Tyrosine Phosphatase Epsilon Is a Positive Regulator of Osteoclast Function in Vitro and In Vivo

Protein tyrosine phosphorylation is a major regulator of bone metabolism. Tyrosine phosphatases participate in regulating phosphorylation, but roles of specific phosphatases in bone metabolism are largely unknown. We demonstrate that young (<12 weeks) female mice lacking tyrosine phosphatase epsilon (PTPϵ) exhibit increased trabecular bone mass due to cell-specific defects in osteoclast function. These defects are manifested in vivo as reduced association of osteoclasts with bone and as reduced serum concentration of C-terminal collagen telopeptides, specific products of osteoclast-mediated bone degradation. Osteoclast-like cells are generated readily from PTPϵ-deficient bone-marrow precursors. However, cultures of these cells contain few mature, polarized cells and perform poorly in bone resorption assays in vitro. Podosomes, structures by which osteoclasts adhere to matrix, are disorganized and tend to form large clusters in these cells, suggesting that lack of PTPϵ adversely affects podosomal arrangement in the final stages of osteoclast polarization. The gender and age specificities of the bone phenotype suggest that it is modulated by hormonal status, despite normal serum levels of estrogen and progesterone in affected mice. Stimulation of bone resorption by RANKL and, surprisingly, Src activity and Pyk2 phosphorylation are normal in PTPϵ-deficient osteoclasts, indicating that loss of PTPϵ does not cause widespread disruption of these signaling pathways. These results establish PTPϵ as a phosphatase required for optimal structure, subcellular organization, and function of osteoclasts in vivo and in vitro.

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Hypoglycemic, hepatoprotective and molecular docking studies of 5-[(4-chlorophenoxy) methyl]-1, 3, 4-oxadiazole-2-thiol

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v13i2.35514 ◽

2018 ◽

Vol 13 (2) ◽

pp. 149 ◽

Cited By ~ 1

Author(s):

Naureen Shehzadi ◽

Khalid Hussain ◽

Nadeem Irfan Bukhari ◽

Muhammad Islam ◽

Muhammad Tanveer Khan ◽

...

Keyword(s):

Molecular Docking ◽

Video Clip ◽

Serum Levels ◽

Docking Studies ◽

Normal Serum ◽

Yeast Cells ◽

Drug Candidate ◽

Molecular Docking Studies

The present study aimed at the evaluation of anti-hyperglycemic and hepatoprotective potential of a new drug candidate, 5-[(4-chlorophenoxy) methyl]-1,3,4-oxadiazole-2-thiol (OXCPM) through in vitro and in vivo assays, respectively. The compound displayed excellent dose-dependent ɑ-amylase (28.0-92.0%), ɑ-glucosidase (40.3-93.1%) and hemoglobin glycosylation (9.0%-54.9%) inhibitory effects and promoted the uptake of glucose by the yeast cells (0.2 to 26.3%). The treatment of the isoniazid- and rifampicin- (p.o., 50 mg/kg of each) intoxicated rats with OXCPM (100 mg/kg, p.o.) resulted in restoring the normal serum levels of the non-enzymatic (total bilirubin, total protein and albumin) and bringing about a remarkable decrease in the levels of enzymatic (alanine transaminases, aspartate transaminases and alkaline phosphatase) biomarkers. The molecular docking studies indicated high binding affinity of the compound for hyperglycemia-related protein targets; fructose-1,6-bisphosphatase, beta2-adrenergic receptors and glucokinase. The results indicate that OXCPM may not only reduce hyperglycemia by enzyme inhibition but also the disease complications through protection of hemoglobin glycosylation and hepatic injury.Video Clip of Methodology:Glucose uptake by yeast cells: 4 min 51 sec <a href="https://www.youtube.com/v/8cJkuMtV0Wc">Full Screen</a> <a href="https://www.youtube.com/watch?v=8cJkuMtV0Wc">Alternate</a>

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Osteogenic growth peptide increases blood and bone marrow cellularity and enhances engraftment of bone marrow transplants in mice

Blood ◽

10.1182/blood.v88.12.4719.bloodjournal88124719 ◽

1996 ◽

Vol 88 (12) ◽

pp. 4719-4724 ◽

Cited By ~ 31

Author(s):

O Gurevitch ◽

S Slavin ◽

A Muhlrad ◽

A Shteyer ◽

D Gazit ◽

...

Keyword(s):

Bone Marrow ◽

Feedback Loop ◽

Serum Levels ◽

High Serum ◽

Normal Serum ◽

Host Cells ◽

Stromal Microenvironment ◽

Marrow Cellularity

The osteogenic growth peptide (OGP) was characterized recently in regenerating bone marrow (BM) and normal serum. In vitro, the OGP regulates stromal-cell proliferation and differentiated functions. In vivo, an increase in serum OGP accompanies the osteogenic phase of postablation BM regeneration. The present results in normal mice show that OGP induces a balanced increase in WBC counts and overall BM cellularity. In mice receiving myeloablative irradiation and syngeneic or semiallogeneic BM transplants, OGP stimulates hematopoietic reconstruction and doubles the survival rate; these effects are dependent on initiating the OGP administration before irradiation. Chimerism measurements in semiallogeneic graft recipients suggest no preferential effect of OGP on residual host cells. The data implicate OGP in the acceleration of hematopoiesis secondary to expansion of the stromal microenvironment and/or enhancement of stroma-derived signals to stem cells. The low-dose effectiveness of OGP is explained by the demonstration of an autocrine positive feedback loop that together with the OGP-binding protein sustains high serum levels of the peptide. A potential OGP-based treatment in combination with chemoradiotherapy is attractive because of the OGP-induced balanced multi-lineage enhancement of hematopoiesis and possible replacement of expensive recombinant cytokines by a readily synthesized peptide.

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Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro

International Journal of Cell Biology ◽

10.1155/2019/6943986 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 4

Author(s):

Sunipa Majumdar ◽

Aniket S. Wadajkar ◽

Hanan Aljohani ◽

Mark A. Reynolds ◽

Anthony J. Kim ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Sustained Release ◽

Double Emulsion ◽

Small Molecular Weight ◽

Rhodamine Phalloidin ◽

Amino Terminal ◽

Osteoclast Function

We have recently demonstrated that a small molecular weight amino-terminal peptide of L-plastin (10 amino acids; “MARGSVSDEE”) suppressed the phosphorylation of endogenous L-plastin. Therefore, the formation of nascent sealing zones (NSZs) and bone resorption are reduced. The aim of this study was to develop a biodegradable and biocompatible PLGA nanocarrier that could be loaded with the L-plastin peptide of interest and determine the efficacy in vitro in osteoclast cultures. L-plastin MARGSVSDEE (P1) and scrambled control (P3) peptide-loaded PLGA-PEG nanoparticles (NP1 and NP3, respectively) were synthesized by double emulsion technique. The biological effect of nanoparticles on osteoclasts was evaluated by immunoprecipitation, immunoblotting, rhodamine-phalloidin staining of actin filaments, and pit forming assays. Physical characterization of well-dispersed NP1 and NP3 demonstrated ~130-150 nm size, < 0.07 polydispersity index, ~-3 mV ζ-potential, and a sustained release of the peptide for three weeks. Biological characterization in osteoclast cultures demonstrated the following: NP1 significantly reduced (a) endogenous L-plastin phosphorylation; (b) formation of NSZs and sealing rings; (c) resorption. However, the assembly of podosomes which are critical for cell adhesion was not affected. L-plastin peptide-loaded PLGA-PEG nanocarriers have promising potential for the treatment of diseases associated with bone loss. Future studies will use this sustained release of peptide strategy to systematically suppress osteoclast bone resorption activity in vivo in mouse models demonstrating bone loss.

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A dual role for semaphorin 4D in bone resorption: Direct on osteoclast function in vitro and dependant on female reproductive function in vivo

Bone ◽

10.1016/j.bone.2010.04.311 ◽

2010 ◽

Vol 47 ◽

pp. S138

Author(s):

R. Dacquin ◽

C. Domenget ◽

P. Jurdic ◽

I. Machuca-Gayet

Keyword(s):

Bone Resorption ◽

Reproductive Function ◽

Dual Role ◽

Semaphorin 4D ◽

Osteoclast Function

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Syk, c-Src, the αvβ3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption

The Journal of Cell Biology ◽

10.1083/jcb.200611083 ◽

2007 ◽

Vol 176 (6) ◽

pp. 877-888 ◽

Cited By ~ 199

Author(s):

Wei Zou ◽

Hideki Kitaura ◽

Jennifer Reeve ◽

Fanxin Long ◽

Victor L.J. Tybulewicz ◽

...

Keyword(s):

Bone Resorption ◽

Αvβ3 Integrin ◽

Chimeric Mice ◽

Signaling Complex ◽

Skeletal Mass ◽

Osteoclast Function ◽

Activation Motif ◽

Bone Resorbing Osteoclast

In this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk−/− osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk−/− embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk deficiency reflects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the αvβ3 integrin and c-Src in a signaling complex, which is generated only when αvβ3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. αvβ3-induced phosphorylation of Syk and the latter's capacity to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRγ. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and αvβ3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.

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Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone

Journal of Cell Science ◽

10.1242/jcs.112.22.3985 ◽

1999 ◽

Vol 112 (22) ◽

pp. 3985-3993 ◽

Cited By ~ 4

Author(s):

I. Nakamura ◽

M.F. Pilkington ◽

P.T. Lakkakorpi ◽

L. Lipfert ◽

S.M. Sims ◽

...

Keyword(s):

Bone Resorption ◽

Cell Attachment ◽

Leading Edge ◽

In Vivo Studies ◽

Sealing Zone ◽

Osteoclast Function ◽

Alpha V Beta 3

The alpha(v)beta(3) integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to alpha(v)beta(3), inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of alpha(v)beta(3) integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC(50)= 0.1 nM) than those required to detach osteoclasts from bone (IC(50) approximately 1 microM); (2) echistatin (IC(50)= 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) alpha(v)beta(3) integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), alpha(v)beta(3) disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC(50)= 13 nM), and alpha(v)beta(3) visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that alpha(v)beta(3) integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC(50)= 0.1 nM) and maintenance of the sealing zone (IC(50) approximately 10 nM).

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Hyperactivation of p21ras and PI3-K Cooperate To Alter Murine and Human NF1 Haploinsufficient Osteoclast Functions.

Blood ◽

10.1182/blood.v108.11.675.675 ◽

2006 ◽

Vol 108 (11) ◽

pp. 675-675

Author(s):

Shi Chen ◽

Alexander Robling ◽

Xiaohong Li ◽

Jin Yuan ◽

Janet Hock ◽

...

Keyword(s):

Bone Resorption ◽

Genetic Disorder ◽

Bone Matrix ◽

Lytic Activity ◽

Mineral Density ◽

Local Bone ◽

Osteoclast Function ◽

K Activity

Abstract Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations of the NF1 tumor suppressor gene that functions as a GTPase activating protein for Ras. Though nullizygous loss of NF1 is associated with the development of malignancies, haploinsufficient phenotypes are now being increasingly recognized to alter cell fates and functions in a number of tissues resulting in nonmalignant disease manifestations. Bone manifestations, including skeletal dysplasias, scoliosis, and osteoporosis occur in 30–60% of all NF1 patients and osteoporosis is an increasingly recognized health problem for women with NF1. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone resorbing cells of the skeleton. Using an established murine model of NF1 developed using homologous recombination, we found that Nf1+/− mice contain elevated numbers of multinucleated osteoclasts and osteoclast progenitors per femur in vivo. Both osteoclasts and osteoclast progenitors from Nf1+/− mice were hyperresponsive to limiting concentrations of M-CSF and RANKL, growth factors that are integral to osteoclast maturation and activation. M-CSF stimulated p21ras-GTP and Akt phosphorylation was elevated in Nf1+/− osteoclasts associated with gains in function in survival and proliferation. Bone resorption by osteoclasts is linked to the migration and adherence of these cells to a local bone surface. Purified populations of Nf1+/− osteoclasts were initially placed in the upper chamber of a transwell coated with vitronectin and haptotaxis to M-CSF was determined. Nf1+/− osteoclasts had a 2–3 fold increase in migration as compared to syngeneic wildtype cells. A similar increase in adhesion of Nf1+/− osteoclasts to the integrin avb3 was also observed. Following adhesion, osteoclasts form a specialized cell-extracellular matrix to initiate degradation of bone matrix by secreting proteinases. Nf1+/− osteoclasts had significantly increased bone resorption as measured by scoring the number and area of individual bone resorbing “pits” on dentine slices and by scoring the total area of resorption. These collective increases in osteoclast function were validated in vivo by the observation that serum TRAP5b activity, a sensitive measure of osteoclast lytic activity was 2.5 fold higher in Nf1+/− mice as compared to WT mice. Furthermore, we performed ovariectomy, an established model of osteoporosis associated with an increase in osteoclast function. In two independent experiments, we found that Nf1+/− mice had a significantly greater reduction in bone mineral density following ovariectomy as compared to syngeneic wildtype mice. We hypothesized that hyperactivation of class1A-PI3-K may contribute to these gains in cellular function. We found that intercrossing Nf1+/− and Class1A PI3-K deficient mice (p85a) restores elevated PI3-K activity, and Nf1+/− osteoclast functions to wildtype levels. Furthermore, in vitro differentiated osteoclasts from NF1 patients also display elevated Ras-PI3-K activity and increased lytic activity analogous to murine Nf1+/− osteoclasts. Collectively, we identify a novel cellular and biochemical NF1 haploinsufficient phenotype in osteoclasts that has potential implications in the pathogenesis of NF1 bone disease.

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GH/IGF-I and bone resorption in vivo and in vitro

Acta Endocrinologica ◽

10.1530/eje.1.01874 ◽

2005 ◽

Vol 152 (3) ◽

pp. 327-332 ◽

Cited By ~ 45

Author(s):

Thor Ueland

Keyword(s):

Bone Resorption ◽

Normal Subjects ◽

Beneficial Effects ◽

Igf Receptors ◽

Osteoclastic Resorption ◽

Igf I ◽

Systemic Factors ◽

Osteoclast Function

IGF-I may act as one of several coupling agents by activating bone formation and bone resorption. In vivo studies in normal subjects, postmenopausal women and patients with excess or diminished GH production (acromegaly and GHD) indicate that both GH and IGF-I activate osteoclasts, but that GH has a more pronounced effect, independently of IGF-I. In vitro, GH and IGF receptors have been demonstrated on osteoclasts and both GH and IGF-I may directly modify osteoclast function and activity. In addition to direct effects on osteoclasts, GH and IGF-I may affect bone resorption indirectly by stimulating release of paracrine mediators that regulate osteoclastic resorption (cytokines). Critical for the bone resorptive process is the balance between OPG and RANKL, which is regulated by many systemic factors. In vivo and in vitro, GH/IGF-I may modulate this balance but these studies are difficult to interpret, reflecting the complexity of this system. Increased OPG expression may possibly protect against GH/IGF-I-induced bone resorption and potentially be important for the long-term beneficial effects of GH replacement. Further studies investigating the OPG/RANKL ratio and system in experimental and transgenic GH/IGF models may clarify these issues.

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