Abstract
Matrix gamma-carboxyglutamic acid protein (MGP) is a mineral-binding extracellular matrix protein synthesized by vascular smooth muscle cells (VSMCs) and chondrocytes that is thought to be a key regulator of tissue calcification. In this study, we identified four polymorphisms in the promoter region of the human MGP gene. Transfection studies showed that the G-7A and T-138C polymorphisms have an important impact on in vitro promoter activity when transiently transfected into VSMCs. We found that one of these polymorphisms (T-138C) is significantly correlated with serum MGP levels in human subjects. Promoter deletion analysis showed that this polymorphism lies in a region of the promoter critical for transcription in VSMCs. This region contains a potential activating protein-1 (AP-1) binding element located between -142 and -136. We have demonstrated that the T-138C polymorphism results in altered binding of an AP-1 complex to this region. The -138T allelic variant binds AP-1 complexes consisting primarily of c-Jun, JunB and its partners Fra-1 and Fra-2 in rat VSMC. Furthermore, the -138T variant form of the promoter was induced following phorbol 12-myristate 13-acetate treatment, while the -138C variant was refractive to phorbol 12-myristate 13-acetate treatment, confirming that AP-1 factors preferentially bind to the -138T variant. This study therefore suggests that a common polymorphism of the MGP promoter influences binding of the AP-1 complex, which may lead to altered transcription and serum levels. This could have important implications for diseases such as atherosclerosis and aortic valve stenosis, since it strongly suggests a genetic basis for regulation of tissue calcification.
Highlights
Extracellular calcification is a common and clinically significant component of a number of important human diseases including atherosclerosis and aortic valve stenosis [1, 2]
We identify a common promoter polymorphism of the matrix glutamyl carboxylase into ␥-carboxyglutamic acid (Gla) protein (MGP) gene that leads to altered binding of an activating protein-1 (AP-1) complex and is associated with variations in in vitro expression and serum levels
Identification of Four Common Polymorphisms in the Promoter Region of MGP—Single-stranded Conformational Polymorphism (SSCP) analysis of 15 overlapping PCR fragments upstream of the MGP gene in a cohort of 40 individuals revealed the presence of four deviant bands (Fig. 1a)
Summary
Subjects—DNA for the initial polymorphism identification was extracted from 40 subjects. The TϪ138C polymorphism was genotyped using a mismatch PCR fragment amplified with the forward primer (5Ј-AAGCATACGATGGCCAAAACTTCTGCA-3Ј) and the reverse primer (5Ј-GAACTAGCATTGGAACTTTTCCCAACC-3Ј), followed by digestion with the restriction enzyme BsrSI. Labeled DNA probe (100,000 cpm) was added to each reaction mixture containing 1 g of double-stranded poly(dI-dC) and 6 g of protein from crude nuclear extract. The same conditions were used except that the specific cold competitor oligonucleotides were added to the reaction mixture 10 min before the addition of the labeled probe. The membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit Ig antibodies (NA934; Amersham Pharmacia Biotech) for 1 h at room temperature and washed four times in PBS with 0.05% Tween 20 for 15 min each. Student’s unpaired t tests were used to compare relative luciferase activities in the transfection experiments
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