Browsing by Author "Prideaux, Matthew"
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Item Age and sex effects on FGF23-mediated response to mild phosphate challenge(Elsevier, 2021) Tippen, Samantha P.; Noonan, Megan L.; Ni, Pu; Metzger, Corinne E.; Swallow, Elizabeth A.; Sacks, Spencer A.; Chen, Neal X.; Thompson, William R.; Prideaux, Matthew; Atkins, Gerald J.; Moe, Sharon M.; Allen, Matthew R.; White, Kenneth E.; Medical and Molecular Genetics, School of MedicineBackground: During aging, there is a normal and mild loss in kidney function that leads to abnormalities of the kidney-bone metabolic axis. In the setting of increased phosphorus intake, hyperphosphatemia can occur despite increased concentrations of the phosphaturic hormone FGF23. This is likely from decreased expression of the FGF23 co-receptor Klotho (KL) with age; however, the roles of age and sex in the homeostatic responses to mild phosphate challenges remain unclear. Methods: Male and female 16-week and 78-week mice were placed on either normal grain-based chow or casein (higher bioavailable phosphate) diets for 8 weeks. Gene expression, serum biochemistries, micro-computed tomography, and skeletal mechanics were used to assess the impact of mild phosphate challenge on multiple organ systems. Cell culture of differentiated osteoblast/osteocytes was used to test mechanisms driving key outcomes. Results: Aging female mice responded to phosphate challenge by significantly elevating serum intact FGF23 (iFGF23) versus control diet; males did not show this response. Male mice, regardless of age, exhibited higher kidney KL mRNA with similar phosphate levels across both sexes. However, males and females had similar blood phosphate, calcium, and creatinine levels irrespective of age, suggesting that female mice upregulated FGF23 to maintain blood phosphorus, and compromised renal function could not explain the increased serum iFGF23. The 17β-estradiol levels were not different between groups, and in vivo bone steroid receptor (estrogen receptor 1 [Esr1], estrogen receptor 2 [Esr2], androgen receptor [Ar]) expression was not different by age, sex, or diet. Trabecular bone volume was higher in males but decreased with both age and phosphate challenge in both sexes. Cortical porosity increased with age in males but not females. In vitro studies demonstrated that 17β-estradiol treatment upregulated FGF23 and Esr2 mRNAs in a dose-dependent manner. Conclusions: Our study demonstrates that aging female mice upregulate FGF23 to a greater degree during a mild phosphate challenge to maintain blood phosphorus versus young female and young/old male mice, potentially due to direct estradiol effects on osteocytes. Thus, the control of phosphate intake during aging could have modifiable outcomes for FGF23-related phenotypes.Item Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models(Wiley, 2020-03) Sego, T. J.; Prideaux, Matthew; Sterner, Jane; McCarthy, Brian Paul; Li, Ping; Bonewald, Lynda F.; Ekser, Burcin; Tovar, Andres; Smith, Lester Jeshua; Anatomy and Cell Biology, School of MedicineNatural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient‐rich oxygenated blood through the vasculature to support cell metabolism within most cell‐dense tissues. Since scaffold‐free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue‐like structures, we generated a generalizable biofabrication method resulting in self‐supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO‐A5 osteoblast‐based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.Item Deletion of the auxiliary α2δ1 voltage sensitive calcium channel subunit in osteocytes and late-stage osteoblasts impairs femur strength and load-induced bone formation in male mice(Oxford, 2024) Wright, Christian S.; Lewis, Karl J.; Semon, Katelyn; Yi, Xin; Reyes Fernandez, Perla C.; Rust, Katie; Prideaux, Matthew; Schneider, Artur; Pederson, Molly; Deosthale, Padmini; Plotkin, Lilian I.; Hum, Julia M.; Sankar, Uma; Farach-Carson, Mary C.; Robling, Alexander G.; Thompson, William R.; Health Sciences, School of Health and Human SciencesOsteocytes sense and respond to mechanical force by controlling the activity of other bone cells. However, the mechanisms by which osteocytes sense mechanical input and transmit biological signals remain unclear. Voltage-sensitive calcium channels (VSCCs) regulate calcium (Ca2+) influx in response to external stimuli. Inhibition or deletion of VSCCs impairs osteogenesis and skeletal responses to mechanical loading. VSCC activity is influenced by its auxiliary subunits, which bind the channel’s α1 pore-forming subunit to alter intracellular Ca2+ concentrations. The α2δ1 auxiliary subunit associates with the pore-forming subunit via a glycosylphosphatidylinositol anchor and regulates the channel’s calcium-gating kinetics. Knockdown of α2δ1 in osteocytes impairs responses to membrane stretch, and global deletion of α2δ1 in mice results in osteopenia and impaired skeletal responses to loading in vivo. Therefore, we hypothesized that the α2δ1 subunit functions as a mechanotransducer, and its deletion in osteocytes would impair skeletal development and load-induced bone formation. Mice (C57BL/6) with LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, were crossed with mice expressing Cre under the control of the Dmp1 promoter (10 kb). Deletion of α2δ1 in osteocytes and late-stage osteoblasts decreased femoral bone quantity (P < .05) by DXA, reduced relative osteoid surface (P < .05), and altered osteoblast and osteocyte regulatory gene expression (P < .01). Cacna2d1f/f, Cre + male mice displayed decreased femoral strength and lower 10-wk cancellous bone in vivo micro-computed tomography measurements at the proximal tibia (P < .01) compared to controls, whereas Cacna2d1f/f, Cre + female mice showed impaired 20-wk cancellous and cortical bone ex vivo micro-computed tomography measurements (P < .05) vs controls. Deletion of α2δ1 in osteocytes and late-stage osteoblasts suppressed load-induced calcium signaling in vivo and decreased anabolic responses to mechanical loading in male mice, demonstrating decreased mechanosensitivity. Collectively, the α2δ1 auxiliary subunit is essential for the regulation of osteoid-formation, femur strength, and load-induced bone formation in male mice.Item Differential Iron Requirements for Osteoblast and Adipocyte Differentiation(Wiley, 2021-07-26) Edwards, Daniel F., III.; Miller, Christopher J.; Quintana-Martinez, Arelis; Wright, Christian S.; Prideaux, Matthew; Atkins, Gerald J.; Thompson, William R.; Clinkenbeard, Erica L.; Medical and Molecular Genetics, School of MedicineBone marrow mesenchymal progenitor cells are precursors for various cell types including osteoblasts, adipocytes, and chondrocytes. The external environment and signals act to direct the pathway of differentiation. Importantly, situations such as aging and chronic kidney disease display alterations in the balance of osteoblast and adipocyte differentiation, adversely affecting bone integrity. Iron deficiency, which can often occur during aging and chronic kidney disease, is associated with reduced bone density. The purpose of this study was to assess the effects of iron deficiency on the capacity of progenitor cell differentiation pathways. Mouse and human progenitor cells, differentiated under standard osteoblast and adipocyte protocols in the presence of the iron chelator deferoxamine (DFO), were used. Under osteogenic conditions, 5μM DFO significantly impaired expression of critical osteoblast genes, including osteocalcin, type 1 collagen, and dentin matrix protein 1. This led to a reduction in alkaline phosphatase activity and impaired mineralization. Despite prolonged exposure to chronic iron deficiency, cells retained viability as well as normal hypoxic responses with significant increases in transferrin receptor and protein accumulation of hypoxia inducible factor 1α. Similar concentrations of DFO were used when cells were maintained in adipogenic conditions. In contrast to osteoblast differentiation, DFO modestly suppressed adipocyte gene expression of peroxisome-proliferating activated receptor gamma, lipoprotein lipase, and adiponectin at earlier time points with normalization at later stages. Lipid accumulation was also similar in all conditions. These data suggest the critical importance of iron in osteoblast differentiation, and as long as the external stimuli are present, iron deficiency does not impede adipogenesis.Item Generation of two multipotent mesenchymal progenitor cell lines capable of osteogenic, mature osteocyte, adipogenic, and chondrogenic differentiation(Springer Nature, 2021-11-19) Prideaux, Matthew; Wright, Christian S.; Noonan, Megan L.; Yi, Xin; Clinkenbeard, Erica L.; Mevel, Elsa; Wheeler, Jonathan A.; Byers, Sharon; Wijenayaka, Asiri R.; Gronthos, Stan; Sankar, Uma; White, Kenneth E.; Atkins, Gerald J.; Thompson, William R.; Physical Therapy, School of Health and Human SciencesMesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with addition of parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C-terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D (1,25D). Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilaginous genes including aggrecan, Sox9, and Comp. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.Item The HIF-PHI BAY 85–3934 (Molidustat) Improves Anemia and Is Associated With Reduced Levels of Circulating FGF23 in a CKD Mouse Model(Wiley, 2021-06) Noonan, Megan L.; Ni, Pu; Agoro, Rafiou; Sacks, Spencer A.; Swallow, Elizabeth A.; Wheeler, Jonathan A.; Clinkenbeard, Erica L.; Capitano, Maegan L.; Prideaux, Matthew; Atkins, Gerald J.; Thompson, William R.; Allen, Matthew R.; Broxmeyer, Hal E.; White, Kenneth E.; Medical and Molecular Genetics, School of MedicineFibroblast growth factor-23 (FGF23) is a critical factor in chronic kidney disease (CKD), with elevated levels causing alterations in mineral metabolism and increased odds for mortality. Patients with CKD develop anemia as the kidneys progressively lose the ability to produce erythropoietin (EPO). Anemia is a potent driver of FGF23 secretion; therefore, a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) currently in clinical trials to elevate endogenous EPO to resolve anemia was tested for effects on iron utilization and FGF23-related parameters in a CKD mouse model. Mice were fed either a casein control diet or an adenine-containing diet to induce CKD. The CKD mice had markedly elevated iFGF23 and blood urea nitrogen (BUN), hyperphosphatemia, and anemia. Cohorts of mice were then treated with a patient-equivalent dose of BAY 85-3934 (BAY; Molidustat), which elevated EPO and completely resolved aberrant complete blood counts (CBCs) in the CKD mice. iFGF23 was elevated in vehicle-treated CKD mice (120-fold), whereas circulating iFGF23 was significantly attenuated (>60%) in the BAY-treated CKD mice. The BAY-treated mice with CKD also had reduced BUN, but there was no effect on renal vitamin D metabolic enzyme expression. Consistent with increased EPO, bone marrow Erfe, Transferrin receptor (Tfrc), and EpoR mRNAs were increased in BAY-treated CKD mice, and in vitro hypoxic marrow cultures increased FGF23 with direct EPO treatment. Liver Bmp-6 and hepcidin expression were downregulated in all BAY-treated groups. Femur trabecular parameters and cortical porosity were not worsened with BAY administration. In vitro, differentiated osteocyte-like cells exposed to an iron chelator to simulate iron depletion/hypoxia increased FGF23; repletion with holo-transferrin completely suppressed FGF23 and normalized Tfrc1. Collectively, these results support that resolving anemia using a HIF-PHI during CKD was associated with lower BUN and reduced FGF23, potentially through direct restoration of iron utilization, thus providing modifiable outcomes beyond improving anemia for this patient population. © 2021 American Society for Bone and Mineral Research (ASBMR).Item A Novel Osteogenic Cell Line that Differentiates into GFP‐Tagged Osteocytes and forms Mineral with a Bone‐like Lacunocanalicular Structure(Wiley, 2019) Wang, Kun; Le, Lisa; Chun, Brad M.; Tiede-Lewis, LeAnn M.; Shiflett, Lora A.; Prideaux, Matthew; Campos, Richard S.; Veno, Patricia A.; Xie, Yixia; Dusevich, Vladimir; Bonewald, Lynda F.; Dallas, Sarah L.; Anatomy and Cell Biology, IU School of MedicineOsteocytes, the most abundant cells in bone, were once thought to be inactive but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane‐targeted GFP driven by the Dmp1‐promoter. One of these clones, OmGFP66, has unique properties compared to previous osteogenic and osteocyte cell models and forms 3‐dimensional mineralized bone‐like structures, containing highly dendritic GFP‐positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone‐like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase, produce a mineralized type‐I‐collagen matrix and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23 and the mature osteocyte marker, Sost. They also express RankL, Opg and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1 and Opg and upregulation of RankL and E11/gp38. Live‐cell imaging revealed the dynamic process by which OmGFP66 bone‐like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule‐like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment.Item Osteocyte RANKL Drives Bone Resorption in Mouse Ligature‐Induced Periodontitis(Oxford University Press, 2023) Kittaka, Mizuho; Yoshimoto, Tetsuya; Levitan, Marcus E.; Urata, Rina; Choi, Roy B.; Teno, Yayoi; Xie, Yixia; Kitase, Yukiko; Prideaux, Matthew; Dallas, Sarah L.; Robling, Alexander G.; Ueki, Yasuyoshi; Biomedical and Applied Sciences, School of DentistryMouse ligature-induced periodontitis (LIP) has been used to study bone loss in periodontitis. However, the role of osteocytes in LIP remains unclear. Furthermore, there is no consensus on the choice of alveolar bone parameters and time points to evaluate LIP. Here, we investigated the dynamics of changes in osteoclastogenesis and bone volume (BV) loss in LIP over 14 days. Time-course analysis revealed that osteoclast induction peaked on days 3 and 5, followed by the peak of BV loss on day 7. Notably, BV was restored by day 14. The bone formation phase after the bone resorption phase was suggested to be responsible for the recovery of bone loss. Electron microscopy identified bacteria in the osteocyte lacunar space beyond the periodontal ligament (PDL) tissue. We investigated how osteocytes affect bone resorption of LIP and found that mice lacking receptor activator of NF-κB ligand (RANKL), predominantly in osteocytes, protected against bone loss in LIP, whereas recombination activating 1 (RAG1)-deficient mice failed to resist it. These results indicate that T/B cells are dispensable for osteoclast induction in LIP and that RANKL from osteocytes and mature osteoblasts regulates bone resorption by LIP. Remarkably, mice lacking the myeloid differentiation primary response gene 88 (MYD88) did not show protection against LIP-induced bone loss. Instead, osteocytic cells expressed nucleotide-binding oligomerization domain containing 1 (NOD1), and primary osteocytes induced significantly higher Rankl than primary osteoblasts when stimulated with a NOD1 agonist. Taken together, LIP induced both bone resorption and bone formation in a stage-dependent manner, suggesting that the selection of time points is critical for quantifying bone loss in mouse LIP. Pathogenetically, the current study suggests that bacterial activation of osteocytes via NOD1 is involved in the mechanism of osteoclastogenesis in LIP. The NOD1-RANKL axis in osteocytes may be a therapeutic target for bone resorption in periodontitis.Item Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection(Springer Nature, 2022-11-04) Yoshimoto, Tetsuya; Kittaka, Mizuho; Doan, Andrew Anh Phuong; Urata, Rina; Prideaux, Matthew; Rojas, Roxana E.; Harding, Clifford V.; Boom, W. Henry; Bonewald, Lynda F.; Greenfield, Edward M.; Ueki, Yasuyoshi; Biomedical Sciences and Comprehensive Care, School of DentistryThe impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.Item Sclerostin Directly Stimulates Osteocyte Synthesis of Fibroblast Growth Factor-23(Springer, 2021) Ito, Nobuaki; Prideaux, Matthew; Wijenayaka, Asiri R.; Yang, Dongqing; Ormsby, Renee T.; Bonewald, Lynda F.; Atkins, Gerald J; Medicine, School of MedicineOsteocyte produced fibroblast growth factor 23 (FGF23) is the key regulator of serum phosphate (Pi) homeostasis. The interplay between parathyroid hormone (PTH), FGF23 and other proteins that regulate FGF23 production and serum Pi levels is complex and incompletely characterised. Evidence suggests that the protein product of the SOST gene, sclerostin (SCL), also a PTH target and also produced by osteocytes, plays a role in FGF23 expression, however the mechanism for this effect is unclear. Part of the problem of understanding the interplay of these mediators is the complex multi-organ system that achieves Pi homeostasis in vivo. In the current study, we sought to address this using a cell line model of the osteocyte, IDG-SW3, known to express FGF23 at both the mRNA and protein levels. In cultures of differentiated IDG-SW3 cells, both PTH1-34 and recombinant human (rh) SCL remarkably induced Fgf23 mRNA expression dose-dependently within 3 h. Both rhPTH1-34 and rhSCL also strongly induced C-terminal FGF23 protein secretion. Secreted intact FGF23 levels remained unchanged, consistent with constitutive post-translational cleavage of FGF23 in this cell model. Both rhPTH1-34 and rhSCL treatments significantly suppressed mRNA levels of Phex, Dmp1 and Enpp1 mRNA, encoding putative negative regulators of FGF23 levels, and induced Galnt3 mRNA expression, encoding N-acetylgalactosaminyl-transferase 3 (GalNAc-T3), which protects FGF23 from furin-like proprotein convertase-mediated cleavage. The effect of both rhPTH1-34 and rhSCL was antagonised by pre-treatment with the NF-κβ signalling inhibitors, BAY11 and TPCK. RhSCL also stimulated FGF23 mRNA expression in ex vivo cultures of human bone. These findings provide evidence for the direct regulation of FGF23 expression by sclerostin. Locally expressed sclerostin via the induction of FGF23 in osteocytes thus has the potential to contribute to the regulation of Pi homeostasis.