Abstract
Aim. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and post-translational. We concentrated our attention on the transcriptional level of regulation. Methods. Transient transfection of Me45 melanoma cells with constructs containing the luciferase gene under the control of complete and truncated GSTP1 promoter was utilized to identify a role of different promoter regions in regulation of the gene transcription in Me45 cells. To identify the transcription factors, interacting with the GSTP1 promoter sites, the competitive EMSA and super shift assay were applied. Results. GSTP1 transcription in Me45 cells is positively regulated by binding NF-κB to the cognate site and ERβin complex with unknown protein to the ARE site; the complex of ERβ with c-Fos negatively regulates the gene expression via CRE site. The interaction of c-Fos/ERβ with GSTP1 CRE site and indirect interaction of ERβ with GSTP1 ARE were identified. Conclusions. The positive regulation of the human GSTP1 gene in Me45 melanoma cells is exerted via NF-κB and ARE sites and the negative one via CRE site of the promoter. ERis indirectly involved in the regulation of GSTP1 transcription. It is bound via c-Fos with CRE site and via unknown protein with ARE site.
Highlights
Glutathione S-transferases comprise a multigene superfamily of enzymes that catalyze the conjugation of electrophilic toxic compounds with glutathione, playing a key role in phase II of detoxification [1]
We utilized truncated promoter constructions to compare the functional role of different cis-acting promoter elements and identified transcription factors binding the responsive elements by competitive electrophoretic mobility shift assay (EMSA) and supershift assay
We suggest that ERβ exerts the similar inhibitory effect at CRE site of GSTP1 promoter
Summary
Glutathione S-transferases comprise a multigene superfamily of enzymes that catalyze the conjugation of electrophilic toxic compounds with glutathione, playing a key role in phase II of detoxification [1]. Besides its typical role in detoxification it possesses other functions, including a ligandin function [3], modulation of signaling pathways [4], conjugation and transport of steroid hormones [5], and. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and post-translational [7]. Despite the vast literature devoted to GSTP1 enzyme the functional characteristics of responsive elements in gene promoter and tissue-specific peculiarities of their regulation are poorly understood. In present research we performed a functional analysis of GSTP1 promoter in human melanoma cells Me45. We utilized truncated promoter constructions to compare the functional role of different cis-acting promoter elements and identified transcription factors binding the responsive elements by competitive EMSA (electrophoretic mobility shift assay) and supershift assay
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