Yi Wen Liu

Functional cooperation of simian virus 40 promoter factor 1 and CCAAT/enhancer-binding protein β and δ in lipopolysaccharide-induced gene activation of IL-10 in mouse macrophages

Previous studies have revealed that LPS can activate transcription of the IL-10 gene promoter through an SV40 promoter factor 1 (Sp1) binding site in mouse macrophage RAW264.7. In this study, we determined that, in addition to Sp1, C/EBPβ and δ were also involved in LPS-induced gene expression of IL-10. By transient transfection with 5′-deletion mutants of the IL-10 promoter, we found that there were two LPS-responsive elements in the promoter of the mouse IL-10 gene. Analysis of these two regions by gel shift assay suggested that Sp1 and C/EBPβ and δ were bound to these two regions, respectively. By site-directed mutagenesis, we found that disruption at both the Sp1 and C/EBP binding sites almost completely blocked the LPS response. By gel shift assay and Western blotting, we found that the DNA binding complex and protein expression of C/EBPβ and δ were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBPβ or C/EBPδ, respectively, activated the promoter of the IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation experiments in intact cells indicated that LPS stimulated interaction between Sp1 and C/EBPβ and δ. These results suggested that the interaction between Sp1 and C/EBPβ and δ induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBPβ and δ proteins and the enhancement of transactivation activity of C/EBPβ and δ by LPS treatment, at least in part, explain the activation of IL-10 gene expression.

Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH(2)-terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea-induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines.

Overexpression of Ha-ras enhances the transcription of human arachidonate 12-lipoxygenase promoter in A431 cells

The effect of transient transfection with expression vectors of Ha-ras on the promoter activity of 12-lipoxygenase in human epidermoid carcinoma A431 cells was studied. Overexpression of Ha-ras increased the promoter activity in a dose- and time-dependent manner, which correlated closely with the cellular expression of Ras protein. Promoters of different gene lengths for human 12-lipoxygenase were used to prepare the luciferase fusion vectors. Following transfection by Ha-ras for 68 h, an approx. 40-fold increase in luciferase reporter activity was observed in plasmids with the 5-flanking region ranging from -951 to -224 bp upstream from translation starting site. There was no obvious stimulation in cells transfected with a vector-bearing promoter with a length of -100 bp. These results indicate that the promoter region ranging from -224 to -100 bp was important for the Ha-ras response. With the aid of additional 5-deletion and site-directed mutagenesis, three Sp1 binding sequences residing at -169 to -161 bp, -158 to -150 bp and -123 to -114 bp were found to be critical for the Ha-ras response of activating the transcription of human 12-lipoxygenase gene.

Induction of 12-lipoxygenase expression by phorbol 12-myristate 13-acetate in human epidermoid carcinoma A431 cells

Phorbol 12-myristate 13-acetate (PMA) increased the expression of 12-lipoxygenase activity and mRNA in a time-dependent manner in human epidermoid carcinoma A431 cells. The increase of 12-lipoxygenase was accompanied by the increase in protein level in microsomes prepared from A431 cells. The PMA-induced expression of 12-lipoxygenase activity and mRNA was inhibited by the treatment of cells with a protein kinase C inhibitor GF 109203X. Promoters of different DNA lengths for human 12-lipoxygenase gene were used to prepare the luciferase fusion vectors. These plasmid constructs were transiently transfected into A431 cells. Following treatment of PMA for 18 h, a 4- to 5-fold increase in luciferase reporter activity was observed in plasmids with the 5-flanking region length of -951 bp and that of -224 bp upstream from translation starting site. A time-dependent induction of luciferase activity by PMA was found to parallel the PMA-induced enzyme activity and mRNA expression. Transient transfection with a series of 5-deletion constructs showed that the 5-flanking region spanning from -224 to -100 bp from translation starting site played an important role for PMA response. Gel mobility shift assay and site-directed mutagenesis indicated that two Sp1 binding sequences residing at -158 to -150 bp and -123 to -114 bp were responsible for the PMA response in activating the transcription of human 12-lipoxygenase gene.

Role of transcriptional factors Sp1, c-Rel, and c-Jun in LPS-induced C/EBPδ gene expression of mouse macrophages

Abstract.Transcription factor C/EBPs are involved in the regulation of various cellular responses. Here, it was suggested that C/EBPδ gene was activated by lipopolysaccharide (LPS) through transcription factors Sp1, c-Rel, and c-Jun. Assay of the luciferase reporter vectors containing a 5′-deletion of the C/EBPδ gene promoter indicated that a LPS-responsive element was positioned between –345 and –35xa0bp of mouse C/EBPδ gene promoter. Transcription factors Sp1, c-Rel, and c-Jun bound to this region were identified using both inxa0vivo chromatin immunoprecipitation and inxa0vitro DNA-protein binding assays. LPS enhanced the proteins and DNA binding capacities of c-Rel and c-Jun, and the downstream Sp1 site was essential for LPS-induced C/EBPδ gene. Treatment of cells with ERK/JNK/p38 inhibitors or NF-κB inhibitor inhibited the LPS-induced C/EBPδ gene expression by inhibiting c-Jun, c-Rel, and p300 binding to DNA. Our findings provide a better understanding of LPS-induced C/EBPδ gene expression.

Epidermal growth factor enhances transcription of human arachidonate 12-lipoxygenase in A431 cells

Epidermal growth factor (EGF), determined by immunoprecipitation and Western blot analysis, increased both enzyme activity and protein level of 12-lipoxygenase in the solubilized microsomes of human epidermoid carcinoma A431 cells, respectively. The EGF-induced expression of 12-lipoxygenase mRNA was inhibited by transcription inhibitors such as actinomycin D and 5,6-dichlorobenzimidazole riboside. Promoters of different lengths for human 12-lipoxygenase gene were used to prepare the luciferase fusion vectors. These construct plasmids were transiently transfected into A431 cells, and the induction of luciferase expression by EGF was examined. A 4- to 6-fold increase in luciferase reporter activity stimulated by EGF for 18 h treatment was observed in plasmids with the 5-flanking region length of -951 bp and that of -224 bp upstream from translation starting site. The time-dependent induction of luciferase activity by EGF paralleled the EGF-induced enzyme activity and expression of 12-lipoxygenase protein. Taken together, the results of this study indicate that EGF enhanced the transcription of the human 12-lipoxygenase gene, resulting in an increase in the amount and activity of 12-lipoxygenase.

Induction of 12-lipoxygenase expression by transforming growth factor-α in human epidermoid carcinoma A431 cells

Transforming growth factor‐α (TGF‐α) increased the expression of 12‐lipoxygenase activity in a time‐dependent manner in human epidermoid carcinoma A431 cells. The increase of 12‐lipoxygenase activity was accompanied by an increase in 12‐lipoxygenase mRNA. The effect of TGF‐α on the promoter activation of 12‐lipoxygenase gene was analyzed by using the luciferase fusion vectors. A dose‐dependent effect of TGF‐α on the reporter activity was observed, which paralleled with its effect on enzyme activity. Transient transfection with a series of 5′‐deleted constructs showed that the 5′‐flanking region spanning from −224 to −100 bp from translation starting site played an important role for TGF‐α response. Site‐directed mutagenesis and gel mobility shift assay indicated that two Sp1 binding sequences residing at −158 to −150 bp and −123 to −114 bp were responsible for the TGF‐α in activation of human 12‐lipoxygenase gene transcription. Expression of Sp1, but not Sp3, stimulated the promoter activity of 12‐lipoxygenase in SL2 cells, indicating that the binding of Sp1 with Sp1 binding sequences played a significant role in the regulation of 12‐lipoxygenase gene.

Regulation of 12-lipoxygenase expression by epidermal growth factor in human epidermoid carcinoma A431 cells.

Arachidonate 12-lipoxygenase in the platelet was the first mammalian lipoxygenase discoverved1. It catalyzes the transformation of arachidonic acid into 12(S)-hydroperoxyeicosatetraenoic acid, which is subsequently converted to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) by a glutathione-dependent peroxidase2. We recently identified a human 12-lipoxygenase as the platelet-type enzyme in human epidermoid carcinoma A431 cells, and found that epidermal growth factor (EGF) increased the 12-lipoxygenase mRNA level by about 2-fold with a lag peroid of 10 h, which was parallel to the increase in enzyme activity3. This was the first evidence indicating the inducibility of a human 12-lipoxygenase gene expression by growth factor. In studying the signal transduction of EGF in the induction of 12-lipoxygenase expression, we recently reported the possible involvement of protein kinase C activation in the expression of EGF-induced 12-lipxoygenase4. The biological activities of 12(S)-HETE induces the expression of glycoprotein Ilb-IIIa on the cell membrane of Lewis lung carcinoma cells5 and may also play a significant role in the pathogenesis of some epidermal and epithelial inflammation6. Takahashi et al.7 identified 12-lipoxygenase in human epidermal cells as a platelet-type enzyme and Hussain et al.8 recently found an overexpression of the human platelet-type 12-lipoxygenase in germinal layer keratinocytes in psoriasis.