title: Pyrophosphates as a major inhibitor of matrix calcification in Pseudoxanthoma elasticum.
abstract: BACKGROUND
  Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by late onset and progressive calcification of elastic fibers in skin, eyes and the cardiovascular system, exemplifying a model for conditions characterized by soft tissue calcification.
  
  
  OBJECTIVE
  The aim of our study was to characterize cellular inorganic pyrophosphate (PPi) homeostasis in PXE.
  
  
  METHODS
  Gene expression of PPi metabolizing enzymes was determined by quantitative real-time PCR after incubation up to 21 days with or without addition of Na2HPO4. Extracellular and cytosolic PPi concentrations were measured by enzyme-linked bioluminescence assay. ALP and ENPP1 activity was determined spectrophotometrically. We further established a human cell culture model suitable for investigating PXE and related disorders without addition of artificial calcification triggers.
  
  
  RESULTS
  Independently of the experimental conditions, PXE fibroblasts revealed a higher degree of matrix calcification. We observed that matrix calcification was associated with altered gene expression of PPi metabolizing enzymes in PXE fibroblasts. In this context, PXE fibroblasts exhibited significantly higher expression of ALP and OPN and reduced mRNA expression and activity of ENPP1. Here, for the first time cytosolic and extracellular PPi levels were shown to be strongly reduced in PXE fibroblasts. We further showed that PPi concentration in bovine and human sera additives had a strong impact on matrix calcification. In a last experimental line, we demonstrated that addition of PPi analogs reduced matrix calcification of PXE fibroblasts most likely by reducing ALP and OPN mRNA expression, restoring ENPP1 activity and subsequently elevating PPi concentrations.
  
  
  CONCLUSIONS
  The results of our study along with recent findings point to the essential role of PPi as the central regulatory metabolites preventing matrix calcification in PXE. But what remains to be determined is the underlying molecular mechanism leading to depletion of PPi in PXE. We further suggest that supplementation of PPi analogs might counteract pathological calcification in PXE and related disorders.
authors.affiliation: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Mareike
authors.initials: M
authors.lastname: Dabisch-Ruthe
authors.affiliation: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Patricia
authors.initials: P
authors.lastname: Kuzaj
authors.affiliation: MVZ Labor Limbach Nürnberg, Nürnberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Götting
authors.affiliation: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Cornelius
authors.initials: C
authors.lastname: Knabbe
authors.affiliation: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany. Electronic address: dhendig@hdz-nrw.de.
authors.forename: Doris
authors.initials: D
authors.lastname: Hendig
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D002114:Calcinosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016022:Case-Control Studies; D002478:Cells, Cultured; D011756:Diphosphates / Q000378:metabolism*; D004547:Elastic Tissue / Q000378:metabolism* / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D011561:Pseudoxanthoma Elasticum / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
chemical_list: D000469:Alkaline Phosphatase; D011756:Diphosphates; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D053495:Osteopontin; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; C102029:ABCC6 protein, human; C506895:SPP1 protein, human; C508960:TGFB1 protein, human; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1
keywords: ABCC6; Bisphosphonate; Calcification; Ectonucleotide pyrophosphatase 1; Pseudoxanthoma elasticum; Pyrophosphate; Tissue nonspecific alkaline phosphate