Browsing by Author "Bidwell, Joseph P."
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Item Disabling the Transcription Factor Nmp4 from Osteogenic Precursors Enhances the Skeleton's Response to the Osteoporosis Drug Parathyroid Hormone(2022-08) Atkinson, Emily Grace; Bidwell, Joseph P.; Robling, Alexander G.; Plotkin, Lilian I.; Wallace, Joseph; Organ, Jason M.; Evans-Molina, CarmellaActivation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis. Yet, FDA-approved osteoanabolics, e.g., parathyroid hormone (PTH) have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice, exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues, therefore, to determine whether the suppression of PTHinduced bone formation is cell autonomous, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4fl/fl) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+ to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+ targeting mature osteoblasts and (iii) Dmp1Cre+ to disable Nmp4 in transitional osteocytes. Virgin female Cre+ and Cre- mice (10wks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1–34 at 30μg/kg, or vehicle for 4wks or 7wks. The skeletal response was assessed using dual-energy X-ray absorptiometry, microcomputed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl;Prx1Cre+ mice recapitulated the global Nmp4-/- skeletal phenotype in the femur, i.e., an enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared to their Nmp4fl/fl;Prx1Cre- controls. This was not observed in the spine, where Prx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl;BglapCre+) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl;Dmp1Cre+) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4-/- MSPCs drive the enhanced response to PTH therapy, and Nmp4 has stage-specific effects on osteoanabolism.Item Dissecting the cellular and molecular mechanisms mediating neurofibromatosis type 1 related bone defects(2014-01-03) Rhodes, Steven David; Yang, Feng-Chun; Clapp, D. Wade; Robling, Alexander G.; Bidwell, Joseph P.Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.Item Genome-Wide Mapping and Interrogation of the Nmp4 Antianabolic Bone Axis(Oxford University Press, 2015-09) Childress, Paul; Stayrook, Keith R.; Alvarez, Marta B.; Wang, Zhiping; Shao, Yu; Hernandez-Buquer, Selene; Mack, Justin K.; Grese, Zachary R.; He, Yongzheng; Horan, Daniel; Pavalko, Fredrick M.; Warden, Stuart J.; Robling, Alexander G.; Yang, Feng-Chun; Allen, Matthew R.; Krishnan, Venkatesh; Liu, Yunlong; Bidwell, Joseph P.; Department of Anatomy & Cell Biology, IU School of MedicinePTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4(-/-) mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8(+) T cells. To determine whether the Nmp4(-/-) phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4(-/-) mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4(-/-) bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4(-/-) mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8(+) T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4(-/-) MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4(-/-) MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.Item THE HAND1 LINEAGE REVEALS DISTINCT ROLES FOR HAND FACTORS DURING CARDIOVASCULAR DEVELOPMENT(2011-03-09) Barnes, Ralston M.; Firulli, Anthony B.; Bidwell, Joseph P.; Conway, Simon J.; Field, Loren J.The basic Helix-Loop-Helix (bHLH) transcription factors Hand1 and Hand2 play critical roles in the development of multiple organ systems during embryogenesis. The dynamic expression patterns of these two factors within developing tissues obfuscates their respective unique and redundant organogenic functions. To define cell lineages potentially dependent upon Hand gene expression, we generated a mutant allele in which the coding region of Hand1 is replaced by Cre recombinase. Subsequent Cre-mediated activation of β-galactosidase or eYFP reporter alleles enabled lineage trace analyses that clearly define the fate of Hand1-expressing cells. Comparisons between Hand1 expression and Hand1-lineage greatly refine our understanding of its dynamic spatio-temporal expression domains and raise the possibility of novel Hand1 functions in structures not thought to be Hand1-dependent. To genetically examine functional overlap between Hand1 and Hand2, we conditionally deleted Hand2 from Hand1-expressing cells. Hand2 conditional knockout mice die midgestation and exhibit cardiovascular and limb defects. Moreover, Hand2 lineage-restricted deletion from the proepicardial organ results in defective epicardialization and failure to form coronary arteries. Together, these novel data demonstrate a hierarchal relationship whereby transient Hand1 expression within the septum transversum defines epicardial precursors that depend upon subsequent Hand2 function.Item High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro(Wiley, 2019-05-20) Davis, Hannah M.; Valdez, Sinai; Gomez, Leland; Malicky, Peter; White, Fletcher A.; Subler, Mark A.; Windle, Jolene J.; Bidwell, Joseph P.; Bruzzaniti, Angela; Plotkin, Lilian I.; Anatomy and Cell Biology, School of MedicineOld age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the pro-inflammatory cytokine, high mobility group box1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/MCSF treated bone marrow cells (BMC). In bone marrow macrophages (BMMs), TLR4 inhibition with LPS-RS, but not RAGE inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly effects differentiation by activating TLR4 in BMMs and RAGE in pre-osteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.Item Improving Combination Osteoporosis Therapy in a Preclinical Model of Heightened Osteoanabolism(Oxford University Press, 2017-09-01) Shao, Yu; Hernandez-Buquer, Selene; Childress, Paul; Stayrook, Keith R.; Alvarez, Marta B.; Davis, Hannah; Plotkin, Lilian I.; He, Yongzheng; Condon, Keith W.; Burr, David B.; Warden, Stuart J.; Robling, Alexander G.; Yang, Feng-Chun; Wek, Ronald C.; Allen, Matthew R.; Bidwell, Joseph P.; Medical and Molecular Genetics, School of MedicineCombining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy. Additionally, we inquired whether Nmp4 interferes with anticatabolic efficacy. Wild-type and Nmp4-/- mice were ovariectomized at 12 weeks of age, followed by therapy regimens, administered from 16 to 24 weeks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Anabolic therapeutic efficacy generally corresponded with PTH + RAL = PTH + ZOL > PTH + ALN = PTH > vehicle control. Loss of Nmp4 enhanced femoral trabecular bone increases under PTH + RAL and PTH + ZOL. RAL and ZOL promoted bone restoration, but unexpectedly, loss of Nmp4 boosted RAL-induced increases in femoral trabecular bone. The combination of PTH, RAL, and loss of Nmp4 significantly increased bone marrow osteoprogenitor number, but did not affect adipogenesis or osteoclastogenesis. RAL, but not ZOL, increased osteoprogenitors in both genotypes. Nmp4 status did not influence bone serum marker responses to treatments, but Nmp4-/- mice as a group showed elevated levels of the bone formation marker osteocalcin. We conclude that the heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, in part by increasing hyperanabolic osteoprogenitors. Nmp4 provides a promising target pathway for identifying barriers to pharmacologically induced bone formation.Item Loss of NMP4 improves diverse osteoporosis therapies in a pre-clinical model : skeletal, cellular, genomic and transcriptomic approaches(2017-06-22) Shao, Yu; Bidwell, Joseph P.; Wek, Ronald C.; Mosley, Amber L.; Liu, Yunlong; White, Kenneth E.We have previously demonstrated that disabling the transcription factor Nuclear Matrix Protein 4 (NMP4) improved parathyroid hormone (PTH)-induced trabecular bone gain in ovariectomized (OVX) and healthy mice. Here we evaluated whether loss of Nmp4 enhanced bone restoration in OVX mice under concurrent PTH combination therapies and anti-catabolic mono-therapies. Wild type (WT) and Nmp4-/- mice were OVX at 12wks of age followed by therapy regimens, administered from 16wks-24wks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Generally the PTH+RAL and PTH+ZOL therapies were more effective in restoring bone than the PTH mono-therapy. Loss of Nmp4 further improved the restoration of femoral trabecular bone under these treatments. RAL and ZOL mono-therapies moderately increased bone volume but unexpectedly the Nmp4-/- mice showed an enhanced RAL-induced increase in femoral trabecular bone. Immunohistochemical and flow cytometry analyses of the bone marrow and serum profiling for markers of bone formation and resorption indicated that the heightened osteoanabolism of the Nmp4-/- mice under these diverse osteoporosis treatments was partially attributed to an expansion of the osteoprogenitor pool. To address whether the enhanced bone formation observed in Nmp4-/- mice produced structurally sound tissue, mechanical testing was conducted on the femurs of healthy mice treated with intermittent PTH, RAL mono-therapy, or PTH+RAL. Nmp4-/- femurs showed modestly improved mechanical and material properties. At the cellular level, loss of Nmp4 accelerated mineralization in differentiating mesenchymal stem/progenitor cells (MSPCs). Transcriptomic and biochemical analyses indicated that loss of Nmp4 elevated ribosome biogenesis and expanded the capacity of the endoplasmic reticulum for processing protein. Preliminary data showed that disabling Nmp4 increased both aerobic glycolysis and oxidative phosphorylation in osteoprogenitors, which is an emerging hallmark of anabolic osteogenic cells. Transcriptomic analysis also suggested NMP4 targeted pathways driving bone formation. These included but not limited to BMP, IGF1, TGFβ and Wnt signaling pathways. Finally, transcriptomic profiling revealed that Nmp4-/- MSPCs showed a significant perturbation in numerous immunomodulatory pathways, particularly in the interleukin system. The heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, providing a promising target pathway for identifying barriers to pharmacologically-induced bone formation.Item Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality(APS, 2019) Shao, Yu; Wichern, Emily; Childress, Paul J.; Adaway, Michele; Misra, Jagannath; Klunk, Angela; Burr, David B.; Wek, Ronald C.; Mosley, Amber L.; Liu, Yunlong; Robling, Alexander G.; Brustovetsky, Nickolay; Hamilton, James; Jacobs, Kylie; Vashishth, Deepak; Stayrook, Keith R.; Allen, Matthew R.; Wallace, Joseph M.; Bidwell, Joseph P.; Anatomy and Cell Biology, IU School of MedicineA goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor Nuclear Matrix Protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared to wild type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyper-anabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion- a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. Expression of matrix genes that contribute to bone material-level mechanical properties were elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.Item Megakaryocytes Enhance Mesenchymal Stromal Cells Proliferation and Inhibit Differentiation(Wiley, 2017) Emmakah, Arbi M.; Arman, Hussain E.; Alvarez, Marta B.; Childress, Paul J.; Bidwell, Joseph P.; Goebel, William S.; Chu, Tien-Min Gabriel; Kacena, Melissa A.; Department of Orthopaedic Surgery, School of MedicineMegakaryocytes (MKs) can induce proliferation of calvarial osteoblasts [Ciovacco et al., 2009], but this same phenomenon has not been reported for bone marrow stromal populations from long bones. Bone marrow contains several types of progenitor cells which can be induced to differentiate into multiple cell types. Herein, we examined mesenchymal stromal cell proliferation and osteoblastic differentiation when rabbit or mouse MK were cultured with i) rabbit bone marrow stromal cells, ii) rabbit dental pulp stromal cells, or iii) mouse bone marrow stromal cells. Our results demonstrated that rabbit and mouse stromal cells co-cultured with rabbit MK or mouse MK, have significant increases in proliferation on day 7 by 52%, 46%, and 24%, respectively, compared to cultures without MK. Conversely, alkaline phosphatase (ALP) activity was lower at various time points in these cells when cultures contain MK. Similarly, calcium deposition observed at day 14 rabbit bone marrow and dental pulp stromal cells and day 21 mouse bone marrow stromal cells was 63%, 69%, and 30% lower respectively, when co-cultured with MK. Gene expression studies reveal transcriptional changes broadly consistent with increased proliferation and decreased differentiation. Transcript levels of c-fos (associated with cell proliferation) trended higher after 3, 7, and 14 days in culture. Also, expression of alkaline phosphatase, osteonectin, osterix, and osteopontin, which are markers for osteoblast differentiation, showed MK-induced decreases in a cell type and time dependent manner. Taken together, these data suggest that MK play a role in stromal cell proliferation and differentiation, from multiple sites/locations in multiple species.Item Modulatory actions of HMGB1 on TLR4 and rage in the primary afferent sensory neuron(2015-04-02) Allette, Yohance Mandela; White, Fletcher A.; Bidwell, Joseph P.; Harrington, Maureen A.; Jones, Kathryn J.Damage Associated Molecular Patterns (DAMPs) act largely as endogenous ligands to initiate and maintain the signaling of both inflammatory processes and the acquired immune response. Prolonged action of these endogenous signals are thought to play a significant role sterile inflammation which may be integral to the development of chronic inflammation pathology. HMGB1 (High Mobility Group Box 1) is a highly conserved non-acetylated protein which is among the most important chromatin proteins and serves to organize DNA and regulate transcription. Following stress or injury to the cell, hyperacetylation of lysine residues causes translocation of HMGB1 and eventual release into the extracellular environment where it can take the form of a DAMP and interact with cell types bearing either the Receptor for Advanced Glycation End-products (RAGE) or Toll-Like Receptor 4 (TLR4). Activation of these surface receptors contribute directly to both acute and chronic inflammation. This project investigated the role of HMGB1 through its receptors Receptor for Advanced Glycation End-products (RAGE) and Toll-Like Receptor 4 (TLR4) as it pertained to the development of chronic inflammation and pathology in small diameter, nociceptive sensory neurons. It was demonstrated that the neuronal signaling associated with exposure to HMGB1 is dependent upon the ligands conformational states, as the state dictates its affinity and types of neuronal response. Neuronal activation by bacterial endotoxin or the disulfide state of HMGB1 is dependent on TLR4 and the associated signaling adapter protein, Myeloid differentiation primary response gene 88 (MYD88). Interruption of the receptor-mediated signaling cascade associated with MyD88 was shown to be sufficient to mitigate ligand-dependent neuronal activation and demonstrated significant behavioral findings. Further downstream signaling of HMGB1 in the neuron has yet to be identified, however important steps have been taken to elucidate the role of chronic neuroinflammation with hopes of eventual translational adaptation for clinical therapeutic modalities.