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1.
Int J Mol Sci ; 25(8)2024 Apr 13.
Article in English | MEDLINE | ID: mdl-38673888

ABSTRACT

Urease, a pivotal enzyme in nitrogen metabolism, plays a crucial role in various microorganisms, including the pathogenic Helicobacter pylori. Inhibiting urease activity offers a promising approach to combating infections and associated ailments, such as chronic kidney diseases and gastric cancer. However, identifying potent urease inhibitors remains challenging due to resistance issues that hinder traditional approaches. Recently, machine learning (ML)-based models have demonstrated the ability to predict the bioactivity of molecules rapidly and effectively. In this study, we present ML models designed to predict urease inhibitors by leveraging essential physicochemical properties. The methodological approach involved constructing a dataset of urease inhibitors through an extensive literature search. Subsequently, these inhibitors were characterized based on physicochemical properties calculations. An exploratory data analysis was then conducted to identify and analyze critical features. Ultimately, 252 classification models were trained, utilizing a combination of seven ML algorithms, three attribute selection methods, and six different strategies for categorizing inhibitory activity. The investigation unveiled discernible trends distinguishing urease inhibitors from non-inhibitors. This differentiation enabled the identification of essential features that are crucial for precise classification. Through a comprehensive comparison of ML algorithms, tree-based methods like random forest, decision tree, and XGBoost exhibited superior performance. Additionally, incorporating the "chemical family type" attribute significantly enhanced model accuracy. Strategies involving a gray-zone categorization demonstrated marked improvements in predictive precision. This research underscores the transformative potential of ML in predicting urease inhibitors. The meticulous methodology outlined herein offers actionable insights for developing robust predictive models within biochemical systems.


Subject(s)
Enzyme Inhibitors , Machine Learning , Urease , Urease/antagonists & inhibitors , Urease/chemistry , Urease/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Helicobacter pylori/enzymology , Helicobacter pylori/drug effects , Algorithms , Humans
2.
Int J Mol Sci ; 25(4)2024 Feb 06.
Article in English | MEDLINE | ID: mdl-38396647

ABSTRACT

Helicobacter pylori (Hp) infections pose a global health challenge demanding innovative therapeutic strategies by which to eradicate them. Urease, a key Hp virulence factor hydrolyzes urea, facilitating bacterial survival in the acidic gastric environment. In this study, a multi-methodological approach combining pharmacophore- and structure-based virtual screening, molecular dynamics simulations, and MM-GBSA calculations was employed to identify novel inhibitors for Hp urease (HpU). A refined dataset of 8,271,505 small molecules from the ZINC15 database underwent pharmacokinetic and physicochemical filtering, resulting in 16% of compounds for pharmacophore-based virtual screening. Molecular docking simulations were performed in successive stages, utilizing HTVS, SP, and XP algorithms. Subsequent energetic re-scoring with MM-GBSA identified promising candidates interacting with distinct urease variants. Lys219, a residue critical for urea catalysis at the urease binding site, can manifest in two forms, neutral (LYN) or carbamylated (KCX). Notably, the evaluated molecules demonstrated different interaction and energetic patterns in both protein variants. Further evaluation through ADMET predictions highlighted compounds with favorable pharmacological profiles, leading to the identification of 15 candidates. Molecular dynamics simulations revealed comparable structural stability to the control DJM, with candidates 5, 8 and 12 (CA5, CA8, and CA12, respectively) exhibiting the lowest binding free energies. These inhibitors suggest a chelating capacity that is crucial for urease inhibition. The analysis underscores the potential of CA5, CA8, and CA12 as novel HpU inhibitors. Finally, we compare our candidates with the chemical space of urease inhibitors finding physicochemical similarities with potent agents such as thiourea.


Subject(s)
Helicobacter pylori , Helicobacter pylori/metabolism , Urease/metabolism , Molecular Dynamics Simulation , Molecular Docking Simulation , Urea/pharmacology
3.
Polymers (Basel) ; 15(14)2023 Jul 12.
Article in English | MEDLINE | ID: mdl-37514411

ABSTRACT

Pesticides have a significant negative impact on the environment, non-target organisms, and human health. To address these issues, sustainable pest management practices and government regulations are necessary. However, biotechnology can provide additional solutions, such as the use of polyelectrolyte complexes to encapsulate and remove pesticides from water sources. We introduce a computational methodology to evaluate the capture capabilities of Calcium-Alginate-Chitosan (CAC) nanoparticles for a broad range of pesticides. By employing ensemble-docking and molecular dynamics simulations, we investigate the intermolecular interactions and absorption/adsorption characteristics between the CAC nanoparticles and selected pesticides. Our findings reveal that charged pesticide molecules exhibit more than double capture rates compared to neutral counterparts, owing to their stronger affinity for the CAC nanoparticles. Non-covalent interactions, such as van der Waals forces, π-π stacking, and hydrogen bonds, are identified as key factors which stabilized the capture and physisorption of pesticides. Density profile analysis confirms the localization of pesticides adsorbed onto the surface or absorbed into the polymer matrix, depending on their chemical nature. The mobility and diffusion behavior of captured compounds within the nanoparticle matrix is assessed using mean square displacement and diffusion coefficients. Compounds with high capture levels exhibit limited mobility, indicative of effective absorption and adsorption. Intermolecular interaction analysis highlights the significance of hydrogen bonds and electrostatic interactions in the pesticide-polymer association. Notably, two promising candidates, an antibiotic derived from tetracycline and a rodenticide, demonstrate a strong affinity for CAC nanoparticles. This computational methodology offers a reliable and efficient screening approach for identifying effective pesticide capture agents, contributing to the development of eco-friendly strategies for pesticide removal.

4.
ACS Omega ; 8(13): 11736-11749, 2023 Apr 04.
Article in English | MEDLINE | ID: mdl-37033853

ABSTRACT

Transient receptor potential (TRP) channels constitute a large group of membrane receptors associated with sensory pathways in vertebrates. One of the most studied is TRPV1, a polymodal receptor tuned for detecting heat and pungent compounds. Specific inhibition of the nociceptive transduction at the peripheral nerve represents a convenient approach to pain relief. While acting as a chemoreceptor, TRPV1 shows high sensitivity and selectivity for capsaicin. In contrast to the drugs available on the market that target the inflammatory system, TRPV1 antagonists act as negative modulators of nociceptive transduction. Therefore, the development of compounds modulating TRPV1 activity has expanded dramatically over time. Experimental data suggest that most agonist and antagonist drugs interact at or near capsaicin's binding site. In particular, the properties of capsaicin's head play an essential role in modulating potency and affinity. Here, we explored a cost-efficient pipeline to predict the effects of introducing chemical modifications into capsaicin's head region. An extensive set of molecules was selected by first considering the geometrical properties of capsaicin's binding site and then molecular docking. Finally, the novel ligands were ranked by combining molecular and pharmacokinetic predictions.

5.
Sensors (Basel) ; 21(23)2021 Dec 02.
Article in English | MEDLINE | ID: mdl-34884063

ABSTRACT

This paper proposes a new theoretical stochastic model based on an abstraction of the opportunistic model for opportunistic networks. The model is capable of systematically computing the network parameters, such as the number of possible routes, the probability of successful transmission, the expected number of broadcast transmissions, and the expected number of receptions. The usual theoretical stochastic model explored in the methodologies available in the literature is based on Markov chains, and the main novelty of this paper is the employment of a percolation stochastic model, whose main benefit is to obtain the network parameters directly. Additionally, the proposed approach is capable to deal with values of probability specified by bounded intervals or by a density function. The model is validated via Monte Carlo simulations, and a computational toolbox (R-packet) is provided to make the reproduction of the results presented in the paper easier. The technique is illustrated through a numerical example where the proposed model is applied to compute the energy consumption when transmitting a packet via an opportunistic network.

6.
J Immunol ; 203(1): 105-116, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31109956

ABSTRACT

We found that protease-activated receptor 1 (PAR1) was transiently induced in cultured osteoclast precursor cells. Therefore, we examined the bone phenotype and response to resorptive stimuli of PAR1-deficient (knockout [KO]) mice. Bones and bone marrow-derived cells from PAR1 KO and wild-type (WT) mice were assessed using microcomputed tomography, histomorphometry, in vitro cultures, and RT-PCR. Osteoclastic responses to TNF-α (TNF) challenge in calvaria were analyzed with and without a specific neutralizing Ab to the Notch2-negative regulatory region (N2-NRR Ab). In vivo under homeostatic conditions, there were minimal differences in bone mass or bone cells between PAR1 KO and WT mice. However, PAR1 KO myeloid cells demonstrated enhanced osteoclastogenesis in response to receptor activator of NF-κB ligand (RANKL) or the combination of RANKL and TNF. Strikingly, in vivo osteoclastogenic responses of PAR1 KO mice to TNF were markedly enhanced. We found that N2-NRR Ab reduced TNF-induced osteoclastogenesis in PAR1 KO mice to WT levels without affecting WT responses. Similarly, in vitro N2-NRR Ab reduced RANKL-induced osteoclastogenesis in PAR1 KO cells to WT levels without altering WT responses. We conclude that PAR1 functions to limit Notch2 signaling in responses to RANKL and TNF and moderates osteoclastogenic response to these cytokines. This effect appears, at least in part, to be cell autonomous because enhanced osteoclastogenesis was seen in highly purified PAR1 KO osteoclast precursor cells. It is likely that this pathway is involved in regulating the response of bone to diseases associated with inflammatory signals.


Subject(s)
Bone Diseases/immunology , Inflammation/immunology , Osteoclasts/physiology , Receptor, Notch2/metabolism , Receptor, PAR-1/metabolism , Animals , Antibodies, Neutralizing/metabolism , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteogenesis/genetics , RANK Ligand/metabolism , Receptor, Notch2/immunology , Receptor, PAR-1/genetics , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism
7.
Nature ; 568(7753): 541-545, 2019 04.
Article in English | MEDLINE | ID: mdl-30971820

ABSTRACT

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1-9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11-18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies that suggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo.


Subject(s)
Hematopoietic Stem Cells/cytology , Osteoclasts/cytology , Osteoclasts/metabolism , Osteopetrosis/genetics , Animals , Animals, Newborn , Bone Development , Female , Genes, Recessive , Male , Mice , Osteopetrosis/pathology , Tooth Eruption
8.
Parasitol Res ; 117(12): 3935-3943, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30298236

ABSTRACT

The potential role of Blastocystis as a pathogen is controversial because it is found in both symptomatic and asymptomatic carriers. Since Cathepsin B has been identified as a main virulence factor that contributes to the pathogenesis of this parasite, the purpose of this study was to analyze the genetic polymorphisms of cathepsin B from Blastocystis from patients with irritable bowel syndrome and from asymptomatic carriers. DNA from fecal samples of both groups, which were previously genotyped by 18S sequencing, was used to amplify a fragment of the cathepsin B gene. Phylogenetic reconstructions were performed and some genetic population indexes were obtained. Amplicons of 27 samples (15 cases, 10 controls, and two commercial ATCC strains) were obtained and analyzed. Phylogenetic reconstructions using nucleotides or inferred amino acid sequences did not separate between cases or controls or among subtypes. Regarding the values of genetic variability, we found that the haplotype and nucleotide diversity indexes of cathepsin B from cases and controls were similar to the values of 18S from controls. By contrast, 18S from cases showed low variability, suggesting that the genetic variability of cathepsin B was not related to the symptomatology of Blastocystis carriers. However, since no polymorphisms related to cases or controls were found, it is logical to assume that the potential damage caused by Blastocystis in situ may be due to unclear mechanisms of Cathepsin B regulation and expression that should be studied in future studies.


Subject(s)
Blastocystis Infections/parasitology , Blastocystis/genetics , Blastocystis/pathogenicity , Cathepsin B/genetics , Irritable Bowel Syndrome/parasitology , Adult , Amino Acid Sequence/genetics , Blastocystis/classification , Feces/parasitology , Female , Genetics, Population , Genotype , Haplotypes , Humans , Male , Middle Aged , Phylogeny , Polymorphism, Genetic , Virulence Factors/genetics
9.
Stat Interface ; 10(3): 471-482, 2017.
Article in English | MEDLINE | ID: mdl-29104713

ABSTRACT

This paper develops a likelihood-based approach to analyze quantile regression (QR) models for continuous longitudinal data via the asymmetric Laplace distribution (ALD). Compared to the conventional mean regression approach, QR can characterize the entire conditional distribution of the outcome variable and is more robust to the presence of outliers and misspecification of the error distribution. Exploiting the nice hierarchical representation of the ALD, our classical approach follows a Stochastic Approximation of the EM (SAEM) algorithm in deriving exact maximum likelihood estimates of the fixed-effects and variance components. We evaluate the finite sample performance of the algorithm and the asymptotic properties of the ML estimates through empirical experiments and applications to two real life datasets. Our empirical results clearly indicate that the SAEM estimates outperforms the estimates obtained via the combination of Gaussian quadrature and non-smooth optimization routines of the Geraci and Bottai (2014) approach in terms of standard errors and mean square error. The proposed SAEM algorithm is implemented in the R package qrLMM().

10.
Nature ; 549(7672): 389-393, 2017 09 21.
Article in English | MEDLINE | ID: mdl-28854169

ABSTRACT

The pathophysiology of neurodegenerative diseases is poorly understood and there are few therapeutic options. Neurodegenerative diseases are characterized by progressive neuronal dysfunction and loss, and chronic glial activation. Whether microglial activation, which is generally viewed as a secondary process, is harmful or protective in neurodegeneration remains unclear. Late-onset neurodegenerative disease observed in patients with histiocytoses, which are clonal myeloid diseases associated with somatic mutations in the RAS-MEK-ERK pathway such as BRAF(V600E), suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet the expression of BRAF(V600E) in the haematopoietic stem cell lineage causes leukaemic and tumoural diseases but not neurodegenerative disease. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk-sac erythro-myeloid progenitors (EMPs) distinct from haematopoietic stem cells. We therefore hypothesized that a somatic BRAF(V600E) mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAF(V600E) in mouse EMPs results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder. This is associated with accumulation of ERK-activated amoeboid microglia in mice, and is also observed in human patients with histiocytoses. In the mouse model, neurobehavioural signs, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses and demonstrate that a somatic mutation in the EMP lineage in mice can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration and this offers opportunities for therapeutic intervention aimed at the prevention of neuronal death in neurodegenerative diseases.


Subject(s)
Erythroid Precursor Cells/pathology , MAP Kinase Signaling System , Mutation , Myeloid Progenitor Cells/pathology , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/pathology , Proto-Oncogene Proteins B-raf/genetics , Animals , Clone Cells/enzymology , Clone Cells/metabolism , Clone Cells/pathology , Disease Models, Animal , Erythroid Precursor Cells/enzymology , Erythroid Precursor Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Histiocytosis/enzymology , Histiocytosis/genetics , Histiocytosis/metabolism , Histiocytosis/pathology , Humans , Macrophages/enzymology , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Microglia/enzymology , Microglia/metabolism , Microglia/pathology , Mosaicism , Myeloid Progenitor Cells/enzymology , Myeloid Progenitor Cells/metabolism , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism
11.
Immunity ; 45(6): 1205-1218, 2016 12 20.
Article in English | MEDLINE | ID: mdl-28002729

ABSTRACT

Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c-MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3-CD11c-MHCII-PU.1lo monocytes differentiated into FcγRIII+PD-L2-CD209a-iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/- mice had reduced Flt3+CD11c-MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs.


Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , Macrophages/immunology , Monocytes/immunology , Adoptive Transfer , Animals , Antigens, Ly/immunology , Cell Separation , Dendritic Cells/cytology , Flow Cytometry , Macrophages/cytology , Mice , Monocytes/cytology , Nitric Oxide Synthase Type II/immunology , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction
12.
Science ; 353(6304)2016 09 09.
Article in English | MEDLINE | ID: mdl-27492475

ABSTRACT

Tissue-resident macrophages support embryonic development and tissue homeostasis and repair. The mechanisms that control their differentiation remain unclear. We report here that erythro-myeloid progenitors in mice generate premacrophages (pMacs) that simultaneously colonize the whole embryo from embryonic day 9.5 in a chemokine-receptor-dependent manner. The core macrophage program initiated in pMacs is rapidly diversified as expression of transcriptional regulators becomes tissue-specific in early macrophages. This process appears essential for macrophage specification and maintenance, as inactivation of Id3 impairs the development of liver macrophages and results in selective Kupffer cell deficiency in adults. We propose that macrophage differentiation is an integral part of organogenesis, as colonization of organ anlagen by pMacs is followed by their specification into tissue macrophages, hereby generating the macrophage diversity observed in postnatal tissues.


Subject(s)
Cell Differentiation/genetics , Embryo, Mammalian/cytology , Gene Expression Regulation, Developmental , Macrophages/cytology , Myeloid Progenitor Cells/cytology , Organogenesis , Animals , CX3C Chemokine Receptor 1 , Embryonic Development , Embryonic Induction , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/metabolism , Female , Hematopoiesis/genetics , Hematopoiesis/physiology , Inhibitor of Differentiation Proteins/metabolism , Kupffer Cells/cytology , Kupffer Cells/metabolism , Macrophages/metabolism , Mice , Mice, Mutant Strains , Myeloid Progenitor Cells/metabolism , Organ Specificity , Receptors, Chemokine/genetics , Transcriptome
13.
J Biol Chem ; 291(8): 3882-94, 2016 Feb 19.
Article in English | MEDLINE | ID: mdl-26703472

ABSTRACT

Continuous parathyroid hormone (PTH) blocks its own osteogenic actions in marrow stromal cell cultures by inducing Cox2 and receptor activator of nuclear factor κB ligand (RANKL) in the osteoblastic lineage cells, which then cause the hematopoietic lineage cells to secrete an inhibitor of PTH-stimulated osteoblast differentiation. To identify this inhibitor, we used bone marrow macrophages (BMMs) and primary osteoblasts (POBs) from WT and Cox2 knock-out (KO) mice. Conditioned medium (CM) from RANKL-treated WT, but not KO, BMMs blocked PTH-stimulated cAMP production in POBs. Inhibition was reversed by pertussis toxin (PTX), which blocks Gαi/o activation. Saa3 was the most highly differentially expressed gene in a microarray comparison of RANKL-treated WT versus Cox2 KO BMMs, and RANKL induced Saa3 protein secretion only from WT BMMs. CM from RANKL-stimulated BMMs with Saa3 knockdown did not inhibit PTH-stimulated responses in POBs. SAA added to POBs inhibited PTH-stimulated cAMP responses, which was reversed by PTX. Selective agonists and antagonists of formyl peptide receptor 2 (Fpr2) suggested that Fpr2 mediated the inhibitory actions of Saa3 on osteoblasts. In BMMs committed to become osteoclasts by RANKL treatment, Saa3 expression peaked prior to appearance of multinucleated cells. Flow sorting of WT marrow revealed that Saa3 was secreted only from the RANKL-stimulated B220(-) CD3(-)CD11b(-/low) CD115(+) preosteoclast population. We conclude that Saa3 secretion from preosteoclasts, induced by RANKL in a Cox2-dependent manner, inhibits PTH-stimulated cAMP signaling and osteoblast differentiation via Gαi/o signaling. The induction of Saa3 by PTH may explain the suppression of bone formation when PTH is applied continuously and may be a new therapeutic target for osteoporosis.


Subject(s)
Cyclic AMP/metabolism , Osteoblasts/metabolism , Osteoclasts/metabolism , Parathyroid Hormone/pharmacology , Second Messenger Systems/drug effects , Serum Amyloid A Protein/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cyclic AMP/genetics , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Mice , Mice, Knockout , Osteoblasts/cytology , Osteoclasts/cytology , Osteogenesis/drug effects , Osteogenesis/genetics , Parathyroid Hormone/metabolism , RANK Ligand/genetics , RANK Ligand/metabolism , Receptors, Formyl Peptide/genetics , Receptors, Formyl Peptide/metabolism , Second Messenger Systems/genetics , Serum Amyloid A Protein/genetics
14.
Mol Endocrinol ; 28(4): 546-53, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24606124

ABSTRACT

Excessive bone resorption is the cause of several metabolic bone diseases including osteoporosis. Thus, identifying factors that can inhibit osteoclast formation and/or activity may define new drug targets that can be used to develop novel therapies for these conditions. Emerging evidence demonstrates that the master regulator of hematopoiesis, Runx1, is expressed in preosteoclasts and may influence skeletal health. To examine the potential role of Runx1 in osteoclast formation and function, we deleted its expression in myeloid osteoclast precursors by crossing Runx1 floxed mice (Runx1(F/F)) with CD11b-Cre transgenic mice. Mice lacking Runx1 in preosteoclasts (CD11b-Cre;Runx1(F/F)) exhibited significant loss of femoral trabecular and cortical bone mass compared with that in Cre-negative mice. In addition, serum levels of collagen type 1 cross-linked C-telopeptide, a biomarker of osteoclast-mediated bone resorption, were significantly elevated in CD11b-Cre;Runx1(F/F) mice compared with those in Runx1(F/F) mice. Tartrate-resistant acid phosphatase-positive osteoclasts that differentiated from bone marrow cells of CD11b-Cre;Runx1(F/F) mice in vitro were larger, were found in greater numbers, and had increased bone resorbing activity than similarly cultured cells from Runx1(F/F) mice. CD11b-Cre;Runx1(F/F) bone marrow cells that were differentiated into osteoclasts in vitro also had elevated mRNA levels of osteoclast-related genes including vacuolar ATPase D2, cathepsin K, matrix metalloproteinase 9, calcitonin receptor, osteoclast-associated receptor, nuclear factor of activated T cells cytoplasmic 1, and cFos. These data indicate that Runx1 expression in preosteoclasts negatively regulates osteoclast formation and activity and contributes to overall bone mass.


Subject(s)
Cell Differentiation , Core Binding Factor Alpha 2 Subunit/metabolism , Osteoclasts/pathology , Animals , Bone Marrow Cells/metabolism , Bone Resorption/metabolism , Bone Resorption/pathology , Bone and Bones/metabolism , Bone and Bones/pathology , CD11b Antigen/metabolism , Gene Deletion , Integrases/metabolism , Mice , Organ Size , Osteoblasts/metabolism , Osteoblasts/pathology , Osteoclasts/metabolism , Osteogenesis
15.
J Cell Biochem ; 115(8): 1449-57, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24590570

ABSTRACT

Cathepsin K (CatK) is a lysosomal cysteine protease necessary for bone resorption by osteoclasts (OCs), which originate from myeloid hematopoietic precursors. CatK-deficient (CatK(-/-) ) mice show osteopetrosis due to defective resorption by OCs, which are increased in number in these mice. We investigated whether genetic ablation of CatK altered the number of hematopoietic stem cells (HSCs) and OC precursor cells (OCPs) using two mouse models: CatK(-/-) mice and a knock-in mouse model in which the CatK gene (ctsk) is replaced by cre recombinase. We found that CatK deletion in mice significantly increased the number of HSCs in the spleen and decreased their number in bone marrow. In contrast, the number of early OCPs was unchanged in the bone marrow. However, the number of committed CD11b(+) OCPs was increased in the bone marrow of CatK(-/-) compared to wild-type (WT) mice. In addition, the percentage but not the number of OCPs was decreased in the spleen of CatK(-/-) mice relative to WT. To understand whether increased commitment to OC lineage in CatK(-/-) mice is influenced by the bone marrow microenvironment, CatK(Cre/+) or CatK(Cre/Cre) red fluorescently labeled OCPs were injected into WT mice, which were also subjected to a mid-diaphyseal femoral fracture. The number of OCs derived from the intravenously injected CatK(Cre/Cre) OCPs was lower in the fracture callus compared to mice injected with CatK(+/Cre) OCPs. Hence, in addition to its other effects, the absence of CatK in OCP limits their ability to engraft in a repairing fracture callus compared to WT OCP.


Subject(s)
Bone Resorption/genetics , Cathepsin K/genetics , Hematopoietic Stem Cells/metabolism , Osteogenesis , Animals , Bone Resorption/pathology , Cathepsin K/metabolism , Fracture Healing/genetics , Hematopoietic Stem Cells/pathology , Mice , Mice, Knockout , Osteoclasts/metabolism , Osteoclasts/pathology , Osteopetrosis/genetics , Osteopetrosis/pathology
16.
J Bone Miner Res ; 28(5): 1203-13, 2013 May.
Article in English | MEDLINE | ID: mdl-23165930

ABSTRACT

Osteoclasts are specialized bone-resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype B220(-) CD3(-) CD11b(-/low) CD115(+) and either CD117(hi), CD117(intermediate), or CD117(low). We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single-cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages, and antigen-presenting dendritic cells in vitro with efficiencies of more than 90%, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages, and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined.


Subject(s)
Bone Marrow Cells/cytology , Dendritic Cells/cytology , Macrophages/cytology , Osteoclasts/cytology , Animals , Male , Mice , Mice, Inbred C57BL
17.
J Bone Miner Res ; 26(6): 1207-16, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21611963

ABSTRACT

Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1-34) (80 µg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115(+) CD117(high) and TN/CD115(+) CD117(int) cells (p < .05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115(+) CD117(low) cells by 39% on day 7 (p < .05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF-κB ligand (RANKL)- and macrophage colony-stimulating factor (M-CSF)-stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115(+) cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin(-) CD117(+) Sca-1(+) ) in the spleen (p < .05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 (p < .05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M-CSF and RANKL.


Subject(s)
Bone Marrow Cells/cytology , Cell Differentiation/drug effects , Hematopoietic Stem Cells/cytology , Osteoclasts/cytology , Osteoclasts/drug effects , Osteogenesis/drug effects , Parathyroid Hormone/pharmacology , Acid Phosphatase/metabolism , Animals , Biological Assay , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Bone Resorption/metabolism , Bone Resorption/pathology , Cattle , Cell Size/drug effects , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Osteoclasts/enzymology , Osteoclasts/pathology , Parathyroid Hormone/administration & dosage , Spleen/drug effects , Tartrate-Resistant Acid Phosphatase
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