cGMP-independent nitric oxide signaling and regulation of the cell cycle
Xiaolin CuiJianhua ZhangPenglin MaDaniela E. MyersIlana G GoldbergKelly J. SittlerJennifer J. BarbPeter J. MunsonAna Del Pilar CintronJ. Philip McCoyShuibang WangRobert L. Danner
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Abstract Background Regulatory functions of nitric oxide (NO • ) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO • on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO • donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt 2 cAMP), and then harvested to extract total RNA for microarray analysis. Bt 2 cAMP was used to block signaling attributable to NO • -induced decreases in cAMP. Results NO • regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt 2 cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO • , Bt 2 cAMP was associated with ARE-containing transcripts. A comparison of NO • and Bt 2 cAMP effects showed that NO • regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO • annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO • , as was E2F1 binding to E2F promoter elements. NO • activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M. Conclusion NO • coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO • may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO • regulates the transcriptome.Keywords:
E2F
Second messenger system
E2F1
E2F
E2F1
Retinoblastoma protein
Cyclin E
Cyclin A
Mesangial cell
Cyclin D
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E2F1
E2F
Ectopic expression
Retinoblastoma
Retinoblastoma protein
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Tumors are serious threats to human health. The transcription factors are regarded as the potential targets for tumor treatment. As an important family of transcription factors, E2F family transcription factors (E2Fs) play vital roles in cell proliferation and regulation. However, the expression feature, gene functions, and molecular interactions of E2Fs in tumorigenesis are not clear. In this study, the transcriptome data, mutation data, and protein-protein interaction data of 10 high-incidence tumors in China from the TCGA database were integrated and analyzed to explore the expression, structure, function, mutation, and phylogenetic characteristics of E2Fs. The results showed that E2F1 and E2F7 were regularly upregulated in the tumor samples. Moreover, E2Fs participated in the regulation of the cell cycle, cell aging, and other signaling pathways. As an important regulator, E2F1 interacted with more proteins than other E2Fs. At the same time, the genetic mutation types of E2Fs varied in tumor type and patient sex, of which gene amplification accounts for the largest proportion. Phylogenetic analysis showed that E2Fs were conserved in 41 species, including fruit flies, nematodes, and humans. Meanwhile, E2Fs had a tendency for gene expansion during evolution. In conclusion, this study clarified the expression pattern, mutation characteristics, and evolutionary trend of E2Fs in high-incidence tumors in China, and suggested that E2F family transcription factors could be novel diagnostic markers for tumor diseases. Furthermore, this work can provide a theoretical basis for the development of anti-tumor-targeted drugs.肿瘤严重威胁人类健康，转录因子是肿瘤治疗的潜在靶点。作为重要的转录因子家族，E2F在细胞增殖与调控进程中发挥重要作用。然而，E2F家族转录因子在肿瘤发生进程中的表达规律、基因功能和分子互作等关键信息尚不清晰。基于此，本研究对TCGA数据库中我国10种高发肿瘤的转录组测序数据、突变数据和蛋白质互作数据进行整合分析，探究E2F家族转录因子的表达、结构、功能、突变和系统发生特征。结果显示，E2F家族转录因子中的E2F1和E2F7基因在多种肿瘤样本中规律性上调表达，参与调控细胞周期、细胞衰老等信号通路；其中，E2F1作为重要的调控因子与其他蛋白的相互作用最多。值得指出的是，E2F家族转录因子的基因突变类型在肿瘤类型和患者性别中均存在差异，基因扩增占比最大。系统发生分析显示，E2F家族转录因子的结构在包括果蝇、线虫和人类在内41个物种中保守，并且它们在物种演化过程中表现出基因扩张倾向。综上所述，本研究阐明了E2F家族转录因子在我国高发肿瘤中的表达规律、突变特征和演化规律，提示E2F家族转录因子是相关肿瘤疾病的新型分子诊断标志物，为抗肿瘤靶向药物研发提供理论依据。.
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Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how E1A affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that E1A is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability.
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E2F1
Retinoblastoma protein
Transcription
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E2F
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E2F1
Retinoblastoma protein
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Abstract The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression by activating growth-related genes and pro-apoptotic tumor suppressor genes, respectively. It is generally accepted that E2F binds to target sequences with its heterodimeric partner DP. Here we show that, while knockdown of DP1 expression inhibited ectopic E2F1- or adenovirus E1a-induced expression of the CDC6 gene and cell proliferation, knockdown of DP1 and DP2 expression did not affect ectopic E2F1- or E1a-induced expression of the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p53, activation of p53 or apoptosis. These observations suggest that growth related and pro-apoptotic E2F targets are regulated by distinct molecular mechanisms and contradict the threshold model, which postulates that E2F activation of pro-apoptotic genes requires a higher total activity of activator E2Fs, above that necessary for E2F-dependent activation of growth-related genes.
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Background/Purpose. The retinoblastoma tumor suppressor protein (pRB) plays a central role in proliferative control and is a frequent target for inactivation in cancer. The G1-Sphase transition of the cell cycle is regulated by pRB, which is capable of interacting with E2F family members and inhibiting the transcription of genesrequired to progress into S-phase. E2F1 is unique from other E2F family members as it can induce both apoptosis and proliferation. To control these contrasting functions of E2F1, a second E2F1 binding site exists in the C-terminus of pRB that can control apoptosis separately from proliferation. This anti-apoptotic function of pRBcan in some circumstances promote tumorigenesis, which leads to the question; is pRB a tumor suppressor or an oncogene? Methods. To investigate this, a gene-targeted mouse model is being engineered to selectively disrupt the ability of pRB to control proliferation through the general E2F binding site while still retaining the ability tocontrol apoptosis through the specific E2F1 site. Results. A series of novel mutants were engineered to selectively disrupt the binding of E2Fs at the general site, and prevent pRB from controlling proliferation. The mutants retain the ability to bind E2F1 and control apoptosis through thespecific binding site, which is not disrupted. Conclusions. By separating the ability of pRB to control proliferation and apoptosis under in vivo conditions, a better understanding into the significance of these two functions in development and tumorigenesis can be gained.
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E2F
Retinoblastoma protein
Retinoblastoma
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Previous work has demonstrated a role for E2F transcription factor activity in the regulation of cell growth during the G0/G1-S phase transition. Indeed, overexpression of E2F proteins, including the E2F1 and E2F2 products, induces DNA synthesis in quiescent fibroblasts. Other experiments have shown that E2F1 expression also induces apoptosis, dependent on p53. Although this could represent a response to aberrant cell cycle progression, we show that only E2F1 induces apoptosis and that this coincides with an ability of E2F1 to induce accumulation of p53 protein. We also find that coexpression of Mdm2, which is known to regulate p53 activity, blocks the E2F1-mediated induction of apoptosis and also blocks the E2F1-mediated accumulation of p53. We propose that E2F1 acts as a specific signal for the induction of apoptosis by affecting the accumulation of p53, which under normal proliferative conditions may be controlled by Mdm2.
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