Abstract 438: P122 Protein, a Positive Regulator for Phospholipase C-δ1, is Upregulated in Human Coronary Spasm by -228G-A Variant for the Promoter
We previously showed that the activity of phospholipase C (PLC)-δ1, a key enzyme for Ca2+ signaling in the vascular smooth muscle cells, was positively correlated with coronary vasomotility and was 3-fold enhanced in patients with coronary spastic angina (CSA), and that 10% of CSA had a structural mutation of 864G-A variant with the amino acid replacement of arginine 257 by histidine in PLC-δ1, which was associated with the increased enzyme activity. PLC-δ1 is negatively regulated by RhoA and positively by p122 protein. We therefore examined the possible roles of RhoA and p122 protein in enhanced PLC-δ1 activity. Protein expression of RhoA was similar between CSA (n=5) and control subjects (n=4), and no structural abnormalities which influence the activity of RhoA were found (n=20). In contrast, the protein expression of p122 was enhanced in CSA (n=11) by three-fold compared with control (n=9) (237±17 vs 85±13 units, p<0.0001). The gene expression of p122, measured by real-time quantitative RT-PCR, was enhanced in CSA (n=7) by 36±9% compared with control (n=7) (p<0.01). Sequence analysis of the genomic DNA coding the promoter region of p122 (−1599 through +1) revealed 8 conversions of the nucleotides at −1466 (C to T), −1319 (T to C), −833 (T to A), −572 (T to G), −276 (G to A), −228 (G to A), −144 (G to C), and −14 (T to C). Of 8 variants, the luciferase activities in the cells transfected with −1466C-T and −228G-A variant promoter construct were significantly increased by 1.34±0.27-fold (p<0.027) and 1.57±0.34-fold (p<0.001), respectively, compared with that of wild type promoter construct. We examined the incidence of the two conversions in 144 CSAs (91 men and 53 women) and 148 controls (62 men and 86 women) by using restriction fragment length polymorphism and mutant allele specific amplification, respectively. The incidence of the −1466C-T conversion was similar between CSA and control, while -228G-A conversion was more frequent in male CSA (8/91) than in male controls (1/62) (p<0.05). Both conversions were similar in female patients. Thus, enhanced p122 protein expression by gene polymorphisms at −228G-A may be involved in the pathogenesis of CSA by enhancing PLC-δ1 activity and intracellular Ca2+ signaling.