Browsing by Author "Noonan, Megan L."
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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 Erythropoietin and a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHDi) lowers FGF23 in a model of chronic kidney disease (CKD)(Wiley, 2020-03-31) Noonan, Megan L.; Clinkenbeard, Erica L.; Ni, Pu; Swallow, Elizabeth A.; Tippen, Samantha P.; Agoro, Rafiou; Allen, Matthew R.; White, Kenneth E.; Medical and Molecular Genetics, School of MedicineIron‐deficiency anemia is a potent stimulator of the phosphaturic hormone Fibroblast growth factor‐23 (FGF23). Anemia, elevated FGF23, and elevated serum phosphate are significant mortality risk factors for patients with chronic kidney disease (CKD). However, the contribution of anemia to overall circulating FGF23 levels in CKD is not understood. Our goal was to investigate the normalization of iron handling in a CKD model using the erythropoiesis stimulating agents (ESAs) Erythropoietin (EPO) and the hypoxia‐inducible factor prolyl hydroxylase inhibitor (HIF‐PHDi) FG‐4592, on the production of, and outcomes associated with, changes in bioactive, intact FGF23 (“iFGF23”). Our hypothesis was that rescuing the prevailing anemia in a model of CKD would reduce circulating FGF23. Wild‐type mice were fed an adenine‐containing diet to induce CKD, then injected with EPO or FG‐4592. The mice with CKD were anemic, and EPO improved red blood cell indices, whereas FG‐4592 increased serum EPO and bone marrow erythroferrone (Erfe), and decreased liver ferritin, bone morphogenic protein‐6 (Bmp‐6), and hepcidin mRNAs. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by >70% after delivery of either ESA, with no changes in serum phosphate. ESA treatment also reduced renal fibrosis markers, as well as increased Cyp27b1 and reduced Cyp24a1 mRNA expression. Thus, improvement of iron utilization in a CKD model using EPO and a HIF‐PHDi significantly reduced iFGF23, demonstrating that anemia is a primary driver of FGF23, and that management of iron utilization in patients with CKD may translate to modifiable outcomes in mineral metabolism.Item FGF23 Synthesis and Activity(Springer, 2019-03) Noonan, Megan L.; White, Kenneth E.; Medical and Molecular Genetics, School of MedicinePurpose of review: The phosphaturic hormone FGF23 is produced primarily in osteoblasts/osteocytes and is known to respond to increases in serum phosphate and 1,25(OH)2 vitamin D (1,25D). Novel regulators of FGF23 were recently identified, and may help explain the pathophysiologies of several diseases. This review will focus on recent studies examining the synthesis and actions of FGF23. Recent findings: The synthesis of FGF23 in response to 1,25D is similar to other steroid hormone targets, but the cellular responses to phosphate remain largely unknown. The activity of intracellular processing genes control FGF23 glycosylation and phosphorylation, providing critical functions in determining the serum levels of bioactive FGF23. The actions of FGF23 largely occur through its co-receptor αKlotho (KL) under normal circumstances, but FGF23 has KL-independent activity during situations of high concentrations. Summary: Recent work regarding FGF23 synthesis and bioactivity, as well as considerations for diseases of altered phosphate balance will be reviewed.Item Gabapentin Disrupts Binding of Perlecan to the α2δ1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation(MDPI, 2022-12-12) Reyes Fernandez, Perla C.; Wright, Christian S.; Masterson, Adrianna N.; Yi, Xin; Tellman, Tristen V.; Bonteanu, Andrei; Rust, Katie; Noonan, Megan L.; White, Kenneth E.; Lewis, Karl J.; Sankar, Uma; Hum, Julia M.; Bix, Gregory; Wu, Danielle; Robling, Alexander G.; Sardar, Rajesh; Farach-Carson, Mary C.; Thompson, William R.; Physical Therapy, School of Health and Human SciencesOur understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified "tethering elements" (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular α2δ1 subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and α2δ1 along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between α2δ1 and PLN domains and demonstrated for the first time that α2δ1 strongly associates with PLN via its domain III. Furthermore, α2δ1 is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLN::α2δ1 binding in vitro, and GBP treatment in vivo results in impaired bone mechanosensation. Our work identified a novel mechanosensory complex within osteocytes composed of PLN and α2δ1, necessary for bone force transmission and sensitive to the drug GBP.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 Increased FGF23 protects against detrimental cardio-renal consequences during elevated blood phosphate in CKD(American Society for Clinical Investigation, 2019-02-21) Clinkenbeard, Erica L.; Noonan, Megan L.; Thomas, Joseph C.; Ni, Pu; Hum, Julia M.; Aref, Mohammad; Swallow, Elizabeth A.; Moe, Sharon M.; Allen, Matthew R.; White, Kenneth E.; Medical and Molecular Genetics, School of MedicineThe phosphaturic hormone FGF23 is elevated in chronic kidney disease (CKD). The risk of premature death is substantially higher in the CKD patient population, with cardiovascular disease (CVD) as the leading mortality cause at all stages of CKD. Elevated FGF23 in CKD has been associated with increased odds for all-cause mortality; however, whether FGF23 is associated with positive adaptation in CKD is unknown. To test the role of FGF23 in CKD phenotypes, a late osteoblast/osteocyte conditional flox-Fgf23 mouse (Fgf23fl/fl/Dmp1-Cre+/-) was placed on an adenine-containing diet to induce CKD. Serum analysis showed casein-fed Cre+ mice had significantly higher serum phosphate and blood urea nitrogen (BUN) versus casein diet and Cre- genotype controls. Adenine significantly induced serum intact FGF23 in the Cre- mice over casein-fed mice, whereas Cre+ mice on adenine had 90% reduction in serum intact FGF23 and C-terminal FGF23 as well as bone Fgf23 mRNA. Parathyroid hormone was significantly elevated in mice fed adenine diet regardless of genotype, which significantly enhanced midshaft cortical porosity. Echocardiographs of the adenine-fed Cre+ hearts revealed profound aortic calcification and cardiac hypertrophy versus diet and genotype controls. Thus, these studies demonstrate that increased bone FGF23, although associated with poor outcomes in CKD, is necessary to protect against the cardio-renal consequences of elevated tissue phosphate.Item Linking Osteocyte Oxygen Sensing and Biomineralization via FGG23: Implications for Chronic Kidney Disease(2022-05) Noonan, Megan L.; White, Kenneth E.; Kota, Janaiah; Graham, Brett H.; Thompson, William R.FGF23 is an osteocyte produced hormone necessary for maintaining systemic phosphate handling, and thus bone structure and function in both rare and common disorders such as chronic kidney disease (CKD). FGF23 is a critical factor in CKD, with elevated levels causing alterations in mineral metabolism and increased odds for mortality. However, the mechanisms directing the production of key modulators of skeletal homeostasis and biomineralization within osteocytes, and how this is altered in chronic kidney disease, remain unclear. The experimental focus of this dissertation was to dissect the molecular systems and role of oxygen sensing in the regulated production of FGF23. In CKD, up to 75% of patients have anemia and concomitant marked elevations in FGF23, increasing mortality odds. Anemia is a potent driver of FGF23 secretion, therefore, current and emerging therapies, including recombinant EPO and the hypoxia inducible factorprolyl hydroxylase inhibitors (HIF-PHI) FG-4592 and BAY 85-3934, were used to improve anemia in the adenine diet-induced mouse model of CKD. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by 65-85% after delivery of EPO or HIF-PHI, with no changes in serum phosphate. This was associated with improved systemic iron utilization and reductions in mRNA markers of renal fibrosis. In osteocyte-like cell cultures treated with HIF-PHI, integrative RNAseq and ATACseq analysis identified candidate genes upregulated in response to mimicked hypoxia, concomitant with elevated Fgf23 expression. These genes were found to be downregulated in CKD bone, therefore, knock-out cells were generated using CRISPR/Cas9 technology. These cells were found to be functionally similar to in vivo conditional knockout models that have enhanced bone mass and elevated FGF23. Taken together, these results further define novel factors involved in the regulation of FGF23 and identify new therapeutic targets.Item Loss of the auxiliary α2δ1 voltage-sensitive calcium channel subunit impairs bone formation and anabolic responses to mechanical loading(Oxford University Press, 2024-01-10) Kelly, Madison M.; Sharma, Karan; Wright, Christian S.; Yi, Xin; Reyes Fernandez, Perla C.; Gegg, Aaron T.; Gorrell, Taylor A.; Noonan, Megan L.; Baghdady, Ahmed; Sieger, Jacob A.; Dolphin, Annette C.; Warden, Stuart J.; Deosthale, Padmini; Plotkin, Lilian I.; Sankar, Uma; Hum, Julia M.; Robling, Alexander G.; Farach-Carson, Mary C.; Thompson, William R.; Physical Therapy, School of Health and Human SciencesVoltage-sensitive calcium channels (VSCCs) influence bone structure and function, including anabolic responses to mechanical loading. While the pore-forming (α1) subunit of VSCCs allows Ca2+ influx, auxiliary subunits regulate the biophysical properties of the pore. The α2δ1 subunit influences gating kinetics of the α1 pore and enables mechanically induced signaling in osteocytes; however, the skeletal function of α2δ1 in vivo remains unknown. In this work, we examined the skeletal consequences of deleting Cacna2d1, the gene encoding α2δ1. Dual-energy X-ray absorptiometry and microcomputed tomography imaging demonstrated that deletion of α2δ1 diminished bone mineral content and density in both male and female C57BL/6 mice. Structural differences manifested in both trabecular and cortical bone for males, while the absence of α2δ1 affected only cortical bone in female mice. Deletion of α2δ1 impaired skeletal mechanical properties in both sexes, as measured by three-point bending to failure. While no changes in osteoblast number or activity were found for either sex, male mice displayed a significant increase in osteoclast number, accompanied by increased eroded bone surface and upregulation of genes that regulate osteoclast differentiation. Deletion of α2δ1 also rendered the skeleton insensitive to exogenous mechanical loading in males. While previous work demonstrates that VSCCs are essential for anabolic responses to mechanical loading, the mechanism by which these channels sense and respond to force remained unclear. Our data demonstrate that the α2δ1 auxiliary VSCC subunit functions to maintain baseline bone mass and strength through regulation of osteoclast activity and also provides skeletal mechanotransduction in male mice. These data reveal a molecular player in our understanding of the mechanisms by which VSCCs influence skeletal adaptation.Item Osteocyte Egln1/Phd2 links oxygen sensing and biomineralization via FGF23(Springer Nature, 2023-01-18) Noonan, Megan L.; Ni, Pu; Solis, Emmanuel; Marambio, Yamil G.; Agoro, Rafiou; Chu, Xiaona; Wang, Yue; Gao, Hongyu; Xuei, Xiaoling; Clinkenbeard, Erica L.; Jiang, Guanglong; Liu, Sheng; Stegen, Steve; Carmeliet, Geert; Thompson, William R.; Liu, Yunlong; Wan, Jun; White, Kenneth E.; Medical and Molecular Genetics, School of MedicineOsteocytes act within a hypoxic environment to control key steps in bone formation. FGF23, a critical phosphate-regulating hormone, is stimulated by low oxygen/iron in acute and chronic diseases, however the molecular mechanisms directing this process remain unclear. Our goal was to identify the osteocyte factors responsible for FGF23 production driven by changes in oxygen/iron utilization. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHI) which stabilize HIF transcription factors, increased Fgf23 in normal mice, as well as in osteocyte-like cells; in mice with conditional osteocyte Fgf23 deletion, circulating iFGF23 was suppressed. An inducible MSC cell line ('MPC2') underwent FG-4592 treatment and ATACseq/RNAseq, and demonstrated that differentiated osteocytes significantly increased HIF genomic accessibility versus progenitor cells. Integrative genomics also revealed increased prolyl hydroxylase Egln1 (Phd2) chromatin accessibility and expression, which was positively associated with osteocyte differentiation. In mice with chronic kidney disease (CKD), Phd1-3 enzymes were suppressed, consistent with FGF23 upregulation in this model. Conditional loss of Phd2 from osteocytes in vivo resulted in upregulated Fgf23, in line with our findings that the MPC2 cell line lacking Phd2 (CRISPR Phd2-KO cells) constitutively activated Fgf23 that was abolished by HIF1α blockade. In vitro, Phd2-KO cells lost iron-mediated suppression of Fgf23 and this activity was not compensated for by Phd1 or -3. In sum, osteocytes become adapted to oxygen/iron sensing during differentiation and are directly sensitive to bioavailable iron. Further, Phd2 is a critical mediator of osteocyte FGF23 production, thus our collective studies may provide new therapeutic targets for skeletal diseases involving disturbed oxygen/iron sensing.