The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, Z.
Right arrow Articles by Bothwell, A. L. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, Z.
Right arrow Articles by Bothwell, A. L. M.
The Journal of Immunology, 2003, 171: 3620-3626.
Copyright © 2003 by The American Association of Immunologists

Receptor Activator of NF-{kappa}B Ligand Stimulates Recruitment of SHP-1 to the Complex Containing TNFR-Associated Factor 6 That Regulates Osteoclastogenesis1

Zhiyuan Zhang, Eijiro Jimi and Alfred L. M. Bothwell2

Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Receptor activator of NF-{kappa}B ligand (RANKL) is essential for differentiation and function of osteoclasts. The negative signaling pathways downstream of RANKL are not well characterized. By retroviral transduction of RAW264.7 cells with a dominant negative Src homology 2 domain-containing phosphatase-1 (SHP-1)(C453S), we studied the role of tyrosine phosphatase SHP-1 in RANKL-induced osteoclastogenesis. Over-expression of SHP-1(C453S) significantly enhanced the number of tartrate-resistant acid phosphatase-positive multinuclear osteoclast-like cells in response to RANKL in a dose-dependent manner. RANKL induced the recruitment of SHP-1 to a complex containing TNFR-associated factor (TRAF)6. GST pull down experiments indicated that the association of SHP-1 with TRAF6 is mediated by SHP-1 lacking the two Src homology 2 domains. RANKL-stimulated I{kappa}B-{alpha} phosphorylation, I{kappa}B-{alpha} degradation and DNA binding ability of NF-{kappa}B were increased after over-expression of SHP-1(C453S). However, RANKL-induced phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, was unchanged. In addition, SHP-1 regulated RANKL-stimulated tyrosine phosphorylation of p85 subunit of phosphatidylinositol 3 kinase and the phosphorylation of Akt. Increased numbers of osteoclasts contribute to severe osteopenia in Mev/Mev mice due to mutation of SHP-1. Like RAW264.7 cells expressing SHP-1(C453S), the bone marrow macrophages of Mev/Mev mice generated much more osteoclast-like cells than that of littermate controls in response to RANKL. Furthermore compared with controls, RANKL induces enhanced association of TRAF6 and RANK in both RAW264.7 cells expressing SHP-1(C453S) and bone marrow macrophages from Mev/Mev mice. Therefore, SHP-1 plays a role in signals downstream of RANKL by recruitment to the complex containing TRAF6 and these observations may help to understand the mechanism of osteoporosis in Mev/Mev mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Src homology 2 domain-containing phosphatase-1 (SHP-1)3 is a cytosolic protein tyrosine phosphatase that is predominantly expressed in hematopoietic cells (1, 2). SHP-1 contains two tandem Src homology 2 (SH2) domains in the N terminus followed by a single phosphatase domain and a C-terminal tail encompassing two sites for tyrosine phosphorylation (3). SHP-1 has now been implicated in the negative regulation of receptor tyrosine kinases such as c-kit, CSF-1 receptor (4, 5), cytokine receptors such as IL-3 (6), IFN-{alpha}{beta} (7), and receptors of the immune system containing the immune receptor tyrosine-based inhibitory motif such as CD22 and Fc{gamma}RIIB (8, 9).

Osteoclasts (OC) are monocyte/macrophage lineage multinucleated cells that play a critical role in bone resorption. OC differentiation is regulated by a variety of hormones, local factors and inflammatory cytokines, such as IL-1 and TNF-{alpha} (10, 11). The receptor activator of NF-{kappa}B ligand (RANKL is also called OPGL, TRANCE, and ODF) is a TNF-related cytokine which is critical for OC differentiation from hematopoietic precursors (12, 13, 14), and regulates OC function and cell survival (15, 16). RANK is an integral membrane protein, which binds to RANKL. Targeted disruption of either RANKL or RANK in mice causes lack of OC and an osteopetrotic phenotype (14, 17).

Similar to other TNF receptor family members, RANK lacks intrinsic catalytic activity. Binding of RANKL to RANK induces the interaction of TNFR-associated factors (TRAFs) with RANK, which then activates NF-{kappa}B through activation of I{kappa}B kinase, and AP-1 through activation of mitogen-activated protein kinases (18). I{kappa}B kinase activation results in the phosphorylation and degradation of I{kappa}B-{alpha}, which then releases the NF-{kappa}B from I{kappa}B inhibition, and allows NF-{kappa}B to translocate to the nucleus. In p50/p52 NF-{kappa}B double knockout mice, mature OC are absent (19), suggesting that NF-{kappa}B activation downstream of RANK mediates RANKL-regulated osteoclastogenesis.

Six members of the TRAF family have been identified. Among them, TRAF6 is critical for activation of NF-{kappa}B by RANK, since deletion of the TRAF6-binding region of RANK completely blocked the RANK-stimulated NF-{kappa}B activity (20, 21). TRAF6-/- mice exhibit severe osteopetrosis and are defective in OC formation (22). However, the mechanism by which TRAF6 activates NF-{kappa}B is unknown. It was reported recently that RANKL activates the anti-apoptotic serine/threonine kinase, Akt, which is mediated by TRAF6 in primary OC and dendritic cells (23). RANKL treatment stimulates RANK recruitment of TRAF6, c-Src, Cbl family-scaffolding proteins, and phospholipid kinase phosphatidylinositol 3-kinase (PI-3 kinase) (23, 24).

A critical role for SHP-1 in regulating development and function of myeloid lineage cells including OC is revealed by the enormous myelo-monocytic expansion found in motheaten (Me/Me) mice and viable motheaten (Mev/Mev) mice which express either no SHP-1 or a catalytically defective SHP-1 protein due to a splice site mutation in the SHP-1 gene (2, 25, 26, 27). To investigate the mechanisms of SHP-1 in regulating RANK signaling pathway in OC precursors, we generated stable transductants of monocyte/macrophage RAW264.7 cells expressing vector alone, wild type (WT), or dominant negative SHP-1(C453S), which lacks phosphatase activity (28, 29, 30, 31). We demonstrated that RANKL induces the complex formation of SHP-1 and TRAF6 by which SHP-1 regulates downstream signals of RANK. The SHP-1(C453S) increases RANKL-induced association of RANK and TRAF6, activation of NF-{kappa}B, and OC differentiation. In addition, SHP-1 regulates RANKL-induced p85 PI-3 kinase and Akt phosphorylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, reagents, and Abs

Six-week-old C57BL/6J-Mev/Mev mice and littermate controls were purchased from The Jackson Laboratory (Bar Harbor, ME). Recombinant mouse RANK ligand and CSF-1 were obtained from R&D Systems (Minneapolis, MN). Anti-TRAF6 (C-20) (for immunoprecipitation), anti-TRAF6 (H-274) (for Western blot), anti-RANK (H-300), anti-I{kappa}B-{alpha}, anti-phospho-c-Jun N-terminal kinase (JNK) Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-I{kappa}B-{alpha} (Ser32), anti-phospho-p44/42 mitogen-activated protein kinase (MAP) (phospho-extracellular signal-regulated kinase 1/2 (ERK 1/2)), anti-phospho-p38 MAP kinase, anti-phospho-Akt Abs were obtained from Cell Signaling Technology (Beverly, MA). Anti-SHP-1, anti-PI-3 kinase p85, and anti-phosphotyrosine mAb, clone 4G10 were obtained from Upstate Biotechnology (Lake Placid, NY). Wortmannin was purchased from BD Biosciences (San Diego, CA), LY294002 was purchased from Calbiochem (La Jolla, CA). Anti-flag M2 and anti-{beta}-actin mAb were obtained from Sigma-Aldrich (St. Louis, MO).

Construction of retroviral vector expressing dominant negative SHP-1

The DNAs of flag-tagged WT SHP-1 and catalytically inactive SHP-1(C453S) in the pEFIII vector were kindly provided by Dr. K. Mizuno (Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan). Using the above DNAs as templates, PCR was first performed to synthesize cDNAs of flag-tagged WT and C453S SHP-1. The primers are: forward, AAC CCC AGG ATG GAC TAC AAG GAC GAC GAT; reverse, TAC CGC GGT CAC TTC CTC TTG AGA GAA CC. The PCR inserts were then subcloned into the pCRII vector, and finally subcloned into the EcoR1 site of pBMN-Z-I-Neo retroviral vector (kindly provided by Dr. G. P. Nolan, Stanford University, Palo Alto, CA). The PA317 packaging cell line was transfected with the recombinant retroviral DNAs and G418-resistant cells were derived as the source of retroviral stocks. Stable retroviral infection of RAW264.7 cells was performed using standard procedures and stable lines selected at 1 mg/ml G418 (32).

Cell culture, immunoprecipitation, and immunoblotting

Bone marrow macrophages (BMMs) were generated as described (33). Briefly, bone marrow cells of Mev/Mev mice and littermate controls were devoid of RBC, and cultured in 10 cm dishes in the presence of 20 ng/ml CSF-1. The media were changed after 48 h of culture, and the cells were cultured for another 48 h. The RAW264.7 monocyte/macrophage cell line was maintained in modified {alpha}-MEM with 10 mM HEPES, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin pH 7.28, at 37°C in a humidified atmosphere. RAW264.7 cells were subcultured into 10 cm dishes at 5 x 106 per dish, and incubated overnight. Before various stimulations, the cells were serum starved for 2 h, processed for various treatments, and lysed in 1% Brij 97, 10% glycerol, 150 mM NaCl, 10 mM Tris, 2 mM EDTA, 20 mM sodium fluoride (NaF), 1 mM sodium vanadate (NaV), 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin (pH 7.3). The lysates were incubated with the appropriate Abs at 4°C for 2 h followed by another 1 h incubation with protein A-Sepharose beads. The immunoprecipitates were washed three times with PBS containing inhibitors described above. Total cell lysates or immunoprecipitates were eluted from the beads by boiling in 2x Laemmli sample buffer and separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes, immunoblotted with appropriate Abs, and detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

GST pull down assay

Bacteria transformed by plasmids of GST fusion proteins containing two SH2 domains of SHP-1 and SHP-1 lacking two SH2 domains in pGEX-2T vector were kindly provided by Dr. T. Yi (Cleveland Clinic Foundation, Cleveland, OH) and Dr. G. Chiang (Salk Institute, La Jolla, CA), respectively. The bacteria was amplified in Luria-Bertani media containing 25 µg/ml ampicillin, and the expression of GST fusion proteins was induced by 0.25 mM IPTG. The bacteria were collected by centrifugation and resuspended in Harvest buffer (40 mM Tris (pH 8.0), 100 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing protease inhibitors, sonicated, and the lysates were centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was incubated with glutathione-Sepharose beads (Amersham Biosciences, Piscataway, NJ), and incubated for 60 min at 4°C. The beads were washed three times with TNTG buffer (40 mM Tris (pH7.5), 100 mM NaCl, 10% glycerol, 1% Triton X-100) and either processed for the GST pull down assay or the beads were added with protein sample buffer, boiled, and the GST fusion proteins were fractionated on SDS-PAGE.

RAW264.7 cells were treated with 100 ng/ml RANKL for 20 min, and lysed in lysis buffer (10 mM HEPES, pH 7.4, 50 mM {beta}-glycerophosphate, 1% Triton X-100, 10% glycerol, 4 mM EDTA, 1 mM DTT, 10 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin), and centrifuged. The supernatant containing ~1 mg protein was precleared with glutathione-Sepharose beads, then added to 5 µg GST alone or GST fusion proteins which were bound to glutathione-Sepharose beads and incubated overnight at 4°C. The precipitates were then washed twice with the lysis buffer, and twice with lysis buffer supplemented with 200 mM NaCl, and boiled in SDS sample buffer.

EMSA assay

Cells were subcultured into 10 cm dishes with 1 x 107 cells/dish, and incubated overnight at 37°C. After various treatments, the cells were suspended in ice-cold PBS and collected by centrifugation. The nuclear extracts were prepared as described elsewhere (34). The sequence of the NF-{kappa}B binding oligonucleotide used as a radioactive DNA probe was 5'-GAT CAG AGG GGA CTT TCC GAG G (35). Equal amounts of extract (10 µg protein) were incubated with 32P-labeled probe and the DNA-protein complex was analyzed on a 4% polyacrylamide gel.

Differentiation of multinuclear osteoclast-like cells (OCL)

RAW264.7 cells were plated in 24-well plates (5,000/well), and incubated with 20 ng/ml RANKL. BMMs of Mev/Mev mice and littermate controls were plated in 24-well plates (1 x 106/well), and incubated with 20 ng/ml RANKL and 20 ng/ml CSF-1. After 5 days of culture, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) as described elsewhere (36).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retroviral infection of SHP-1(C453S) into RAW264.7 cells enhances RANKL-induced osteoclastogenesis

To study the mechanisms of tyrosine phosphatase SHP-1 in regulating OC differentiation, we over-expressed a dominant negative mutant of SHP-1 which is deficient in catalytic activity, SHP-1(C453S). Comparisons were made with RAW264.7 cells transduced with the empty vector or WT SHP-1. SHP-1 constructs were tagged with the FLAG epitope to facilitate detection. Comparable levels of expression of transduced WT SHP-1 and SHP-1(C453S) in RAW264.7 cells were observed (Fig. 1A). RAW264.7 cells express high levels of endogenous SHP-1. Exogenous expression of SHP-1 constructs enhanced the total SHP-1 expression in this cell (Fig. 1B). Multinucleated OC were detected by TRAP staining after treatment of RAW264.7 cells with RANKL for 5 days (data not shown). Over-expression of SHP-1(C453S) significantly increased the OC formation compared with either empty vector transductants or WT SHP-1 (Fig. 1, C and D). Over-expression of WT SHP-1 did not show significant inhibition of OC generation by RANKL (Fig. 1D), probably due to the high expression of endogenous SHP-1 in the cell. This model mimics the increased osteoclastogenesis in Mev/Mev mice (Fig. 1, E and F). Treatment of BMMs with RANKL plus CSF-1 induced formation of OCL, with much higher levels in Mev/Mev mice compared with littermate controls.



View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 1. Over-expression of SHP-1(C453S) enhances RANKL-induced osteoclastogenesis which mimics that in Mev/Mev mice. Total cell lysates from RAW264.7 cells and RAW264.7 cells over-expressing vector, WT SHP-1, and SHP-1(C453S) were separated by SDS-PAGE and immunoblotted with anti-flag Ab. (A, upper panel) and anti-SHP-1 Ab (B, left upper panel). The membranes were also blotted with anti-actin Ab to determine the amount of proteins (lower panels of A and B). Densitometry was performed for B, left panel, and the data was shown in B, right panel. C, RAW264.7 cells expressing vector and C453S mutant were treated with 20 ng/ml RANKL followed by TRAP staining after 5 days of culture. Magnification: x200. D, RAW264.7 cells expressing vector, WT SHP-1, and C453S mutant were treated with 10, 20, 40, 80, 160 ng/ml RANKL, and the cells were processed as described in C. The number of TRAP-positive multinucleated cells were counted. The data is representative of three independent experiments, each in triplicate. E, BMMs of Mev/Mev mice and control littermates were cultured in the presence of 20 ng/ml CSF-1 and 20 ng/ml RANKL. After 5 days of culture, cells were processed for TRAP staining. Magnification is x200. F, The number of TRAP-positive multinucleated cells in (E) were counted each in triplicate.

 
RANKL induces association of SHP-1 and TRAF6 mediated by SHP-1 lacking the SH2 domains

TRAF6 interacts with RANK in response to RANKL and then TRAF6 regulates downstream signals of RANK (20, 21). We demonstrated that in RAW264.7 cells, RANKL treatment induced the association of SHP-1 and TRAF6 by coimmunoprecipitation, whereas no association of these two proteins was detected in untreated RAW264.7 cells (Fig. 2A).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. RANKL stimulates complex formation of SHP-1 and TRAF6 mediated by SHP-1 lacking SH2 domains. A, RAW264.7 cells were treated with or without 100 ng/ml RANKL for 20 min. The lysates were immunoprecipitated with normal serum (NS, lane 2) and anti-TRAF6 (lanes 3 and 4) Abs. Total cell lysates and the immune complexes were separated by SDS-PAGE and immunoblotted with anti-SHP-1 Ab (upper panel). The membrane was stripped and reblotted with anti-TRAF6 Ab to determine the amount of protein (lower panel). B, A summary diagram describing the GST fusion proteins of SHP-1 used in C. C, The GST or GST fusion proteins containing two SH2 domains of SHP-1 (SHP-1, SH2) and SHP-1 lacking two SH2 domains (SHP-1, PTP-C) prepared as described in Materials and Methods were then fractionated by SDS-PAGE and the gels were stained in GelCode Blue Staining Reagent (left panel). RAW264.7 cells were treated with 100 ng/ml RANKL for 20 min. The lysates were incubated with GST alone or different GST fusion proteins bound to beads. The released protein complexes were processed for electrophoresis and immunoblotted with anti-TRAF6 Ab (right panel).

 
To substantiate these findings and determine which domain of SHP-1 mediates the association with TRAF6, we used chimeric GST fusion proteins containing either the two amino-terminal SH2 domains only (SHP-1, SH2) or a chimera that lacks these domains but contains the PTP domain and the carboxyl-terminal tail (SHP-1, PTP-C) (Fig. 2, B and C). Lysates of RANKL-stimulated RAW264.7 cells were incubated with purified GST-fusion proteins consisting of different domains of SHP-1. TRAF6 was only pulled down by SHP-1 and PTP-C, whereas SHP-1 and TRAF6 association was not detected by SHP-1, SH2. Incubation of lysates with GST alone served as a negative control.

Over-expression of SHP-1(C453S) enhances the activation of NF-{kappa}B in response to RANKL

NF-{kappa}B activation in response to RANKL regulates the differentiation of OC (19, 20, 37, 38). To investigate the potential role of SHP-1 in regulating RANKL-induced NF-{kappa}B activation, we examined the RAW264.7 cell lines expressing either an empty vector or the mutant SHP-1(C453S). Cells were treated with RANKL for 15 or 30 min with 100 ng/ml RANKL. Gel shift analysis showed that NF-{kappa}B DNA-binding activity was increased after treatment for 15 min and this activity was further enhanced after 30 min of treatment. Over-expression of SHP-1(C453S) increased the basal activity of NF-{kappa}B, as well as the RANKL-stimulated NF-{kappa}B activity at 15 and 30 min (Fig. 3A).



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 3. SHP-1(C453S) over-expression enhances the RANKL-induced activation of NF-{kappa}B. A, RAW264.7 cells expressing vector and C453S mutant were treated with 100 ng/ml RANKL for indicated times. The nuclear extracts were prepared and EMSA was performed using 32P-labeled NF-{kappa}B oligonucleotide. B, RAW-vector and RAW-SHP-1(C453S) were treated with 100 ng/ml RANKL for indicated times. Total cell lysates were immunoblotted with anti-phospho-I{kappa}B-{alpha} (panel 1), anti-I{kappa}B-{alpha} (panel 2), anti-phospho-ERK1/2 (panel 3), anti-phospho-JNK (panel 4), and anti-phospho-p38 MAP kinase (panel 5) Abs. The same lysates were also blotted with anti-{beta}-actin (panel 6) Ab to quantitate the amount of proteins.

 
RANKL has been shown to stimulate phosphorylation of I{kappa}B-{alpha} and ERK1/2 phosphorylation in primary OC and DC (23). In the vector transduced RAW264.7 cell line, treatment with 100 ng/ml RANKL stimulated I{kappa}B-{alpha} phosphorylation by 10 min, which was maintained up to 40 min. I{kappa}B-{alpha} degradation was not detected up to 10 min of treatment. At 40 min, the degradation was 40% determined by densitometry. Our result of slight I{kappa}B-{alpha} degradation by RANKL in vector-transduced RAW264.7 cells was consistent with other published results using mature OC and dendritic cells (23, 24). SHP-1(C453S) over-expression significantly increased the RANKL-stimulated I{kappa}B-{alpha} phosphorylation and degradation. The phosphorylation of I{kappa}B-{alpha} was detected at 5 min, quickly decreased at 10 min, and was undetectable after 20 min of treatment. I{kappa}B-{alpha} was 61% degraded by densitometry at 5 min of RANKL treatment, and I{kappa}B-{alpha} was completed degraded at 20 min. (Fig. 3B). RANKL also stimulated phosphorylation of MAP kinases ERK1/2, JNK, and p38 MAPK. In contrast to effects on I{kappa}B-{alpha}, over-expression of SHP-1(C453S) has no effect on RANKL-induced MAP kinase phosphorylation (Fig. 3B).

SHP-1 regulates RANKL-stimulated tyrosine phosphorylation of p85 and Akt phosphorylation

To further characterize candidate targets of SHP-1 regulation, we hypothesized that SHP-1 may alter particular tyrosine phosphorylated proteins that might be recruited to the TRAF6/RANK complex. The kinase, p85 PI-3 kinase, was reported to be recruited to the complexes containing TRAF6 after RANKL treatment in primary OC and DC (23, 24). In T cells, SHP-1 regulates PI-3 kinase phosphorylation and activity (39). We then characterized the effect of SHP-1 on phosphorylation of p85 PI-3 kinase in response to RANKL (Fig. 4A). RANKL induced tyrosine phosphorylation of p85 PI-3 kinase at 10 min, and this stimulation was further enhanced at 20 min. In WT SHP-1-over-expressing cells, there was no detectable p85 phosphorylation at 10 min. At 20 min, RANKL-induced p85 phosphorylation was slightly decreased compared with the vector-transduced cells. In contrast, in cells over-expressing SHP-1(C453S), RANKL stimulated p85 phosphorylation at 5 min, and the phosphorylation of p85 was significantly increased at 10 and 20 min compared with the vector-transduced cells.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 4. SHP-1 regulates RANKL-stimulated tyrosine phosphorylation of p85 PI-3 kinase and Akt phosphorylation. A, RAW264.7 cells expressing vector, WT SHP-1 and C453S mutant, were treated with 100 ng/ml RANKL for indicated times, and lysates were immunoprecipitated with anti-p85 and immunoblotted with anti-phosphotyrosine Ab (upper panel). The membrane was then reblotted with anti-p85 Ab to determine the amount of proteins (lower panel). B, RAW264.7 cells expressing vector and C453S mutant were treated with or without 100 ng/ml RANKL for 20 min. Total cell lysates were immunoblotted with anti-phospho-Akt Ab (upper panel) and anti-{beta}-actin Ab to determine the amount of protein (lower panel). C, RAW264.7 cells were treated with or without 100 nM wortmannin for 30 min, or 10 µM LY294002 for 30 min in the presence or absence of 100 ng/ml RANKL. Nuclear extracts were prepared and EMSA was performed using 32P-labeled NF-{kappa}B oligonucleotide.

 
It was reported that PI-3 kinase mediates RANKL-induced phosphorylation of the anti-apoptotic serine/threonine kinase, Akt, in primary OC and DC (23). The RANKL-induced phosphorylation of Akt in RAW264.7 cells was also repressed after inhibition of PI-3 kinase activity (data not shown). The role of SHP-1 in RANKL-stimulated Akt phosphorylation was then studied. SHP-1(C453S) over-expression increased the basal level of Akt phosphorylation, and the phosphorylation of Akt induced by RANKL was also enhanced compared with that of vector control (Fig. 4B). However, gel shift analysis indicated that inhibition of PI-3 kinase activity by either wortmannin or LY294002 failed to inhibit RANKL-induced NF-{kappa}B activation (Fig. 4C) and differentiation of OC from RAW264.7 cells (data not shown).

The increased Akt phosphorylation after over-expressing SHP-1(C453S) suggested that this SHP-1 mutant may regulate survival of RAW264.7 cells, which contributed to the increases in TRAP-positive OCL. We then performed annexin V staining to test the effect of SHP-1(C453S) expression on apoptosis of the cells. Although serum starvation-induced apoptosis was slightly inhibited (15% decrease in SHP-1(C453S), expressing cells compared with vector control), over-expression of SHP-1(C453S) did not show a significant effect on unstimulated or RANKL-stimulated cells without serum starvation (data not shown). In addition, expression of C453S mutant has no effect on cell proliferation detected by counting the cell numbers after 1, 2, and 3 days in the presence or absence of RANKL (data not shown). These suggest that the increased number of OCL in response to RANKL after expressing C453S mutant is due to increased cell differentiation.

SHP-1 regulates the RANKL-induced association of RANK and TRAF6

Association of TRAF6 with RANK plays a key role in RANKL-induced NF-{kappa}B activation and OC differentiation (20, 21). We then analyzed the possible role of SHP-1 in the complex formation of RANK and TRAF6 by coimmunoprecipitation. The RAW264.7 cell lines over-expressing vector alone, WT SHP-1, or SHP-1(C453S) were incubated with or without RANKL. The cell lysates were immunoprecipitated with Ab to RANK and then blotted with anti-TRAF6 Ab (Fig. 5A). RANKL induced association of RANK with TRAF6 in cells expressing vector alone or WT SHP-1 and there were increased levels in both untreated and RANKL-stimulated cell extracts over-expressing SHP-1(C453S).



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5. Inactivation of SHP-1 in both RAW264.7 cells expressing SHP-1(C453S) and BMMs of Mev/Mev mice enhances RANKL-induced TRAF6 and RANK association. RAW264.7 cells expressing vector, WT SHP-1 and C453S mutant (A), and BMMs of Mev/Mev mice and littermate controls (B), were treated with or without 100 ng/ml RANKL for 20 min. The lysates were immunoprecipitated with anti-RANK Ab and the proteins were processed for immunoblotting with anti-TRAF6 Ab (upper panel). The membrane was stripped and reblotted with anti-RANK Ab (lower panel) to determine the amount of protein.

 
We then further examined the association of RANK and TRAF6 in the BMMs of Mev/Mev mice (Fig. 5B). Treatment with RANKL induced the association of RANK and TRAF6 in BMMs of littermate controls. The RANKL-induced association of RANK and TRAF6 was enhanced in BMMs of Mev/Mev mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Retroviral transduction of RAW264.7 cells with dominant negative SHP-1(C453S) significantly enhanced RANKL-induced osteoclastogenesis. Using this model, we demonstrated that RANKL induces the recruitment of SHP-1 to the TRAF6-containing complex. Over-expression of SHP-1(C453S) resulted in significantly greater NF-{kappa}B activation and RANK/TRAF6 association than that observed in vector alone- or WT-SHP-1-transduced cells. These results suggest that SHP-1 may serve as a negative regulator of RANKL-induced intracellular signals and osteoclastogenesis.

To maintain normal cellular response to stimuli, the balance of positive and negative regulators of receptor signaling is important. Recently, negative regulatory mechanisms concerning RANKL-induced osteoclastogenesis that maintain bone homeostasis have been reported. Osteoprotegerin, a decoy receptor for RANKL, inhibits RANKL-induced osteoclastogenesis by competing with RANK to bind RANKL (40). Moreover, IFN-{gamma} produced by activated T cells induces TRAF6 degradation, thereby preventing OC formation (41). It has been recently reported that RANKL induces IFN-{beta} gene expression in OC precursors. IFN-{beta} inhibits OC differentiation by interfering with the expression of RANKL-induced c-Fos, an essential transcriptional factor for osteoclastogenesis (42), suggesting that RANKL signaling itself activates a negative regulator of gene expression. We report here another new negative regulatory mechanism that SHP-1 modulates association of RANK with TRAF6 to maintain bone homeostasis.

It has been shown that TRAF6-deficient mice exhibit severe osteopetrosis due to lack of osteoclast formation (22). By binding to cell surface receptor RANK, RANKL induces the association of TRAF6 with RANK, which then stimulates the NF-{kappa}B activity and osteoclastogenesis (20, 21). Kobayashi et al.(43) recently demonstrated that the second and third zinc fingers of TRAF6 play an essential role in differentiation of osteoclasts, whereas, the RING finger is responsible for osteoclast maturation. Our results indicate that SHP-1 is recruited to the TRAF6-containing complex in response to RANKL. The TRAF6 zinc finger domain may play a role in the SHP-1 recruitment, and may mediate the effect of SHP-1 on inhibition of RANKL-stimulated NF-{kappa}B activity and osteoclast formation.

In resting cells such as bone marrow macrophages and lymphocytes, the SH2 domains of SHP-1 interact with its catalytic domain and inhibit the catalytic activity of SHP-1 (44, 45). Upon cell activation, SHP-1 binds to tyrosine-phosphorylated immunoreceptor tyrosine-based inhibitory motifs (e.g., CD22) by the SH2 domain of SHP-1 (3, 46), and this binding relieves the steric inhibition and activates the phosphatase (44). Our GST pull down assay indicated that RANKL-induced recruitment of SHP-1 to the TRAF6 complex in RAW264.7 cells is mediated by SHP-1 lacking the two SH2 domains. It was reported that the carboxyl-terminal region of SHP-1 is required for interaction with the p32/p30 proteins. In a DA3 erythropoietin receptor cell line expressing a SHP-1 mutant containing the c terminus of SHP-1, the p32/p30 proteins are hyperphosphorylated (47). P32/p30 are also expressed in motheaten hematopoietic cells, suggesting a possible association between hyperphosphorylation of p32/30 and the phenotype of the mice. In addition, a high-affinity binding site for phosphatidic acid and phosphatidylinositol 3,4,5-trisphosphate is mapped to the 41 carboxyl-terminal amino acids of SHP-1 (48). Further characterization of the mechanisms of SHP-1 recruitment to TRAF6 complex in response to RANKL needs to be undertaken.

A possible mechanism regulating Akt/PKB activation by RANKL stimulation is that PI-3 kinase is involved in RANKL signaling pathway (23, 24). We showed that RANKL stimulated tyrosine phosphorylation of p85 subunit of PI-3 kinase in a time-dependent manner. Inhibition of SHP-1 activity by over-expression of dominant negative SHP-1(C453S) significantly enhanced the RANKL-stimulated p85 and Akt phosphorylation. In dendritic cells, tyrosine kinase c-Src and adaptor protein c-Cbl are also recruited to RANK, forming a complex with TRAF6 upon RANKL stimulation. TRAF6 then further enhances the kinase activity of c-Src and tyrosine phosphorylation of c-Cbl (23, 24). These results strongly suggest that SHP-1 associated with TRAF6 after the stimulation of RANKL regulates Src family kinase and PI-3 kinase activation and subsequently Akt phosphorylation. However, Wong et al. (23) and our study also shows here that inhibition of PI-3K has no effect on RANKL-induced NF-{kappa}B activation and osteoclastogenesis. Although, NF-{kappa}B regulates several cellular functions including cell survival by other TNFR family members, activation of PI-3 kinase-Akt pathway by RANKL may be involved in the survival of osteoclast precursors and osteoclasts.

Our results suggest a new negative regulatory pathway downstream of RANKL. SHP-1 regulates RANKL-induced osteoclastogenesis by recruiting to the complex containing TRAF6. These may help to identify novel approaches to treat diseases characterized by increased osteoclastic bone resorption, such as osteoporosis associated with rheumatoid arthritis and osteoarthritis.


   Acknowledgments
 
We thank Dr. Peijun Zhang and Dr. Yalai Bai for very helpful discussions, Nancy Troiano for image processing, and Stephen E. Maher and Claire Wickens for technical assistance.


   Footnotes
 
1 This work was supported by a fellowship from the Arthritis Foundation (to Z.Z.) and National Institutes of Health Grant GM40924 (to A.B). Back

2 Address correspondence and reprint requests to Dr. Alfred L. M. Bothwell, Section of Immunobiology, Yale University School of Medicine, P.O. Box 208011, 300 Cedar Street, New Haven, CT 06520-8011. E-mail address: alfred.bothwell{at}yale.edu Back

3 Abbreviations used in this paper: SHP-1, Src homology 2 domain-containing phosphatase-1; SH2, Src homology 2; OC, osteoclast; RANKL, NF-{kappa}B ligand; TRAF, TNFR-associated factor; PI-3, phosphatidylinositol 3; WT, wild type; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; BMM, bone marrow macrophage; OCL, osteoclast-like cell; TRAP, tartrate-resistant acid phosphatase. Back

Received for publication October 16, 2002. Accepted for publication July 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Matthews, R. J., D. B. Bowne, E. Flores, M. L. Thomas. 1992. Characterization of hematopoietic intracellular protein tyrosine phosphatases: description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences. Mol. Cell. Biol. 12:2396.[Abstract/Free Full Text]
  2. Kozlowski, M., I. Mlinaric-Rascan, G. S. Feng, R. Shen, T. Pawson, K. A. Siminovitch. 1993. Expression and catalytic activity of the tyrosine phosphatase PTP1C is severely impaired in motheaten and viable motheaten mice. J. Exp. Med. 178:2157.[Abstract/Free Full Text]
  3. Neel, B. G., N. K. Tonks. 1997. Protein tyrosine phosphatases in signal transduction. Curr. Opin. Cell Biol. 9:193.[Medline]
  4. Paulson, R. F., S. Vesely, K. A. Siminovitch, A. Bernstein. 1996. Signalling by the W/Kit receptor tyrosine kinase is negatively regulated in vivo by the protein tyrosine phosphatase Shp1. Nat. Genet. 13:309.[Medline]
  5. Chen, H. E., S. Chang, T. Trub, B. G. Neel. 1996. Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1. Mol. Cell. Biol. 16:3685.[Abstract]
  6. Yi, T., A. L. Mui, G. Krystal, J. N. Ihle. 1993. Hematopoietic cell phosphatase associates with the interleukin-3 (IL-3) receptor {beta}-chain and down-regulates IL-3-induced tyrosine phosphorylation and mitogenesis. Mol. Cell. Biol. 13:7577.[Abstract/Free Full Text]
  7. David, M., H. E. Chen, S. Goelz, A. C. Larner, B. G. Neel. 1995. Differential regulation of the {alpha}/{beta} interferon-stimulated Jak/Stat pathway by the SH2 domain-containing tyrosine phosphatase SHPTP1. Mol. Cell. Biol. 15:7050.[Abstract]
  8. Law, C. L., S. P. Sidorenko, K. A. Chandran, Z. Zhao, S. H. Shen, E. H. Fischer, E. A. Clark. 1996. CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-{gamma}(1) upon B cell activation. J. Exp. Med. 183:547.[Abstract/Free Full Text]
  9. Nadler, M. J., B. Chen, J. S. Anderson, H. H. Wortis, B. G. Neel. 1997. Protein-tyrosine phosphatase SHP-1 is dispensable for Fc{gamma}RIIB-mediated inhibition of B cell antigen receptor activation. J. Biol. Chem. 272:20038.[Abstract/Free Full Text]
  10. Jimi, E., I. Nakamura, L. T. Duong, T. Ikebe, N. Takahashi, G. A. Rodan, T. Suda. 1999. Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells. Exp. Cell Res. 247:84.[Medline]
  11. van der Pluijm, G., W. Most, L. van der Wee-Pals, H. de Groot, S. Papapoulos, C. Lowik. 1991. Two distinct effects of recombinant human tumor necrosis factor-{alpha} on osteoclast development and subsequent resorption of mineralized matrix. Endocrinology 129:1596.[Abstract/Free Full Text]
  12. Lacey, D. L., E. Timms, H. L. Tan, M. J. Kelley, C. R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al 1998. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165.[Medline]
  13. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, et al 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95:3597.[Abstract/Free Full Text]
  14. Kong, Y. Y., H. Yoshida, I. Sarosi, H. L. Tan, E. Timms, C. Capparelli, S. Morony, A. J. Oliveira-dos-Santos, G. Van, A. Itie, et al 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development, and lymph-node organogenesis. Nature 397:315.[Medline]
  15. Jimi, E., S. Akiyama, T. Tsurukai, N. Okahashi, K. Kobayashi, N. Udagawa, T. Nishihara, N. Takahashi, T. Suda. 1999. Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function. J. Immunol. 163:434.[Abstract/Free Full Text]
  16. Fuller, K., B. Wong, S. Fox, Y. Choi, T. J. Chambers. 1998. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J. Exp. Med. 188:997.[Abstract/Free Full Text]
  17. Li, J., I. Sarosi, X. Q. Yan, S. Morony, C. Capparelli, H. L. Tan, S. McCabe, R. Elliott, S. Scully, G. Van, et al 2000. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc. Natl. Acad. Sci. USA 97:1566.[Abstract/Free Full Text]
  18. Arch, R. H., R. W. Gedrich, C. B. Thompson. 1998. Tumor necrosis factor receptor-associated factors (TRAFs) - a family of adapter proteins that regulates life and death. Genes Dev. 12:2821.[Free Full Text]
  19. Franzoso, G., L. Carlson, L. Xing, L. Poljak, E. W. Shores, K. D. Brown, A. Leonardi, T. Tran, B. F. Boyce, U. Siebenlist. 1997. Requirement for NF-{kappa}B in osteoclast and B cell development. Genes Dev. 11:3482.[Abstract/Free Full Text]
  20. Darnay, B. G., J. Ni, P. A. Moore, B. B. Aggarwal. 1999. Activation of NF-{kappa}B by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-{kappa}B-inducing kinase: identification of a novel TRAF6 interaction motif. J. Biol. Chem. 274:7724.[Abstract/Free Full Text]
  21. Galibert, L., M. E. Tometsko, D. M. Anderson, D. Cosman, W. C. Dougall. 1998. The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-{kappa}B, a member of the TNFR superfamily. J. Biol. Chem. 273:34120.[Abstract/Free Full Text]
  22. Naito, A., S. Azuma, S. Tanaka, T. Miyazaki, S. Takaki, K. Takatsu, K. Nakao, K. Nakamura, M. Katsuki, J. Inoue. 1999. Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4:353.[Abstract]
  23. Wong, B. R., D. Besser, N. Kim, J. R. Arron, M. Vologodskaia, H. Hanafusa, Y. Choi. 1999. TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-src. Mol. Cell. 4:1041.[Medline]
  24. Arron, J. R., M. Vologodskaia, B. R. Wong, M. Naramura, N. Kim, H. Gu, Y. Choi. 2001. A positive regulatory role for Cbl family proteins in tumor necrosis factor-related activation-induced cytokine (TRANCE) and CD40L-mediated Akt activation. J. Biol. Chem. 276:30011.[Abstract/Free Full Text]
  25. Tsui, H. W., K. A. Siminovitch, L. de Souza, F. W. Tsui. 1993. Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene. Nat. Genet. 4:124.[Medline]
  26. Aoki, K., E. Didomenico, N. A. Sims, K. Mukhopadhyay, L. Neff, A. Houghton, M. Amling, J. B. Levy, W. C. Horne, R. Baron. 1999. The tyrosine phosphatase SHP-1 is a negative regulator of osteoclastogenesis and osteoclast resorbing activity: increased resorption and osteopenia in MEv/MEv mutant mice. Bone 25:261.[Medline]
  27. Umeda, S., W. G. Beamer, K. Takagi, M. Naito, S. Hayashi, H. Yonemitsu, T. Yi, L. D. Shultz. 1999. Deficiency of SHP-1 protein-tyrosine phosphatase activity results in heightened osteoclast function and decreased bone density. Am. J. Pathol. 155:223.[Abstract/Free Full Text]
  28. Battaglino, R., D. Kim, J. Fu, B. Vaage, X.-Y. Fu, P. Stashenko. 2002. c-myc is required for osteoclast differentiation. J. Bone Miner. Res. 17:763.[Medline]
  29. Horwood, N. J., J. Elliott, T. J. Martin, M. T. Gillespie. 2001. IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro. J. Immunol. 166:4915.[Abstract/Free Full Text]
  30. Dong, Q., K. A. Siminovitch, L. Fialkow, T. Fukushima, G. P. Downey. 1999. Negative regulation of myeloid cell proliferation and function by the SH2 domain-containing tyrosine phosphatase-1. J. Immunol. 162:3220.[Abstract/Free Full Text]
  31. Dustin, L. B., D. R. Plas, J. Wong, Y. T. Hu, C. Soto, A. C. Chan, M. L. Thomas. 1999. Expression of dominant-negative Src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation. J. Immunol. 162:2717.[Abstract/Free Full Text]
  32. Zheng, L., T. J. Dengler, M. S. Kluger, L. A. Madge, J. S. Schechner, S. E. Maher, J. S. Pober, A. L. M. Bothwell. 2000. Cytoprotection of HUVEC against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses. J. Immunol. 164:4665.[Abstract/Free Full Text]
  33. Kobayashi, K., N. Takahashi, E. Jimi, N. Udagawa, M. Takami, S. Kotake, N. Nakagawa, M. Kinosaki, K. Yamaguchi, N. Shima, et al 2000. Tumor necosis factor {alpha} stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J. Exp. Med. 191:275.[Abstract/Free Full Text]
  34. Voll, R. E., E. Jimi, R. J. Phillips, D. F. Barber, M. Rincon, A. C. Hayday, R. A. Flavell, S. Ghosh. 2000. NF-{kappa}B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development. Immunity 13:677.[Medline]
  35. Ghosh, S., D. Baltimore. 1990. Activation in vitro of NF-{kappa}B by phosphorylation of its inhibitor I{kappa}B. Nature 344:678.[Medline]
  36. Takahashi, N., H. Yamana, S. Yoshiki, G. D. Roodman, G. R. Mundy, S. J. Jones, A. Boyde, T. Suda. 1988. Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122:1373.[Abstract/Free Full Text]
  37. Wong, B. R., R. Josien, S. Y. Lee, M. Vologodskaia, R. M. Steinman, Y. Choi. 1998. The TRAF family of signal transducers mediates NK-{kappa}B activation by the TRANCE receptor. J. Biol. Chem. 273:28355.[Abstract/Free Full Text]
  38. Lomaga, M. A., W. C. Yeh, I. Sarosi, G. S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al 1999. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13:1015.[Abstract/Free Full Text]
  39. Cuevas, B., Y. Lu, S. Watt, R. Kumar, J. Zhang, K. A. Siminovitch, G. B. Mills. 1999. SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase phosphorylation and activity. J. Biol. Chem. 274:27583.[Abstract/Free Full Text]
  40. Udagawa, N., N. Takahashi, H. Yasuda, A. Mizuno, K. Itoh, Y. Ueno, T. Shinki, M. T. Gillespie, T. J. Martin, K. Higashio, T. Suda. 2000. Osteoprotegrin produced by osteoblasts is an important regulator in osteoclast development and function. Endocrinology 141:3478.[Abstract/Free Full Text]
  41. Takayanagi, H., K. Ogasawara, S. Hida, T. Chiba, S. Murata, K. Sato, A. Takaoka, T. Yokochi, H. Oda, K. Tanaka, et al 2000. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature 408:600.[Medline]
  42. Takayanagi, H., S. Kim, K. Matsuo, H. Suzuki, T. Suzuki, K. Sato, T. Yokochi, H. Oda, K. Nakamura, N. Ida, et al 2002. RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-{beta}. Nature 416:744.[Medline]
  43. Kobayashi, N., Y. Kadono, A. Naito, K. Matsumoto, T. Yamamoto, S. Tanaka, J.-i. Inoue. 2001. Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis. EMBO J. 20:1271.[Medline]
  44. Pei, D., U. Lorenz, U. Klingmuller, B. G. Beel, C. T. Walsh. 1994. Intramolecular regulation of protein tyrosine phosphatase SH-PTP1: a new function for Src homology 2 domains. Biochemistry 33:15483.[Medline]
  45. Martin, A., H. W. Tsui, M. J. Shulman, D. Isenman, F. W. Tsui. 1999. Murine SHP-1 splice variants with altered Src homology 2 (SH2) domains: implications for the SH2-mediated intramolecular regulation of AHP-1. J. Biol. Chem. 274:21725.[Abstract/Free Full Text]
  46. DAmbrosio, D., K. L. Hippen, S. A. Minskoff, I. Mellman, G. Pani, K. A. Siminovitch, J. C. Cambier. 1995. Recruitment and activation of PTP1C in negative regulation of antigen receptor by Fc{gamma}RIIB1. Science 268:293.[Abstract/Free Full Text]
  47. Yang, W., M. Tabrizi, K. Berrda, T. Yi. 1998. SHP-1 phosphatase C terminus interacts with novel substrates p32/p30 during erythropoietin and interleukin-3 mitogenic responses. Blood 91:3746.[Abstract/Free Full Text]
  48. Frank, C., H. Keilhack, F. Opitz, O. Zshornig, F. D. Bohmer. 1999. Binding of phosphatidic acid to the protein-tyrosine phosphatase SHP-1 as basis for activity modulation. Biochem. 38:11993.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. R. Kiesel, Z. S. Buchwald, and R. Aurora
Cross-Presentation by Osteoclasts Induces FoxP3 in CD8+ T Cells
J. Immunol., May 1, 2009; 182(9): 5477 - 5487.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H.-J. Kim, K. Zhang, L. Zhang, F. P. Ross, S. L. Teitelbaum, and R. Faccio
The Src family kinase, Lyn, suppresses osteoclastogenesis in vitro and in vivo
PNAS, February 17, 2009; 106(7): 2325 - 2330.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Mori, S. Tsuji, M. Inui, Y. Sakamoto, S. Endo, Y. Ito, S. Fujimura, T. Koga, A. Nakamura, H. Takayanagi, et al.
Inhibitory Immunoglobulin-Like Receptors LILRB and PIR-B Negatively Regulate Osteoclast Development
J. Immunol., October 1, 2008; 181(7): 4742 - 4751.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
C.-H. Liu, F. S. Machado, R. Guo, K. E. Nichols, A. W. Burks, J. C. Aliberti, and X.-P. Zhong
Diacylglycerol kinase {zeta} regulates microbial recognition and host resistance to Toxoplasma gondii
J. Exp. Med., April 16, 2007; 204(4): 781 - 792.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
M. Muthukuru and C. W. Cutler
Upregulation of Immunoregulatory Src Homology 2 Molecule Containing Inositol Phosphatase and Mononuclear Cell Hyporesponsiveness in Oral Mucosa during Chronic Periodontitis
Infect. Immun., February 1, 2006; 74(2): 1431 - 1435.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. K. Manna and G. T. Ramesh
Interleukin-8 Induces Nuclear Transcription Factor-{kappa}B through a TRAF6-dependent Pathway
J. Biol. Chem., February 25, 2005; 280(8): 7010 - 7021.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S.-I. Hayashi, M. Tsuneto, T. Yamada, M. Nose, M. Yoshino, L. D. Shultz, and H. Yamazaki
Lipopolysaccharide-Induced Osteoclastogenesis in Src Homology 2-Domain Phosphatase-1-Deficient Viable Motheaten Mice
Endocrinology, June 1, 2004; 145(6): 2721 - 2729.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Saito, N. Ohara, H. Hotokezaka, S. Fukumoto, K. Yuasa, M. Naito, T. Fujiwara, and K. Nakayama
Infection-induced Up-regulation of the Costimulatory Molecule 4-1BB in Osteoblastic Cells and Its Inhibitory Effect on M-CSF/RANKL-induced in Vitro Osteoclastogenesis
J. Biol. Chem., April 2, 2004; 279(14): 13555 - 13563.
[Abstract] [Full Text] [PDF]

Home page
 Rheumatology (Oxford)Home page
Robin Goodfellow (42-12)
Rheumatology, December 1, 2003; 42(12): 1580 - 1580.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, Z.
Right arrow Articles by Bothwell, A. L. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, Z.
Right arrow Articles by Bothwell, A. L. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS