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

BACKGROUND: 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 (TGFbeta) family (TGFbeta1-3 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. METHODS: The role of the pro-osteogenic pathways BMP2-SMADs-RUNX2, TGFbeta-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. RESULTS: We demonstrate the upregulation of the BMP2-SMADs-RUNX2 and TGFbeta-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 TGFbetas nor endoplasmic reticulum stress pathways seem to be perturbed in PXE. CONCLUSIONS: 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 PX

Novel deletions causing pseudoxanthoma elasticum underscore the genomic instability of the ABCC6 region

Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a heritable disease that affects elastic fibers. Thus far, >200 mutations have been characterized by various PCR-based techniques (primarily direct sequencing), identifying up to 90% of PXE-causing alleles. This study wanted to assess the importance of deletions and insertions in the ABCC6 genomic region, which is known to have a high recombinational potential. To detect ABCC6 deletions/insertions, which can be missed by direct sequencing, multiplex ligation-dependent probe amplification (MLPA) was applied in PXE patients with an incomplete genotype. MLPA was performed in 35 PXE patients with at least one unidentified mutant allele after exonic sequencing and exclusion of the recurrent exon 23-29 deletion. Six multi-exon deletions and four single-exon deletions were detected. Using MLPA in addition to sequencing, we expanded the ABCC6 mutation spectrum with 9 novel deletions and characterized 25% of unidentified disease alleles. Our results further illustrate the instability of the ABCC6 genomic region and stress the importance of screening for deletions in the molecular diagnosis of PXE. Journal of Human Genetics (2010) 55, 112-117; doi: 10.1038/jhg.2009.132; published online 15 January 201

Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence

Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein. © 2014 Xue et al

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice

Supraphysiological levels of the osteoblast‐enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase‐1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast‐specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6‐week‐old mice lacking osteoblast NPP1 expression (osteoblast‐specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast‐specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast‐specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin–sensitizing under‐carboxylated osteocalcin (195% increase; p < .05). However, following high‐fat‐diet challenge, osteoblast‐specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity

Mise en évidence de protéines révélant une réponse au stress adaptative divergente entre les espèces Dreissena polymorpha et Dreissena rostriformis bugensis

International audienceZebra mussels Dreissena polymorpha, are bivalve molluscs used in ecotoxicology, as a sentinel species with, among other things, a strong bioaccumulation capacity. The quagga mussel, Dreissena rostriformis bugensis, has more recently colonized Western Europe. Also invasive, it competes with zebra mussels for habitats and, in some areas, has completely replaced it. Its use as a sentinel species is envisaged but requires understanding the mechanisms involved during the adaptive stress response and comparing them to those of the zebra mussel, which are better characterized. With this in mind, an exposure of the two species to a classical contaminant was performed to compare their responses. The individuals were exposed to a concentration of 100 μg / L of cadmium for 7 days, and then the gill proteins were separated by two-dimensional electrophoresis and the variable abundance proteoforms were identified by mass spectrometry. The functional analysis reveals promising elements for the study of the adaptive response in both species. A difference of expression of actors of the energy metabolism pathways points a physiological difference, with a reallocation of energy as well as the appearance of truncated proteins.Les moules zébrées, Dreissena polymorpha sont des mollusques bivalves utilisés en écotoxicologie comme espèces sentinelles du fait, entre autre, de leur capacité de bioaccumulation. La moule quagga, Dreissena rostriformis bugensis, a colonisé plus récemment l'Europe occidentale. Egalement invasive, elle est en compétition avec la moule zébrée pour les habitats et, dans certaines zones, l'a complètement remplacée. Son utilisation en tant qu'espèce sentinelle est envisagée mais nécessite de comprendre les mécanismes mis en jeu au cours de la réponse adaptative au stress et de les comparer à ceux de la moule zébrée, mieux caractérisés. Dans cette optique, une exposition des deux espèces à un contaminant classique a été réalisée afin de comparer leurs réponses. Les individus ont été exposés à une concentration de 100μg/L de cadmium pendant 7 jours, puis les protéines des branchies ont été séparées par électrophorèse bidimensionnelle et les protéoformes d'abondance variable ont été identifiées par spectrométrie de masse. L'analyse fonctionnelle révèle des éléments prometteurs pour l'étude de la réponse adaptative chez les deux espèces. Une différence d'expression d'acteurs des voies du métabolisme énergétique pointe une différence physiologique, avec une réallocation de l'énergie ainsi que l'apparition de protéines tronquées

Multilocus variable number of tandem repeat analysis reveals multiple introductions in Spain of Xanthomonas arboricola pv. Pruni, the causal agent of bacterial spot disease of stone fruits and almond

Xanthomonas arboricola pv. pruni is the causal agent of the bacterial spot disease of stone fruits, almond and some ornamental Prunus species. In Spain it was first detected in 2002 and since then, several outbreaks have occurred in different regions affecting mainly Japanese plum, peach and almond, both in commercial orchards and nurseries. As the origin of the introduction(s) was unknown, we have assessed the genetic diversity of 239 X. arboricola pv. pruni strains collected from 11 Spanish provinces from 2002 to 2013 and 25 reference strains from international collections. We have developed an optimized multilocus variable number of tandem repeat analysis (MLVA) scheme targeting 18 microsatellites and five minisatellites. A high discriminatory power was achieved since almost 50% of the Spanish strains were distinguishable, confirming the usefulness of this genotyping technique at small spatio-temporal scales. Spanish strains grouped in 18 genetic clusters (conservatively delineated so that each cluster contained haplotype networks linked by up to quadruple-locus variations). Furthermore, pairwise comparisons among populations from different provinces showed a strong genetic differentiation. Our results suggest multiple introductions of this pathogen in Spain and redistribution through contaminated nursery propagative plant material