NO-dependent osteoclast motility: reliance on cGMP-dependent protein kinase I and VASP

Beatrice B. Yaroslavskiy1, Yongjun Zhang1, Sara E. Kalla1, Verónica García Palacios1, Allison C. Sharrow1, Yanan Li1, Mone Zaidi2, Chuanyue Wu1 and Harry C. Blair1,*

1 Departments of Pathology and of Cell Biology and Physiology, University of Pittsburgh and Veteran's Affairs Medical Center, Pittsburgh, PA 15243, USA
2 Mount Sinai Bone Program, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA

* Author for correspondence (e-mail: hcblair{at}imap.pitt.edu)

Accepted 22 August 2005


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The osteoclast degrades bone in cycles; between cycles, the cell is motile. Resorption occurs by acid transport into an extracellular compartment defined by an {alpha}vß3 integrin ring. NO has been implicated in the regulation of bone turnover due to stretch or via estrogen signals, but a specific mechanism linking NO to osteoclastic activity has not been described. NO stimulates osteoclast motility, and at high concentrations NO causes detachment and terminates resorption. Here we demonstrate that NO regulates attachment through the cGMP-dependent protein kinase I (PKG I) via phosphorylation of the intermediate protein VASP. VASP colocalized with the {alpha}vß3 ring in stationary cells, but alternating bands of VASP and {alpha}vß3 occurred when motility was induced by NO donors or cGMP. Redistribution of VASP correlated with its phosphorylation. Dependency of NO-induced motility on PKG I and on VASP was shown by siRNA knockdown of each protein. VASP knockdown also altered distribution of {alpha}vß3 at the attachment site. We conclude that PKG I and VASP are essential for reorganization of attachment and cytoplasmic proteins in motility induced by NO or by cGMP.

Key words: Osteoclast, Nitric oxide, Cyclic GMP-dependent protein kinase I, Integrin, Vasodilator-stimulated phosphoprotein, Migfilin


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) regulates attachment, motility and survival in many cell systems; it is a major regulator of bone turnover. Transgenic studies showed that NO is vital to bone development (van't Hof and Ralston, 2001Go), and that NO mediates responses to estrogen and mechanical stretch (Armour et al., 2001Go; Nomura and Takano-Yamamoto, 2000Go). In bone, NO is most clearly understood as a signal that regulates osteoclastic activity (Blair et al., 2002Go), and bone-losing states respond to NO donor treatment (Hukkanen et al., 2003Go; Jamal et al., 1998Go).

NO is a membrane-permeant gas that acts by stimulating kinase activity or ionic (often Ca2+) channels. The primary receptor is typically a guanylyl cyclase that converts GTP into cGMP. The cyclase is a heterodimer of ~70 kDa subunits and includes an NO-reactive heme group (Lincoln and Cornwell, 1993Go; Hofmann et al., 2000Go). If it does not react with a specific target such as the heme group of guanylyl cyclase, NO decays in milliseconds to produce the free radical NO* and peroxynitrite, which also produce physiological effects. In the osteoclast, whether free radical or cGMP-mediated pathways are predominant is controversial. A series of studies showed that peroxynitrite metabolites of NO inhibit bone resorption (MacIntyre et al., 1991Go; Mancini et al., 1998Go), although there are also data supporting a cGMP-related mechanism (Van Epps-Fung et al., 1994Go; Mancini et al., 2000Go; Yaroslavskiy et al., 2004Go). Although it is not clear to what extent the free radical and cGMP pathways operate, the pathways are not mutually exclusive, and several studies have found biphasic responses. This suggests that NO may support cell activity at low concentrations but cause apoptosis at higher concentrations (van't Hof and Ralston, 1997Go; Brandi et al., 1995Go), where free radical reactions would be more likely.

Our purpose in this study is to determine mechanisms involved in the osteoclast activity cycle under conditions with continuing cellular activity, in particular to determine whether osteoclast motility and activity are regulated by cGMP-dependent protein kinase activity or by alternative mechanisms such as peroxynitrite reactions that are independent of cGMP. There are two cGMP-dependent protein kinases; they are both found in bone and these kinases are the predominant proteins activated by cGMP (Lincoln and Cornwell, 1993Go). PKG I (78 kDa) is found in mature bone, and particularly in the osteoclast (Van Epps-Fung et al., 1994Go); PKG II (86 kDa) is expressed in chondrocytes and is vital to skeletal development (Miyazawa et al., 2002Go). PKG I has two isoforms, transcribed from the same gene, differing only in the first exon. It acts as a dimer (Lincoln et al., 1993Go), activated by cGMP at 0.05-1.00 µM, but responding to cAMP at ~20-fold higher concentrations. The cGMP-dependent protein kinases recognize R/K-R/K-X-S/T sites that overlap with cAMP-dependent protein kinase substrates, but many consensus sites are not phosphorylated and there are many atypical phosphorylation sites (Lincoln and Cornwell, 1993Go).

Our work regarding the effects of NO on osteoclasts at concentrations permitting cell survival pointed to a probable primary effect on cell attachment (Yaroslavskiy et al., 2004Go). This is in keeping with work in other organs showing that actin- and integrin-linked adhesion protein complexes respond to PKG I. Investigation of the role of focal adhesions in the regulation of platelets and endothelial cells led to the discovery of the vasodilator-stimulated phosphoprotein (VASP), a key element in complexes of intermediate proteins. VASP is phosphorylated by PKG I at Ser239 (Meinecke et al., 1994Go; Butt et al., 1994Go; Reinhard et al., 1995bGo) and the phosphorylated protein can be identified with a monoclonal antibody (Smolenski et al., 1998Go). Preliminary analysis of human osteoclasts suggested that VASP was abundantly expressed. Thus, we hypothesized that, in the osteoclast, NO-induced motility is initiated by cGMP activating PKG I and PKG I modifying the complex ring-like integrin attachment and attached cytoskeletal structure via VASP. This was studied by immunolocalization and immunoprecipitation of key proteins, by micro-cinematography of osteoclast motility, and by cellular response after siRNA suppression of key proteins in the putative pathway. The studies support a role for NO in initiating motility by a mechanism in which PKG I and VASP are essential, with VASP allowing the cell to organize cytoskeletal complexes that include or exclude {alpha}vß3 depending on the VASP phosphorylation state.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reagents
S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) were from Sigma (St Louis, MO). The hydrolysis-resistant cGMP analogs 8-Br-guanosine-3',5'-cyclic monophosphate (8-Br-cGMP) or 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) and the hydrolysis-resistant antagonist 8-(Rp-4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-cGMPS) were from Biolog (Bremen, Germany). NG-Monomethyl-L-arginine acetate (L-NMMA) and 6-Anilinoquinoline-5,8-guinone (Ly83583) were from Biomol (Plymouth Meeting, PA). Cytokines, from RDI (Flanders, NJ) unless indicated, were human recombinant ligand domains, produced in bacteria. Rhodamine-phalloidin and LysoTracker, used at 1:250 or 5 µM, respectively, were from Molecular Probes (Eugene, OR).

Osteoclasts
Human cells were from citrate-anticoagulated blood, obtained with approval of the institutional review board. Mononuclear cells were recovered on ficoll; CD14+ cells were enriched using anti-CD14-magnetic microbeads (Miltenyi Biotec, Auburn CA) (Blair et al., 2004Go). The CD14+ cells were plated at 1x106/cm2 with human CSF-1, 25 ng/ml and human RANKL, 30 ng/ml, on tissue culture substrate (glass or plastic) or on dentine slices, for 16-21 days, to generate osteoclasts. Motility comparisons also used rat osteoclasts, produced by lavage of tibial bone as described (Zaidi et al., 1992Go).

Antibodies and in situ labeling
Anti-{alpha}vß3 was monoclonal anti-human clone 23C6 from Santa Cruz (Santa Cruz, CA), used at 1:50 for in situ labeling; Anti-{alpha}v (Santa Cruz) was used for blots at 1:200. Anti-filamin monoclonal MAB1678 was from Chemicon (Temecula, CA), used at 1:3000. Anti-migfilin monoclonal, as described (Tu et al., 2003Go), was used at 1:500. Anti-PKG rabbit anti-human PKG I (Stressgen; Victoria BC), was used at 1:1000 for western blots and 1:500 for in situ localization. Rabbit anti-human VASP (Calbiochem) was used at 1:500 for in situ labeling or blots, or 1:125 for immunoprecipitation. Anti-p-VASP Ser239 clone 16C2 (Upstate, Lake Placid, NY) was used at 2 µg/ml for blots or 20 µg/ml to label cells in situ. Anti-vinculin (Santa Cruz) was used at 1:200 for western blots. Anti-zyxin (BD Biosciences, San Jose, CA) was used at 1:250 for in situ labeling and 1:2500 for blots. Secondary antibodies were Al-488-conjugated goat anti-mouse (Molecular Probes) at 1:500, Cy-3-conjugated goat anti-mouse (Jackson Labs, West Grove, PA) at 1:500, or goat anti-mouse or rabbit-HRP (Bio-Rad, Hercules CA) at 1:10,000. In situ labeling was carried out as described (Yaroslavskiy et al., 2004Go), with cells fixed in 1% paraformaldehyde on ice for 25 minutes. Permeabilization used 0.25% Triton X-100 and 40 µg/ml digitonin for 5 minutes. Wash solutions included 0.5% bovine serum albumin and 2% nonimmune serum to reduce nonspecific reactions. Primary antibody reactions and labeling reactions were each carried out for 1 hour at room temperature unless specified.

Western analysis and immunoprecipitation
Cells were lysed in 0.5% polyglycol ether (NP-40, Sigma) with phosphatase and proteinase inhibitors. Proteins were separated using Laemmli buffers using 9% polyacrylamide gels, and transferred to polyvinylidine membranes for immunolabeling (Yaroslavskiy et al., 2004Go). For immunoprecipitation, lysates were centrifuged to remove debris and pre-cleared with protein A/G plus (Santa Cruz). Lysates were incubated overnight at 4°C with antibody, and bound proteins were recovered using protein A/G agarose (Miltenyi Biotech, Auburn, CA) by centrifugation after a 2 hour incubation. The precipitated beads were washed five times and eluted in lysis buffer for western blots. Where blots were reprobed, they were stripped of antibodies using Restore (Pierce, Rockford IL). Blots were developed using enhanced chemiluminescence ECL (Amersham, Piscataway, NJ).

siRNAs
Cells were transfected with siRNA targeting two PKG I sequences or four VASP sequences, using mock transfection with scrambled sequence (noncoding) siRNA in controls. siRNAs were labeled with Cy3 for demonstration of transfection efficiency. Target sequences were 21 bp stretches in early exons starting with AA, with G/C content less than 60%. These were screened to eliminate homology to other proteins using BLAST (www.ncbi.nlm.nih.gov/BLAST). For PKG I (GenBank accession number Z92867), target sequences were (+109 from the start codon) AAGAGGAAACTCCACAAATGC and (+124) AAATGCCAGCGGTGCTCCCAGT. For VASP (accession number Z46389) target sequences were (+121) AACCCCACGGCCAATTCCTTT; (+274) AACTTCGGCAGCAAGGAGGAT; (+700) AAACTCAGGAAAGTCAGCAAG; and (+847) AAAACCCCCAAGGATGAATCT. From these sequences, siRNA 29-mer oligonucleotides (sense and antisense) were manufactured (Integrated DNA Technologies, Coralville, IA) with the 8 bp leader sequence complementary to the T7 promoter primer. Templates were hybridized to T7 promoter primers and extended with Klenow DNA polymerase. The double-stranded template was transcribed by T7 RNA polymerase and the transcripts were hybridized to create dsRNA. RNA was digested to remove the single-strand leaders, resulting in ds-siRNA. Transfection used mixtures of siRNAs with 100 nM total siRNA (50 nM each of two siRNAs for PKG, 25 nM each of four for VASP). To determine transfection efficiency, Cy3 was covalently attached to the duplex siRNA (Silencer siRNA labeling kit, Ambion, Austin, TX). Cells were transfected with siRNA using siPORT Amine transfection reagent (Ambion), a blend of polyamines.

Image acquisition and motility
Images were obtained on a Nikon TE2000 inverted phase-fluorescence microscope equipped with a 37°C stage heater, using a 12-bit 1600x1200 pixel monochrome CCD camera (Spot, Diagnostic Instruments, Sterling Heights, MI); color where shown reflects filter color. Filters for green fluorescence were: excitation 450-490 nm, 510 nm dichroic mirror, 520 nm barrier filter; and for red fluorescence: excitation 536-556 nm, 580 nm dichroic mirror, 590 nm barrier filter. For phase-contrast photomicrographs of living cells a NA 0.95 long working distance 40x objective was used; fluorescence-labeled proteins were photographed using 1.3 NA 40x or 100x oil objectives. Cell movement was studied by time-lapse microcinematography with movement determined by difference in location between frames, as described (Yaroslavskiy et al., 2004Go; Zaidi et al., 1992Go; Bear et al., 2002Go).


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclast motility assays
Osteoclast motility was measured using microcinematography with analysis by published methods (Zaidi et al., 1992Go). A summary of rate of motion (µ/t) is shown in Fig. 1A. The raw cellular outlines and geometric cell center used to show motility in the rat osteoclast are illustrated in Fig. S1A in supplementary material, and the calculated positions for the cell at 1 minute intervals are shown in Fig. S1B. Movie 1 shows similar effects on human osteoclasts on glass, but over a longer period (21 exposures at 2 minute intervals). Motility was inhibited using 50 µM of the hydrolysis-resistant cGMP-blocking agent Rp-cGMPS (first series of frames in the movie) or activated using 100 µM of the hydrolysis-resistant cGMP activator 8-pCPT-cGMP (second series of frames in the movie). Similar activation by the NO donor sodium nitroprusside is shown in a third series of frames. Note that, in addition to net cell movement, the spread area of the cells is reduced by either cGMP or NO activation.



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 1. Osteoclast motility is accelerated by cGMP. (A) Motility in human osteoclasts with cGMP activation. This is shown as superimposed frames at 30 minute intervals of osteoclast preparations on glass with the blocking cGMP analog Rp-cGMPS (50 µM, top) or with the cGMP agonist 8-pCPT-cGMP (100 µM, bottom). Two phase images were subtracted with the first image deleting the red channel and the second deleting the green channel, so that net motion can be seen as red and green double images (yellow where moving cells overlap). There was only minor movement when cGMP was blocked, mainly redistribution of nuclei and vesicles within the cells but also some slight variation of membrane spreading. The cGMP agonist in contrast causes net cell motion. The fields shown are 225 by 290 µm. The NO donor sodium nitroprusside (100 µM) produced motility at essentially the same rate, with a rapid time course. For phase-contrast microcinematography of these fields showing the comparison with the NO donor sodium nitroprusside, see supplementary material. (B) High-resolution measurements of motility in rat osteoclasts. This was measured using the attached area, summing new attachment and retracted area as a function of time as described (Zaidi et al., 1992Go) over 1 minute frames for 10 minutes before (left bar, mean±s.d. of eight frames) and after (right bar, mean±s.d. of 20 frames) addition of 250 µM 8-Br-cGMP. The difference in the rate of motility was statistically significant (P<0.01) even over this short time frame. Cellular motility by geometric center of attachment showed that motion was accelerated by cGMP activation (shown in supplementary material).

 
Osteoclast motility induced by cGMP and NO donors
Either cGMP agonists or NO donors greatly increased osteoclast movement on tissue-culture substrates, in rat and human osteoclasts. When cGMP was blocked, net osteoclast motion was minimal over ~1 hour (Fig. 1A, top panel). In contrast, in the presence of a cGMP donor, osteoclasts spent more time in a rounded morphology, and typically moved approximately one-half of the cell diameter in 30 minutes (Fig. 1A, bottom panel). This motion had no apparent direction, but included back and forth or circular motion with periods of 0.5-1 hour. This, and comparison of motion after cGMP agonist and an NO donor, which were essentially the same, are shown as Movie 1 in supplementary material. Response in the rate of change of osteoclast area was within 5 minutes (Fig. 1B). The mechanism modifying cell attachment was unknown, but we had established that NO-induced detachment correlated with integrin rearrangement (Yaroslavskiy et al., 2004Go), so we searched for proteins that link cGMP and integrin function.

cGMP-induced motility separates VASP and {alpha}vß3
Non-amplified mRNA gene screening showed that the PKG-I-modified protein VASP was expressed in osteoclasts (not shown). In platelet or endothelial cell response to NO, VASP rearranges the integrin-associated cytoskeleton, which regulates endothelial cell retraction or platelet adhesion. As these NO responses are distinctly different from osteoclast motility, we queried whether osteoclast VASP plays a role in the integrin-cytoskeletal arrangement that may initiate motion. We examined VASP and {alpha}vß3 distribution in human osteoclasts. Osteoclast VASP overlapped on the integrin ring when PKG I was inactive (Fig. 2A,C). Activating cGMP analogs, which induce motility, led to a separation of the labels (red and green bands, Fig. 2A,D), whether cells were on (Fig. 2A,B) or off bone (Fig. 2C,D). In motile cells, separation of the integrin and VASP labels was consistent in images of numerous cells.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2. VASP and {alpha}vß3 overlap in stationary cells but distribute separately in motile cells. Cells were incubated for 1 hour with 50 µM Rp-cGMPS to inactivate endogenous cGMP activity (A,C) or for 1 hour with 100 µM 8-pCPT-cGMP to induce maximal motility (B,D). (A,B) When cGMP was blocked (top panels), osteoclasts on bone labeled for VASP (red) and {alpha}vß3 (green) showed distribution consistent with the annular attachment site; merged images confirmed this (yellow labeling). After cGMP activation (bottom panels), VASP distribution was partially separated from {alpha}vß3. It is seen in parallel rings (arrows, bottom merged image) on one side of the cell. Under these conditions, the cells are highly motile (see Fig. 1). (C,D) When grown on glass, the cells spread uniformly, facilitating labeling, and an analogous labeling pattern to that on bone was clearly seen. With PKG I suppressed by Rp-cGMPS (top), VASP was associated with the integrin forming a yellow, double-labeled ring in the merged image. With the nonhydrolyzable cGMP analog 8-pCPT-cGMP (bottom panels), {alpha}vß3 and VASP were disassociated at one edge of the cell, now occurring in discrete rings. This polarization was consistent with cell motility. Bar, 10 µm (A,B); 20 µm (C,D).

 

PKG I knockdown eliminates motility in response to cGMP
We studied dependency of the NO motility on the cGMP-dependent protein kinase I by siRNA knockdown. This is crucial to the PKG-I-VASP motility hypothesis versus possible free radical mechanisms, because free radical reactions are not mediated by cGMP (MacIntyre et al., 1991Go). When PKG I was eliminated, effects of NO on motility were effectively eliminated (Fig. 3). Western blotting showed that control cells transfected with noncoding siRNA, but not cells transfected with siRNA targeting PKG I, expressed the enzyme. Motility after cGMP activation was monitored by time-lapse photography (as in Fig. 1) using cells 48 hours after transfection. Osteoclasts transfected with noncoding siRNA had good motility whereas <10% of cells transfected with siRNA targeting PKG I moved measurably. Non-motile PKG I knockdown cells did spread slightly (inset, Fig. 3B). The small number of motile cells was consistent with the number of nontransfected cells, determined by Cy3 labeling of siRNAs (not shown). In osteoclasts on bone slices transfected with siRNA PKG-Cy3, actin distribution resembled that in cells treated with cGMP blockers (Fig. 3C).



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 3. Eliminating PKG I from human osteoclasts prevents cGMP-induced motility and eliminates cGMP-dependent reorganization of the attachment ring. (A) Gene silencing at 48 hours post siRNA transfection reduced PKG I ~95% by densitometry of western analysis. Real-time PCR gave a similar result (tenfold reduction, not shown). The western blot shows osteoclast lysates from cells treated with siRNA for PKG I compared to lysate from cells after mock transfection using a noncoding siRNA sequence. (B) Effect on motility in transfected cells. Means±s.d. of eight cells are shown. Time-lapse photography over 2 hours after addition of 8-pCPT-cGMP (see Fig. 1). Inset, motion over 20 frames by digital difference (lighter image, outlined in yellow, new position; darker image, outlined in green, old position). Note that the siRNA-treated cell has spread slightly, without moving, whereas the mock-transfected cell has moved about one cell diameter in this period. The variable diameter in the moving mock-transfected cell is typical of motile osteoclasts (see also Movie 1 in supplementary material). (C) Effect on cGMP-induced detachment. In each frame, a single osteoclast on bone is shown, with actin labeled with Alexa-488 phalloidin (green) and siRNA labeled with Cy3 (red). In frames 1 and 2, the transfected siRNA is specific for PKG I. The cell in frame 2 was also treated with 8-pCPT-cGMP, 100 µM for 1 hour, but its attachment ring was unaffected. The cell in frame 3 (bottom frame) was also transfected, but with a noncoding siRNA. In this case, its response to 8-pCPT-cGMP was normal, and the attachment has broken up into discrete clumps (arrows). Each field is 80 µm square.

 



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Western blot studies of VASP in human osteoclasts, VASP-associated proteins in VASP precipitates, the effect of PKG I suppression on VASP phosphorylation, and the time-course of VASP phosphorylation after cGMP or NO activation. (A) Osteoclast lysates probed for VASP. VASP is abundant and occurs in cGMP-inhibited (Ctl, 50 µM Rp-cGMPS) and cGMP-stimulated (cGMP, 100 µM 8-pCPT-cGMP) osteoclasts in similar amounts. Fifteen µg lysates of osteoclasts were separated on 10% polyacrylamide in Laemmli buffer, and transferred to PVDF for labeling. Std, 100 ng of recombinant VASP. (B) VASP immunoprecipitates in cGMP-inhibited and -activated osteoclasts, probed for migfilin, {alpha}v integrin and VASP. Immunoprecipitation for VASP was done using 900 µg lysates of osteoclasts after treatment with 50 µM Rp-cGMPS for 1 hour (Inhib) or with 100 µM 8-pCPT-cGMP for 1 hour (cGMP). These conditions essentially eliminate (Rp-cGMPS) or strongly promote (8-pCPT-cGMP) PKG I activity. Note that VASP is phosphorylated by activated PKG I. This also changed the relative abundance of its associated proteins. Migfilin was co-precipitated with VASP mainly in cGMP-activated cells (top panel), whereas {alpha}v integrin precipitation by anti-VASP was reduced by cGMP. Phospho-Vasp (p-VASP), as expected, was greatly increased by cGMP activation relative to VASP. Two or more blots gave similar results. (C) Western blots of PKG I, phospho-VASP, VASP and actin in human osteoclasts after siRNA transfection targeting PKG I relative to mock-transfected control cells. Each lane is a 15 µg osteoclast lysate separated as in A, using cells either transfected with siRNA targeting PKG I as in Fig. 3 (left lane), or a mock-transfected control (right lane). The efficiency of siRNA targeting (~75%) was lower than in the motility study shown in Fig. 3, but nonetheless it markedly reduced phospho-VASP relative to the mock-transfected control cells. (D) Phospho-VASP in VASP immunoprecipitates as a function of time after treatment of osteoclasts with 8-pCPT-cGMP or S-nitroso-N-acetylpenicillamine. Polyclonal anti-VASP was used for immunoprecipitation of protein from 900 µg osteoclast lysates, either without treatment or after 100 µM 8-pCPT-cGMP or 60 µM S-nitroso-N-acetylpenicillamine for the times indicated. Each lane (except an isoimmune control for immunoprecipitation by the antibody and a medium control with no cell lysate) represents an osteoclast immunoprecipitate separated on 9% SDS-PAGE as in A, with immunolabeling using antibody to phospho-VASP (p-VASP) compared to total VASP. Phospho-VASP was detected using anti phospho-Ser239-VASP monoclonal antibody. Increases in phospho-VASP using 8-pCPT-cGMP typically peaked by 1 hour, whereas S-nitroso-N-acetylpenicillamine (SNAP, lower left) peaked at 2-3 hours, in keeping with the half-life of that agent. Sodium nitroprusside gave a similar response but p-VASP increased within 10 minutes in keeping with the short half life of sodium nitroprusside (not illustrated).

 
PKG I regulates VASP phosphorylation and VASP-protein associations
In other cell types, VASP was phosphorylated by PKG I and then redistributed. Therefore, we studied VASP phosphorylation in response to cGMP and the effect of cGMP on association of VASP with key attachment-associated proteins (Fig. 4). Western analysis and immunoprecipitation showed that VASP is a major protein in osteoclasts and did not vary significantly in quantity with cGMP activation (Fig. 4A). An antibody specific for the VASP phosphorylated at Ser239 (Smolenski et al., 1998Go) showed that PKG I modifies this site in VASP in osteoclasts stimulated by cGMP analogs (Fig. 4B). This effect was essentially eliminated by siRNAs targeting PKG I expression (Fig. 4C). Additional analysis of immunoprecipitates (Fig. 4B) showed that the integrin {alpha}vß3 was precipitated mainly by nonphosphorylated VASP, in keeping with the immunolocalization results. Migfilin, which secondarily organizes proteins including adhesion-related kinases, was precipitated strongly when VASP was activated, but was barely detectible in precipitates with blocking cGMP analogs. Migfilin regulates cytoplasmic connections at cell-matrix adhesions for actin remodeling (Tu et al., 2003Go). NO donors and cGMP analogs led to similar VASP phosphorylation, the outcome differing only in time course, which was in keeping with the half-lives of the stimuli (Fig. 4D). Other proteins that were studied by western blotting and immunoprecipitation included PKG I, zyxin and filamin. PKG I was easily detected in lysates on western blots (Fig. 3) but it was weakly seen in VASP precipitates, with or without cGMP, indicating that association of PKG I with the VASP complex was weak or transient. Zyxin distribution overlapped on the attachment proteins by immunofluorescence, as expected, but also labeled nuclei and cytoplasmic structures. On western blots, the quantity of zyxin did not vary with cGMP activation, although cGMP activation increased the amount of cytoplasmic labeling (not shown). Filamin was weakly detected in VASP precipitates and did not show a consistent response to cGMP activation (not shown).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 5. Distribution of PKG I, VASP and migfilin in cGMP-inhibited and cGMP-activated osteoclasts. In osteoclasts grown on glass transfected with siRNA specific for PKG 1 (A), VASP (green) was partially distributed in a peripheral ring (arrows) similar to {alpha}vß3 (see Fig. 2) whereas migfilin (red) was cytoplasmic. In contrast, in cGMP-stimulated cells (B), migfilin clearly colocalized with the peripheral VASP (arrows). Migfilin distribution in cells on bone was consistent with that in A and B, but is seen better in the higher resolution possible on glass substrate, and only glass-attached cells are shown. (C) In osteoclasts on bone with the cGMP inhibitor Rp-cGMPS at 50 µM, PKG I (green) was distributed in the cytoplasm; very little PKG I occurred in the cell periphery, marked by phalloidin (red) to label actin (arrows). (D) With the addition of 100 µM 8-pCPT cGMP, a nonhydrolysable cGMP analog, PKG I and actin were redistributed and there was some overlap (arrows). In cGMP agonists at high concentrations over longer times, nuclear localization of PKG I also occurred (not illustrated); extensive nuclear labeling correlated with complete detachment and may be associated with cell death (see text). Bar, 20 µm (A,B); 10 µm (C,D).

 
PKG I mediates reorganization of the osteoclast attachment via VASP and migfilin
Results from immunoprecipitation included cGMP-dependent association of VASP with migfilin. Migfilin was studied further because this intermediate protein can organize downstream motility response including membrane kinase activity. VASP and migfilin were distributed in peripheral and cytoplasmic patterns when cGMP activity was inhibited. After cGMP activation, the proteins were partially associated (Fig. 5A,B). VASP was phosphorylated by PKG I as expected (Fig. 4), but PKG I distribution did not show a clear pattern relative to the cytoskeleton (Fig. 5C,D), although PKG I in osteoclasts on bone after cGMP activation did include peripheral distribution consistent with its action on VASP (Fig. 5D). This, and poor immunoprecipitation of PKG I by anti-VASP (not shown), suggest that PKG I is at most transiently associated with the membrane attachment ring and its associated proteins. Another noteworthy finding from labeling PKG I was that, after prolonged stimulation with nonhydrolyzable cGMP analogs, PKG I frequently labeled the nuclei (not shown); these cells detached from substrate, suggesting that this is a toxic or pre-apoptotic response. In contrast, surprisingly large concentrations of short-term acting NO donors (e.g. 100 µM sodium nitroprusside) did not cause permanent detachment or cell death.

VASP knockdown impairs organization of the attachment site and prevents motility
The results to this point were consistent with a role for VASP in mediating rearrangement of the osteoclast cytoskeleton during motility. However, there are many actin-organizing proteins and redundant mechanisms often regulate important processes such as motility. Thus, these data were not sufficient to demonstrate that VASP is necessary for detachment or motility in response to NO or cGMP. Therefore, we performed siRNA gene silencing targeting VASP. Reduction of VASP protein (Fig. 6A) allowed cells to attach but modified the appearance of the actin ring. There were complex changes in the appearance of the cell attachment and the cells no longer responded to cGMP stimulation (Fig. 6B, right panel). These changes suggest that VASP may function in attachment aside from the response to NO/cGMP. This is not unexpected, as VASP is an intermediate protein with multiple domains, but further investigation of VASP in osteoclast cytoskeletal organization is beyond the scope of this work. However, in keeping with a central role of VASP in the NO/cGMP response, motility in response to cGMP activation was effectively eliminated in VASP-knockdown cells (Fig. 6C).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6. Reducing VASP with siRNA impairs formation of the osteoclast attachment and eliminates motility in response to NO. (A) Western blot showing reduction of VASP in cells with VASP-specific sequences versus a noncoding siRNA construct. This transfection used a mixture of four siRNAs. The knockdown efficiency (80-90%) was sufficient to show clear differences. (B) Impaired attachment with altered {alpha}vß3 distribution in VASP-targeted siRNA-treated cells on bone. The siRNA was labeled with Cy3 to allow identification of transfected cells. Approximately 85% of attached cells were labelled with Cy3 (red). In contrast to the effect of PKG I knockdown, where the attachment ring was intact (Fig. 3), VASP-knockdown cells had an abnormal attachment with a fragmented {alpha}vß3 ring (green). After VASP knockdown, the attachment structure did not change significantly with the cGMP agonist 8-pCPT-cGMP (100 µM, 1 hour). Each field is 40 µm horizontally. (C) Effect of a cGMP agonist on motility in mock-transfected and VASP-inhibited cells. There was no measurable motility in VASP-transfected cells when cells were exposed to 50 µM 8-pCPT-cGMP. The effect on motility was essentially the same as the effect of PKG I knockdown, but effects on the cell attachment were not observed with PKG I knockdown. Results are the means±s.d. of ten separate experiments.

 

    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclasts are motile cells that resorb bone by extracellular acid secretion via a specialized attachment to the extracellular matrix. Matrix binding is mediated by integrins, chiefly the {alpha}vß3 integrin heterodimer, which is directly linked to matrix degradation (Ross et al., 1993Go). Earlier work showed that activation of cGMP-dependent protein kinase I was inversely related to acid secretion by osteoclasts and matrix degradation (Van Epps-Fung et al., 1994Go; Dong et al., 1999Go), but high resolution studies of living osteoclasts on bone showed that the initial effect of NO donors or cGMP analogs on human osteoclasts involves redistribution of {alpha}vß3 and actin, which is tied directly to NO or cGMP-induced detachment (Yaroslavskiy et al., 2004Go). This suggested that the mechanism of action of NO depends chiefly on PKG I and that PKG I acts by regulating attachment and motility. There remained, however, significant controversies, including the fact that NO also inhibits osteoclasts via non-PKG-I-dependent mechanisms (MacIntyre et al., 1991Go; Mancini et al., 1998Go) and the lack of a mechanism for PKG-I-responsive rearrangement of the osteoclast attachment. Indeed, whether osteoclasts survive detachment in response to NO has been questioned. Some studies point to inactivation or apoptosis in osteoclasts or osteoclast precursors exposed to high levels of NO donors or cGMP analogs (van't Hof and Ralston, 1997Go). Time-lapse cinematography showed that cGMP or NO do not simply cause osteoclast detachment, but greatly increase motility in human or rodent osteoclasts (Fig. 1 and Movie 1 in supplementary material).

The vasodilator-stimulated phosphoprotein, VASP modifies actin organization and is a key PKG I target linked to integrin attachments; VASP was discovered in the circulatory system and has also been shown to regulate platelet aggregation (Meinecke et al., 1994Go; Butt et al., 1994Go; Reinhard et al., 1995bGo; Smolenski et al., 1998Go). VASP is widely distributed and involved in intracellular movement including macrophage phagocytosis (Coppolino et al., 2001Go), but VASP in osteoclasts had not been studied. We found mRNA for VASP in osteoclasts on gene screens and verified its presence by immunolocalization, western blotting and immunoprecipitation. We showed by motility studies after siRNA knockdown of PKG I and VASP that a NO/cGMP/PKG I/VASP pathway exists in the osteoclast, and that this is required for NO or cGMP-dependent osteoclast motility.

VASP is a member of the Ena/VASP protein family, which regulates temporal and spatial control of actin-filament dynamics. These proteins localize to the sites of actin assembly, such as focal adhesion, membrane ruffles and neuronal growth cones. Ena/VASP protein structure is conserved with a proline-rich core flanked by EVH1 and EVH2 domains. The EVH1 domain is essential for targeting of Ena/VASP proteins to focal adhesions (Sanjay et al., 2001Go; Niebuhr et al., 1997Go). The proline-rich region contains sites that bind to profilin (Ball et al., 2000Go). The EVH2 domain also contains binding sites for actin; in vitro it induces actin filament nucleation, promotes filament bundling and mediates oligomerization (Reinhard et al., 1995aGo; Walders-Harbeck et al., 1995Go; Bachmann et al., 1999Go).

VASP contains several Thr/Ser phosphorylation sites that modify its function and make it particularly important in cells with high cAMP or cGMP-dependent protein kinase activity. Active sites include Ser157 in the proline-rich region and Ser239 and Thr278 in the EVH2 domain (Butt et al., 1994Go). Ser157 is phosphorylated preferentially by the cAMP-dependent protein kinase, whereas Ser239 is preferred by the cGMP-dependent protein kinase. The Ser239 site is of particular importance in cytoskeletal rearrangements mediated by NO and cGMP in other cells, and a specific monoclonal antibody has been developed to characterize its phosphorylation state. We used this antibody to study cGMP-dependent VASP phosphorylation in human osteoclasts, with results showing that VASP phosphorylation at this site occurs in the osteoclast in response to NO donors or cGMP. Under basal conditions, VASP was partially phosphorylated (Fig. 4). Phosphorylation and motility were greatly increased by strong cGMP activation, exceeding by far the level of motility during normal osteoclast activity, where cells make approximately one resorption pit per day. Very strong cGMP activation is also associated with nuclear PKG I localization and apoptosis, in keeping with changes observed with NO or cGMP stimuli in other studies (Brandi et al., 1995Go; van't Hof and Ralston, 1997Go; Mancini et al., 2000Go). However, our data are most consistent with NO and cGMP being major regulators of osteoclast attachment and motility, with apoptosis requiring long periods of high-level stimulation.

Our studies demonstrate a requirement for VASP in cGMP-dependent osteoclast motility and provide clues as to the mechanism, but associations between osteoclast integrin attachment and motility will require further characterization. These include determination of the meaning of the sequential arcs of VASP and {alpha}vß3 in motile osteoclasts. These may reflect sequential organization of membrane complexes during membrane spreading. Further study of the role of organizing protein migfilin will also be important. Migfilin is associated with VASP only when VASP is phosphorylated by PKG I (see Fig. 4). An additional caveat is that motility subsequent to VASP-mediated reorganization undoubtedly involves additional downstream steps. In particular, although VASP was essential for NO/cGMP induced motility, it remains uncertain how this mechanism is related to motility induced by other stimuli such as CSF-1 and integrin-associated kinases. Both CSF-1 and fms stimulate src and phosphoinositydyl-3-kinase (Insogna et al., 1997Go; Pilkington et al., 1998Go; Teti et al., 1998Go; Grey et al., 2000Go). The phosphoinositydyl-3-kinase is likely to be activated during NO-induced motility because integrin-related complexes are rearranged. The phosphoinositydyl-3-kinase mediates a calcium signal, so calcium signals are likely after VASP activation. Local increases in calcium were observed in NO- or cGMP-stimulated osteoclasts (data not shown). These could represent NO/cGMP effects via a calcium channel or secondary effects of integrin rearrangement. As integrin-related phosphoinositydyl-3-kinase-dependent calcium signals are established, an indirect downstream calcium signal is likely. On the other hand, although calcium causes retraction in human osteoclasts as in rat cells (Datta et al., 1989Go), we have been unable to detach human osteoclasts with ionomycin or by increasing supernatant calcium to 20 mM (data not shown), suggesting that calcium cannot initiate detachment without a second signal, such as the NO/cGMP/PKG I/VASP pathway.

In conclusion, we demonstrate using time-lapse photography, immunolocalization, western analysis and siRNA gene knockdown that rapid motility in human osteoclasts induced by NO or cGMP depends on the cGMP-dependent protein kinase I and on the intermediate protein VASP. VASP is phosphorylated at Ser239 by the kinase in the osteoclasts just as it is modified by PKG I in endothelial cell relaxation. Downstream to VASP phosphorylation, the {alpha}vß3 integrin ring and other intermediate proteins, including migfilin, are redistributed. Redistribution of the attachment complex is highly likely to induce further attachment-related signals, which remain to be characterized.


    Acknowledgments
 
This work was supported in part by US National Institutes of Health AG12951, AR47700, GM65188, DK70526, AG23176 and by the Department of Veteran's Affairs (USA).


    Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/118/23/5479/DC1


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Armour, K. E., Armour, K. J., Gallagher, M. E., Godecke, A., Helfrich, M. H., Reid, D. M. and Ralston, S. H. (2001). Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase. Endocrinology 142, 760-766.[Abstract/Free Full Text]

Bachmann, C., Fischer, L., Walter, U. and Reinhard, M. (1999). The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation. J. Biol. Chem. 274, 23549-23557.[Abstract/Free Full Text]

Ball, L., Kuhne, J., R., Hoffmann, B., Hafner, A., Schmieder, P., Volkmer- Engert, R., Hof, M., Wahl, M., Schneider-Mergener, J., Walter, U., et al. (2000). Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity. EMBO J. 19, 4903-4914.[Abstract/Free Full Text]

Bear, J. E., Svitkina, T. M., Krause, M., Schafer, D. A., Loureiro, J. J., Strasser, G. A., Maly, I., V, Chaga, O. Y., Cooper, J. A., Borisy, G. G., et al. (2002). Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109, 509-521.[CrossRef][Medline]

Blair, H. C., Zaidi, M. and Schlesinger, P. H. (2002). Mechanisms balancing skeletal matrix synthesis and degradation. Biochem. J. 364, 329-341.[CrossRef][Medline]

Blair, H. C., Borysenko, C. W., Villa, A., Schlesinger, P. H., Kalla, S. E., Yaroslavskiy, B. B., Garcia-Palacios, V., Oakley, J. I. and Orchard, P. J. (2004). In vitro differentiation of CD14 cells from osteopetrotic subjects: contrasting phenotypes with TCIRG1, CLCN7, and attachment defects. J. Bone Mineral Res. 19, 1329-1338.[Medline]

Brandi, M., Hukkanen, L. M., Umeda, T. N., Bianchi, S., Gross, S. S., Polak, J. M. and MacIntyre, I. (1995). Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms. Proc. Natl. Acad. Sci. USA 92, 2954-2958.[Abstract/Free Full Text]

Butt, E., Abel, K., Krieger, M., Palm, D., Hoppe, V., Hoppe, J. and Walter, U. (1994). cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. J. Biol. Chem. 269, 14509-14517.[Abstract/Free Full Text]

Coppolino, M., Krause, G. M., Hagendorff, P., Monner, D. A., Trimble, W., Grinstein, S., Wehland, J. and Sechi, A. S. (2001). Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fc{gamma} receptor signalling during phagocytosis. J. Cell Sci. 114, 4307-4318.[Abstract/Free Full Text]

Datta, H. K., MacIntyre, I. and Zaidi, M. (1989). The effect of extracellular calcium elevation on morphology and function of isolated rat osteoclasts. Biosci. Rep. 9, 747-751.[CrossRef][Medline]

Dong, S.-S., Williams, J. P., Jordan, S. E., Cornwell, T. and Blair, H. C. (1999). Nitric oxide regulation of cGMP production in osteoclasts. J. Cell. Biochem. 73, 478-487.[CrossRef][Medline]

Grey, A., Chen, Y., Paliwal, I., Carlberg, K. and Insogna, K. (2000). Evidence for a functional association between phosphatidylinositol 3-kinase and c-src in the spreading response of osteoclasts to colony-stimulating factor-1. Endocrinology 141, 2129-2138.[Abstract/Free Full Text]

Hofmann, F., Ammendola, A. and Schlossmann, J. (2000). Rising behind NO: cGMP-dependent protein kinases. J. Cell Sci. 113, 1671-1676.[Abstract/Free Full Text]

Hukkanen, M., Platts, L. A., Lawes, T., Girgis, S. I., Konttinen, Y. T., Goodship, A. E., MacIntyre, I. and Polak, J. M. (2003). Effect of nitric oxide donor nitroglycerin on bone mineral density in a rat model of estrogen deficiency-induced osteopenia. Bone 32, 142-149.[CrossRef][Medline]

Insogna, K., Sahni, L. M., Grey, A. B., Tanaka, S., Horne, W. C., Neff, L., Mitnick, M., Levy, J. B. and Baron, R. (1997). Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts. J. Clin. Invest. 100, 2476-2485.[Abstract/Free Full Text]

Jamal, S. A., Browner, W. S., Bauer, D. C. and Cummings, S. R. (1998). Intermittent use of nitrates increases bone mineral density: the study of osteoporotic fractures. J. Bone Mineral Res. 13, 1755-1759.[Medline]

Lincoln, T. M. and Cornwell, T. L. (1993). Intracellular cyclic GMP receptor proteins. FASEB J. 7, 328-338.[Abstract/Free Full Text]

Lincoln, T. M., Pryzwansky, P., B, Cornwell, T. L., Wyatt, T. A. and MacMillan, L. A. (1993). cGMP-dependent protein kinase in smooth muscle and neutrophils. Adv. Second Messenger Phosphoprotein Res. 28, 121-132.[Medline]

MacIntyre, I., Zaidi, M., Alam, A. S., Datta, H. K., Moonga, B. S., Lidbury, P. S., Hecker, M. and Vane, J. R. (1991). Osteoclastic inhibition: an action of nitric oxide not mediated by cyclic GMP. Proc. Natl. Acad. Sci. USA 88, 2936-2940.[Abstract/Free Full Text]

Mancini, L., Moradi-Bidhendi, N., Brandi, M. L. and MacIntyre, I. (1998). Nitric oxide superoxide and peroxynitrite modulate osteoclast activity. Biochem. Biophys. Res. Commun. 243, 785-790.[CrossRef][Medline]

Mancini, L., Moradi-Bidhendi, N., Becherini, L., Martineti, V. and MacIntyre, I. (2000). The biphasic effects of nitric oxide in primary rat osteoblasts are cGMP dependent. Biochem. Biophys. Res. Commun. 274, 477-481.[CrossRef][Medline]

Meinecke, M., Geiger, J., Butt, E., Sandberg, M., Jahnsen, T., Chakraborty, T., Walter, U., Jarchau, T. and Lohmann, S. M. (1994). Human cyclic GMP-dependent protein kinase I beta overexpression increases phosphorylation of an endogenous focal contact-associated vasodilator-stimulated phosphoprotein without altering the thrombin-evoked calcium response. Mol. Pharmacol. 46, 283-290.[Abstract/Free Full Text]

Miyazawa, T., Ogawa, Y., Chusho, H., Yasoda, A., Tamura, N., Komatsu, Y., Pfeifer, A., Hofmann, F. and Nakao, K. (2002). Cyclic GMP-dependent protein kinase II plays a critical role in C-type natriuretic peptide-mediated endochondral ossification. Endocrinology 143, 3604-3610.[Abstract/Free Full Text]

Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U., Gertler, F. B., Wehland, J. and Chakraborty, T. (1997). A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J. 16, 5433-5444.[Abstract/Free Full Text]

Nomura, S. and Takano-Yamamoto, T. (2000). Molecular events caused by mechanical stress in bone. Matrix Biol. 19, 91-96.[CrossRef][Medline]

Pilkington, M. F., Sims, S. M. and Dixon, S. J. (1998). Wortmannin inhibits spreading and chemotaxis of rat osteoclasts in vitro. J. Bone Mineral Res. 13, 688-694.[Medline]

Reinhard, M., Giehl, K., Abel, K., Haffner, C., Jarchau, T., Hoppe, V., Jockusch, B. M. and Walter, U. (1995a). The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins. EMBO J. 14, 1583-1589.[Abstract]

Reinhard, M., Jouvenal, K., Tripier, D. and Walter, U. (1995b). Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein). Proc. Natl. Acad. Sci. USA 92, 7956-7960.[Abstract/Free Full Text]

Ross, F. P., Chappel, J., Alvarez, J. I., Sander, D., Butler, W. T., Farach- Carson, M. C., Mintz, K. A., Robey, P. G., Teitelbaum, S. L. and Cheresh, D. A. (1993). Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin {alpha}vß3 potentiate bone resorption. J. Biol. Chem. 268, 9901-9907.[Abstract/Free Full Text]

Sanjay, A., Houghton, A., Neff, L., DiDomenico, E., Bardelay, C., Antoine, E., Levy, J., Gailit, J., Bowtell, D., Horne, W. C. et al. (2001). Cbl associates with Pyk2 and Src to regulate Src kinase activity, {alpha}vß3 integrin-mediated signaling, cell adhesion, and osteoclast motility. J. Cell Biol. 152, 181-195.[Abstract/Free Full Text]

Smolenski, A., Bachmann, C., Reinhard, K., Honig-Liedl, P., Jarchau, T., Hoschuetzky, H. and Walter, U. (1998). Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody. J. Biol. Chem. 273, 20029-20035.[Abstract/Free Full Text]

Teti, A., Taranta, A., Migliaccio, S., Degiorgi, A., Santandrea, E., Villanova, I., Faraggiana, T., Chellaiah, M. and Hruska, K. A. (1998). Colony stimulating factor-1-induced osteoclast spreading depends on substrate and requires the vitronectin receptor and the c-src proto-oncogene. J. Bone Mineral Res. 13, 50-58.[Medline]

Tu, Y., Wu, S., Shi, X., Chen, K. and Wu, C. (2003). Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 113, 37-47.[CrossRef][Medline]

Van Epps-Fung, C., Williams, J., Cornwell, T., Lincoln, T., McDonald, J. M., Radding, W. and Blair, H. C. (1994). Regulation of osteoclastic acid secretion by cGMP-dependent protein kinase. Biochem. Biophys. Res. Comm. 204, 565-571.[CrossRef][Medline]

van't Hof, R. J. and Ralston, S. H. (1997). Cytokine-induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity. J. Bone Mineral Res. 12, 1797-1804.[Medline]

van't Hof, R. J. and Ralston, S. H. (2001). Nitric oxide and bone. Immunology 103, 255-261.[CrossRef][Medline]

Walders-Harbeck, B., Khaitlina, S. Y., Hinssen, H., Jockusch, B. M. and Illenberger, S. (1995). The vasodilator-stimulated phosphoprotein promotes actin polymerisation through direct binding to monomeric actin. FEBS Lett. 529, 275-280.[CrossRef]

Yaroslavskiy, B. B., Li, Y., Ferguson, D. J. P., Kalla, S. E., Oakley, J. I. and Blair, H. C. (2004). Autocrine and Paracrine Nitric Oxide Regulate Attachment of Human Osteoclasts. J. Cell. Biochem. 91, 962-972.[CrossRef][Medline]

Zaidi, M., Alam, A. S., Shankar, V. S., Bax, B. E, Moonga, B. S., Bevis, P. J., Pazianas. M. and Huang. C. L. (1992). A quantitative description of components of in vitro morphometric change in the rat osteoclast model: relationships with cellular function. Eur. Biophys. J. 21, 349-355.[Medline]