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Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

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Affinity of the ATF to HMOX1 enhancer. Dual-luciferase reporter assay was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS-AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1- azp*-p65-flag (azp*-p65). For each of the vectors, four dose gradients (0, 50 ng, 100 ng, and 150 ng) were used. The firefly-luciferase reporters were (a) hHO4.9luc and (b) pGL3-control, respectively. The data represent the means ± S.D.
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fig5: Affinity of the ATF to HMOX1 enhancer. Dual-luciferase reporter assay was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS-AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1- azp*-p65-flag (azp*-p65). For each of the vectors, four dose gradients (0, 50 ng, 100 ng, and 150 ng) were used. The firefly-luciferase reporters were (a) hHO4.9luc and (b) pGL3-control, respectively. The data represent the means ± S.D.

Mentions: In order to assess the affinity of the ATF to HMOX1 enhancer, a dual-luciferase reporter assay was performed. HHO4.9luc and pGL3-control were used as the firefly-luciferase reporter. In the hHO4.9luc group, for the ATF vectors, a larger dose resulted in a higher luminescence intensity ratio (LIR). At 50 ng, 100 ng, and 150 ng vector dose, the LIR of pcDNA3.1-AZP-p65-flag was 1.88-, 2.71-, and 4.00-fold above control, while that of pcDNA3.1-NLS-AZP-p65-flag was 2.04-, 2.67-, and 3.88-fold above control (Figure 5(a)). Here, the “fold above control” represented ratios of the LIR of the ECV304 cells transfected with different candidate vectors to that of the ECV304 cells transfected with pcDNA3.1 (control). There were no significant differences between the two ATF vector groups, indicating that the ATF without NLS could also enter the nuclei (which was consistent with the immunofluorescence results) and upregulate the reporter gene expression. This may be attributed to the N′-terminal of the p65 functional domain, which acts as an NLS in NF-κB. For the three control vectors, LIR showed no significant difference among each dose gradient. In the pGL3-control group, LIR showed no significant difference among each dose gradient for the five candidate vectors (Figure 5(b)).


Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

Affinity of the ATF to HMOX1 enhancer. Dual-luciferase reporter assay was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS-AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1- azp*-p65-flag (azp*-p65). For each of the vectors, four dose gradients (0, 50 ng, 100 ng, and 150 ng) were used. The firefly-luciferase reporters were (a) hHO4.9luc and (b) pGL3-control, respectively. The data represent the means ± S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2913762&req=5

fig5: Affinity of the ATF to HMOX1 enhancer. Dual-luciferase reporter assay was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS-AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1- azp*-p65-flag (azp*-p65). For each of the vectors, four dose gradients (0, 50 ng, 100 ng, and 150 ng) were used. The firefly-luciferase reporters were (a) hHO4.9luc and (b) pGL3-control, respectively. The data represent the means ± S.D.
Mentions: In order to assess the affinity of the ATF to HMOX1 enhancer, a dual-luciferase reporter assay was performed. HHO4.9luc and pGL3-control were used as the firefly-luciferase reporter. In the hHO4.9luc group, for the ATF vectors, a larger dose resulted in a higher luminescence intensity ratio (LIR). At 50 ng, 100 ng, and 150 ng vector dose, the LIR of pcDNA3.1-AZP-p65-flag was 1.88-, 2.71-, and 4.00-fold above control, while that of pcDNA3.1-NLS-AZP-p65-flag was 2.04-, 2.67-, and 3.88-fold above control (Figure 5(a)). Here, the “fold above control” represented ratios of the LIR of the ECV304 cells transfected with different candidate vectors to that of the ECV304 cells transfected with pcDNA3.1 (control). There were no significant differences between the two ATF vector groups, indicating that the ATF without NLS could also enter the nuclei (which was consistent with the immunofluorescence results) and upregulate the reporter gene expression. This may be attributed to the N′-terminal of the p65 functional domain, which acts as an NLS in NF-κB. For the three control vectors, LIR showed no significant difference among each dose gradient. In the pGL3-control group, LIR showed no significant difference among each dose gradient for the five candidate vectors (Figure 5(b)).

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

Show MeSH
Related in: MedlinePlus