Pseudoxanthoma elasticum: reduced gamma-glutamyl carboxylation of matrix gla protein in a mouse model (Abcc6-/-)

Abstract

Pseudoxanthoma elasticum (PXE), a heritable multi-system disorder manifesting with ectopic mineralization of soft connective tissues, is caused by mutations in the ABCC6/MRP6 gene/protein system, but the mechanisms how the ABCC6 mutations lead to aberrant mineralization are currently unknown. In this study, we utilized a transgenic mouse model, Abcc6-/-, to examine the mineralization processes. We focused on matrix gla protein (MGP) which has been shown to be critical, when activated by gamma-carboxylation of glutamyl residues, for prevention of unwanted mineralization. The concentration of MGP in the serum of Abcc6-/- mice was significantly reduced when compared to wild-type controls (p<0.004). More importantly, MGP isolated from the liver of Abcc6-/- mice was largely under-carboxylated and therefore possesses no activity. Finally, examination of the Abcc6-/- mice revealed association of total and under-carboxylated forms of MGP with ectopic mineralization while the gamma-carboxylated form was essentially absent. These results suggest that MGP in Abcc6-/- mice is largely in inactive form and is unable to prevent the unwanted mineralization of connective tissues in PXE.

Figures

Figure 1

Concentration of serum MGP of wild-type (WT) and Abcc6−/− knock-out (KO) mice, as determined by an ELISA using an antibody recognizing the N-terminal 3–15 amino acids of MGP. The standard curve with human recombinant MGP, provided with the ELISA kit, is shown in the inset. The antibody shows 29% cross reactivity with a corresponding mouse protein [22], and the values are corrected accordingly. The values are expressed as mean ± SE; n = 3–6 animals per group; *p<0.04 (Mann-Whitney); **p<0.03 (Student’s t-test).

Figure 2

Demonstration of MGP mRNA and assay of total (tMGP) and carboxylated (cMGP) forms of MGP in the liver in WT and KO mice. RT-PCR demonstrated the presence of MGP mRNA both in WT and KO liver RNA samples (panel a; NC, negative control). Protein extracts were examined by Western analysis using anti-tMGP and anti-cMGP antibodies. The equal protein loading was verified by anti-β-actin antibody (panel b). There was no difference in the relative amount of tMGP, while the amount of cMGP and consequently the c/tMGP ratio were reduced in the knock-out mice. The values are mean ± SE; n=3 (panel c).

Figure 3

Localization of total (tMGP), under-carboxylated (ucMGP) and carboxylated (cMGP) forms of the protein detected by using specific antibodies in the vibrissae of Abcc6−/−mice. Muzzle skin containing the vibrissae was prepared for histopathology and stained for mineral deposits with Alizarin Red. Distinct foci of mineralization were noted in the connective tissue capsule surrounding the vibrissae (open arrows, panels a–c). Adjacent sections were stained with specific antibodies and the antibody localization was visualized by Texas Red-conjugated secondary antibodies. Co-localization of tMGP and ucMGP with the mineral deposits was noted (white arrows; panels d and e) while staining for cMGP was entirely negative (*) (panel f).