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01.12.2014 | Research | Ausgabe 1/2014 Open Access

Orphanet Journal of Rare Diseases 1/2014

Perturbation of specific pro-mineralizing signalling pathways in human and murine pseudoxanthoma elasticum

Orphanet Journal of Rare Diseases > Ausgabe 1/2014
Mohammad J Hosen, Paul J Coucke, Olivier Le Saux, Anne De Paepe, Olivier M Vanakker
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1750-1172-9-66) contains supplementary material, which is available to authorized users.

Competing interests

The authors’ declare that they have no competing interests.

Authors’ contribution

MJH and OMV contributed to the experimental design, data collection and interpretation. PJC contributed to the data collection and interpretation. OLS provided the mouse specimens and contributed to the data interpretation. ADP was involved in study design and data interpretation. All authors were involved in writing the paper and had final approval of the submitted and published versions. MJH, ADP and OMV accept responsibility for the integrity of the data analysis.



Pseudoxanthoma elasticum (PXE) is characterized by skin (papular lesions), ocular (subretinal neovascularisation) and cardiovascular manifestations (peripheral artery disease), due to mineralization and fragmentation of elastic fibres in the extracellular matrix (ECM). Caused by mutations in the ABCC6 gene, the mechanisms underlying this disease remain unknown. The knowledge on the molecular background of soft tissue mineralization largely comes from insights in vascular calcification, with involvement of the osteoinductive Transforming Growth Factor beta (TGFβ) family (TGFβ1-3 and Bone Morphogenetic Proteins [BMP]), together with ectonucleotides (ENPP1), Wnt signalling and a variety of local and systemic calcification inhibitors. In this study, we have investigated the relevance of the signalling pathways described in vascular soft tissue mineralization in the PXE knock-out mouse model and in PXE patients.


The role of the pro-osteogenic pathways BMP2-SMADs-RUNX2, TGFβ-SMAD2/3 and Wnt-MSX2, apoptosis and ER stress was evaluated using immunohistochemistry, mRNA expression profiling and immune-co-staining in dermal tissues and fibroblast cultures of PXE patients and the eyes and whiskers of the PXE knock-out mouse. Apoptosis was further evaluated by TUNEL staining and siRNA mediated gene knockdown. ALPL activity in PXE fibroblasts was studied using ALPL stains.


We demonstrate the upregulation of the BMP2-SMADs-RUNX2 and TGFβ-2-SMAD2/3 pathway, co-localizing with the mineralization sites, and the involvement of MSX2-canonical Wnt signalling. Further, we show that apoptosis is also involved in PXE with activation of Caspases and BCL-2. In contrast to vascular calcification, neither the other BMPs and TGFβs nor endoplasmic reticulum stress pathways seem to be perturbed in PXE.


Our study shows that we cannot simply extrapolate knowledge on cell signalling in vascular soft tissue calcification to a multisystem ectopic mineralisation disease as PXE. Contrary, we demonstrate a specific set of perturbed signalling pathways in PXE patients and the knock-out mouse model. Based on our findings and previously reported data, we propose a preliminary cell model of ECM calcification in PXE.
Additional file 1:List of primers used in qPCR experiments.(TIFF 16 KB)
Additional file 2:Rate of apoptosis (%) in PXE fibroblasts compared to controls. PXE fibroblasts showed 3× more apoptosis compared to controls. (n = 8 and 5 for patients and controls respectively). (TIFF 432 KB)
Additional file 3:Magnification of TUNEL staining in native PXE fibroblasts (×40). Positive labelling in the cytoplasm of the cells is arrowed. Scale bar = 50 μm. (TIFF 38 KB)
Additional file 4:Relative expression of RUNX2 in PXE fibroblasts transfected with anti-RUNX2 siRNA after 24, 48 and 72 hours. siRNA silencing causes a downregulation of RUNX2 expression of 65% at 24 hours, 54% at 48 hours and 28% at 72 hours. (n = 6 each). (TIFF 32 KB)
Additional file 5:Quantification of the effect of RUNX2 knockdown on apoptosis in PXE fibroblasts (n = 2 cultures). Results are shown after 24, 48 and 72 hours respectively and demonstrate a 13 to 20% reduction of apoptosis, though results differ between different cell lines. After 72 hours, no reducing effect of siRNA silencing of RUNX2 on apoptosis can be seen anymore. (DOC 44 KB)
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