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The inorganic pyrophosphate transporter ANK preserves the differentiated phenotype of articular chondrocyte.

ABSTRACT: The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1beta challenge. Wnt proteins and the inorganic pyrophosphate (PP(i)) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PP(i) to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1beta challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PP(i) transport were strongly reduced by IL-1beta, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1beta effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1beta. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of beta-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PP(i) compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1beta and Ank knock-down conditions. Phenotype changes induced by IL-1beta were also supported by activation of the JNK pathway, but this latter was not sensitive to PP(i) supplementation. Altogether our data demonstrate that the transport of PP(i) by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner.

SUBMITTER: Cailotto F

PROVIDER: S-EPMC2856265 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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The inorganic pyrophosphate transporter ANK preserves the differentiated phenotype of articular chondrocyte.

Cailotto Frederic F Sebillaud Sylvie S Netter Patrick P Jouzeau Jean-Yves JY Bianchi Arnaud A

The Journal of biological chemistry 20100203 14

The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1beta challenge. Wnt proteins and the inorganic pyrophosphate (PP(i)) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PP(i) to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1beta challenge, with cessatio ...[more]

PMID: 20133941

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Project description:IntroductionBiomaterial-based tissue engineering has not successfully reproduced the structural architecture or functional mechanical properties of native articular cartilage. In scaffold-free tissue engineering systems, cells secrete and organize the entire extracellular matrix over time in response to environmental signals such as oxygen level. In this study, we investigated the effect of oxygen on the formation of neocartilage from human-derived chondrogenic cells.Materials and methodsArticular chondrocytes (ACs) and articular cartilage progenitor cells (ACPs) derived from healthy human adults were guided toward cell condensation by centrifugation onto plate inserts that were uncoated or coated with either agarose or fibronectin. Neocartilage discs were cultured at hyperoxic (20%) or physioxic (5%) oxygen levels, and biochemical, biomechanical, and molecular analyses were used to compare the cartilage produced by ACs versus ACPs.ResultsFibronectin-coated inserts proved optimal for growing cartilaginous discs from both cell types. In comparison with culture in hyperoxia, AC neocartilage cultured at physioxia exhibited a significant increase in chondrogenic gene expression, proteoglycan production, and mechanical properties with a concomitant decrease in collagen content. At both oxygen levels, ACP-derived neocartilage produced tissue with significantly enhanced mechanical properties and collagen content relative to AC-derived neocartilage. Both ACs and ACPs produced substantial collagen II and reduced levels of collagens I and X in physioxia relative to hyperoxia. Neocartilage from ACPs exhibited anisotropic organization characteristic of native cartilage with respect to collagen VI of the pericellular matrix when compared with AC-derived neocartilage; however, only ACs produced abundant surface-localized lubricin.Discussion and conclusionsGuiding human-derived cells toward condensation and subsequent culture in physioxia promoted the articular cartilage tissue phenotype for ACs and ACPs. Unlike ACs, ACPs are clonable and highly expandable while retaining chondrogenicity. The ability to generate large tissues utilizing a scaffold-free approach from a single autologous progenitor cell may represent a promising source of neocartilage destined for cartilage repair.

Project description:ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-beta1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-beta1. TGF-beta1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed to affect the inducing effect of TGF-beta1 on Ank mRNA level. These data show that TGF-beta1 increases ePPi levels, mainly by the induction of the Ank gene, which requires activation of Ras, Raf-1, ERK, and Ca2+-dependent PKC pathways in chondrocytes.

Project description:During the development of the appendicular skeleton, the cartilaginous templates undergo hypertrophic differentiation and remodels into bone, except for the cartilage most adjacent to joint cavities where hypertrophic differentiation and endochondral bone formation are prevented, and chondrocytes instead form articular cartilage. The mechanisms that prevent hypertrophic differentiation and endochondral bone formation of the articular cartilage have not been elucidated. To explore the role of the synovial microenvironment in chondrocyte differentiation, osteochondral allografts consisting of articular cartilage, epiphyseal bone, and growth plate cartilage from distal femoral epiphyses of inbred Lewis rats expressing enhanced green fluorescent protein from a ubiquitous promoter were transplanted either in inverted or original (control) orientation to matching sites in wildtype littermates, thereby allowing for tracing of transplanted cells and their progenies. We found that no hypertrophic differentiation occurred in the growth plate cartilage ectopically placed at the joint surface. Instead, the transplanted growth plate cartilage, with time, remodeled into articular cartilage. This finding suggests that the microenvironment at the articular surface inhibits hypertrophic differentiation and supports articular cartilage formation. To explore this hypothesis, rat chondrocyte pellets were cultured with and without synoviocyte-conditioned media. Consistent with the hypothesis, hypertrophic differentiation was inhibited and expression of the articular surface marker lubricin (Prg4) was dramatically induced when chondrocyte pellets were exposed to synovium- or synoviocyte-conditioned media, but not to chondrocyte- or osteoblast-conditioned media. Taken together, we present evidence for a novel mechanism by which synoviocytes, through the secretion of a factor or factors, act directly on chondrocytes to inhibit hypertrophic differentiation and endochondral bone formation and promote articular cartilage formation. This mechanism may have important implications for articular cartilage development, maintenance, and regeneration.

Project description:Excess PPi triggers developmental defects in a cell-autonomous manner. The level of inorganic pyrophosphate (PPi) must be tightly regulated in all kingdoms for the proper execution of cellular functions. In plants, the vacuolar proton pyrophosphatase (H+-PPase) has a pivotal role in PPi homeostasis. We previously demonstrated that the excess cytosolic PPi in the H+-PPase loss-of-function fugu5 mutant inhibits gluconeogenesis from seed storage lipids, arrests cell division in cotyledonary palisade tissue, and triggers a compensated cell enlargement (CCE). Moreover, PPi alters pavement cell (PC) shape, stomatal patterning, and functioning, supporting specific yet broad inhibitory effects of PPi on leaf morphogenesis. Whereas these developmental defects were totally rescued by the expression of the yeast soluble pyrophosphatase IPP1, sucrose supply alone canceled CCE in the palisade tissue but not the epidermal developmental defects. Hence, we postulated that the latter are likely triggered by excess PPi rather than a sucrose deficit. To formally test this hypothesis, we adopted a spatiotemporal approach by constructing and analyzing fugu5-1 PDF1 pro ::IPP1, fugu5-1 CLV1 pro ::IPP1, and fugu5-1 ICL pro ::IPP1, whereby PPi was removed specifically from the epidermis, palisade tissue cells, or during the 4 days following seed imbibition, respectively. It is important to note that whereas PC defects in fugu5-1 PDF1 pro ::IPP1 were completely recovered, those in fugu5-1 CLV1 pro ::IPP1 were not. In addition, phenotypic analyses of fugu5-1 ICL pro ::IPP1 lines demonstrated that the immediate removal of PPi after seed imbibition markedly improved overall plant growth, abolished CCE, but only partially restored the epidermal developmental defects. Next, the impact of spatial and temporal removal of PPi was investigated by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). Our analysis revealed that the metabolic profiles are differentially affected among all the above transgenic lines, and consistent with an axial role of central metabolism of gluconeogenesis in CCE. Taken together, this study provides a conceptual framework to unveil metabolic fluctuations within leaf tissues with high spatio-temporal resolution. Finally, our findings suggest that excess PPi exerts its inhibitory effect in planta in the early stages of seedling establishment in a tissue- and cell-autonomous manner.

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The inorganic pyrophosphate transporter ANK preserves the differentiated phenotype of articular chondrocyte.

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The inorganic pyrophosphate transporter ANK preserves the differentiated phenotype of articular chondrocyte.

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