Browsing by Author "Plotkin, Lilian"

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    Examining the Role of Hypothalamus-Derived Neuromedin-U (NMU) in Bone Remodeling of Rats
    (MDPI, 2023-03-31) Born-Evers, Gabriella; Orr, Ashley L.; Hulsey, Elizabeth Q.; Squire, Maria E.; Hum, Julia M.; Plotkin, Lilian; Sampson, Catherine; Hommel, Jonathan; Lowery, Jonathan W.; Anatomy, Cell Biology and Physiology, School of Medicine
    Global loss of the neuropeptide Neuromedin-U (NMU) is associated with increased bone formation and high bone mass in male and female mice by twelve weeks of age, suggesting that NMU suppresses osteoblast differentiation and/or activity in vivo. NMU is highly expressed in numerous anatomical locations including the skeleton and the hypothalamus. This raises the possibility that NMU exerts indirect effects on bone remodeling from an extra-skeletal location such as the brain. Thus, in the present study we used microinjection to deliver viruses carrying short-hairpin RNA designed to knockdown Nmu expression in the hypothalamus of 8-week-old male rats and evaluated the effects on bone mass in the peripheral skeleton. Quantitative RT-PCR confirmed approximately 92% knockdown of Nmu in the hypothalamus. However, after six weeks, micro computed tomography on tibiae from Nmu-knockdown rats demonstrated no significant change in trabecular or cortical bone mass as compared to controls. These findings are corroborated by histomorphometric analyses which indicate no differences in osteoblast or osteoclast parameters between controls and Nmu-knockdown samples. Collectively, these data suggest that hypothalamus-derived NMU does not regulate bone remodeling in the postnatal skeleton. Future studies are necessary to delineate the direct versus indirect effects of NMU on bone remodeling.
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    High glucose alters the secretome of mechanically stimulated osteocyte-like cells affecting osteoclast precursor recruitment and differentiation
    (Wiley, 2017-12) Maycas, Marta; Portolés, Maria Teresa; Matesanz, María Concepción; Buendía, Irene; Linares, Javier; Feito, María José; Arcos, Daniel; Vallet-Regí, María; Plotkin, Lilian; Esbrit, Pedro; Gortázar, Arancha R.; Anatomy and Cell Biology, School of Medicine
    Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and β-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1β MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication.
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    Mechanisms of Palmitate-Induced Lipotoxicity in Osteocytes
    (Elsevier, 2019-10) Al Saedi, Ahmed; Bermeo, Sandra; Plotkin, Lilian; Myers, Damian E.; Duque, Gustavo; Anatomy and Cell Biology, IU School of Medicine
    Background Lipotoxicity is defined as cellular toxicity observed in the presence of an abnormal accumulation of fat and adipocyte-derived factors in non-fat tissues. Palmitic acid (PA), an abundant fatty acid in the bone marrow and particularly in osteoporotic bones, affects osteoblastogenesis and osteoblast function, decreasing their survival through induction of apoptosis and dysfunctional autophagy. In this study, we hypothesized that PA also has a lipotoxic effect on osteocytes in vitro. Methods Initially, we tested the effect of PA on osteocyte-derived factors DKK1, sclerostin and RANKL. Then, we tested whether PA affects survival and causes apoptosis in osteocytes. Subsequently, we investigated the effect of PA on autophagy by detecting the membrane component LC3-II (Western blot) and staining it and lysosomes with Lysotracker Red dye. Results PA decreases RANKL, DKK1 and sclerostin expression in osteocytes. In addition, we found that PA induces apoptosis and reduces osteocyte survival. PA also caused autophagy failure identified by a significant increase in LC3-II and a reduced number of autophagosomes/lysosomes in the cytoplasm. Conclusion In addition to the effects of PA on RANKL, DKK1 and sclerostin expression, which could have significant deleterious impact on bone cell coupling and bone turnover, PA also induced apoptosis and reduced autophagy in osteocytes. Considering that apoptosis and cell dysfunction are two common changes occurring in the osteocytes of osteoporotic bone, our findings suggest that PA could play a role in the pathogenesis of the disease. Suppression of these effects could bring new potential targets for therapeutic interventions in the future.
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    Nmp4 Suppresses Osteoanabolic Potency
    (2023-07) Heim, Crystal Noelle; Bidwell, Joseph; Wek, Ronald; White, Kenneth; Robling, Alexander; Plotkin, Lilian
    Treating severe osteoporosis is limited to two strategies: 1. Stimulation of the parathyroid hormone receptor with analogs for parathyroid hormone (PTH) or parathyroid hormone related peptide, and 2. Stimulation of Wnt signaling via neutralization of sclerostin, a natural inhibitor of this pathway, with a monoclonal antibody (romosozumab-aqqg, Scl-mAb). Despite mobilizing distinct molecular and cellular pathways to stimulate bone gain, all their efficacies rapidly diminish. Identifying the barrier to enhancing potency is a clinical priority. We recently reported that mice harboring the conditional loss of the transcription factor Nmp4 (Nuclear Matrix Protein 4) in mesenchymal stem/progenitor cells (MSPCs) exhibited no measurable baseline effect on the skeleton but showed a significantly enhanced increase in bone formation during PTH therapy. Remarkably, (and unexpectedly) skeletal response to PTH therapy was not improved when Nmp4 was conditionally disabled at the osteoblast or osteocyte stages. For the present study, we hypothesized that the potency of any osteoanabolic drug is pre-programmed (and can be re-programmed) in osteoprogenitors. To test this hypothesis, we treated our global Nmp4-/- mice, various conditional knockout mice, and their controls with Scl-mAb. We observed a similar pattern of improved bone response in our mouse models, which we previously observed with the PTH therapy. That is, removal of Nmp4 early in osteoblast differentiation (MSPC) was required for an exaggerated bone-formation response to Scl-mAb therapy. Disabling Nmp4 later in osteogenic differentiation did not increase the potency of Scl-mAb. These data suggest that Nmp4 is part of a common barrier to improving the efficacy of any osteoanabolic. Potential pathways and actors that comprise the re-programming of Nmp4-/- MSPCs to support the exaggerated osteoanabolic effect on the skeleton are discussed.
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    The Role of Wnt Signaling in Bone Mechanotransduction
    (2019-11) Bullock, Whitney Ann; Robling, Alexander; Bidwell, Joseph; Plotkin, Lilian; Sankar, Uma; White, Kenneth
    The aging US population is experiencing a growing incidence of osteoporosis, characterized by increased fracture risk and low bone mass. In skeletal tissue, canonical Wnt signaling is a critical regulator of bone mass, and dysregulation of the Wnt pathway has been implicated in numerous skeletal displasias. Some components of the Wnt signaling pathway have a clear role in bone homeostasis, particularly in the response of bone to altered mechanical environment. Other pathway components are more poorly defined. One important intracellular signal transduction node in the Wnt cascade is β- catenin, which modulates gene expression and cell-cell junctions, among other functions. During periods of disuse, β-catenin is degraded, leading to inhibition of Wnt targets. Here, I characterize the role of β-catenin in bone during a disuse challenge, using a genetic mouse model expressing an inducible constitively-active mutant form of β- catenin in the osteocyte population. I hypothesize that prevention of β-catenin degradation during disuse will prevent the bone wasting effects of mechanodeprivation. As a second goal, I focus on upstream (membrane-bound) modulation of Wnt. Here, I investigate the low-density lipoprotein receptor-related receptor 4 (Lrp4), in the regulation of bone mass and mechanotransduction. I generated an Lrp4 knockin mouse model harboring a missense mutation found among human patients with abnormally high bone mass. I hypothesize that the mutation compromises sclerostin action on bone cells. Understanding how each of these components of the Wnt signaling pathway interact, may lead to novel therapeutic targets for treatment of bone diseases.