Transcriptional factor E2F1 inhibits expression of the cellular repressor of E1A-stimulated genes and regulates VSMCs differentiation in vitro

The human cellular repressor of EIA-stimulated genes (hCREG), originally cloned from the cDNA library of HeLa cell line, was found to rapidly induce the differentiation of diverse type cells such as pluripotent mouse EC cells, monocytes U937 and myeloid cells EML. It was identified in previous study that not only could overexpression of hCREG regulate and hold the differentiation vascular smooth muscle cells (VSMCs) in vitro, but inhibit the neointima formation in rat carotid artery after balloon injury. These properties suggest that hCREG might have played an important role in antagonizing restenosis of vessel by inhibiting phenotypic modulation of VSMCs. In order to further elucidate the biological functions of hCREG in VSMCs, the upstream molecule mechanisms in regulating its expression were analyzed. At first, bioinformatics was used to predict the hCREG promoter and the binding sites of transcriptional factors. According to bioinformatic results, the pEGFP-hCREG-P945 reporter gene vector was constructed successfully which contained upstream 945 bp of hCREG where two binding sites of E2FI were determined. Subsequently, the hCREG promoter activity was identified directly by detecting the expression of reporter protein-GFP with fluorescence microscope and Western blot analysis when the vector was transfected into human VSMCs-HITASY. Secondly, both the expression of hCREG and smooth muscle alpha-actin(SM alpha-actin) was detected to increase in differentiation HITASY cells cultured for 72 h with serum deprivation, in which the expression of E2171 was reduced significantly. Inversely, the increase of E2FI expression was detected in dedifferentiation cells cultured with 10% FBS medium accompanied with the reduction of hCREG and SM alpha-actin. It is suggested that E2171 maybe inhibit the expression of hCREG by binding to the hCREG promoter. Furthermore, the E21`1 oligodeoxynucleotide (ODN) and mismatch E2FI ODN were designed and used to block the binding of E2F1 to hCREG promoter by transcriptional factor "decoy strategy". Western blot analysis showed that expression of hCR-EG, GFP and SM alpha-actin was increased obviously when the E2FI ODN was transfected in dedifferentiation HITASY cells. The result identified that transcription factor E2F1 inhibited the expression of hCREG and promote dedifterentiation of VSMCs by binding to the sites of hCREG promoter. It can be concluded that E2FI, as a transcriptional regulation factor of hCREG, can repress the expression of hCREG and involve in a pivotal role in the process of VSMCs phenotypic modulation.