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µCT analysis and TRAP staining of femurs and tibias of 4-week-old ank/ank mice and wild-type littermates. (A) 2D µCT reconstruction of the sagittal plane of the femoral metaphysis of wild-type (WT) and ank/ank mice. Bar = 1 mm. (B) 2D µCT reconstruction of the axial plane of the femoral metaphysis of wild-type and ank/ank mice. Bar = 1 mm. (C) Trabecular bone volume/tissue volume (BV/TV), trabecular number, trabecular thickness, and trabecular separation were calculated. (D) Cortical thickness was calculated. Cortical thickness in each sample was obtained at three points around the perimeter of each bone. Data presented in panels C and D are presented as mean ± SD for five male mice in each genotype group (ap < .01 versus wild-type littermates; bp < .05 versus wild-type littermates). (E) TRAP staining of sections of tibias of 4-week-old ank/ank mice and wild-type littermates. Bar = 200 µm. The osteoclast number is expressed per millimeter of bone surface; osteoclast surface is expressed as percent of bone surface. Data are presented as mean ± SD. for five male mice in each genotype group (ap < .01 versus wild-type littermates).
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The progressive ankylosis gene (ank) encodes a transmembrane protein that transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu. ank/ank mice, which express a truncated nonfunctional ANK, showed a markedly reduced bone mass, bone-formation rate, and number of tartrate-resistant acid phosphatase-positive (TRAP(+)) mult...
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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‑associ...
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... Similar to its role in cartilage, ANK is regarded as an important regulator of calcification and development of bone tissue. While Ank knockdown decreased the expression of osteoblast marker genes Alpl, Ibsp, and Sp7, its overexpression increased these markers in preosteoblastic MC3T3-E1 cells [13,14]. In ank mice, parameters of trabecular bone quality in the femur and tibia are affected [14] with a decrease in bone volume and trabecular thickness with increased trabecular separation, without an effect on cortical thickness [14]. ...
... While Ank knockdown decreased the expression of osteoblast marker genes Alpl, Ibsp, and Sp7, its overexpression increased these markers in preosteoblastic MC3T3-E1 cells [13,14]. In ank mice, parameters of trabecular bone quality in the femur and tibia are affected [14] with a decrease in bone volume and trabecular thickness with increased trabecular separation, without an effect on cortical thickness [14]. These studies indicate that on the one hand, ANK prevents mineralization in the cartilage while on the other hand acts as a pro-mineralization factor in the bone. ...
... While Ank knockdown decreased the expression of osteoblast marker genes Alpl, Ibsp, and Sp7, its overexpression increased these markers in preosteoblastic MC3T3-E1 cells [13,14]. In ank mice, parameters of trabecular bone quality in the femur and tibia are affected [14] with a decrease in bone volume and trabecular thickness with increased trabecular separation, without an effect on cortical thickness [14]. These studies indicate that on the one hand, ANK prevents mineralization in the cartilage while on the other hand acts as a pro-mineralization factor in the bone. ...
Pathological mineralization of intervertebral disc is debilitating and painful and linked to disc degeneration in a subset of human patients. An adenosine triphosphate efflux transporter, progressive ankylosis (ANK) is a regulator of extracellular inorganic pyrophosphate levels and plays an important role in tissue mineralization. However, the function of ANK in intervertebral disc has not been fully explored. Herein we analyzed the spinal phenotype of Ank mutant mice (ank/ank) with attenuated ANK function. Micro-computed tomography and histological analysis showed that loss of ANK function results in the aberrant annulus fibrosus mineralization and peripheral disc fusions with cranial to caudal progression in the spine. Vertebrae in ank mice exhibit elevated cortical bone mass and increased tissue non-specific alkaline phosphatase-positive endplate chondrocytes with decreased subchondral endplate porosity. The acellular dystrophic mineral inclusions in the annulus fibrosus were localized adjacent to apoptotic cells and cells that acquired osteoblast-like phenotype. Fourier transform infrared spectral imaging showed that the apatite mineral in the outer annulus fibrosus had similar chemical composition to that of vertebral bone. Transcriptomic analysis of annulus fibrosus and nucleus pulposus tissues showed changes in several biological themes with a prominent dysregulation of BMAL1/CLOCK circadian regulation. The present study provides new insights into the role of ANK in the disc tissue compartments and highlights the importance of local inorganic pyrophosphate metabolism in inhibiting the mineralization of this important connective tissue.
... cleavage of the membrane-bound component, was demonstrated. The molecular signatures associated with the co-exposure-mediated osteogenesis inhibition included the enhanced downregulation of OPG, OCN, collagen, and cellular alkaline phosphatase mRNA transcription coupled with the decreased levels of mineral deposition conceivably due to the inhibition of the progressive ankylosis protein (ANK) signaling pathway, whose deficiency is known to impair osteoblastogenesis and bone formation [29]. The mineralization inhibition of bone nodules, coupled with the downregulation of bone sialoprotein mRNA and protein levels, was also found by Kamel and Yee [30] after continuous and intermittent exposure of primary rat calvarial cells to N-terminal PTHrP. ...
Parathyroid-hormone-related protein (PTHrP) is encoded by the PTHLH gene which, via alternative promoter usage and splicing mechanisms, can give rise to at least three isoforms of 139, 141, and 173 amino acids with distinct C-terminals. PTHrP is subjected to different post-translational processing that generates smaller bioactive forms, comprising amino terminus, mid-region (containing a nuclear/nucleolar targeting signal), and carboxy terminus peptides. Both the full-length protein and the discrete peptides are key controllers of viability, proliferation, differentiation, and apoptosis in diverse normal and pathological biological systems via the reprogramming of gene expression and remodulation of PKA or PKC-mediated signalization mechanisms. The aim of this review is to pick up selected studies on PTHrP-associated signatures as revealed by molecular profiling assays, focusing on the available data about exemplary differentiating, differentiated, or nontumoral cell and tissue models. In particular, the data presented relate to adipose, bone, dental, cartilaginous, and skin tissues, as well as intestinal, renal, hepatic, pulmonary, and pancreatic epithelia, with a focus on hepatic fibrosis-, pancreatitis-, and diabetes-related changes as diseased states. When reported, the biochemical and/or physiological aspects associated with the specific molecular modulation of gene expression and signal transduction pathways in the target model systems under examination are also briefly described.
... CST3 is an osteoclast coupling factor regulating osteoblast differentiation [94,111]. ANK2and SRCIN are regulators of osteoblast and osteoclast differentiation, and deficiency of the former has been associated with reduced bone mass and calcification [112][113][114]. ...
Benzo[a]pyrene (BaP) is ubiquitously present in the aquatic environment and has been identified as a bone toxicant. Previous studies have demonstrated that ancestral BaP exposure can cause transgenerational bone deformities in fish. Transgenerational effects are thought to be caused by heritable epigenetic changes, such as DNA methylation, histone modification, and non-coding RNAs. To investigate the role of DNA methylation in BaP-induced transgenerational skeletal deformities and the related transcriptomic changes in deformed vertebrae, we examined the vertebrae of male F1 and F3 medaka fish using high-throughput RNA sequencing (RNA-seq) and whole-genome bisulphite sequencing (WGBS). The histological results revealed that osteoblast numbers at the vertebral bone decreased in the BaP-derived F1 and F3 adult males in comparison with the control group. Differentially methylated genes (DMGs) associated with osteoblastogenesis (F1 and F3), chondrogenesis (F1 and F3), and osteoclastogenesis (F3) were identified. However, RNA-seq data did not support the role of DNA methylation in the regulation of genes involved in skeletogenesis since there was very little correlation between the level of differential methylation and gene expression profiles related to skeletogenesis. Although DNA methylation plays a major role in the epigenetic regulation of gene expression, the dysregulation of vertebral gene expression patterns observed in the current study is most likely to be mediated by histone modification and miRNAs. Notably, RNA-seq and WGBS data indicated that genes related to nervous system development are more sensitive to ancestral BaP exposure, indicating a more complex transgenerational phenotype in response to ancestral BaP exposure.
... Interestingly, in the study by Elango et al. [28] on the osteo-differentiation of mesenchymal stem cells (MSCs), the cooperative inhibitory effect of PTHrP and soluble RANKL (sRANKL), a circulating form released in vivo by disintegrin metalloproteinase-mediated cleavage of the membrane-bound component, was demonstrated. The molecular signatures associated to the coexposure-mediated osteogenesis inhibition included the enhanced down-regulation of OPG, OC, collagen, and cellular alkaline phosphatase mRNA transcription coupled with the decreased levels of mineral deposition conceivably due to the inhibition of the progressive ankylosis protein (ANK) signaling pathway, whose deficiency is known to impair osteoblastogenesis and bone formation [29]. The mineralization inhibition of bone nodules, coupled with the down-regulation of bone sialoprotein mRNA and protein levels, was also found by Kamel and Yee [30] after continuous and intermittent exposure of primary rat calvarial cells to N-terminal PTHrP. ...
Parathyroid-hormone-related protein (PTHrP) is encoded by PTHLH gene which, by alternative promoter usage and splicing mechanisms, can give rise to at least three isoforms of 139, 141 and 173 amino acids with distinct C-terminals. PTHrP is subjected to different post-translational processing that generates smaller bioactive forms, comprising amino terminus, midregion (containing a nuclear/nucleolar targeting signal) and carboxy terminus peptides. Both the full-length protein and the discrete peptides are key controllers of viability, proliferation, differentiation and apoptosis in diverse normal and pathological biological systems via the reprogramming of gene expression and remodulation of PKA or PKC-mediated signalization mechanisms. The aim of this review is to pick up selected studies on PTHrP-associated signatures as revealed by molecular profiling assays, focusing on the available data about exemplary differentiating, differentiated or non-tumoral cell and tissue models. In particular, the data presented relate to adipose, bone, dental, cartilaginous and skin tissues, and also intestinal, renal, hepatic, pulmonary and pancreatic epithelia, with a focus on hepatic fibrosis-, pancreatitis- and diabetes-related changes as diseased states. Whether reported, the biochemical and/or physiological aspects associated with the specific molecular modulation of gene expression and signal transduction pathways in the target model systems under examination will be also briefly commented.
... Pyrophosphatase phosphodiesterase 1 (NPP1, encoded by the gene ENPP1) and ankylosis protein (ANK) are involved in inhibiting mineralization. NPP1 generates pyrophosphate, and the transmembrane protein ANK allows pyrophosphate to pass through the plasma membrane to the extracellular matrix; thus, HA formation is inhibited in the extracellular vesicles [97,98]. 1α,25-OH 2 D 3 stimulates mineralization via direct action on osteoblasts [68,88,99]. ...
Despite its rigid structure, the bone is a dynamic organ, and is highly regulated by endocrine factors. One of the major bone regulatory hormones is vitamin D. Its renal metabolite 1α,25-OH2D3 has both direct and indirect effects on the maintenance of bone structure in health and disease. In this review, we describe the underlying processes that are directed by bone-forming cells, the osteoblasts. During the bone formation process, osteoblasts undergo different stages which play a central role in the signaling pathways that are activated via the vitamin D receptor. Vitamin D is involved in directing the osteoblasts towards proliferation or apoptosis, regulates their differentiation to bone matrix producing cells, and controls the subsequent mineralization of the bone matrix. The stage of differentiation/mineralization in osteoblasts is important for the vitamin D effect on gene transcription and the cellular response, and many genes are uniquely regulated either before or during mineralization. Moreover, osteoblasts contain the complete machinery to metabolize active 1α,25-OH2D3 to ensure a direct local effect. The enzyme 1α-hydroxylase (CYP27B1) that synthesizes the active 1α,25-OH2D3 metabolite is functional in osteoblasts, as well as the enzyme 24-hydroxylase (CYP24A1) that degrades 1α,25-OH2D3. This shows that in the past 100 years of vitamin D research, 1α,25-OH2D3 has evolved from an endocrine regulator into an autocrine/paracrine regulator of osteoblasts and bone formation.
... In the post-proliferation period, the extracellular matrix and its components are arranged and changed in the direction of mineralization. In the mineralization medium, mRNA levels of the alkaline phosphatase decreased and the expression of other genes responsible for proteins such as sialoprotein, osteopontin, and osteocalcin increased with matrix mineralization [44]. ...
A new generation of scaffolds capable of acting not only as support for cells but also as a source of biological cues to promote tissue regeneration is currently a hot topic in bone Tissue Engineering (TE) research. The controlled release of the Growth Factors (GFs) from the scaffolds is a suitable strategy to achieve such a goal. Platelet Rich Plasma (PRP) is an autologous source of GFs, providing several bioactive agents known to act on bone regeneration. In this study, citric acid-modified starch nanoparticles loaded with PRP will be included in a gelatin scaffold. Scaffolds were evaluated in vitro with respect to compressive strength, water uptake, and protein release. The size of the pores in the production scaffolds was evaluated to be around 150 microns which is suitable for bone regeneration. The scaffolds were then seeded with human adipose-derived stem cells (hASCs) and cultured in vitro. The osteogenic behavior of the seeded hASCs was evaluated by alkaline phosphatase activity (ALP activity) and calcium assay analysis. The results show the promotion of morphological, mechanical, and biological properties of the scaffolds by adding PRP-loaded nanoparticles. Our results suggest that the PRP-loaded starch nanoparticles enhance the biological and bone regeneration of the gelatin-based scaffolds.
... Similar to its role in the cartilage, ANK is regarded as an important regulator of calci cation and Page 3/29 development of the bone tissue. While Ank knockdown decreased the expression of osteoblast marker genes ALPL, Ibsp, and Sp7, its overexpression increased these markers in preosteoblastic MC3T3-E1 cells 12,13 . In ank mice, parameters of trabecular bone quality in the femur and tibia are affected 13 with a pronounced decrease in bone volume and trabecular thickness with increased trabecular separation, without an effect on cortical thickness 13 . ...
... While Ank knockdown decreased the expression of osteoblast marker genes ALPL, Ibsp, and Sp7, its overexpression increased these markers in preosteoblastic MC3T3-E1 cells 12,13 . In ank mice, parameters of trabecular bone quality in the femur and tibia are affected 13 with a pronounced decrease in bone volume and trabecular thickness with increased trabecular separation, without an effect on cortical thickness 13 . These studies indicate dual roles of ANK as a mineralization inhibitor in the cartilage, whereas, a pro-mineralization factor in the bone. ...
... While Ank knockdown decreased the expression of osteoblast marker genes ALPL, Ibsp, and Sp7, its overexpression increased these markers in preosteoblastic MC3T3-E1 cells 12,13 . In ank mice, parameters of trabecular bone quality in the femur and tibia are affected 13 with a pronounced decrease in bone volume and trabecular thickness with increased trabecular separation, without an effect on cortical thickness 13 . These studies indicate dual roles of ANK as a mineralization inhibitor in the cartilage, whereas, a pro-mineralization factor in the bone. ...
Pathological mineralization of intervertebral disc is debilitating and painful and linked to disc degeneration in a subset of human patients. An adenosine triphosphate transporter, progressive ankylosis (ANK) is a regulator of extracellular inorganic pyrophosphate levels and plays an important role in tissue mineralization. However, the function of ANK in intervertebral disc has not been fully explored. Herein we analyzed the spinal phenotype of Ank mutant mice ( ank / ank ) with attenuated ANK function. Micro-computed tomography and histological analysis showed that loss of ANK function results in aberrant annulus fibrosus mineralization and peripheral disc fusions with cranial to caudal progression in the spine. Vertebrae in ank / ank mice exhibit elevated cortical bone mass and increased tissue non-specific alkaline phosphatase-positive endplate chondrocytes with decreased subchondral endplate porosity. The acellular dystrophic mineral inclusions in the annulus fibrosus were localized adjacent to apoptotic cells and cells that acquired osteoblast-like phenotype. Fourier transform infrared spectral imaging showed that the mineral in the outer annulus fibrosus had similar chemical composition to that of vertebral bone. Microarray-based transcriptomic analysis of annulus fibrosus and nucleus pulposus tissues showed changes in several pathways associated with mineralization including transforming growth factor β and mitogen-activated protein kinase signaling. The present study provides new insights into the role of ANK in the disc tissue compartments, and highlights the importance of local inorganic pyrophosphate metabolism in inhibiting mineralization of this important connective tissue.
... Inorganic pyrophosphate accumulates both intracellularly and extracellularly, with the intracellular inorganic pyrophosphate being transported out of the cell through the progressive ankylosis protein (ANKH), which is a multi-pass pyrophosphate transporter [57,58]. The combined actions of ANKH and ENPP-1 result in an increased extracellular pyrophosphate concentration, which is inhibitory to the formation of the hydroxyapatite crystals [59,60]. For mineralisation to proceed, the elevated extracellular pyrophosphate concentration must be reduced. ...
Tissue-nonspecific alkaline phosphatase (TNAP) is one of four isozymes, which include germ cell, placental and intestinal alkaline phosphatases. The TNAP isozyme has 3 isoforms (liver, bone and kidney) which differ by tissue expression and glycosylation pattern. Despite a long history of investigation, the exact function of TNAP in many tissues is largely unknown. Only the bone isoform has been well characterised during mineralization where the enzyme hydrolyses pyrophosphate to inorganic phosphate, which combines with calcium to form hydroxyapatite crystals deposited as new bone. The inorganic phosphate also increases gene expression of proteins that support tissue mineralization. Recent studies have shown that TNAP is expressed in preadipocytes from several species, and that inhibition of TNAP activity causes attenuation of intracellular lipid accumulation in these and other lipid-storing cells. The mechanism by which TNAP stimulates lipid accumulation is not known; however, proteins that are important for controlling phosphate levels in bone are also expressed in adipocytes. This review examines the evidence that inorganic phosphate generated by TNAP promotes transcription that enhances the expression of the regulators of lipid storage and consequently, that TNAP has a major function of lipid metabolism.
... The evidence of accelerated bone formation by the substantial volume that is populated by the host suggests that the CaP implant itself is recognized as an endogenous tissue. Additionally, the pyrophosphate content of the CaP implant could have a positive biological effect as previous results have shown that initial exposure of pyrophosphate to non-mineralizing osteoblast initiates mineralization in vitro (Kim et al., 2010). Even though pyrophosphate is known as a key regulator in biomineralization in vivo, inhibiting spontaneous mineralization, acting as the body's own softener and being part of normal metabolism (Fleisch & Bisaz, 1962;Millán, 2013;Orriss et al., 2016), we hypothesize that the complex crosstalk and feedback loops involved with pyrophosphate in the process of biomineralization could be central to the mechanism of which our CaP material is recognized and replaced by bone. ...
The restoration of cranio‐maxillofacial deformities often requires complex reconstructive surgery in a challenging anatomical region, with abnormal soft tissue structures and bony deficits. In this proof‐of‐concept, the possibility of vertical bone augmentation was explored by suspending hemispherically shaped titanium‐reinforced porous calcium phosphate (CaP) implants (n = 12) over the frontal bone in a sheep model (n = 6). The animals were euthanized after week 13 and the specimens were subject to micro‐computed tomography (μCT) and comprehensive histological analysis. Histology showed that the space between implant and the recipient bone was filled with a higher percentage of newly formed bone (NFB) versus soft tissue with a median of 53% and 47%, respectively. Similar results were obtained from the μ‐CT analysis, with a median of 56% NFB and 44% soft tissue filling the void. Noteworthy, significantly higher bone‐implant contact was found for the CaP (78%, range 14%–94%) versus the Titanium (29%, range 0%–75%) portion of the implant exposed to the surrounding bone. The histological analysis indicates that the CaP replacement by bone is driven by macrophages over time, emphasized by material‐filled macrophages found in close vicinity to the CaP with only a small number of single osteoclasts found actively remodeling the NFB. This study shows that CaP based implants can be assembled with the help of additive manufacturing to guide vertical bone formation without decortification or administration of growth factors. Furthermore, it highlights the potential disadvantage of a seamless fit between the implant and the recipient's bone.
... In theory, a compensatory upregulation of TNAP activity in bone could have reduced PPi incorporation in hydroxyapatite of ank mice. However, the reported reduced ALPL expression in ank osteoblasts (22) indicates that it is very unlikely that increased TNAP activity has contributed to the reduction in the amounts of PPi that incorporated into the mineral phase of bone of the mutant mice. In conclusion, analysis of bones of Enpp1 À/À and Ank mice recapitulates our results obtained in HEK293 cells, and shows that also in intact animals, Ank does not mediate release of substantial amounts of PPi from cells. ...
The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question if PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1‐deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1‐proficient cells. Ank‐activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1−/− mice contained <2.5% of the PPi found in bones of wild type mice, showing that Enpp1‐activity is also a prerequisite for Ank‐dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1‐mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60–70% of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself. This article is protected by copyright. All rights reserved.
