 at the K292 residue, which prevented Gfi1‐p53 binding and subsequent enrichment of p53 at Noxa, PUMA and p21 promoters in MM cells. Since SphK1 activity can also prevent apoptosis of MM cells, we next determine if Gfi1 regulated SphK1 in MM cells. CD138+ cells from MM patients showed increased levels of SphK1 mRNA compared with normal donors and SphK1 mRNA levels and protein activity were further increased in MM cells by exogenous IL‐6 and S1P. Further, direct co‐culture of MM cells with BMSC enhanced Gfi1, IL6 (3 fold) and SphK1 (2.5 fold) mRNA levels in MM cells. Importantly, Gfi1 KD in MM cells profoundly downregulated SphK1 mRNA levels and reduced expression of phospho‐SphK1, suggesting that Gfi1 enhances MM growth in part via increasing expression and activity of SphK1.ConclusionTaken together, our results suggest that Gfi1 may act as a key regulator of MM growth and survival through its regulation of p53 and SphK1 activity, and that targeting Gfi1 may be a novel therapeutic strategy for MM patients.Support or Funding InformationVA Merit Review funds to GDR

Sphingosine kinase 1 is upregulated with lysophosphatidic acid receptor 2 in human colorectal cancer.

To examine the expression of SphK1, an oncogenic kinase that produces sphingosine 1-phosphate (S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid (LPA) receptor overexpressed in various cancers, in human colorectal cancer. Real-time reverse-transcription polymerase chain reaction was used to measure the mRNA expression of SphK1, LPAR2, and the three major S1P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of SphK1 and LPAR2. Colorectal cancer tissue in 22 of 27 patients had higher levels of SphK1 mRNA than in normal tissue. In two-thirds of the samples, SphK1 mRNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 mRNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between SphK1 and LPAR2 expression [Pearson's correlation coefficient (r) = 0.784 and P < 0.01]. The mRNA levels of SphK1 and LPAR2 did not correlate with TNM stage. Our findings suggest that S1P and LPA may play important roles in the development of colorectal cancer via the upregulation of SphK1 and LPAR2, both of which could serve as new therapeutic targets in the treatment of colorectal cancer.

Abstract 4103: Sphingosine kinase-1 mediates LPA-induced gastric cancer cell proliferation

Abstract The lysophospholipids, lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are important mediators of a variety of biological processes important for maintenance and progression of cancer. Autotaxin and sphingosine kinase 1 (SphK1), the enzymes responsible for synthesis of LPA and S1P, respectively, are elevated in many cancer types. We previously reported that LPA markedly enhanced SphK1 and S1P3 receptor mRNA levels, transactivated EGFR, and caused migration in MKN-1 gastric cancer cells [Shida et al., Cancer Res. 68:6569, 2008]. We have now shown that LPA potently stimulates proliferation of MKN-1 cells and downregulation of SphK1 or treatment with the isozyme-specific SphK1 inhibitor, SK1-I, markedly reduced LPA-mediated proliferation. The signaling pathways responsible for LPA-induced SphK1 upregulation were also investigated. U0126, a MEK1/2 inhibitor, completely abrogated LPA-induced elevation of SphK1 mRNA, while inhibitors of PI3K, protein kinase C, p38, JNK or NF-κB did not have an effect. Additionally, siRNA mediated downregulation of ERK2, but not AKT 1 or Akt2, blocked LPA-mediated SphK1 mRNA increase. These results were further confirmed by adenoviral expression of dominant negative MEK1, which inhibited LPA-induced SphK1 upregulation, while constitutively active MEK1 increased basal SphK1 mRNA levels. Furthermore, adenoviral expression of dominant negative or constitutively active AKT1 also did not change SphK1 mRNA levels. Our data implicate ERK2 as an important mediator of LPA signaling and upregulation of SphK1 and additionally, point to SphK1 and S1P production as potential therapeutic targets in gastric cancer. This work was supported by NCI grant R01CA61774 to SS and NIH grant 5K12HD055881 and Susan G. Komen for the Cure Research Foundation Career Catalyst Research Grant KG090510 to KT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4103.

Interleukin-1 Regulates the Expression of Sphingosine Kinase 1 in Glioblastoma Cells

Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells.

Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow.

Atherosclerosis is a focal inflammatory disease and preferentially occurs in areas of low fluid shear stress and oscillatory flow, whereas the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow. Sphingosine kinase-1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1 phosphate (S1P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, we demonstrated that exposure of human aortic endothelial cells to oscillatory flow (shear stress, +/-5 dyn/cm(2) for 48 h) resulted in a marked increase in SphK1 mRNA levels compared with endothelial cells kept in static culture. In contrast, laminar flow (shear stress, 20 dyn/cm(2) for 48 h) decreased SphK1 mRNA levels. We further investigated the role of SphK1 in TNF-alpha-induced expression of inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and VCAM-1 by using small interfering RNA (siRNA) specifically for SphK1. Treatment of endothelial cells with SphK1 siRNA suppressed TNF-alpha-induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF-alpha-induced cell surface expression of VCAM-1, but not ICAM-1, protein. Exposure of endothelial cells to S1P led to an increase in MCP-1 protein secretion and MCP-1 mRNA levels and activation of NF-kappaB-mediated transcriptional activity. Treatment of endothelial cells with the p38 MAPK inhibitor SB-203580 suppressed S1P-induced MCP-1 protein secretion. These data suggest that SphK1 mediates TNF-alpha-induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature.