Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia

Kanaujiya, Jitendra Kumar; Bastow, Edward; Luxmi, Raj; Zou, Hao; Zattas, Dimitrios; Hochstrasser, Mark; Reichenberger, Ernst; Chen, I‐Ping

doi:10.1038/s41598-018-34157-5

Cited by 12 publications

(14 citation statements)

References 44 publications

(47 reference statements)

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“…P5L and G389R both localised more slowly to the cell membrane, E490del showed some cell membrane localisation but was mostly cytoplasmic and S375del did not at any time. A recent paper on novel mutant ANKH-F377del, also shows evidence of the mutated protein to be dispersed in the cytoplasm 18 . Although we see similar wt.ANKH localisation as reported by Kanaujiya, J. et al 18 and many other non-bone cell lines, we cannot discount the effects that this may be specific to bone.…”

Section: Discussionmentioning

confidence: 95%

“…A recent paper on novel mutant ANKH-F377del, also shows evidence of the mutated protein to be dispersed in the cytoplasm 18 . Although we see similar wt.ANKH localisation as reported by Kanaujiya, J. et al 18 and many other non-bone cell lines, we cannot discount the effects that this may be specific to bone. These very different localisation dynamics, even between mutants causing similar clinical features, highlights the complex and diverse function of ANKH in the pathogenesis of CPPDD and CMD.…”

Section: Discussionmentioning

confidence: 95%

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Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia

Vijen

Hawes

Runions

et al. 2020

Sci Rep

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ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1. Under normal conditions cartilage and bone calcification involves hydroxyapatite (HA) deposition requiring the non-enzymatic reaction of extracellular calcium and inorganic phosphate (ePi). In rheumatologic conditions like calcium pyrophosphate dihydrate deposition disease (CPPDD) calcium pyrophosphate dihydrate (CPPD) can deposit contributed by Inorganic pyrophosphate (PPi), a regulator of calcification, inhibiting HA deposition. At least four proteins influence PPi homeostasis in the extracellular matrix (ECM): these include the transmembrane pyrophosphate transporter ANKH (the human homologue to the mouse progressive ankylosis protein), ENPP1 (ectonucleotide pyrophosphatase 1), TNAP (tissue nonspecific alkaline phosphatase, one of the four forms of alkaline phosphatase ALPL) and PIT-1 (sodium-dependent phosphate transport 1, also named as SLC20A1); ENPP1 readily hydrolyses extracellular adenosine triphosphate (eATP) into extracellular PPi (ePPi) and extracellular AMP (eAMP), whilst ANKH exports intracellular PPi (iPPi) into the ECM, TNAP hydrolyses ePPi into extracellular Pi(ePi), and PIT-1 imports ePi into the cell via a sodium exchange channel to be used in metabolic pathways 1,2. ANKH is mainly expressed at the cell membrane and mutations in ANKH cause two distinct conditions-CPPDD [MIM118600] and craniometaphyseal dysplasia (CMD [MIM123000]) 3,4. CPPDD typically presents

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 95%

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia

Vijen

Hawes

Runions

et al. 2020

Sci Rep

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“…( 9 ) We showed that mutant ANK/ANKH protein levels were significantly reduced due to rapid protein degradation whereas the expression of mutant Ank/ANKH mRNA transcripts was comparable to wild‐type Ank/ANKH . ( 5 ) Although Ank KI/KI mice showed a tendency of decreased serum Pi, the difference was not statistically significant in comparison to Ank +/+ mice. ( 10 ) Other Pi regulating hormones such as PTH, 25‐OHD, and intact and cleaved FGF23, were unexpectedly normal in the CMD mouse model as well as in patients with CMD.…”

Section: Introductionmentioning

confidence: 93%

“…(3,4) ANK is a transmembrane protein localized on plasma membranes, endoplasmic reticulum, and Golgi apparatus. (5,6) ANKH/ANK is highly conserved within vertebrates and ubiquitously expressed in skeletal and nonskeletal tissues to regulate or prevent tissue mineralization by transporting intracellular pyrophosphate (PPi) to the extracellular matrix. (3,7) In vitro experiments in oocytes showed that CMD mutant ANK transports less PPi.…”

Section: Introductionmentioning

confidence: 99%

Restriction of Dietary Phosphate Ameliorates Skeletal Abnormalities in a Mouse Model for Craniometaphyseal Dysplasia

Fujii

Kozák

Dutra

et al. 2020

Journal of Bone and Mineral Research

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Craniometaphyseal dysplasia (CMD), a rare genetic bone disorder, is characterized by lifelong progressive thickening of craniofacial bones and metaphyseal flaring of long bones. The autosomal dominant form of CMD is caused by mutations in the progressive ankylosis gene ANKH (mouse ortholog Ank), encoding a pyrophosphate (PPi) transporter. We previously reported reduced formation and function of osteoblasts and osteoclasts in a knockin (KI) mouse model for CMD (Ank KI/KI) and in CMD patients. We also showed rapid protein degradation of mutant ANK/ANKH. Mutant ANK protein displays reduced PPi transport, which may alter the inorganic phosphate (Pi) and PPi ratio, an important regulatory mechanism for bone mineralization. Here we investigate whether reducing dietary Pi intake can ameliorate the CMD-like skeletal phenotype by comparing male and female Ank +/+ and Ank KI/KI mice exposed to a low (0.3%) and normal (0.7%) Pi diet for 13 weeks from birth. Serum Pi and calcium (Ca) levels were not significantly changed by diet, whereas PTH and 25-hydroxy vitamin D (25-OHD) were decreased by low Pi diet but only in male Ank +/+ mice. Importantly, the 0.3% Pi diet significantly ameliorated mandibular hyperostosis in both sexes of Ank KI/KI mice. A tendency of decreased femoral trabeculation was observed in male and female Ank +/+ mice as well as in male Ank KI/KI mice fed with the 0.3% Pi diet. In contrast, in female Ank KI/KI mice the 0.3% Pi diet resulted in increased metaphyseal trabeculation. This was also the only group that showed increased bone formation rate. Low Pi diet led to increased osteoclast numbers and increased bone resorption in all mice. We conclude that lowering but not depleting dietary Pi delays the development of craniofacial hyperostosis in CMD mice without severely compromising serum levels of Pi, Ca, PTH, and 25-OHD. These findings may have implications for better clinical care of patients with CMD.

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“…ankylosis progressive homolog (anKH), an amino acid of 492 base pairs in length, is a multichannel transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu (13). Extracellular PPi was discovered to be a regulator of pathological calcification and a potential inhibitor of calcium phosphate mineralization (14)(15)(16). ANKH is the human homolog of the murine ANK gene (15).…”

Section: Introductionmentioning

confidence: 99%

Ankylosis progressive homolog upregulation inhibits cell viability and mineralization during fibroblast ossification by regulating the Wnt/β‑catenin signaling pathway

He¹,

Dong²

2020

Mol Med Rep

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ankylosis progressive homolog (anKH) is associated with fibroblast ossification in ankylosing spondylitis (AS). as the human anKH gene is poorly characterized relative to its murine counterpart, the aim of the present study was to examine anKH expression in ligament tissue isolated from patients with AS and the role played by this gene in AS-associated fibroblast ossification. Fibroblasts were isolated from ligament tissue collected from patients with aS and ligament tissue from individuals with spinal cord fractures, then cultured. Fibroblasts from patients with AS were subsequently transfected with an ANKH overexpression vector, while those collected from individuals with spinal cord fractures were transfected with small interfering rna specific for ANKH. Cell viability, apoptosis and mineralization were analyzed using MTT assays, flow cytometry and Alizarin Red staining, respectively. Furthermore, ANKH mrna and protein expression levels were analyzed using reverse transcription-quantitative PCR and western blotting analysis, respectively. The expression levels of osteogenesis markers, including alkaline phosphatase, osteocalcin, runt-related transcription factor 2, c-Myc, as well as the β-catenin signaling protein, were also determined using western blotting. The results of the present study revealed that anKH protein expression levels were downregulated in aS total ligament tissue extract, compared with spinal fracture ligament. Moreover, the fibroblasts derived from patients with AS exhibited an increased viability and reduced apoptosis rates, compared with the fibroblasts from patients with spinal fracture. Notably, ANKH overexpression inhibited viability, mineralization and ossification, increased the phosphorylation of β-catenin and downregulated β-catenin and c-Myc protein expression levels in fibroblasts from patients with AS. In addition, ANKH overexpression increased the ratio of p-β-catenin/β-catenin in fibroblasts from patients with AS. By contrast, ANKH silencing in fibroblasts from patients with spinal fracture resulted in the opposite effect. In conclusion, the findings of the present study suggested that ANKH may inhibit fibroblast viability, mineralization and ossification, possibly by regulating the Wnt/β-catenin signaling pathway.

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Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia

Cited by 12 publications

References 44 publications

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia

Restriction of Dietary Phosphate Ameliorates Skeletal Abnormalities in a Mouse Model for Craniometaphyseal Dysplasia

Ankylosis progressive homolog upregulation inhibits cell viability and mineralization during fibroblast ossification by regulating the Wnt/β‑catenin signaling pathway

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