The Higher Isoform of G6PD Directly Binds to the Myocardin Promoter and Regulates its Expression in Vascular Smooth Muscle Cells

Smooth muscle cell (SMC) plasticity is regulated by several transcription factors, such as Serum response factor (Srf) and co-activator Myocardin (MYOCD) and krüppel-like factors 4 and 5 (KLF4 and KLF5). SRF-MYOCD invokes the expression of SMC-restricted genes and controls differentiated phenotype of the vascular smooth muscle cells (VSMCs). Recently we demonstrated that the novel high molecular weight (HMW) isoform of glucose-6-phosphate dehydrogenase (G6PD 545 ) suppresses the transcription of Myocardin ( Myocd ) gene, in VSMCs. However, the underlying mechanism through which G6PD 545 regulates the transcription of the Myocd gene is unclear. We hypothesized that G6PD 545 directly binds to the Myocd promoter and transcriptionally suppresses its gene expression. First, we subjected VSMCs to fractionation studies using ultracentrifugation and observed the presence of the enzymatically active G6PD 545 protein in the nuclear fraction. Next, we performed CHIP analysis in human coronary artery smooth muscle cells using anti-G6PD antibody and conducted PCR to amplify 3 fragments within the 700 bp of the Myocd promoter, upstream of the TSS. We found that G6PD 545 was bound to a DNA fragment between -166 to +57 bp of the Myocd promoter, upstream of TSS, based on the amplicon obtained in the PCR reaction. Furthermore, we synthesized 4 Biotin-labeled oligos encompassing the region -166 to +57 of the Myocd promoter and conducted EMSA assays to narrow down the G6PD 545 binding site on the Myocd promoter. We observed that Oligo#2 was robustly bound by G6PD 545 and harbored a SP1 like DNA recognition sequence –“GGGGAGGCG”. Deletion of this 9-mer from Oligo#2 resulted in the complete inhibition of G6PD 545 binding to the Oligo#2, suggesting that G6PD 545 recognized a specific/cognate DNA binding sequence on the Myocd promoter similar to that observed in other transcription factors. Furthermore, we cloned the 700 bp Myocd promoter in the pGL3-Basic Luciferase vector and performed the Dual Luciferase assays ± G6PD 545 -wild type (G6PD hi -wt) protein, that was cloned in the pDsRed-Express-N1 Vector. We found that co-transfection of the Myocd promoter with G6PD 545 -wt, in HEK293T17 cells, significantly decreased the Myocd promoter activity, suggesting that G6PD 545 -wt may be functioning as a repressor of the Myocd promoter activity. Moreover, enzymatically inactive G6PD 545 mutant was did not suppress Myocd promoter activity, suggesting that the DNA binding activity of G6PD 545 is independent of its enzymatic activity. Taken together, this data indicates that G6PD 545 may function as a novel transcription factor and regulate the expression of proteins involved in the differentiation of VSMCs. NHLBI R01HL166546 and R01HL132574 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.