Extracellular matrix complexity in biomarker studies: a novel assay detecting total serum tenascin-C reveals different distribution to isoform-specific assays.

Serum biomarkers are the gold standard in non-invasive disease diagnosis and have tremendous potential as prognostic and theranostic tools for patient stratification. Circulating levels of extracellular matrix molecules are gaining traction as an easily accessible means to assess tissue pathology. However, matrix molecules are large, multimodular proteins that are subject to a vast array of post-transcriptional and post-translational modifications. These modifications often occur in a tissue- and/or disease-specific manner, generating hundreds of different variants, each with distinct biological roles. Whilst this complexity can offer unique insight into disease processes, it also has the potential to confound biomarker studies. Tenascin-C is a pro-inflammatory matrix protein expressed at low levels in most healthy tissues but elevated in, and associated with the pathogenesis of, a wide range of autoimmune diseases, fibrosis, and cancer. Analysis of circulating tenascin-C has been widely explored as a disease biomarker. Hundreds of different tenascin-C isoforms can be generated by alternative splicing, and this protein is also modified by glycosylation and citrullination. Current enzyme-linked immunosorbent assays (ELISA) are used to measure serum tenascin-C using antibodies, recognising sites within domains that are alternatively spliced. These studies, therefore, report only levels of specific isoforms that contain these domains, and studies on the detection of total tenascin-C are lacking. As such, circulating tenascin-C levels may be underestimated and/or biologically relevant isoforms overlooked. We developed a highly specific and sensitive ELISA measuring total tenascin-C down to 0.78ng/ml, using antibodies that recognise sites in constitutively expressed domains. In cohorts of people with different inflammatory and musculoskeletal diseases, levels of splice-specific tenascin-C variants were lower than and distributed differently from total tenascin-C. Neither total nor splice-specific tenascin-C levels correlated with the presence of autoantibodies to citrullinated tenascin-C in rheumatoid arthritis (RA) patients. Elevated tenascin-C was not restricted to any one disease and levels were heterogeneous amongst patients with the same disease. These data confirm that its upregulation is not disease-specific, instead suggest that different molecular endotypes or disease stages exist in which pathology is associated with, or independent of, tenascin-C. This immunoassay provides a novel tool for the detection of total tenascin-C that is critical for further biomarker studies. Differences between the distribution of tenascin-C variants and total tenascin-C have implications for the interpretation of studies using isoform-targeted assays. These data highlight the importance of assay design for the detection of multimodular matrix molecules and reveal that there is still much to learn about the intriguingly complex biological roles of distinct matrix proteoforms.

Anti-RANKL Therapy Prevents Glucocorticoid-Induced Bone Loss and Promotes Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy.

Bisphosphonates prevent bone loss in glucocorticoid (GC)-treated boys with Duchenne muscular dystrophy (DMD) and are recommended as standard of care. Targeting receptor activator of nuclear factor kappa-B ligand (RANKL) may have advantages in DMD by ameliorating dystrophic skeletal muscle function in addition to their bone anti-resorptive properties. However, the potential effects of anti-RANKL treatment upon discontinuation in GC-induced animal models of DMD are unknown and need further investigation prior to exploration in the clinical research setting. In the first study, the effects of anti-RANKL and deflazacort (DFZ) on dystrophic skeletal muscle function and bone microstructure were assessed in mdx mice treated with DFZ or anti-RANKL, or both for 8 weeks. Anti-RANKL and DFZ improved grip force performance of mdx mice but an additive effect was not noted. However, anti-RANKL but not DFZ improved ex vivo contractile properties of dystrophic muscles. This functional improvement was associated with a reduction in muscle damage and fibrosis, and inflammatory cell number. Anti-RANKL treatment, with or without DFZ, also improved trabecular bone structure of mdx mice. In a second study, intravenous zoledronate (Zol) administration (1 or 2 doses) following 2 months of discontinuation of anti-RANKL treatment was mostly required to record an improvement in bone microarchitecture and biomechanical properties in DFZ-treated mdx mice. In conclusion, the ability of anti-RANKL therapy to restore muscle function has profound implications for DMD patients as it offers the possibility of improving skeletal muscle function without the steroid-related skeletal side effects.

Spatial Lipidomic Profiling of Mouse Joint Tissue Demonstrates the Essential Role of PHOSPHO1 in Growth Plate Homeostasis.

Lipids play a crucial role in signaling and metabolism, regulating the development and maintenance of the skeleton. Membrane lipids have been hypothesized to act as intermediates upstream of orphan phosphatase 1 (PHOSPHO1), a major contributor to phosphate generation required for bone mineralization. Here, we spatially resolve the lipid atlas of the healthy mouse knee and demonstrate the effects of PHOSPHO1 ablation on the growth plate lipidome. Lipids spanning 17 subclasses were mapped across the knee joints of healthy juvenile and adult mice using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), with annotation supported by shotgun lipidomics. Multivariate analysis identified 96 and 80 lipid ions with differential abundances across joint tissues in juvenile and adult mice, respectively. In both ages, marrow was enriched in phospholipid platelet activating factors (PAFs) and related metabolites, cortical bone had a low lipid content, whereas lysophospholipids were strikingly enriched in the growth plate, an active site of mineralization and PHOSPHO1 activity. Spatially-resolved profiling of PHOSPHO1-knockout (KO) mice across the resting, proliferating, and hypertrophic growth plate zones revealed 272, 306, and 296 significantly upregulated, and 155, 220, and 190 significantly downregulated features, respectively, relative to wild-type (WT) controls. Of note, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, lysophosphatidylethanolamine, and phosphatidylethanolamine derived lipid ions were upregulated in PHOSPHO1-KO versus WT. Our imaging pipeline has established a spatially-resolved lipid signature of joint tissues and has demonstrated that PHOSPHO1 ablation significantly alters the growth plate lipidome, highlighting an essential role of the PHOSPHO1-mediated membrane phospholipid metabolism in lipid and bone homeostasis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Gene Therapy Using Recombinant AAV Type 8 Vector Encoding TNAP-D10 Improves the Skeletal Phenotypes in Murine Models of Osteomalacia.

Hypophosphatasia (HPP), caused by loss-of-function mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNAP), is characterized by skeletal and dental hypomineralization that can vary in severity from life-threatening to milder manifestations only in adulthood. PHOSPHO1 deficiency leads to early-onset scoliosis, osteomalacia, and fractures that mimic pseudo-HPP. Asfotase alfa, a life-saving enzyme replacement therapy approved for pediatric-onset HPP, requires subcutaneous injections 3 to 6 times per week. We recently showed that a single injection of an adeno-associated virus vector serotype 8 harboring TNAP-D10 (AAV8-TNAP-D10) effectively prevented skeletal disease and prolonged life in Alpl -/- mice phenocopying infantile HPP. Here, we aimed to determine the efficacy of AAV8-TNAP-D10 in improving the skeletal and dental phenotype in the Alpl Prx1/Prx1 and Phospho1 -/- mouse models of late-onset (adult) HPP and pseudo-HPP, respectively. A single dose of 3 × 1011 vector genomes per body (vg/b) was injected intramuscularly into 8-week-old Alpl Prx1/Prx1 and wild-type (WT) littermates, or into 3-day-old Phospho1 -/- and WT mice, and treatment efficacy was evaluated after 60 days for late-onset HPP mice and after 90 days for Phospho1 -/- mice. Biochemical analysis showed sustained serum alkaline phosphatase activity and reduced plasma PPi levels, and radiographic images, micro-computed tomography (micro-CT) analysis, and hematoxylin and eosin (H&E) staining showed improvements in the long bones in the late-onset HPP mice and corrected scoliosis in the Phospho1 -/- mice. Micro-CT analysis of the dentoalveolar complex did not reveal significant changes in the phenotype of late-onset HPP and pseudo-HPP models. Moreover, alizarin red staining analysis showed that AAV8-TNAP-D10 treatment did not promote ectopic calcification of soft organs in adult HPP mice after 60 days of treatment, even after inducing chronic kidney disease. Overall, the AAV8-TNAP-D10 treatment improved the skeletal phenotype in both the adult HPP and pseudo-HPP mouse models. This preclinical study will contribute to the advancement of gene therapy for the improvement of skeletal disease in patients with heritable forms of osteomalacia. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Increased PHOSPHO1 and alkaline phosphatase expression during the anabolic bone response to intermittent parathyroid hormone delivery.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.

Perspective on Dentoalveolar Manifestations Resulting From PHOSPHO1 Loss-of-Function: A Form of Pseudohypophosphatasia?

Mineralization of the skeleton occurs by several physicochemical and biochemical processes and mechanisms that facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Two key phosphatases, phosphatase, orphan 1 (PHOSPHO1) and tissue-non-specific alkaline phosphatase (TNAP), play complementary roles in the mineralization process. The actions of PHOSPHO1 on phosphocholine and phosphoethanolamine in matrix vesicles (MVs) produce inorganic phosphate (Pi) for the initiation of HA mineral formation within MVs. TNAP hydrolyzes adenosine triphosphate (ATP) and the mineralization inhibitor, inorganic pyrophosphate (PPi), to generate Pi that is incorporated into MVs. Genetic mutations in the ALPL gene-encoding TNAP lead to hypophosphatasia (HPP), characterized by low circulating TNAP levels (ALP), rickets in children and/or osteomalacia in adults, and a spectrum of dentoalveolar defects, the most prevalent being lack of acellular cementum leading to premature tooth loss. Given that the skeletal manifestations of genetic ablation of the Phospho1 gene in mice resemble many of the manifestations of HPP, we propose that Phospho1 gene mutations may underlie some cases of "pseudo-HPP" where ALP may be normal to subnormal, but ALPL mutation(s) have not been identified. The goal of this perspective article is to compare and contrast the loss-of-function effects of TNAP and PHOSPHO1 on the dentoalveolar complex to predict the likely dental phenotype in humans that may result from PHOSPHO1 mutations. Potential cases of pseudo-HPP associated with PHOSPHO1 mutations may resist diagnosis, and the dental manifestations could be a key criterion for consideration.

Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy.

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through 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 WT 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 expressions to be downregulated in response to phosphate and PTH. 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. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

Tenascin-C is a driver of inflammation in the DSS model of colitis.

Inflammatory Bowel Disease (IBD) is a grouping of chronic inflammatory disorders of the gut. Tenascin-C is a pro-inflammatory, extracellular matrix protein found upregulated in IBD patients and whilst a pathological driver of chronic inflammation, its precise role in the etiology of IBD is unknown. To study tenascin-C's role in colitis pathology we investigated its expression in a murine model of IBD. Wild-type (WT) or tenascin-C knockout (KO) male mice were left untreated or treated with dextran sodium sulphate (DSS) in their drinking water. Tenascin-C was upregulated at the mRNA level in the colitic distal colon of day eight DSS treated mice, coinciding with significant increases in gross and histological pathology. Immunohistochemistry localized this increase in tenascin-C to areas of inflammation and ulceration in the mucosa. Tenascin-C KO mice exhibited reduced gross pathology in comparison. These differences also extended to the histopathological level where reduced colonic inflammation and tissue damage were found in KO compared to WT mice. Furthermore, the severity of the distal colon lesions were less in the KO mice after 17 days of recovery from DSS treatment. This study demonstrates a role for tenascin-C as a driver of inflammatory pathology in a murine model of IBD and thus suggests neutralizing its pro-inflammatory activity could be explored as a therapeutic strategy for treating IBD.

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

AIMS: Osteoarthritis (OA) is the most prevalent systemic musculoskeletal disorder, characterized by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here, we sought to examine the contribution of accelerated growth to OA 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. METHODS: We examined vulnerability of Socs2 -/- mice to OA following surgical induction of disease (destabilization of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. RESULTS: We observed a significant increase in mean number (wild-type (WT) DMM: 532 (SD 56); WT sham: 495 (SD 45); knockout (KO) DMM: 169 (SD 49); KO sham: 187 (SD 56); p < 0.001) and density (WT DMM: 2.2 (SD 0.9); WT sham: 1.2 (SD 0.5); KO DMM: 13.0 (SD 0.5); KO sham: 14.4 (SD 0.7)) of growth plate bridges in Socs2 -/- in comparison with WT. Histological examination of WT and Socs2 -/- knees revealed articular cartilage damage with DMM in comparison to sham. 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 with WT (p < 0.001). DMM had no effect on the SCB thickness in comparison with sham in either genotype. CONCLUSION: Together, these data suggest that enhanced GH signalling through SOCS2 deletion accelerates growth plate fusion, however this has no effect on OA vulnerability in this model. Cite this article: Bone Joint Res 2022;11(3):162-170.

Combined growth hormone and insulin-like growth factor-1 rescues growth retardation in glucocorticoid-treated mdxmice but does not prevent osteopenia.

Short stature and osteoporosis are common in Duchenne muscular dystrophy (DMD) and its pathophysiology may include an abnormality of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis, which is further exacerbated by long-term glucocorticoid (GC) treatment. Hence, an agent that has anabolic properties and may improve linear growth would be beneficial in this setting and therefore requires further exploration. A 5-week-old x-linked muscular dystrophy (mdx) mice were used as a model of DMD. They were treated with prednisolone ± GH + IGF-1 for 4 weeks and then compared to control mdx mice to allow the study of both growth and skeletal structure. GC reduced cortical bone area, bone fraction, tissue area and volume and cortical bone volume, as assessed by micro computed tomography (CT) In addition, GC caused somatic and skeletal growth retardation but improved grip strength. The addition of GH + IGF-1 therapy rescued the somatic growth retardation and induced additional improvements in grip strength (16.9% increase, P < 0.05 compared to control). There was no improvement in bone microarchitecture (assessed by micro-CT and static histomorphometry) or biomechanical properties (assessed by three-point bending). Serum bone turnover markers (Serum procollagen 1 intact N-terminal propeptide (P1NP), alpha C-terminal telopeptide (αCTX)) also remained unaffected. Further work is needed to maximise these gains before proceeding to clinical trials in boys with DMD.

miR-29b inhibits TGF-ß1-induced cell proliferation in articular chondrocytes.

Transforming growth factor ß1 (TGF-ß1) is a known regulator of chondrocyte proliferation and promotes cartilage repair in osteoarthritis (OA). microRNA-29b-3p (miR-29b-3p) is downregulated by TGF-ß1 and overexpressed in OA cartilage. However, the ability of miR-29b-3p to mediate the chondrocyte pro-proliferative effects of TGF-ß1 is not yet understood. This current study aimed to investigate the effect of miR-29b-3p on TGF-ß1-induced cell proliferation in murine articular chondrocytes. The stimulation of chondrocytes by TGF-ß1 for 24 h resulted in the downregulation of miR-29b-3p expression. The ratio of G0/G1 phase cells decreased in response to TGF-ß1 whereas the ratio of S phase cells was increased. Consistent with this observation, miR-29b-3p overexpression inhibited TGF-ß1's ability to promote the ratio of S phase cells and downregulate the ratio of G0/G1 phase cells. These findings suggest that the downregulation of miR-29b-3p is a likely requirement for TGF-ß1-mediated proliferation of murine articular chondrocytes. Furthermore, implying that miR-29b-3p expression may be involved in reduced chondrocyte proliferation in OA.

A Systems-Level Analysis of Total-Body PET Data Reveals Complex Skeletal 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. 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 in bones. In this report we show that different bones within the skeleton have a unique glucose metabolism and form a complex metabolic network, which could not be identified using single tissue simplistic PET standard uptake values analysis. The application of our approach could reveal new physiological and pathological tissue interactions beyond skeletal metabolism, due to PET radiotracers diversity and the advent of clinical total-body PET systems.

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 PPIs tested inhibited the activity of PHOSPHO1 with IC50 ranging between 0.73 µM for esomeprazole to 19.27 µM for pantoprazole. In contrast, these PPIs did not inhibit TNAP activity. We also found that mineralisation of bone matrix in primary osteoblast cultures was inhibited by several PPIs 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 show for the first time that PPIs 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.

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.

The role of miR-29 family in disease.

MicroRNAs are small noncoding RNAs that can bind to the target sites in the 3'-untranslated region of messenger RNA to regulate posttranscriptional gene expression. Increasing evidence has identified the miR-29 family, consisting of miR-29a, miR-29b-1, miR-29b-2, and miR-29c, as key regulators of a number of biological processes. Moreover, their abnormal expression contributes to the etiology of numerous diseases. In the current review, we aimed to summarize the differential expression patterns and functional roles of the miR-29 family in the etiology of diseases including osteoarthritis, osteoporosis, cardiorenal, and immune disease. Furthermore, we highlight the therapeutic potential of targeting members of miR-29 family in these diseases. We present miR-29s as promoters of osteoblast differentiation and apoptosis but suppressors of chondrogenic and osteoclast differentiation, fibrosis, and T cell differentiation, with clear avenues for therapeutic manipulation. Further research will be crucial to identify the precise mechanism of miR-29 family in these diseases and their full potential in therapeutics.

Ablation of Enpp6 Results in Transient Bone Hypomineralization.

Biomineralization is a fundamental process key to the development of the skeleton. The phosphatase orphan phosphatase 1 (PHOSPHO1), which likely functions within extracellular matrix vesicles, has emerged as a critical regulator of biomineralization. However, the biochemical pathways that generate intravesicular PHOSPHO1 substrates are currently unknown. We hypothesized that the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6) is an upstream source of the PHOSPHO1 substrate. To test this, we characterized skeletal phenotypes of mice homozygous for a targeted deletion of Enpp6 (Enpp6 -/- ). Micro-computed tomography of the trabecular compartment revealed transient hypomineralization in Enpp6 -/- tibias (p < 0.05) that normalized by 12 weeks of age. Whole-bone cortical analysis also revealed significantly hypomineralized proximal bone in 4- but not 12-week-old Enpp6 -/- mice (p < 0.05) compared with WT animals. Back-scattered SEM revealed a failure in 4-week-old trabecular bone of mineralization foci to propagate. Static histomorphometry revealed increased osteoid volume (p > 0.01) and osteoid surface (p < 0.05), which recovered by 12 weeks but was not accompanied by changes in osteoblast or osteoclast number. This study is the first to characterize the skeletal phenotype of Enpp6 -/- mice, revealing transient hypomineralization in young animals compared with WT controls. These data suggest that ENPP6 is important for bone mineralization and may function upstream of PHOSPHO1 as a novel means of generating its substrates inside matrix vesicles. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

Magnetic imaging of subseafloor hydrothermal fluid circulation pathways.

Hydrothermal fluid circulation beneath the seafloor is an important process for chemical and heat transfer between the solid Earth and overlying oceans. Discharge of hydrothermal fluids at the seafloor supports unique biological communities and can produce potentially valuable mineral deposits. Our understanding of the scale and geometry of subseafloor hydrothermal circulation has been limited to numerical simulations and their manifestations on the seafloor. Here, we use magnetic inverse modeling to generate the first three-dimensional empirical model of a hydrothermal convection system. High-temperature fluid-rock reactions associated with fluid circulation destroy magnetic minerals in the Earth's crust, thus allowing magnetic models to trace the fluid's pathways through the seafloor. We present an application of this modeling at a hydrothermally active region of the East Manus Basin.

PHOSPHO1 is a skeletal regulator of insulin resistance and obesity.

BACKGROUND: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear. Here, we probe the mechanism underlying metabolic regulation by analysing Phospho1 mutant mice. RESULTS: Phospho1-/- mice 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 normalised by feeding a 2% choline rich diet resulting in a normalisation in insulin sensitivity and fat mass. CONCLUSION: We show that mice lacking the bone mineralisation enzyme PHOSPHO1 exhibit improved basal glucose homeostasis and resist high-fat-diet-induced weight gain and diabetes. This study identifies PHOSPHO1 as a potential bone-derived therapeutic target for the treatment of obesity and diabetes.