Interleukin-1 regulates the expression of sphingosine kinase 1 in glioblastoma cells

Abstract

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.

FIGURE 1.

Effect of IL-1 on proliferation, invasion, and expression of SphK1. RNA was isolated from untreated cultures of the indicated glioblastoma cell lines or primary human astrocytes, reverse transcribed, and the expression of IL-1β, SphK1, and SphK2 was analyzed by quantitative PCR (A). Data are expressed as a -fold induction after normalization to 18 S RNA. B, primary human astrocytes were stimulated with 10 ng/ml IL-1 or 100 nm PMA for 18 h, and the expression of SphK1 and SphK2 was analyzed as described above. C, U373 cells were transfected with either control or SphK1 siRNA. Subsequently, cells were treated with 10 ng/ml IL-1 as indicated, and the number of viable cells was monitored using a WST-1 assay as described under “Experimental Procedures.” The efficiency of SphK1 down-regulation was analyzed by quantitative PCR (inset). D and E, U373 cells were incubated with 5 μg/ml control or anti-IL-1 neutralizing antibodies for 24 h. The proliferation (D) and the in vitro invasion into Matrigel (E) were analyzed as described under “Experimental Procedures.”

FIGURE 2.

IL-1 up-regulates SphK1 expression and activity in glioblastoma cells. U373 cells were stimulated with 10 ng/ml IL-1 or 5 μm S1P for 18 h. A, SphK1 or SphK2 mRNA expression was analyzed using real-time PCR. B, A172 and U373 cells were transfected with either control or SphK1 siRNA. 48 h later, protein lysates were prepared and down-regulation of SphK1 protein expression was measured by Western blotting. C, lysates were prepared, and SphK1 protein level was determined by Western blotting. D, sphingosine kinase assays were performed in whole cell lysates, and bands corresponding to [³²P]S1P were separated and quantitated. E, U373 cells were treated with 10 ng/ml IL-1 for the indicated times or for 18 h with indicated concentration of IL-1. F, subsequently, RNA was isolated, reverse-transcribed, and SphK1 mRNA expression was analyzed using real-time PCR (TaqMan). Data shown in the A, E, and F are expressed as -fold induction after normalization to 18 S rRNA.

FIGURE 3.

IL-1 regulates expression of both isoforms SphK1a and SphK1c expressed in U373 cells. A, structure of the human sphk1 gene and putative spliced isoforms: SphK1a, SphK1b, and SphK1c. Specific primers, located on the boundaries of the exons, were designed, and the expression of SphK1 isoforms was analyzed by PCR. B, ethidium bromide-stained PCR products are shown with the table indicating the primers used, and the expected size of the products. C, U373 cells were stimulated with 10 ng/ml IL-1 for 18 h, and expression of both SphK1a and SphK1c isoforms was determined by real-time PCR using isoform-specific primer sets (numbers 1 and 3, respectively). Data are expressed as -fold induction after normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA.

FIGURE 4.

IL-1 activates multiple signaling pathways in glioblastoma cells. U373 cells were stimulated with 10 ng/ml IL-1 for the indicated time periods, and protein lysates were prepared. The activation of signaling molecules was analyzed by Western blotting using specific antibodies. Tubulin is included as a loading control.

FIGURE 5.

IL-1 regulates SphK1 expression via NF-κB-independent pathway. A, U373 cells were pretreated with 5 μm BAY11–7082, 1 μm CAY10470, or 5 μm parthenolide for 1 h, and subsequently stimulated with 10 ng/ml IL-1 for 18 h. SphK1 mRNA expression was measured using real-time PCR. Data are expressed as -fold induction after normalization to 18 S rRNA. B, U373 cells were transfected with either control siRNA or SMART-pool siRNA to p65, incubated for 48 h, and then stimulated with 10 ng/ml IL-1 for 18 h. RNA was isolated and SphK1 mRNA expression was analyzed as described above. ACT mRNA expression is shown as a positive control for p65 regulation.

FIGURE 6.

IL-1 activates SphK1 expression via JNK and c-Jun. A, U373 cells were pretreated with 1 μm SP600125, 10 μm SB202190, 1 μm U0126, or 10μm LY294002 for 1 h, and subsequently stimulated with 10 ng/ml IL-1 for 18 h. SphK1 mRNA expression was measured using real-time PCR. Data are expressed as -fold induction after normalization to 18S rRNA. B, cells were pretreated with either 10 μm JNK peptide inhibitor or 10 μm control peptide for 1 h, and then stimulated with 10 ng/ml IL-1 or 5 μm S1P for 18 h. SphK1 mRNA expression was analyzed as described above. C, cells were transfected with either control or c-jun SMART pool siRNA, incubated for 48 h, and then stimulated with 10 ng/ml IL-1 for 18 h. SphK1 mRNA expression was analyzed as described above. D, U373-TAM67 cells expressing inducible dominant-negative c-Jun(TAM-67) were cultured in the presence of tetracycline for 24 h and then stimulated with 10 ng/ml IL-1 or 100 nm PMA. SphK1 mRNA expression was analyzed as described above. E, U373 cells were pretreated with 10 μm SK1-I for 1 h, and then stimulated with 10 ng/ml IL-1 for 18 h. SphK1 mRNA expression was analyzed as described above.

FIGURE 7.

Identification of the critical AP-1 binding element within the first intron of the sphk1 gene. A, U373 cells were left untreated or preincubated with 10 ng/ml IL-1 for 18 h, and then treated with 5μg/ml actinomycin D. RNA was isolated at indicated times, reverse-transcribed, and SphK1 mRNA expression was analyzed using real-time PCR. Data are expressed as -fold induction after normalization to 18 S rRNA. B, primary human astrocytes were transiently transfected with the indicated reporter plasmids, and aβ-galactosiordase expression vector. One day after transfection, cells were stimulated with 10 ng/ml IL-1, 5μm S1P, or 100 nm PMA, cultured for an additional 24 h, and harvested. CAT activities were normalized to β-galactosidase activities to account for transfection efficiency. Results are expressed as -fold induction. C, point mutation were introduced into AP-1 and CRE binding sites located in the promoter of SphK1. Primary human astrocytes were transfected with the obtained plasmids and analyzed as described above. D, nuclear extracts were prepared from control and IL-1-treated U373 cells as indicated. DNA binding activity of AP-1 was than analyzed by EMSA using the ³²P-labeled oligonucleotide probe (left panel). Specific antibodies or normal rabbit serum (NRS) were added to the binding reaction, and binding was analyzed as described above in control nuclear extract (middle panel) or extract from 4-h IL-1-treated cells (right panel).