Despite of numerous data about the specific expression and transcriptional regulatory mechanisms of human sialyltransferase genes involved in ganglioside biosynthesis, such as human GM3 synthase (hST3Gal V) and human GD3 synthase (hST8Sia I) genes [30–48], the molecular mechanism of regulation of hST8Sia V gene expression remains elusive. In the present study, we have elucidated for the first time the regulatory mechanism for the human glioblastoma cell-specific expression of the human GD1c/GT1a/GQ1b synthase (hST8Sia V) gene. RT-PCR result demonstrated that hST8Sia V gene is specifically expressed only in human malignant glioblastoma cell line U87MG. Moreover, immunofluorescent confocal microscopy also clarified that the expression levels of the ganglioside GQ1b specifically increase in human glioblastoma U87MG cells. Considering previous reports that the differential alterations to glycan structures on cell surface which are strictly controlled by the expression patterns of glycosyltransferase genes are a hallmark of neoplastic cell transformation [55] and individual glycosyltransferases responsible for changes in glycan structures of cancer cells could be cancer biomarkers [56], our results implicate that hST8Sia V, a subset of glycosyltransferases, could be human glioblastoma-specific biomarker.
Previous studies revealed that human sialyltrasnferase genes have multiple transcription start sites and their transcriptions occur in cell type-specific manner [31, 42, 43, 57]. In the present study, we firstly determined the TSS of hST8Sia V gene by 5’-RACE experiment to explore the promoter region responsible for the human glioblastoma-specific transcription of hST8Sia V gene. Based on the sequence analysis of 5’-RACE product, the promoter region with 2502 bp upstream of the TSS of the hST8Sia V gene was isolated and characterized by functional analysis using luciferase reporter assay. Similar with RT-PCR result, we demonstrated that the 2.5 kb promoter region of hST8Sia V gene shows strong promoter activity in only human glioblastoma U87MG cells, but not in other tissue-derived cancer cells. Furthermore, we also proved by the deletion mutant analysis that the region between − 1140 to -949 is functionally the core promoter region crucial for transcription of hST8Sia V gene in human glioblastoma U87MG cells.
Recently we have reported that transcription of human sialyltransferase genes are regulated by MAPK or AMPK signaling pathway [47, 50, 52]. In the current study, we demonstrated that transcription of hST8Sia V gene in human glioblastoma U87MG cells is modulated by JNK signaling pathway, as shown in promoter assay using specific inhibitors of protein kinases.
The c-Jun N-terminal kinase (JNK), part of the mitogen-activated protein kinase (MAPK) family, is a protein kinase that has crucial functions in various cellular processes, including cell growth, differentiation, tumorigenesis, apoptosis, inflammation, and stress responses [58, 59]. Previous studies showed that the levels of JNK activity was elevated in human glioblastoma tumor samples and in human glioblastoma cell lines including U87 MG [60] and the histological grade of glioblastoma strongly correlated with constitutive activation of JNK [61]. In addition, the JNK pathway is known to be activated in glioblastoma cell and promotes progression and invasion of glioblastoma [62].
To elucidate the regulatory mechanism for the human glioblastoma cell-specific expression of the hST8Sia V gene, it is important to find out the transcription factor as the downstream effector of the JNK pathway that is vital for the transcription of the hST8Sia V gene in human glioblastoma U87MG cells. Our present result clearly proved that the AP-1 binding site located at nucleotide − 1043/-1037 of hST8Sia V promoter region, which is completely matched with the known AP-1 consensus sequence (5’-TGACTCA-3’) [54], is necessary for transcription of hST8Sia V gene in human glioblastoma U87MG cells, as revealed by site-directed mutagenesis and promoter assay.
AP-1 transcription factor, which is mainly composed of c-Jun and c-Fos proteins, regulates various biological processes, including cell proliferation, cell differentiation, apoptosis, and tumorigenesis [63, 64]. It is well established that AP-1 is be the main target of JNK downstream and is activated by the phosphorylation of the N-terminal domain of c-Jun by JNK, which leads to promote transcription of downstream genes after translocation into the nucleus [58–64].
In conclusion, we have proved for the first time human glioblastoma cell-specific expression of the hST8Sia V gene and the transcriptional regulation of hST8Sia V by JNK/AP-1 signaling pathway in human glioblastoma cell.