Sp1 and Sp3 regulate basal transcription of the survivin gene

Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. In this study, we cloned and identified the proximal 269 bp promoter of survivin gene, which exhibited strong promoter activity in HeLa cells. The TATA-less, GC-rich promoter contains 7 putative binding sites for Sp1, two of which (one at position -148 to -153, the other at position -127 to -140) are essential in regulating basal survivin promoter activity. Not only Sp1 but also Sp3 can activate the survivin promoter, which were proven by EMSA, blocking Sp1 or Sp3 using RNAi or mithramycin treatment of HeLa cells, and overexpression of Sp1 or Sp3. Our results collectively suggest that Sp1 cooperates with Sp3 to regulate survivin promoter activity.     

Sp1 and Sp3 transcription factors upregulate the proximal promoter of the human prostate-specific antigen gene in prostate cancer cells

The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.     

Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway

Background

H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.

Results

Our results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.

Conclusions

We made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.     

Sp1和Sp3介导的转录调控

基本转录因子Sp1和Sp3对转录调控区GC盒有很强的亲和力,参与几乎所有细胞功能,包括细胞增殖、凋亡、分化和新生物的转化.但在同一细胞中Sp1和Sp3对不同基因的作用并不相同,二者对基因特异性的转录调控是Sp1和Sp3研究领域的重要问题.近年来发现,Sp1和Sp3自身表达水平、结合的靶序列、磷酸化、糖基化等翻译后修饰,其他蛋白质的结合以及染色质结构与修饰等方面均可影响Sp1和Sp3的转录活性.本文从Sp1和Sp3蛋白参与转录调节的机制以及影响其基因特异性转录活性的诸方面因素这两大侧面,介绍了近年来的最新进展.     

Dual effects of histone deacetylase inhibition by trichostatin A on endothelial nitric oxide synthase expression in endothelial cells

Inhibition of histone deacetylases by trichostatin A (TSA) has pleiotropic effects on gene expression. We demonstrated that at low dose (0.1 microg) TSA increased the eNOS mRNA levels, which was followed by a time- and dose-dependent down-regulation. Cycloheximide, a protein synthesis inhibitor, completely abolished TSA-induced decrease in eNOS expression, indicating that new protein synthesis is required for the inhibiting effect. Mevastatin--an inhibitor HMG-CoA reductase and geranylgeranylation reaction dose-dependently antagonized TSA-induced reduction. This mevastatin-mediated antagonism was completely abolished by geranylgeranylpyrophosphate, suggesting that geranylgeranyl modification is needed to activate the eNOS mRNA destabilizing factor--a mechanism responsible for statin-mediated eNOS upregulation.