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Farquharson, C.

Publications and source records attributed to Farquharson, C..

11 recordsLinked to original sources

A frameshift mutation in PRICKLE1 causes skeletal dysplasia in Cheviot sheep

Skeletal dysplasias can occur in animals, including humans, and are attributed to both environmental and genetic causes. As our understanding of the distinguishing phenotypes of different skeletal dysplasias improves, alongside elucidation of the mechanisms and genetic networks that contribute to skeletal development, identifying and controlling genetic mutations in livestock populations has become increasingly possible, including approaches beyond selective breeding alone. Within Scotland an 'achondroplasia' syndrome that was first described in the South Country Cheviot breed of sheep (OMIA 000004-9940), has continued to occur periodically within the same geographical locations, despite culling of affected lambs over many decades. Affected lambs exhibit rounded heads, short legs, a loss of hooves, abnormal stance and short twisted tails. As the condition has been observed in flocks and as progeny from specific ram and ewe matings, and cases have been co-twinned to normal siblings, this suggests a syndrome caused by a recessive genetic condition rather than environmental factors. Through homozygosity mapping and whole genome sequence analyses, we identified a 10 bp deletion in the open reading frame of the PRICKLE1 gene that encodes prickle planar cell polarity protein 1 and is essential for normal vertebrate embryonic development. We identified additional phenotypes present in affected lambs, including a loss of phalanges, neural tube defects, vertebral and articular chondrocyte defects, hair orientation differences and polycystic kidneys, consistent with a loss of PRICKLE1 function based on comparisons to mouse mutant lines. These findings enabled the development of a simple DNA test for the identified PRICKLE1 mutation that was made commercially available to facilitate the elimination of this genetic disease from sheep flocks.

genetics↗

TNAP and PHOSPHO1 function synergistically to afford critical control over the mineralisation of the postnatal murine skeleton

Biomineralisation is essential for skeletal integrity, yet the synergistic roles of tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 in postnatal bone mineralisation remain poorly defined. To decipher this, we generated a novel murine model in which Alpl was deleted in Prx1- expressing cells (AlplPrx1/Prx1) in mice with a global Phospho1-/- deficiency to overcome the perinatal lethality that arises upon dual global deletion. Using a multi-modal approach to spatially phenotype the limbs of these animals, we reveal mice lacking both TNAP and PHOSPHO1 exhibit a distinct lack of mineralisation and altered anatomical structure at postnatal day 1 (PN1) and 3-weeks of age. Although viable, these mice did not thrive due to their reduced size, thus further investigations were conducted on mice with a heterozygous deletion of TNAP (Alplwt/Prx1;Phospho1-/-). Although smaller than wild-types at PN1 and 3 weeks old, these mice did not display the gross limb deformations observed in the homozygous animals and the single, functioning Alpl allele rescued the loss of biomineralisation observed following dual phosphatase deletion. At 6-weeks of age, compromised epiphyses and metaphyses were only seen in AlplPrx1/Prx1 animals. Further, we found that tibial geometry and porosity was significantly altered by Phospho1 deletion (Phospho1-/-), which was compounded in the Alplwt/Prx1;Phospho1-/- mice and linked to alterations in collagen configuration, matrix mineralisation and growth plate deformities. Together, our findings establish the mechanistic framework for TNAP and PHOSPHO1 in permissive biomineralisation, providing critical insights into this fundamental process. Significance StatementBiomineralisation is essential for skeletal development and is critically dependent on phosphatases that release inorganic phosphate for hydroxyapatite formation. Our study investigates the dual role of PHOSPHO1 and TNAP in this process, using a novel murine knockout model. Deletion of both enzymes results in complete loss of bone mineralisation, demonstrating their critical synergistic function. Further, we show that PHOSPHO1 and TNAP exhibit distinct, spatially-restricted functions in the tibia and thus enhances our understanding of the fundamentals processes underpinning biomineralisation. These findings also have clinical relevance as they have the potential to inform on treatment strategies for hypo- and hyper-mineralised pathologies.

developmental biology↗

Proteomic characterisation of matrix vesicles from primary osteoblasts indicates a mixed population with both exosomal and ectosomal routes of biogenesis.

Matrix Vesicles are a crucial step in producing a mineralised, healthy skeleton. Released from chondrocytes and osteoblasts, they concentrate calcium and phosphate to establish the deposition of hydroxyapatite around and within the collagen fibrils of the extracellular matrix, becoming embedded in the matrix in the process. In the 55 years since H. Clarke Anderson first described them, blebbing from the surface of chondrocytes, a consensus on their role, contents and their biogenesis, has yet to be reached. This is in part due to the range of cell types from which they are released and the multitude techniques that can be employed to isolate them. In this study, we, for the first time, characterise the proteome of matrix vesicles isolated from primary osteoblasts. By focussing on those vesicles that have become embedded within the matrix, we are able to avoid bias for specific modes of biogenesis and focus only on osteoblast-released vesicles that are associated with the matrix. Moreover, by studying these vesicles over a time course of mineralising activity, we are able to identify changes in the properties of these vesicles, and develop a more accurate picture of which proteins are involved specifically in mineralisation. In particular we identify the presence of markers associated with the ectosomal and exosomal release of matrix vesicles, as well as identifying proteins required for the maintenance and mineralisation of the extracellular matrix. These data portray a heterogeneous population of matrix vesicles, with different roles to play in bone development.

cell biology↗

Polyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone

Non-collagenous proteins (NCPs) are specialized biomacromolecules within the extracellular matrix (ECM) that regulate the mineralization of calcified tissues, such as bone and dentin. Numerous in vitro studies have demonstrated that natural polyanionic NCPs and their analogues can mediate intrafibrillar mineralization, characterized by the infiltration of apatite minerals into collagen fibrils. However, these studies primarily utilize self-assembled collagen fibrils or demineralized mature tissues, leaving it unclear whether pristine embryonic bone ECM at a developmental stage permissive to mineral deposition can regulate intrafibrillar mineralization independently or requires polyanionic NCP substitutes to promote the process artificially. To address this, we employed an ex vivo model of endochondral ossification using metatarsals isolated from 15-day-old embryonic mice (E15). In addition to a supersaturated calcium (Ca) and inorganic phosphate (Pi) medium, we introduced fetuin-A, a native polyanionic NCP or poly-DL-aspartic acid (pAsp), commonly used as an NCP substitute. The incorporation of either additive was essential for the effective mineralization of embryonic metatarsals. Both fetuin-A and pAsp played a direct role in facilitating the infiltration of Ca-Pi precursors into the avascular cartilaginous matrix. Raman spectroscopy and electron microscopy confirmed the formation of hydroxyapatite (HAp) exhibiting diverse levels of crystallinity, with fetuin-A supplementation resulting in the greatest HAp accumulation within the rudiments. HAp was localized in the perichondrium, a region conducive to initial mineralization and enriched with a fibrillar network of collagen types I and II. Three-dimensional reconstructions implementing Dijkstras algorithm revealed the association between HAp and collagen fibrils either organized in an intrafibrillar, extrafibrillar, or combined arrangement.

biophysics↗

Mitochondrial dysfunction and mitophagy blockade contribute to renal osteodystrophy in chronic kidney disease-mineral bone disorder

Chronic kidney disease-mineral and bone disorder (CKD-MBD) presents with extra-skeletal calcification and renal osteodystrophy (ROD). The origins of ROD likely lie with elevated uremic toxins and/or an altered hormonal profile but the cellular events responsible remain unclear. Here, we report that stalled mitophagy contributes to mitochondrial dysfunction in bones of a CKD-MBD mouse model, and also human CKD-MBD patients. RNA-seq analysis exposed an altered expression of genes associated with mitophagy and mitochondrial function in tibia of CKD-MBD mice. The accumulation of damaged osteocyte mitochondria and the expression of mitophagy regulators, p62/SQSTM1, ATG7 and LC3 was inconsistent with functional mitophagy, and in mito-QC reporter mice with CKD-MBD, there was a 2.3-fold increase in osteocyte mitolysosomes. Altered expression of mitophagy regulators in human CKD-MBD bones was also observed. To determine if uremic toxins were possibly responsible for these observations, indoxyl sulfate treatment of osteoblasts revealed mitochondria with distorted morphology and whose membrane potential and oxidative phosphorylation were decreased, and oxygen-free radical production increased. The altered p62/SQSTM1 and LC3-II expression was consistent with impaired mitophagy machinery and the effects of indoxyl sulfate were reversible by rapamycin. In conclusion, mitolysosome accumulation from impaired clearance of damaged mitochondria may contribute to the skeletal complications, characteristic of ROD. Targeting mitochondria and the mitophagy process may therefore offer novel routes for intervention to preserve bone health in patients with ROD. Such approaches would be timely as our current armamentarium of anti-fracture medications has not been developed for, or adequately studied in, patients with severe CKD-MBD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/573355v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f23caforg.highwire.dtl.DTLVardef@6e3e94org.highwire.dtl.DTLVardef@1ce6adeorg.highwire.dtl.DTLVardef@ca5558_HPS_FORMAT_FIGEXP M_FIG C_FIG TRANSLATIONAL STATEMENTRenal osteodystrophy (ROD) remains the major skeletal complication of chronic kidney disease-mineral and bone disorder (CKD-MBD). As a disease characterised by biochemical and hormone abnormalities, ROD is exacerbated by osteocyte mitochondrial dysfunction. Advances in our understanding of the mitophagy pathway are vital to improving the clinical management of ROD. The dysregulation of mitophagy in murine and human CKD-MBD bone provided evidence of delayed clearance of damaged mitochondria, which was also observed in uremic toxin-treated-osteoblasts but reversible upon rapamycin treatment. This study reveals the therapeutic potential of managing ROD by restoring defective mitophagy in osteocytes.

genomics↗

Differential Expression of Alkaline Phosphatase and PHOSPHO1 in Bone from a Murine Model of Chronic Kidney Disease

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities; a condition known as renal osteodystrophy (ROD). While Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are recognized to be critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both wild-type and Phospho1 knockout (P1KO) mice using dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (p < 0.05) but decreased in trabecular bone (p < 0.05). These changes were accompanied by decreased TNAP (p < 0.01) and increased PHOSPHO1 (p < 0.001) expression in wild-type CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expression to be down-regulated in response to phosphate and PTH. While matrix mineralization was increased with phosphate (Pi), it decreased with PTH and FGF23 had no effect. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism in an attempt to protect the bone from hypomineralization. We propose that increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

cell biology↗

The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability

Osteoarthritis is the most prevalent systemic musculoskeletal disorder characterised by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here we sought to examine the contribution of accelerated growth to osteoarthritis development using a murine model of excessive longitudinal growth. Suppressor of cytokine signalling 2 (SOCS2) is a negative regulator of growth hormone (GH) signalling, thus mice deficient in SOCS2 (Socs2-/-) display accelerated bone growth. We examined vulnerability of Socs2-/- mice to osteoarthritis following surgical induction of disease (destabilisation of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. We observed significant increase in number (WT DMM: 532{+/-}56; WT sham: 495{+/-}45; KO DMM: 169{+/-}49; KO sham: 187{+/-}56; P<0.01) and density (WT DMM: 2.2{+/-}0.9; WT sham: 1.2{+/-}0.5; KO DMM: 13.0{+/-}0.5; KO sham: 14.4{+/-}0.7) of growth plate bridges in Socs2-/- in comparison to wild-type (WT). Histological examination of WT and Socs2-/- knees revealed articular cartilage damage with DMM in comparison to sham (WT DMM: 3.4{+/-}0.4; WT sham: 0.3{+/-}0.05 (P<0.05); KO DMM: 3.2{+/-}0.8; KO sham: 0.8{+/-}0.3). Articular cartilage lesion severity scores (mean and maximum) were similar in WT and Socs2-/- mice with either DMM, or with ageing. Micro-CT analysis revealed significant decreases in SCB thickness, epiphyseal trabecular number and thickness in the medial compartment of Socs2-/-, in comparison to WT (P<0.001). DMM had no effect on the SCB thickness in comparison to sham in either genotype. Together these data suggest that enhanced GH signalling through SOCS2 deletion accelerates growth plate fusion, however this has no effect on osteoarthritis vulnerability in this model. Summary statementDeletion of SOCS2 results in accelerated growth plate fusion, however this has no effect on osteoarthritis vulnerability.

pathology↗

Proton pump inhibitors inhibit PHOSPHO1 activity and matrix mineralisation in vitro

Proton pump inhibitors (PPIs) have been associated with an increased risk of fragility fractures in pharmaco-epidemiological studies. The mechanism is unclear but it has been speculated that by neutralising gastric acid, they may reduce intestinal calcium absorption, causing secondary hyperparathyroidism and bone loss. Here we investigated that hypothesis that the skeletal effects of PPI might be mediated by inhibitory effects on the bone-specific phosphatase PHOSPHO1. We found that the all PPI tested potential inhibited the activity of PHOSPHO1 with IC50 ranging between 0.73M for esomeprazole to 19.27M for pantoprazole. In contrast, these PPIs did not inhibit TNAP activity. We also found that mineralisation of bone matrix in primary osteoblast cultures inhibited by several PPI in a concentration dependent manner. In contrast, the histamine-2 receptor antagonists (H2RA) nizatidine, famotidine, cimetidine and ranitidine had no inhibitory effects on PHOSPHO1 activity. Our experiments shown for the first time that PPI inhibit PHOSPHO1 activity and matrix mineralisation in vitro revealing a potential mechanism by which these widely used drugs are associated with the risk of fractures.

cell biology↗

A systems-level analysis of dynamic total-body PET data reveals complex skeletal energy metabolism networks in vivo

Bone is now regarded to be a key regulator of a number of metabolic processes, in addition to the regulation of mineral metabolism. However, our understanding of complex bone metabolic interactions at a systems level remains rudimentary, limiting our ability to assess systemic mechanisms underlying diseases and develop novel therapeutics. In vitro molecular biology and bioinformatics approaches have frequently been used to understand the mechanistic changes underlying disease at the cell level, however, these approaches lack the capability to interrogate dynamic multi-bone metabolic interactions in vivo. Here we present a novel and integrative approach to understand complex bone metabolic interactions in vivo using total-body positron emission tomography (PET) network analysis of murine 18F-FDG scans, as a biomarker of glucose metabolism signature in bones. In this report we show that different bones within the skeleton have a unique glucose metabolism and form a complex metabolic network. These data could have important therapeutic implications in the management of the metabolic syndrome and skeletal disease. The application of our approach to clinical and preclinical total-body PET studies promises to reveal further physiological and pathological tissue interactions, which simplistic PET standard uptake values analysis fail to interrogate, extending beyond skeletal metabolism, due to the diversity of PET radiotracers available and under development as well as the advent of clinical total-body PET systems. One Sentence SummaryBones form a complex metabolic network.

physiology↗

Ubiquitin-protein ligase Ubr5 cooperates with Hedgehog signalling to promote skeletal tissue homeostasis

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway. Author SummaryUbiquitin ligases modify proteins post-translationally which is essential for a variety of cellular processes. UBR5 is an E3 ubiquitin ligase and in Drosophila is a regulator of Hedgehog signaling. In mammals, the Hedgehog (HH) signalling pathway, among many other roles, plays an essential role in tissue maintenance, a process called homeostasis. A murine genetic system was developed to specifically eliminate UBR5 function from embryonic limb tissue that subsequently forms bone and connective tissue (ligaments and tendons). This approach revealed that UBR5 operates as a potent suppressor of excessive growth of normal cartilage and bone and prevents formation of bone in ectopic sites in connective tissue near the knees and ankle joints. In contrast to abnormal growth, UBR5 inhibits degradation of the articular cartilage that cushions the knee joint leading to extensive exposure of underlying bone. Furthermore, Ubr5 interacts with smoothened, a component of the HH pathway, identifying UBR5 as a regulator of mammalian HH signaling in the postnatal musculoskeletal system. In summary, this work shows that UBR5 interacts with the HH pathway to regulate skeletal homeostasis in and around joints of the legs and identifies targets that may be harnessed for biomedical engineering and clinical applications.

genetics↗

PHOSPHO1, a novel skeletal regulator of insulin resistance and obesity

The skeleton is recognised as a key endocrine regulator of metabolism. Here we show that mice lacking the bone mineralization enzyme PHOSPHO1 (Phospho1-/-) exhibited improved basal glucose homeostasis and resisted high-fat-diet induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalized by feeding a 2% choline rich diet resulting in a normalization in insulin sensitivity and fat mass. This study identifies PHOSPHO1 as a potential therapeutic target for the treatment of obesity and diabetes.

physiology↗