Abstract
Human erthroleukemia cells (HEL) differentiate towards megakaryocytic (MK) phenotype when stimulated with phorbol 12-myristate-13-acetate (PMA). We observed that the expression of Gq, a protein that plays a major role in platelet signal transduction, is increased in PMA-treated HEL cells. Western blotting revealed that Gq is upregulated in PMA-treated cells relative to untreated cells. Gq gene induction by PMA treatment was investigated with respect to transcriptional control. Serial 5′-truncations of the upstream region (upto 2727 bp from the ATG) of Gq gene were fused to a luciferase (Luc) reporter gene vector, PGL-3 Basic, and were transiently transfected into HEL cells in the absence and presence of PMA (10 nM). After 24 h, reporter gene activities were measured using Dual Luciferase Reporter Assay System (Promega). A reporter plasmid −1042 bp-Luc with a genomic region −1042/−1 showed a 12 fold activity in PMA treated cells and 4 fold activity in untreated cells. Its truncated plasmid with the genomic region −1036/−1 showed a decrease in luciferase activity by 50% in treated cells; and the activity became identical to that in untreated cells. Further truncation between −1036 and −1011 caused a complete loss of activity in both the cells. Thus, a PMA responsive element was localized to a region between −1042 and −1037 bp. Transcription factor data base search (TFSEARCH) predicted two consensus sites for early growth response factor EGR-1 at -1042/−1031 and −1026/−1015. Gel shift studies were performed with two oligos, −1042/−1012 and −1036/−1012, and nuclear extracts from PMA- treated and untreated cells. The studies with −1042/−1012 probe and extracts from treated cells showed that there was nuclear protein binding, which was abolished by competition with the consensus EGR-1 sequence. In extracts from untreated cells, the protein binding was observed but was not competed with consensus EGR-1 sequence. This suggests EGR-1 binding to the region −1042/−1012 in PMA-treated cells and role for this transcription factor in inducing Gq promoter activity. Moreover, studies on the region −1036/−1012 showed nuclear protein binding that was identical between extracts of untreated and treated cells, and it was not competed with consensus EGR-1 sequence. These findings suggest that, EGR-1 binding is localized to −1042/−1037, but not to −1036/−1012.
Conclusion: A PMA responsive sequence (−1042/−1037) was identified in the Gq promoter. Our studies suggest that EGR-1 binding to this sequence confers the PMA responsive activity. These studies provide further evidence that EGR-1 plays an important role in the upregulation of Gq expression during PMA induced megakaryocytic differentiation.
The Reporter System for GPCR Assay with the Fission YeastSchizosaccharomyces pombe
