Regulation of Platelet PLC-β2 Expression by NF-κB: Studies in Human Platelet PLC-β2 Deficiency

• Published in: Blood 2006-11, Vol.108 (11), p.699-699
• Main Authors: Mao, Guangfen, Kunapuli, Satya P., Rao, A. Koneti
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V108.11.699.699

We have previously described a patient with platelet phospholipase C (PLC)-β2 deficiency characterized by impaired platelet responses to activation with multiple G-protein coupled receptor agonists. The PLC-β2 coding sequence was normal and platelet PLC-β2 mRNA levels were decreased in the patient (Blood , 2002, 99:905). Very little is currently known regarding the transcriptional regulation of PLC-β2. PCR-amplification of patient leukocyte DNA and sequencing of the PLC-β2 5′-upstream region revealed a heterozygous 13-bp deletion (−1645 to −1633 bp from ATG) that encompasses a consensus binding site (GGGAATTCCC) for nuclear factor-κB, NF-κB. This deletion was present in the propositus and her affected son, but not in control subjects. PCR amplification of region −1791 to −1606 bp of genomic DNA revealed one band in 5 control subjects (size ~186 bp) on agarose gel electrophoresis but 2 bands in the patient and her son, consistent with a heterozygous defect. Luciferase reporter gene studies were performed in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA, 30 nM) to induce megakaryocytic transformation. Genomic fragment (−1648/−23 nt) of PLC-β2 5′-upstream sequence and its truncated form without the 13 nt region (−1633/−23 nt) were inserted upstream of luciferase gene in a promoterless expression vector PGL3-basic (Promega) and transiently transfected into HEL cells. Truncation of the wild-type −1648/−23 fragment at 1631 bp resulted in a consistent decrease in promoter activity by ~ 25% (6 experiments, p