Wortmannin Inhibits Spreading and Chemotaxis of Rat Osteoclasts In Vitro

Pilkington, Mary F.; Sims, Stephen M.; Dixon, S. Jeffrey

doi:10.1359/jbmr.1998.13.4.688

Cited by 44 publications

(32 citation statements)

References 29 publications

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“…It has also been demonstrated by others that wortmannin inhibits spreading and chemotaxis of rat osteoclasts (33). Using immunostaining we demonstrated that PI 3-K and proteins phosphorylated on tyrosine residues were localized at the periphery of spreading osteoclasts treated with CSF-1.…”

Section: Discussionsupporting

confidence: 77%

The role of phosphoinositide 3-kinase in spreading osteoclasts induced by colony-stimulating factor-1

Palacio

Félix

2001

European Journal of Endocrinology

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Background: Colony-stimulating factor-1 (CSF-1), a growth and survival factor for osteoclasts, stimulates these cells to spread and migrate towards a gradient of CSF-1. This may support the translocation of osteoclasts to new sites on the bone surface to be resorbed. Phosphoinositide 3-kinase (PI 3-K) is a lipid kinase participating in various signal transduction pathways. Objective: To investigate the role of PI 3-K in the CSF-1-induced spreading of osteoclasts. Methods: In isolated rat osteoclasts treated with or without CSF-1, the distribution of PI 3-K and proteins phosphorylated on tyrosine were investigated using immunofluorescence. In murine osteoclast-like cells grown from bone marrow cells co-cultured with osteoblasts, the activation of the PI 3-K by CSF-1 was determined both in vivo and in vitro. In vivo, the enzyme product in the cell was determined after extraction and separation with thin layer chromatography; in vitro, PI 3-K activity was measured in the pellet immunoprecipitated from the cell lysate. Results: Inhibition of PI 3-K blocked the CSF-1-induced spreading of osteoclasts. In spreading osteoclasts, a portion of PI 3-K was translocated to the periphery where proteins phosphorylated on tyrosine appeared simultaneously. In osteoclast-like cells, CSF-1 stimulated PI 3-K activity. This activity could be immunoprecipitated with antibody against phophotyrosine residues. Conclusion: PI 3-K participates in the CSF-1-induced spreading of osteoclasts. The activated PI 3-K may induce the reorganization of the cytoskeleton resulting in spreading and migration.

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Section: Discussionsupporting

confidence: 77%

The role of phosphoinositide 3-kinase in spreading osteoclasts induced by colony-stimulating factor-1

Palacio

Félix

2001

European Journal of Endocrinology

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“…The essential role of PI3K in chemotaxis of different cell types (Pilkington et al, 1998;Sadhu et al, 2003) is determined by its ability to activate Rac and Cdc42 (Benard et al, 1999;Hawkins et al, 1995;Nakagawa et al, 2001). In neurons, too, α7 nAChR transduces signals to PI3K (Kihara et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

Chernyavsky

Arredondo

Marubio

et al. 2004

Journal of Cell Science

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Nicotinergic agents can act as both chemokines and chemoattractants for cell migration. Epidermal keratinocytes both synthesize acetylcholine and use it as a paracrine and autocrine regulator of cell motility. To gain a mechanistic insight into nicotinergic control of keratinocyte motility, we determined types of nicotinic acetylcholine receptors and signaling pathways regulating keratinocyte chemokinesis and chemotaxis, using respective modifications of the agarose gel keratinocyte outgrowth assay. Random migration of keratinocytes was significantly (P<0.05) inhibited by hemicholinum-3, a metabolic inhibitor of acetylcholine synthesis, as well as by the α-conotoxins MII and AuIB, preferentially blocking α3-containing nicotinic acetylcholine receptors. The use of antisense oligonucleotides specific for nicotinic-acetylcholine-receptor subunits and knockout mice demonstrated pivotal role for the α3β2 channel in mediating acetylcholine-dependent chemokinesis. Signaling pathways downstream of α3β2 included activation of the protein-kinase-C isoform δ and RhoA-dependent events. The nicotinergic chemotaxis of keratinocytes was most pronounced towards the concentration gradient of choline, a potent agonist of α7 nicotinic acetylcholine receptor. The α7-preferring antagonist α-bungarotoxin significantly (P<0.05) diminished keratinocyte chemotaxis, further suggesting a central role for the α7 nicotinic acetylcholine receptor. This hypothesis was confirmed in experiments with anti-α7 antisense oligonucleotides and α7-knockout mice. The signaling pathway mediating α7-dependent keratinocyte chemotaxis included intracellular calcium, activation of calcium/calmodulin-dependent protein-kinase II, conventional isoforms of protein-kinase C, phosphatidylinositol-3-kinase and engagement of Rac/Cdc42. Redistribution of α7 immunoreactivity to the leading edge of keratinocytes upon exposure to a chemoattractant preceded crescent shape formation and directional migration. Application of high-resolution deconvolution microscopy demonstrated that, on the cell membrane of keratinocytes, the nicotinic acetylcholine receptor subunits localize with the integrin β1. The obtained results demonstrate for the first time that α3 and α7 nicotinic acetylcholine receptors regulate keratinocyte chemokinesis and chemotaxis, respectively, and identify signaling pathways mediating these functions, which has clinical implications for wound healing and control of cancer metastases.

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“…Importantly, all three molecules and their downstream substrates have been identified as candidate therapeutic targets for treatment of OC-related bone disorders. In OCs, once activated by M-CSF, PI3-K influences survival as well as actin remodeling (38,39). Pharmacologic inhibitors of PI3-K, including LY294002 and wortmannin, show a dramatic reduction in the development of OCs in cultures treated with M-CSF and RANKL.…”

Section: Microarray As Well As Quantitative Real-time Pcr Analysis Ofmentioning

confidence: 99%

The p85α Subunit of Class I_A Phosphatidylinositol 3-Kinase Regulates the Expression of Multiple Genes Involved in Osteoclast Maturation and Migration

Munugalavadla

Vemula

Sims

et al. 2008

Molecular and Cellular Biology

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Intracellular signals involved in the maturation and function of osteoclasts are poorly understood. Here, we demonstrate that osteoclasts express multiple regulatory subunits of class I A phosphatidylinositol 3-kinase (PI3-K) although the expression of the full-length form of p85␣ is most abundant. In vivo, deficiency of p85␣ results in a significantly greater number of trabeculae and significantly lower spacing between trabeculae as well as increased bone mass in both males and females compared to their sex-matched wild-type controls. Consistently, p85␣؊/؊ osteoclast progenitors show impaired growth and differentiation, which is associated with reduced activation of Akt and mitogen-activated protein kinase extracellular signal-regulated kinase 1 (Erk1)/Erk2 in vitro. Furthermore, a significant reduction in the ability of p85␣ ؊/؊ osteoclasts to adhere to as well as to migrate via integrin ␣v␤3 was observed, which was associated with reduced bone resorption. Microarray as well as quantitative real-time PCR analysis of p85␣؊/؊ osteoclasts revealed a significant reduction in the expression of several genes associated with the maturation and migration of osteoclasts, including microphathalmia-associated transcription factor, tartrate-resistant acid phosphatase, cathepsin K, and ␤3 integrin. Restoring the expression of the full-length form of p85␣ but not the version with a deletion of the Src homology-3 domain restored the maturation of p85␣ ؊/؊ osteoclasts to wild-type levels. These results highlight the importance of the full-length version of the p85␣ subunit of class I A PI3-K in controlling multiple aspects of osteoclast functions.Osteoclasts (OCs) are derived from precursors of monocyte/ macrophage lineage, whose growth and maturation are mainly dependent on two osteoblast/stromal cell-derived cytokines, including macrophage colony stimulating factor (M-CSF) and receptor activator of NF-B ligand (RANKL) (22,30,35,63). The critical role for these two cytokines in OC growth and differentiation has been further illustrated by studying mice lacking the expression of 64). These mice show severe osteopetrosis and lack mature OCs. M-CSF and RANKL regulate OC progenitor (OCp) growth and function in part by regulating the expression of several OC genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, calcitonin receptor, and integrin ␤3 (15, 29). Stimulation of OC precursors by RANKL and M-CSF results in the activation of a number of signaling molecules, including Gab2, Grb2, Vav, Src homology-2 (SH2)-containing inositol-5-

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Wortmannin Inhibits Spreading and Chemotaxis of Rat Osteoclasts In Vitro

Cited by 44 publications

References 29 publications

The role of phosphoinositide 3-kinase in spreading osteoclasts induced by colony-stimulating factor-1

The role of phosphoinositide 3-kinase in spreading osteoclasts induced by colony-stimulating factor-1

Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

The p85α Subunit of Class I_A Phosphatidylinositol 3-Kinase Regulates the Expression of Multiple Genes Involved in Osteoclast Maturation and Migration

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Wortmannin Inhibits Spreading and Chemotaxis of Rat Osteoclasts In Vitro

Cited by 44 publications

References 29 publications

The role of phosphoinositide 3-kinase in spreading osteoclasts induced by colony-stimulating factor-1

The role of phosphoinositide 3-kinase in spreading osteoclasts induced by colony-stimulating factor-1

Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

The p85α Subunit of Class IA Phosphatidylinositol 3-Kinase Regulates the Expression of Multiple Genes Involved in Osteoclast Maturation and Migration

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The p85α Subunit of Class I_A Phosphatidylinositol 3-Kinase Regulates the Expression of Multiple Genes Involved in Osteoclast Maturation and Migration