Matrix Gla protein is involved in elastic fiber calcification in the dermis of pseudoxanthoma elasticum patients

Dealba Gheduzzi,Federica Boraldi,Giulia Annovi,Chiara Paolinelli Devincenzi,Leon J Schurgers,Cees Vermeer,Daniela Quaglino,Ivonne Pasquali Ronchetti

doi:10.1038/labinvest.3700667

Abstract

Mature MGP (Matrix γ-carboxyglutamic acid protein) is known to inhibit soft connective tissues calcification. We investigated its possible involvement in pseudoxanthoma elasticum (PXE), a genetic disorder whose clinical manifestations are due to mineralization of elastic fibers. PXE patients have lower serum concentration of total MGP compared to controls (P<0.001). Antibodies specific for the noncarboxylated (Glu-MGP) and for the γ-carboxylated (Gla-MGP) forms of MGP were assayed on ultrathin sections of dermis from controls and PXE patients. Normal elastic fibers in controls and patients were slightly positive for both forms of MGP, whereas Gla-MGP was more abundant within control's than within patient's elastic fibers (P<0.001). In patients' calcified elastic fibers, Glu-MGP intensively colocalized with mineral precipitates, whereas Gla-MGP precisely localized at the mineralization front. Data suggest that MGP is present within elastic fibers and is associated with calcification of dermal elastic fibers in PXE. To investigate whether local cells produce MGP, dermal fibroblasts were cultured in vitro and MGP was assayed at mRNA and protein levels. In spite of very similar MGP mRNA expression, cells from PXE patients produced 30% less of Gla-MGP compared to controls. Data were confirmed by immunocytochemistry on ultrathin sections. Normal fibroblasts in vitro were positive for both forms of MGP. PXE fibroblasts were positive for Glu-MGP and only barely positive for Gla-MGP (P<0.001). In conclusion, MGP is involved in elastic fiber calcification in PXE. The lower ratio of Gla-MGP over Glu-MGP in pathological fibroblasts compared to controls suggests these cells may play an important role in the ectopic calcification in PXE.

Highlights

The high concentration of calcium and phosphate in the extracellular space would lead to tissue calcification unless efficiently inhibited by a series of proteins and glycoproteins that have been found to operate in soft connective tissues to prevent calcium precipitation.[1]
Immunolocalization of matrix Gla protein (MGP) in the Human Dermis In order to investigate whether MGP is involved in elastic fiber calcification in pseudoxanthoma elasticum (PXE), ultrathin sections of skin biopsies from normal subjects and from patients affected by PXE were immunostained in parallel by using antibodies specific for the noncarboxylated (Glu-MGP) and for the g-carboxylated (Gla-MGP) forms of MGP.[32,33]
Relevant findings of this work are that human dermal fibroblasts express and produce MGP, and that MGP is present within normal elastic fibers in the human dermis

Summary

Introduction

The high concentration of calcium and phosphate in the extracellular space would lead to tissue calcification unless efficiently inhibited by a series of proteins and glycoproteins that have been found to operate in soft connective tissues to prevent calcium precipitation.[1]. A significant decrease in fetuin was measured in the serum of patients with pseudoxanthoma elasticum (PXE),[6] a genetic disorder characterized by mineralization of elastic fibers,[7,8,9] and it has been suggested that fetuin could have been removed from serum and ‘bound to the mineral deposits’.5,10. Another protein involved in inhibition of calcium precipitation in soft connective tissues is matrix Gla protein (MGP), a 10-kDa secreted protein containing 5-glutamic acid residues that must be g-carboxylated by a vitamin K-. Dependent g-carboxylase in order to acquire calcium-binding properties.[11,12] MGP-deficient mice manifest extensive calcification of the aorta and articular cartilage,[12] similar to what has been observed in Keutel syndrome, which is due to mutations in the human MGP gene.[13,14]

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Results

Conclusion