Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, and Departments of Medicine and Biochemistry, Loma Linda University, Loma Linda, California 92357
Submitted 10 July 2003 ; accepted in final form 20 May 2004
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ABSTRACT |
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osteoclast; resorption; nuclear factor-; c-Src
The objective of this study was threefold: 1) to determine whether PTP-oc plays a regulatory role in the functional activity, differentiation, proliferation, and/or apoptosis of precursors of osteoclastic cells; 2) to determine whether the enzymatic activity of PTP-oc is essential for these processes; and 3) if so, to gain insights into the molecular mechanisms by which PTP-oc acts to regulate osteoclast formation and/or activity. Our experimental approach was to determine and compare the effects of overexpression of wild-type (WT)-PTP-oc and phosphatase-deficient (PD)-PTP-oc in "osteoclast-like" cells on their differentiation, apoptosis, and bone resorption activity. Primary osteoclasts are terminally differentiated and do not proliferate, and they are not suitable for transgenic overexpression studies. Thus this study used human promyelomonocytic leukemic U-937 cells as the cell model, because 1) these cells express PTP-oc (30); 2) they have been used in the past in a number of laboratories as the cell model of osteoclast precursors (6, 7, 10, 12, 25); 3) we have developed a protocol to induce the differentiation of these cells into osteoclast-like cells, which stained strongly for tartrate-resistant acid phosphatase (TRAP) and expressed known osteoclastic marker genes, i.e., TRAP, calcitonin receptor, receptor activator of NF- (RANK), cathepsin K (CatK), and matrix metalloproteinase (MMP)-9, and the derived osteoclast-like cells were capable of resorbing bone in vitro; and 4) at the time this study was initiated several years ago, there were no convenient preosteoclast cell line models available or suitable for this work.
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MATERIALS AND METHODS |
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Resorption pit formation assay. U-937 cells (5,000 cells) were plated on a circular (70 mm in diameter) dentin disk in 48-well plates. 12-O-tetradecanoylphorbol 12-myristate 13-acetate (TPA) (107 M; Sigma, St. Louis, MO) was added 24 h later, and the cells were incubated for 48 h. The cell medium was then replaced with fresh RPMI 1640 containing 108 M 1,25(OH)2D3 (Biomol Biomolecules Research Laboratory, Plymouth Meeting, PA). The cells were incubated for 25 days with medium containing 1,25(OH)2D3 changed every 3 days. This TPA/1,25(OH)2D3 sequential treatment converted U-937 cells into osteoclast-like cells. The medium was then changed to fresh RPMI 1640 without effectors, and the resorption pit formation was measured 48 h later as previously described (31). The resorption activity per osteoclast (an index of mean osteoclast activity) was determined by dividing the total resorption pit area by the number of resorption pits.
The average resorption pit depth was determined by confocal microscopy using an Olympus IX70 inverted confocal microscope workstation (Olympus, Melville, NY). Briefly, after the total area and number of acid hematoxylin-stained resorption pits on the dentin disk were measured, each dentin disk was mounted on a glass slide in 50% glycerin (vol/vol). The coverslip was sealed with nail polish and dried for 72 h, and the dried slide was mounted upside down on the microscope. The resorption pits were viewed on the Olympus microscope fitted with a confocal argon-krypton mixed gas laser. Serial images of the pits at 2-µm intervals from the top surface of the dentin disk to the bottom of the pit were obtained using a z-plane motorized substage. Each image was obtained using appropriate excitation and emission filters. Six randomly selected pits per disc and four replicate discs per group were analyzed. The average depth is calculated from the number of 2-µm slices needed to reach the bottom of the pit.
Stable U-937 cell pools overexpressing PTP-oc protein. The pcDNA 3.1(+) expression vector (Invitrogen, Carlsbad, CA) was initially used to produce stable U-937 cells. The full-length rabbit PTP-oc cDNA was cloned into the pcDNA 3.1(+) vector. The PD-PTP-oc expression construct was prepared by polymerase chain reaction (PCR)-based, site-directed mutagenesis with WT-PTP-oc pcDNA 3.1(+) as the template using the QuickChange kit (Stratagene, La Jolla, CA), in which the catalytic cysteine (Cys325) was mutated to serine. The mutant lacked appreciable PTP activity (data not shown). U-937 cells were transfected with each PTP-oc expression vector or empty vector with Effectene (Qiagen, Valencia, CA). However, "stable cells" generated with these constructs were unstable (apparently because of gene silencing) and did not overexpress PTP-oc after 23 mo.
We subsequently used our recently developed transposon-based "Prince Charming" (pPC) gene transfer vector (13) for this work. This vector system has two important advantages: 1) cell clones produced with the pPC vector are extremely stable (13, 40), and 2) the maintenance of the resulting stable cell clones does not require continuous selection pressure, which allows experimentation with these stable cells in the absence of antibiotics (13). To construct the pPC-PTP-oc expression vector, linkers corresponding to the NotI and PmeI restriction sites were added in frame to the 5' and 3' ends, respectively, of the full-length WT- and PD-PTP-oc cDNA. The resulting plasmids were cloned into the pPC cassette at the NotI-PmeI sites to generate respective pPC-PTP-oc expression vector. The U-937 cells were then transfected with each pPC-PTP-oc expression vector as well as the pPC-RFP vector (a control vector) (13) using Effectene. After selection and expansion in G418 for 4 wk, stable cell pools overexpressing WT- or PD-PTP-oc were isolated. Three different stable cell pools for each group were used in these studies. The cell pools produced with the pPC-PTP-oc vectors were extremely stable: the PTP-oc protein expression level in each cell pool has not changed for more than 1 year, even in the absence of G418 (data not shown).
Measurement of cellular PTP-oc protein levels. The cellular PTP-oc protein level was measured by Western immunoblot assay using a guinea pig polyclonal antibody against the unique region of the NH2 terminus of rabbit PTP-oc as described previously (9). The blot was stripped and reblotted against an anti-actin antibody for normalization against protein loading.
Differentiation of U-937 cells into osteoclast-like cells. U-937 cell differentiation was monitored by counting the number of TRAP-positive, multinucleated (i.e., 2 or more nuclei) osteoclast-like cells formed after the TPA/1,25(OH)2D3 treatment. Briefly, U-937 cells (10,000 cells/well) were plated in RPMI 1640 medium. The cells were treated with 107 M TPA for 2 days, which converted a significant number of suspension cells into attached cells. The unattached cells were removed, and the attached cells were rinsed once with RPMI 1640 medium. 1,25(OH)2D3 (108 M) was then added for 26 days with changes of medium containing 1,25(OH)2D3 every 23 days. A large number of attached cells were then fused to form TRAP-positive, multinucleated cells. The number of TRAP-positive, multinucleated cells was counted in eight representative fields with a bright-field microscope.
U-937 cell differentiation was also quantitated by measuring the TRAP specific activity. Briefly, U-937 cells (50,000 cells/well) were plated in 24-well plates in serum-free RPMI 1640 medium. After the TPA/1,25(OH)2D3 treatment, the attached cells were extracted with 0.5 ml of 0.1% Triton X-100. The TRAP activity in the extract was measured with a TRAP assay kit (Sigma) and normalized against cellular protein determined with the bicinchoninic acid protein assay (Pierce, Rockford, IL).
Expression of osteoclastic genes in the U-937 cell-derived, osteoclast-like cells.
Total RNA was extracted from parental U-937 cells or U-937 cell-derived, osteoclast-like cells using the RNeasy kit (Qiagen). The RNA was digested with 0.4 U/µl DNase I (Invitrogen), and the DNase was heat-inactivated at 80°C for 10 min. Complementary DNA (cDNA) was synthesized from the DNase-treated RNA sample using Superscript II reverse transcriptase (GIBCO-BRL, Grand Island, NY). PCR amplification of calcitonin receptor (CTR), TRAP, RANK, CatK, MMP-9, and -actin (cDNA loading control) was performed individually with HotStar Taq DNA polymerase using the primer sets shown in Table 1. The reaction was subjected to a hot start at 95°C, followed by 35 amplification cycles comprising denaturing for 1 min at 95°C, annealing for 1 min at 52°C, and extending for 1 min at 72°C per cycle. The PCR products were analyzed on 1.2% agarose gels.
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Assays of cell proliferation, cell cycle, and viable cell number. U-937 cell proliferation was assessed by [3H]thymidine incorporation. Briefly, U-937 cells (10,000 cells in 1 ml of serum-free RPMI) were plated in 24-well plates. After 24 h, 1.5 µCi of [3H]thymidine (Research Products International, Mount Prospect, IL) was added for 6 h and the cells were transferred to a test tube, collected by centrifugation, and washed three times with ice-cold phosphate-buffered saline. After the final wash, the cells were obtained with a cotton swab moistened in 12.5% trichloroacetic acid to trap the cellular material in the cotton fibers. The cotton tips were then washed three times with 12.5% trichloroacetic acid, once with 95% ethanol, and air dried. [3H]thymidine incorporation was determined by liquid scintillation counting.
Cell cycle analysis was performed using fluorescence-activated cell sorting (FACSCalibur; BD Biosciences, San Jose, CA) with propidium iodide to label cell DNA. The relative number of viable cells was assessed with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-based assay (Roche Diagnostics, Penzberg, Germany). In this assay, MTT is taken up and reduced only by metabolically active cells, in part by the action of dehydrogenases.
Measurement of c-Src Tyr527 phosphorylation level. The relative levels of Tyr527-phosphorylated (PY-527) c-Src and total c-Src level were measured by performing the Western immunoblot assay as described previously (34) using a phospho-specific polyclonal antibody against PY-527 of c-Src (Biosource International, Camarillo, CA) and an anti-c-Src antibody (Upstate Biotechnology, Lake Placid, NY), respectively.
Measurement of I degradation.
The I
degradation was assessed by determining the amounts of cellular I
in each cell pool. A decrease in cellular I
level compared with control indicates an increase in degradation. Briefly, equal amounts of cell extracts, prepared by extraction in the radioimmunoprecipitation assay (RIPA) buffer, were separated by 10% SDS-PAGE and transblotted onto nitrocellulose membrane. The cellular I
level was determined by Western immunoblot assay using a specific anti-I
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and normalized against actin content for protein loading.
NF- nuclear translocation assay.
Nuclear translocation of NF-
is an index of NF-
activation (4). To assess NF-
nuclear translocation, the nuclei of U-937 cells were isolated as described previously (17), washed, and extracted with the RIPA buffer. The same amount of nuclear proteins from each extract was fractionated on 10% SDS-PAGE and transblotted onto a nitrocellulose membrane. The nuclear NF-
level was measured by Western immunoblots using a specific anti-p65 RelA antibody (BD Transduction Laboratories, San Diego, CA). There is no convenient nuclear housekeeping protein suitable for normalization of protein loading. Thus no reblotting against a housekeeping protein was done.
NF- transcriptional activation assay.
A firefly luciferase (luc) reporter gene-based pNF-
-transcription activity assay kit (BD Bioscience, Palo Alto, CA), which uses a construct (pNF-
-luc) containing multiple copies of NF-
consensus sequence fused to the luc reporter gene, was used to assess NF-
transcriptional activity. Briefly, cells were transiently transfected with 1 µg of pNF-
-luc plasmid or with the promoterless pTAL-Luc (control) with Effectene. Two days later, the luc activity in the cell extract was measured with an assay kit from Promega (Madison, WI). To control for transfection efficiency, cells in each experiment were also transfected with pGL3-control plasmids. The NF-
activation was normalized against SV-40 promoter activity and reported as relative changes of parental cells.
Statistical analysis. Results are shown as means ± SE or SD with three to six replicates or repeat measurements. Statistical significance was determined with two-tailed Student's t-test, and the difference was significant when P < 0.05.
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RESULTS |
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Effects of PTP-oc overexpression on PY-527 level of c-Src in U-937 cells.
Because we have preliminary evidence that PTP-oc acts to stimulate osteoclast activity by activating the c-Src PTK activity through the PTP-oc-mediated dephosphorylation of PY-527 of c-Src (19, 34), we measured the steady-state c-Src PY-527 level in each test cell pool. Figure 9 shows that the steady-state c-Src PY-527 level (normalized against either total c-Src or actin) in cells stably overexpressing WT-PTP-oc was reduced to 25% (P < 0.01) of that of the vector control cells. Cells overexpressing PD-PTP-oc had a 25% (P < 0.05) increase in c-Src PY-527 levels. The c-Src PY-527 level correlated negatively with the resorption activity of the derived osteoclast-like cells (r = 0.998, P < 0.01 for pit area/pit; r = 0.961, P < 0.05 for pit depth), suggesting that the bone resorption activity of U-937 cell-derived, osteoclast-like cells is associated with the PTP-oc-mediated c-Src activation. The c-Src PY-527 level also correlated negatively with osteoclast-like cells formed in response to the TPA/1,25(OH)2D3 sequential treatment (r = 0.996, P < 0.01 for osteoclast-like cell number).
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DISCUSSION |
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This study presents strong evidence that PTP-oc may have some regulatory roles in the formation and resorption activity of osteoclast-like cells in the U-937 cell model. To that end, we have demonstrated that overexpression of WT-PTP-oc increased the bone resorption activity and the formation of osteoclast-like cells in response to the TPA/1,25(OH)2D3 sequential treatment. By contrast, overexpression of PD-PTP-oc in U-937 cells suppressed differentiation into osteoclast-like cells in response to the TPA-1,25(OH)2D3 sequential treatment and the bone resorption activity of the derived osteoclast-like cells. Importantly, the fact that overexpression of PD-PTP-oc not only did not enhance, but also significantly suppressed, the bone resorption activity and the differentiation of U-937 cell-derived, osteoclast-like cells led us to conclude that the enzymatic activity (but not the protein level) of PTP-oc is important for these processes. This further led us to speculate that PTP-oc functions in osteoclasts and precursor cells to dephosphorylate key tyrosyl-phosphorylated proteins rather than to serve as docking proteins. The fact that PTP-oc overexpression stimulates the differentiation of U-937 cells is not unexpected, because there is circumstantial evidence that members of GLEPP1/PTP-oc family have enhancing effects on the differentiation of several cell types, including U-937 cells (9, 28, 29, 36).
Consistent with the previous finding that overexpression of WT-GLEPP1 in U-937 cells induced apoptosis of the terminally differentiated monocytes/macrophages (30), we found that overexpression of WT-PTP-oc in U-937 cells also increased apoptosis. Similarly to PD-GLEPP1 (30), overexpression of PD-PTP-oc had no significant effect on U-937 cell apoptosis. Although the reason for the lack of an effect of PD-PTP-oc on apoptosis is unclear, these findings raise the interesting possibility that high, ultraphysiological levels of cellular enzyme activity of PTP-oc (or GLEPP1) enhanced the apoptosis of U-937 cells. Consistent with this speculation are our previous findings that suppression of PTP-oc expression with PTP-oc antisense oligodeoxynucleotide did not appear to affect the life span of mature rabbit osteoclasts (34). Moreover, we have preliminary evidence that the enhancing effect of PTP-oc on cell apoptosis might be unique to U-937 cells, because WT-PTP-oc overexpression in murine RAW267.4 cells (an osteoclast precursor cell model) did not enhance apoptosis (unpublished observation).
The mechanism whereby high levels of active PTP-oc (or GLEPP1) induce apoptosis is unknown. While we suspect that some of the PTP-oc actions are associated with the PTP-oc-mediated NF- activation (see below), we do not think that the induced U-937 cell apoptosis involved NF-
activation, for the following three reasons: 1) activation of NF-
in other cell types (including RAW264.7 cells) has antiapoptotic effects; 2) overexpression of PD-PTP-oc, which led to a marked inhibition of NF-
, did not show a corresponding inhibition of U-937 cell apoptosis; and 3) while the actions of NF-
on apoptosis are mediated largely through caspase-dependent pathways, a specific caspase inhibitor could only slightly inhibit the GLEPP1-induced apoptosis in U-937 cells (30). Thus it is possible that GLEPP1 and/or PTP-oc acts primarily through caspase-independent pathways to induce apoptosis in U-937 cells.
One of the most surprising findings of this study is that overexpression of WT-PTP-oc also led to the stimulation of cell proliferation in U-937 cells as reflected by an increase in [3H]thymidine incorporation and an increase in the number of cells at the S phase, with a corresponding decrease in the number of cells at the G0/G1 phase. Because there was not a large difference in the number of viable cells between the test cell populations, the increase in apoptosis in response to WT-PTP-oc overexpression might have been counterbalanced by the PTP-oc-associated stimulation in cell proliferation. On the other hand, although PD-PTP-oc overexpression did not affect apoptosis, it appeared to reduce cell proliferation as reflected by a reduction in [3H]thymidine incorporation and a significant increase in the number of cells at G2/M phase without a corresponding reduction in the number of cells at G0/G1 phase. However, the significance of this finding is unknown at this time.
Regarding the potential mechanisms whereby PTP-oc regulates the bone resorption activity of osteoclasts, we have strong circumstantial evidence in rabbit osteoclasts that the PY-527 of c-Src could be a substrate of PTP-oc and that PTP-oc may act through activation of the c-Src signaling pathway by dephosphorylating the PY-527 residue of c-Src to activate bone resorption activity (19, 34). In this study, we have shown that overexpression of WT-PTP-oc in U-937 cells, which led to an increase in bone resorption activity, also activated c-Src PTK activity by reducing c-Src PY-527 levels, whereas overexpression of PD-PTP-oc, which caused an inhibition of bone resorption activity, also reduced c-Src PTK activity by increasing c-Src PY-527 levels. That the c-Src PY-527 level correlated strongly with the bone resorption activity of the U-937 cell-derived, osteoclast-like cells provided additional associative evidence for the involvement of c-Src activation in the PTP-oc-mediated activation of osteoclast-like cells.
The findings that overexpression of WT-PTP-oc resulted in the activation of NF- and that the bone resorption activity of the derived osteoclast-like cells also correlated with NF-
activation raise the interesting possibility that PTP-oc-mediated osteoclast activation may involve NF-
activation. The mechanism whereby PTP-oc activates the NF-
pathway and the relationship between PTP-oc-mediated c-Src activation and the associated NF-
activation is largely unknown. Under unstimulated conditions, NF-
is retained in the cytoplasm as a heterodimer with an inhibitory protein, I
(41). I
is the most important I
member in cells of hematopoietic origin, including osteoclasts (8). Activation of NF-
is usually initiated by phosphorylation of I
at Ser32 and/or Ser36, which prompts the dissociation of I
from NF-
, marking it for ubiquitination and rapid degradation via the ubiquitin-proteasome pathway (5). The liberated NF-
is, in turn, rapidly phosphorylated and translocated to the nucleus to function as a transcriptional complex to activate specific gene expression. An important discovery relevant to the relationship between NF-
activation and c-Src activation is that I
is also phosphorylated at Tyr42 in certain cell populations (e.g., osteoclasts, lymphocytes) (1, 2, 14, 21, 32). The protein-tyrosine phosphorylation of I
is mediated by members of the c-Src PTK family, such as c-Lck in T lymphocytes (1) and c-Src in cells of the osteoclast lineage (2). The protein-tyrosine phosphorylation of I
also leads to the rapid degradation of I
(21, 32) and nuclear translocation of NF-
for specific gene transcription. Consistent with the premise that phosphorylation of I
at Tyr42 in osteoclasts plays a regulatory role in the activation of NF-
, administration of a dominant negative I
mutant protein in which Tyr42 was deleted blocked both NF-
activation and osteoclast activation (1). Therefore, we postulate that PTP-oc dephosphorylates Tyr527 of c-Src in osteoclasts, resulting in the activation of its PTK activity, which in turn phosphorylates I
at Tyr42, leading to degradation of I
and nuclear translocation and activation of NF-
pathways.
Inasmuch as our proposed mechanism focuses only on PTP-oc-mediated NF- activation, we by no means conclude that NF-
activation is the primary mechanism by which the PTP-oc-c-Src pathway acts to regulate osteoclast activity. To the contrary, there is abundant evidence that c-Src regulates osteoclast activity through multiple mechanisms. Accordingly, the SH2 domain of c-Src has been shown to function as an adaptor protein to regulate c-Src trafficking and membrane localization, to mediate interactions with downstream signaling proteins, and/or to recruit key signaling proteins to membrane receptor PTKs for phosphorylation (26). Activated c-Src in osteoclasts has also been reported to migrate to mitochondria to phosphorylate cytochrome c oxidase, which is required for osteoclast function (20). There is also evidence for the involvement of PYK2 in the c-Src-induced spreading and activation of osteoclasts (18). Thus it is clear that the mechanism whereby the PTP-oc-c-Src pathway regulates osteoclast activity is complex and involves multiple signaling pathways. Further work is needed to delineate the relationship between these pathways and PTP-oc-mediated NF-
activation.
Finally, we are somewhat surprised to note the highly significant correlation between PTP-oc-dependent c-Src activation and the differentiation of U-937 cells into osteoclast-like cells. There is compelling evidence in the literature against a role for c-Src in osteoclast differentiation (33). Consequently, we think that the PTP-oc-dependent osteoclast differentiation probably does not involve the c-Src pathway. Much additional work is needed to determine the exact mechanism by which PTP-oc enhances osteoclast differentiation.
In summary, we have shown in U-937 cells that 1) PTP-oc stimulated the bone resorption activity of the U-937 cell-derived, osteoclast-like cells and enhanced the differentiation of U-937 cells into osteoclast-like cells; 2) the enzymatic activity (but not the protein level) of PTP-oc was essential for these processes; 3) high levels of enzymatically active PTP-oc led to an increase in apoptosis as well as in proliferation; and 4) PTP-oc stimulated the bone resorption activity and differentiation of U-937 cell-derived, osteoclast-like cells through c-Src-dependent and c-Src-independent NF- activation, respectively. However, because U-937 cells are not bona fide osteoclast precursors, these findings must be confirmed with primary osteoclasts and/or precursors before the role of PTP-oc in osteoclast activity and differentiation can be ascertained.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of S.-M. Suhr: Dept. of Biochemistry, Yonsei Univ., Seoul, Korea.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* M. Amoui and S.-M. Suhr contributed equally to this work.
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