N-acetylcysteine treatment ameliorates the skeletal phenotype of a mouse model of diastrophic dysplasia.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in SLC26A2, a cell membrane sulfate-chloride antiporter. Sulfate uptake impairment results in low cytosolic sulfate, leading to cartilage proteoglycan (PG) undersulfation. In this work, we used the dtd mouse model to study the role of N-acetyl-l-cysteine (NAC), a well-known drug with antioxidant properties, as an intracellular sulfate source for macromolecular sulfation. Because of the important pre-natal phase of skeletal development and growth, we administered 30 g/l NAC in the drinking water to pregnant mice to explore a possible transplacental effect on the fetuses. When cartilage PG sulfation was evaluated by high-performance liquid chromatography disaccharide analysis in dtd newborn mice, a marked increase in PG sulfation was observed in newborns from NAC-treated pregnancies when compared with the placebo group. Morphometric studies of the femur, tibia and ilium after skeletal staining with alcian blue and alizarin red indicated a partial rescue of abnormal bone morphology in dtd newborns from treated females, compared with pups from untreated females. The beneficial effect of increased macromolecular sulfation was confirmed by chondrocyte proliferation studies in cryosections of the tibial epiphysis by proliferating cell nuclear antigen immunohistochemistry: the percentage of proliferating cells, significantly reduced in the placebo group, reached normal values in dtd newborns from NAC-treated females. In conclusion, NAC is a useful source of sulfate for macromolecular sulfation in vivo when extracellular sulfate supply is reduced, confirming the potential of therapeutic approaches with thiol compounds to improve skeletal deformity and short stature in human DTD and related disorders.

Altered signaling in the G1 phase deregulates chondrocyte growth in a mouse model with proteoglycan undersulfation.

In several skeletal dysplasias defects in extracellular matrix molecules affect not only the structural and mechanical properties of cartilage, but also the complex network of signaling pathways involved in cell proliferation and differentiation. Sulfated proteoglycans, besides playing an important structural role in cartilage, are crucial in modulating the transport, diffusion, and interactions of growth factors with their specific targets, taking part in the regulation of signaling pathways involved in skeletal development and growth. In this work, we investigated by real time PCR and Western blots of the microdissected growth plate and by immunohistochemistry the molecular basis of reduced chondrocyte proliferation in the growth plate of the dtd mouse, a chondrodysplastic model with defective chondroitin sulfate proteoglycan sulfation of articular and growth plate cartilage. We detected activation of the Wnt pathway, leading to an increase in the non-phosphorylated form of nuclear ß-catenin and subsequent up-regulation of cyclin D1 expression in the G1 phase of the cell cycle. ß-Catenin was further stabilized by up-regulation of Smad3 expression through TGF-ß pathway synergistic activation. We demonstrate that notwithstanding cyclin D1 expression increase, cell cycle progression is compromised in the G1 phase due to reduced phosphorylation of the pocket protein p130 leading to inhibition of transcription factors of the E2F family which are crucial for cell cycle progression and DNA replication. These data, together with altered Indian hedgehox signaling detected previously, explain at the molecular level the reduced chondrocyte proliferation rate of the dtd growth plate leading to reduced skeletal growth.

Diastrophic dysplasia sulfate transporter (SLC26A2) is expressed in the adrenal cortex and regulates aldosterone secretion.

Elucidation of the molecular mechanisms leading to autonomous aldosterone secretion is a prerequisite to define potential targets and biomarkers in the context of primary aldosteronism. After a genome-wide association study with subjects from the population-based Cooperative Health Research in the Region of Augsburg F4 survey, we observed a highly significant association (P=6.78×10(-11)) between the aldosterone to renin ratio and a locus at 5q32. Hypothesizing that this locus may contain genes of relevance for the pathogenesis of primary aldosteronism, we investigated solute carrier family 26 member 2 (SLC26A2), a protein with known transport activity for sulfate and other cations. Within murine tissues, adrenal glands showed the highest expression levels for SLC26A2, which was significantly downregulated on in vivo stimulation with angiotensin II and potassium. SLC26A2 expression was found to be significantly lower in aldosterone-producing adenomas in comparison with normal adrenal glands. In adrenocortical NCI-H295R cells, specific knockdown of SLC26A2 resulted in a highly significant increase in aldosterone secretion. Concomitantly, expression of steroidogenic enzymes, as well as upstream effectors including transcription factors such as NR4A1, CAMK1, and intracellular Ca(2+) content, was upregulated in knockdown cells. To substantiate further these findings in an SLC26A2 mutant mouse model, aldosterone output proved to be increased in a sex-specific manner. In summary, these findings point toward a possible effect of SLC26A2 in the regulation of aldosterone secretion potentially involved in the pathogenesis of primary aldosteronism.

Alteration of proteoglycan sulfation affects bone growth and remodeling.

Diastrophic dysplasia (DTD) is a chondrodysplasia caused by mutations in the SLC26A2 gene, leading to reduced intracellular sulfate pool in chondrocytes, osteoblasts and fibroblasts. Hence, proteoglycans are undersulfated in the cartilage and bone of DTD patients. To characterize the bone phenotype of this skeletal dysplasia we used the Slc26a2 knock-in mouse (dtd mouse), that was previously validated as an animal model of DTD in humans. X-rays, bone densitometry, static and dynamic histomorphometry, and in vitro studies revealed a primary bone defect in the dtd mouse model. We showed in vivo that this primary bone defect in dtd mice is due to decreased bone accrual associated with a decreased trabecular and periosteal appositional rate at the cell level in one month-old mice. Although the osteoclast number evaluated by histomorphometry was not different in dtd compared to wild-type mice, urine analysis of deoxypyridinoline cross-links and serum levels of type I collagen C-terminal telopeptides showed a higher resorption rate in dtd mice compared to wild-type littermates. Electron microscopy studies showed that collagen fibrils in bone were thinner and less organized in dtd compared to wild-type mice. These data suggest that the low bone mass observed in mutant mice could possibly be linked to the different bone matrix compositions/organizations in dtd mice triggering changes in osteoblast and osteoclast activities. Overall, these results suggest that proteoglycan undersulfation not only affects the properties of hyaline cartilage, but can also lead to unbalanced bone modeling and remodeling activities, demonstrating the importance of proteoglycan sulfation in bone homeostasis.

Matrix disruptions, growth, and degradation of cartilage with impaired sulfation.

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-µm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.

Further delineation of CANT1 phenotypic spectrum and demonstration of its role in proteoglycan synthesis.

Desbuquois dysplasia (DD) is characterized by antenatal and postnatal short stature, multiple dislocations, and advanced carpal ossification. Two forms have been distinguished on the basis of the presence (type 1) or the absence (type 2) of characteristic hand anomalies. We have identified mutations in calcium activated nucleotidase 1 gene (CANT1) in DD type 1. Recently, CANT1 mutations have been reported in the Kim variant of DD, characterized by short metacarpals and elongated phalanges. DD has overlapping features with spondyloepiphyseal dysplasia with congenital joint dislocations (SDCD) due to Carbohydrate (chondroitin 6) Sulfotransferase 3 (CHST3) mutations. We screened CANT1 and CHST3 in 38 DD cases (6 type 1 patients, 1 Kim variant, and 31 type 2 patients) and found CANT1 mutations in all DD type 1 cases, the Kim variant and in one atypical DD type 2 expanding the clinical spectrum of hand anomalies observed with CANT1 mutations. We also identified in one DD type 2 case CHST3 mutation supporting the phenotype overlap with SDCD. To further define function of CANT1, we studied proteoglycan synthesis in CANT1 mutated patient fibroblasts, and found significant reduced GAG synthesis in presence of ß-D-xyloside, suggesting that CANT1 plays a role in proteoglycan metabolism.

Defective proteoglycan sulfation of the growth plate zones causes reduced chondrocyte proliferation via an altered Indian hedgehog signalling.

Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis. A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation. These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth.

Recruitment of casein kinase 2 is involved in AbetaPP processing following cholinergic stimulation.

The amyloid-beta protein precursor (AbetaPP) is an integral membrane protein subjected to constitutive and regulated proteolytic processing. We have previously demonstrated that protein kinase C epsilon (PKCepsilon) plays a key role in the regulation of AbetaPP metabolism via cholinergic receptors. The purpose of the present work is to clarify whether other putative signaling systems are involved in the same pharmacological pathway. We focused particularly on casein kinase 2 (CK2), demonstrating a direct interaction between PKCepsilon and CK2 following cholinergic stimulation. Treatment of human neuroblastoma SH-SY5Y cells with a selective inhibitor of CK2 reduced the effect of carbachol on the release of sAbetaPPalpha. This treatment did not influence the activation and translocation of PKCepsilon suggesting that the latter is located upstream of CK2. On the basis of our results, we add another player to the complex cellular mechanisms regulating non-amyloidogenic processing of AbetaPP.