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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

Several lines of evidence suggest that the c-Src tyrosine kinase has a specific role in bone-resorbing osteoclasts. To investigate this further, we examined the expression of c-Src, its kinase family members, and their putative substrates in the human leukemia cell line FLG 29.1. Western blot analysis with specific antibodies against Src family members showed expression of Src, Fyn, and Lyn, lower levels of Yes and Hck, and the absence of Lck tyrosine kinase. During a 3-day treatment with phorbol 12-myristate, 13-acetate (PMA), which induces differentiation of FLG 29.1 cells toward an osteoclast-like phenotype, the levels of Src and Fyn increased and the levels of Lyn decreased. In a similar leukemia cell line, HL-60, Src protein was not constitutively expressed and not induced by PMA treatment, which leads to monocytic differentiation. PMA treatment of FLG 29.1 cells induced a strong increase in the expression of p120 Cbl and Pyk2 kinase, which are putative Src substrates. Pyk2 phosphorylation increased upon adherence of FLG 29.1 cells to fibronectin and to ST2 stromal cells. The expression of other Src substrates and interacting proteins, such as p120 Cas, p130 Cas, vinculin, Fak kinase, and the p85 phosphatidylinositol 3-kinase subunit either did not change or slightly increased during PMA treatment. The elevated total protein tyrosine phosphorylation in PMA-treated FLG 29.1 cells was abolished by herbimycin A, a Src inhibitor. These data are consistent with the proposed role of Src in the osteoclastic function and support the use of FLG 29.1 cells as a model to study Src substrates in the cells of the osteoclastic lineage.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

C-Src is a cytoplasmic tyrosine kinase that represents a prototype for a kinase family currently consisting of nine members: Src, Yes, Fyn, Yrk, Fgr, Hck, Lyn, Lck, and Blk.(1) Src, Fyn, and Yes are ubiquitously expressed, while the expression of other family members is more restricted, especially to cells of the hematopoietic lineage. The osteopetrotic phenotype of Src-deficient mice revealed a crucial role of Src in osteoclast-mediated bone resorption.(2) The bone phenotype in Src-deficient mice is due to an autonomous defect in the formation of the ruffled border, which is necessary for bone resorption by osteoclasts.(3,4) On the basis of these findings, Src is thought to have an unique role in osteoclasts, since other Src family members could apparently not substitute for its role in Src-deficient mice.

Bone-resorbing osteoclasts are cells of hematopoietic origin, most likely of the monocyte-macrophage family.(5) To understand better the role of Src in osteoclasts and the basis for the lack of redundancy with other Src family kinases, it is important to determine the expression of Src and its family members during osteoclastic differentiation and in mature osteoclasts. It is particularly interesting to determine whether expression of Src and its family members is different in cells of osteoclast lineage, as compared with related cells of the monocyte/macrophage lineage. These studies are complicated by the difficulty in isolating large quantities of purified osteoclasts and by the lack of model osteoclast-like cell lines. We have taken advantage of a recently described human leukemic cell line, FLG 29.1, which upon treatment with phorbol 12-myristate, 13-acetate (PMA) exhibits some features of an osteoclast phenotype and therefore may serve as an in vitro model for osteoclastogenesis.(6–9) In FLG 29.1 cells, we analyzed the expression of Src family kinases, their putative substrates, as well as total tyrosine phosphorylation of cellular proteins. The results in FLG 29.1 cells were compared with those of HL-60 cells, a human leukemic cell line that can differentiate into monocytes/macrophages.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

Antibodies

The following polyclonal antibodies (PAb's) and monoclonal antibodies (MAb's) were used: antiphosphotyrosine (4G10) MAb (#05–321; Upstate Biotechnology Inc., Lake Placid, NY, U.S.A.); anti-Src MAbs (LA074; Quality Technology, Camden, NJ, U.S.A.); anti-Fyn PAb (sc-16; Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.); anti-Lyn MAb (#L05620; Transduction Laboratories); anti-Fgr PAb (#F33930; Transduction Laboratories, Lexington, KY, U.S.A.); anti-Hck PAb (sc-72; Santa Cruz Biotechnologies); anti-Lck PAb (NT, #06–136; Upstate Biotechnology Inc.); anti-Yes PAb (#Y35330; Transduction Laboratories); anti-Fak MAb (#05–182; Upstate Biotechnology Inc.); anti-Pyk2 PAb (sc-1515; Santa Cruz Biotechnologies); anti-Cbl PAb (sc-170; Santa Cruz Biotechnologies); anti-p120 Cas MAb (#P17920; Transduction Laboratories,); anti-p130 Cas MAb (#P27820; Transduction Laboratories); anti-PI 3-kinase PAb (#06–195; Upstate Biotechnology Inc.); anti-vinculin PAb (V-9131; Sigma Chemical Co., St. Louis, MO, U.S.A.); and anti-S6 kinase PAb (kindly provided by P. Dennis and G. Thomas, FMI, Basel, Switzerland). The horseradish peroxidase-coupled secondary antibodies were purchased from CAPPEL Research Products (Durham, NC, U.S.A.).

Cell culture

FLG 29.1 and HL-60 cells were cultured in RPMI supplemented with 10% heat-inactivated fetal calf serum (GIBCO BRL, Basel, Switzerland) in 75 cm2 tissue culture flasks. For experiments, cells were placed in either 75 cm2 or 175 cm2 tissue culture flasks and cultured for 3 days. The cells were harvested by centrifugation in 50 ml Falcon tubes (Falcon Labware, Lincoln Park, NJ, U.S.A.) and resuspended in fresh culture medium and placed at equal density into new flasks. After ∼20 h, the cells were induced to differentiate by addition of PMA (dissolved in acetone; Precision Biochemicals, Inc., Vancouver, BC, Canada) to a final concentration of 0.1 μM. Cultures taken as negative controls were treated with acetone alone at the same concentration as in the treated cultures.

For study of cell adhesion, FLG 29.1 cells were differentiated by 48 h treatment with PMA, transferred into medium without serum, and plated on fibronectin-coated plates or untreated tissue culture plastic plates. As a control, exponentially growing suspension culture of FLG 29.1 was taken. Fibronectin coating of plates was done by applying diluted fibronectin in phosphate-buffered saline (PBS, without calcium and magnesium) to the plates and incubating for 4 h in a tissue culture incubator. Fibronectin (0.1% suspension form bovine plasma; Sigma) was diluted in PBS so that final amount applied was 1 μg/cm2 and the volume of PBS was 5 ml/10-cm plate. After incubation, plates were washed twice with PBS and the cells were seeded and let to adhere for different time periods. For adhesion experiments with stromal cells, the ST2 cells (a kind gift of Prof. Gregory Mundy) were grown in alpha minimum essential medium to about 80% confluency and overlaid with PMA-treated FLG 29.1 cells in medium without serum, as in experiments with fibronectin.

Lysates of murine osteoclasts were kindly provided by Prof. Anna Teti. The cells were obtained in a murine coculture system with primary murine bone marrow stromal cells, and, after tartrate-resistant acid phosphatase (TRAP) staining, judged to be >80% pure. About 30–40% of cells were multinucleated, the remaining were mononuclear preosteoclasts.

Cell extraction

Upon differentiation of FLG 29.1 cultures for the indicated time periods (0–72 h), cells were harvested by centrifugation (4 minutes at 1000g) and washed twice with ice-cold PBS. The cell pellet was subsequently extracted with NP-40 lysis buffer (25 mM Tris, pH 7.4, 10% glycerol, 1% NP-40, 50 mM NaF, 1 mM sodium vanadate, 1 mM phenylmethylsulfonylfluoride, and 10 μg/ml aprotinin, leupeptin, and pepstatin A) on ice for about 15 minutes. Cleared cell lysates were obtained by subsequent centrifugation at 14,000 rpm for 5 minutes at 4°C in a microcentrifuge. Protein concentrations were determined using a Coomassie Blue–based method (Bradford micro method; Bio-Rad, Laboratories AG, Glattbrugg, Switzerland). For SDS-PAGE analysis, samples containing 1 mg/ml protein were prepared in SDS sample buffer and incubated for 5 minutes at 95°C.

SDS-PAGE and Western blotting

Cellular proteins (20 μg) and immunoprecipitates were resolved on 8, 10, 12, or 15% polyacrylamide gels (acrylamide-bisacrylamide ratio 37.5:1 and 150:1) at 25 mA using a Bio-Rad Mini gel apparatus. Electroblotting of proteins onto a polyvinyliden fluoride (PVDF) membrane (Millipore Corp., Bedford, MA, U.S.A.) was performed overnight at 25 V and 4°C (Mini Trans-Blot unit; Bio-Rad). The nonspecific binding to the membranes was blocked by incubation in PBS containing 3% gelatin. The detection of immunoreactive bands was performed after incubation with primary and secondary antibodies and visualized with the enhanced chemiluminescence (ECL) kit from Amersham Switzerland (Zurich, Switzerland) according to the manufacturer's instructions.

For quantitation of Western blots, the signals on developed films were scanned with a HP DeskScan II (Hewlett Packard SA, Geneva, Switzerland), and the bands of interest were quantitated with the ImageQuant program (Molecular Dynamics, Sunnyvale, CA, U.S.A.). The data were corrected for background signal.

Immunoprecipitation

Cleared cellular lysates were diluted with NP-40 lysis buffer to a final protein concentration of 2 mg/ml. Equal amounts of protein from PMA-treated cells (1 mg) and 3 mg of untreated suspension cells (which have much lower Pyk levels) were precipitated with 4 μg of a Pyk-specific antibody prebound to protein G Sepharose. After incubation for 2 h on ice in an orbital shaker, the beads were washed three times with 1 ml of lysis buffer buffer/500 mM NaCl (the third wash step without protease inhibitors) and once with 50 mM Tris, pH 7.4. The antigens were released from the beads by boiling in 50 μl of SDS loading buffer and analyzed by 8% SDS-PAGE and Western blotting as described above.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

Expression of Src family kinases

To test the expression of Src family members in human leukemia FLG 29.1 cells, we first optimized the conditions for their immunoblot detection with specific antibodies using lysates from cells that expressed specific Src family members as appropriate positive controls (data not shown). Subsequently, FLG 29.1 cells were extracted into detergent-containing buffer, and the same amounts of cellular protein were analyzed by SDS-PAGE and immunoblotting. The p60 Src, p59 Fyn, and p53/p56 Lyn were detected in the lysates of undifferentiated FLG 29.1 cells (Fig. 1A, zero time). The p56 Lck was not detected in FLG 29.1 cells (Fig. 1B, upper panel), which is in agreement with its reported T-cell restricted expression. The p59 Hck was detected (Fig. 1B, middle panel), as well as a very weak signal for p56 Hck and p62 Yes. The expression of p56 Hck and p62 Yes appeared to be rather low, since the signal was readily detected in positive controls and not in FLG 29.1 cells, where it was hardly distinguishable from the background (Fig. 1B, middle and bottom panels). The p59 Fgr was not detected by Western blotting, but a kinase activity was immunoprecipitated with anti-Fgr antibody, suggesting some expression of this kinase (data not shown). A B cell–specific Src kinase family member, p55 Blk, was not detected in FLG 29.1 cells (data not shown). The expression of Yrk, which has been cloned only in chickens, but not yet in mammals, was not analyzed.

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Figure FIG. 1.. Expression of Src family members in osteoclast-like FLG 29.1 cells. FLG 29.1 cells were cultured in RPMI medium containing 10% heat-inactivated fetal calf serum for 3 days and placed in fresh medium. After 24 h, differentiation was induced by the addition of 0.1 μM PMA from a 10 mM stock solution dissolved in acetone. The cells were incubated for the indicated periods of time and harvested by centrifugation. Vehicle-treated control cells (“0 h PMA”) were collected after 19 h. Whole cellular lysates were prepared as described in the Materials and Methods. Equal amounts of protein (30 μg) were separated on a 10% polyacrylamide gel and transferred to a PVDF membrane. Expression of cytosolic tyrosine kinases of the Src family was examined by incubation with specific antibodies for 1 h. (A) p60 Src, p59 Fyn, and p53/56 Lyn; (B) p56 Lck, p56/59 Hck, and p62 Yes. Immunoreactive bands were detected with horseradish peroxidase-labeled secondary antibodies and ECL substrates. Positive controls (pc) were: Jurkat cells (Lck); Hck-transfected NIH-3T3 cells (Hck), and human endothelial cells (Yes). As a negative control (nc) a BALB/c 3T3 cell lysate was used. For all controls, an equal amount of protein was analyzed (30 μg). The exposure times in (A) were 2–5 minutes and in (B) 5–10 minutes.

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Treatment of FLG 29.1 cells with PMA for 2–3 days induces a number of morphological and phenotypic changes that indicate differentiation toward the osteoclast phenotype.(6–9) We examined expression of Src family members upon treatment of cells with PMA. The expression of Src increased, starting at 19 h, and continued to rise up to 64 h after the addition of PMA (Fig. 1A, upper panel). The expression of p59 Fyn also increased, but with kinetics different from that of Src: it reached maximum expression already at 40 h and it remained at the same level up to 64 h of PMA treatment (Fig. 1A, middle panel). In contrast, p53/p56 Lyn expression dramatically decreased after 19 h and dropped further after 48 h and 64 h of PMA treatment (Fig. 1A, lower panel). The low expression of p59 Hck decreased after 48–64 h of PMA treatment, while very low levels of p56 Hck and p62 Yes expression did not change during PMA treatment (Fig. 1B, middle and lower panel). These data show that FLG 29.1 cells express several members of the Src family and that their expression changes in different directions upon induction of differentiation with PMA.

Similar to FLG 29.1, HL-60 is also a human leukemia cell line, but with the potential to differentiate toward a macrophage/monocyte phenotype upon PMA treatment.(10) To further investigate the specific presence of Src on FLG 29.1 cells and its PMA-induced increase during differentiation toward the osteoclastic phenotype, we tested in parallel Src expression in PMA-treated HL-60 and FLG 29.1 cells. HL-60 cells did not have detectable Src and Src did not appear after treatment with PMA (Fig. 2, upper panel). In the same experiment, Src levels strongly increased in FLG 29.1 cells upon PMA treatment (Fig. 2, lower panel), in agreement with data shown above (Fig. 1A). We conclude that Src is increasingly expressed during differentiation of a cell line of osteoclastic, but not of myeloid lineage. This is consistent with a crucial role of Src in osteoclasts, but not in monocytes and macrophages.

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Figure FIG. 2.. Src is not expressed in HL-60 cells of the monocyte/macrophage lineage. Cellular extracts of PMA-treated FLG 29.1 and HL-60 cells were prepared as described in Fig. 1, and equal amounts of protein (30 μg) were fractionated on a 10% SDS-PAGE. Src expression in both cell lines was compared by Western blotting using a Src-specific MAb. The blots were done in parallel. As a negative control (nc) 30 μg of protein from Src-deficient fibroblasts (src15 cells) was used; as a positive control (pc) 30 μg of protein from chicken Src-transfected BALB/c 3T3 fibroblasts was analyzed.

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Expression of putative Src substrates

The expression of a number of protein kinase substrates for Src was examined in FLG 29.1 osteoclast-like cells. C-Cbl (cellular homolog of v-Cbl, Casitas B-lineage lymphoma NS-1 retrovirus) was first identified in cells transformed by the BCR-abl tyrosine kinase oncogene as a major tyrosine-phosphorylated protein.(11) Cbl is also phosphorylated and associated with Src family members, such as Src, Fyn, and Lyn in T and B lymphocytes and macrophages.(12–14) Data from Baron and colleagues suggest that Cbl is a Src substrate in osteoclasts and that it is important for bone resorption by these cells.(15) In FLG 29.1 cells, expression of Cbl strongly increased after treatment with PMA, reaching a maximum at 40 h, and remained at that level up to 64 h of treatment (Fig. 3A). p120 Cas (cadherin-associated Src substrate) is a membrane-associated protein thought to be a c-Src substrate and shown to associate with cadherins, proteins mediating cell–cell adhesion.(16) In FLG 29.1 cells, p120 Cas was expressed, but its levels did not change after PMA treatment (Fig. 3A). p130 Cas (for Crk-associated substrate) is phosphorylated in v-Src transformed cells and during integrin-mediated cell adhesion in a c-Src–dependent manner.(17,18) p130 Cas was detected in FLG 29.1 cells, and its expression increased after treatment with PMA, although with faster kinetics and to a lesser extent than for Cbl (Fig. 3A). Vinculin is a cytoskeletal protein associated with focal adhesion plaques and involved in integrin-mediated signaling.(19) In FLG 29.1 cells, vinculin was expressed and its levels slightly increased after PMA treatment (Fig. 3A). In conclusion, the expression of Cbl increased strongly, the expression of p130 Cas and vinculin increased weakly, whereas the expression of p120 Cas did not change in PMA-treated FLG 29.1 cells.

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Figure FIG. 3.. Expression of Src family substrates in osteoclast-like FLG 29.1 cells. (A) Cbl, p120 Cas, p130 Cas, and vinculin. Cellular extracts of PMA-treated FLG 29.1 cells (see legend to Fig. 1) were subjected to 10% SDS-PAGE followed by electroblotting onto a PVDF membrane. The time-dependent expression of the potential Src substrates Cbl, p130 Cas, p120 Cas, and vinculin was analyzed by Western blotting with specific antibodies. (B) Pyk2 and Fak. FLG 29.1 cells were allowed to differentiate in the presence of 0.1 μM PMA for the indicated periods of time and harvested by centrifugation. Equal amounts of precleared cellular lysates (25 μg) were separated by 10% SDS-PAGE. The proteins were transferred to a membrane, and expression of Pyk2 and Fak was analyzed by immunoblotting with specific antibodies. (C) P70 S6 kinase and p85 PI 3-kinase. Cellular extracts of differentiating FLG 29.1 cells were prepared as described in the legend to Fig. 1. To separate differentially phosphorylated forms of p70 S6 kinase, cell extracts (40 μg protein) were resolved by 12% SDS-PAGE (acrylamide/bisacrylamide ratio, 150:1) and p70 S6 kinase analyzed by Western blotting. As a positive control (pc) for the mobility shift of active p70 S6 kinase, 40 μg of protein from fluoroaluminate-activated MC3T3-E1 cells was analyzed. For Western blotting analysis, 20 μg of P85 PI3-kinase proteins per lane were fractionated by 10% SDS-PAGE.

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Fak (focal adhesion kinase) is a cytoplasmic tyrosine kinase that does not belong to the Src family. It consists of a central kinase domain, surrounded by N- and C-terminal domains of unknown function. Fak is phosphorylated by Src in fibroblasts and plays a role in cell adhesion and integrin-mediated signaling.(20) Pyk2 (proline-rich tyrosine kinase 2) is a recently identified, Fak-related cytoplasmic tyrosine kinase that is also regulated by Src.(21,22) We examined the expression of Fak and Pyk2 in FLG 29.1 cells. Specific antibodies were used that did not show cross-reactivity between Fak and Pyk2. Untreated cells expressed both Fak and Pyk2 kinases (Fig. 3B, zero time). The expression of both Fak and Pyk2 increased after treatment with PMA, starting at 15 h. Pyk2 expression reached a maximum at 40 h and remained at the same level up to 71 h after PMA treatment (Fig. 3B). Fak expression increased slowly up to 71 h of PMA treatment (Fig. 3B). The increase in Pyk2 expression was more prominent than the increase in Fak expression (estimated by densitometry as 12-fold vs. 3-fold, respectively). Thus, the expression of these two related kinases was differently affected during FLG 29.1 differentiation, suggesting possibly distinct roles. This is the first report on the expression of Pyk2 in osteoclastic cells and it would be interesting to analyze its expression in primary osteoclasts.

We have also tested the expression of two proteins that are not substrates for Src but may be involved in Src-induced cellular signaling. The serine/threonine protein kinase p70 S6K is activated by many mitogens, as well as by v-Src expression.(23) The activated, phosphorylated forms of p70 S6K have retarded mobility in SDS polyacrylamide gels and may generate up to five distinct bands. The slowest migration is characteristic for the most active forms of the kinase. In undifferentiated FLG 29.1 cells, p70 S6K was detected in its moderately active state, indicated by the presence of several forms of phosphorylated kinase (Fig. 3C, see positive control for strongly activated kinase). Phosphorylation of p70 S6K decreased during PMA treatment of FLG 29.1 cells (Fig. 3C), indicating inactivation of the kinase activity. This is consistent with the notion that p70 S6K plays a crucial role in cell proliferation and a report indicating induction of cell differentiation after inhibition of p70 S6K.(23,24) Phosphatidylinositol 3-kinase (PI 3-kinase) is a phospholipid kinase involved in several cellular responses, including vesicle trafficking.(25) Wortmannin, a specific inhibitor of PI 3-kinase, inhibits ruffled border formation and bone resorption by osteoclasts in a pit assay in vitro(26,27) and in animal models in vivo.(28) Thus, similar to Src, PI 3-kinase may be crucial for osteoclast function. In FLG 29.1 cells, p85 was detected in undifferentiated cultures, and its levels of expression did not change after PMA treatment (Fig. 3C). This finding is consistent with the view that PI 3-kinase activity and not its levels are more readily regulated during cell differentiation.(29)

To evaluate whether the results obtained with FLG 29.1 cells are representative for primary osteoclasts, we compared the expression of Src and two interesting potential Src substrates, Pyk2 and Cbl. As a source of primary osteoclasts, we used murine bone marrow–derived cells, differentiated in vitro in a coculture system with stromal cells. The preparation of osteoclasts was >80% pure, as judged by TRAP staining, with 30–40% multinucleated cells. In murine osteoclasts, Src is expressed at levels about 2-fold higher than in PMA-treated FLG 29.1 cells (Fig. 4). Both Pyk2 and Cbl were detected in primary murine osteoclasts, but at levels lower than in PMA-treated FLG 29.1 cells (Fig. 4). These data confirm the results obtained with the FLG 29.1 cell line, and, in addition, suggest that a high level of Src expression is more tightly associated with the mature osteoclast phenotype than the expression of Pyk2 and Cbl. Nevertheless, high levels of active Src could, via phosphorylation, confer an osteoclast-specific role to these two proteins.

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Figure FIG. 4.. Expression of Src, Pyk2, and Cbl in primary murine osteoclasts: comparison with FLG 29.1 cells. In vitro differentiated murine osteoclasts were obtained after coculture with bone marrow stromal cells and assessed to be >80% pure. FLG 29.1 cells were cultured with or without PMA for 51 h. The expression of Src, Pyk2, and Cbl was analyzed in cellular extracts by Western blotting. Slightly different migration of Pyk2 and Cbl in murine osteoclasts than in human FLG 29.1 cells likely results from species differences.

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Tyrosine phosphorylation of proteins in FLG 29.1 cells

Since Src is a tyrosine kinase and its expression increases after PMA-induced differentiation of osteoclast-like FLG 29.1 cells, we have tested whether the phosphorylation of cellular proteins on tyrosine changed during PMA treatment of cells. The Src kinase activity in immunoprecipitates increased 4-fold after PMA treatment (data not shown). The same amounts of total detergent-soluble cellular proteins were resolved by SDS-PAGE, blotted, and probed with the antiphosphotyrosine MAb 4G10. After PMA stimulation of FLG 29.1 cells, there was a prominent increase in tyrosine phosphorylation of many cellular proteins at molecular weights of 45, 55, 63, 95, 160, and 190 kDa (Fig. 5A). The time course of protein tyrosine phosphorylation correlated with the time course of Src expression (compare Figs. 5A and 1A). Tyrosine phosphorylation of one protein at about 120 kDa decreased in PMA-treated cells (Fig. 5A). Interestingly, the molecular weights of major tyrosine phosphorylated proteins in whole cell lysates of PMA-treated FLG 29.1 cells did not correspond to the known Src substrates, whose expression is shown above. It is worth noting that the majority of Src substrates have so far been identified in fibroblastic cells and that the substrates in osteoclasts may be different. We conclude that protein tyrosine phosphorylation increases during differentiation of FLG 29.1 cells and that the Src family, as well as Fak and Pyk2 tyrosine kinases, may contribute to this increase.

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Figure FIG. 5.. Tyrosine phosphorylation increases in partly different sets of proteins during differentiation of FLG 29.1 and HL-60 cells and is sensitive to Src inhibitor herbimycin A. The cells were cultured in the presence of 0.1 μM PMA for the indicated periods of time and harvested. Equal amounts of protein were separated on 12% (A, B) or 15% (C) SDS-PAGE and transferred onto membranes. Tyrosine-phosphorylated proteins were detected with antiphosphotyrosine MAb 4G10. (A) The time course of tyrosine phosphorylation after PMA treatment of FLG 29.1 cells. Molecular weight markers (BioRad prestained marker, high range) are indicated on the left. Estimated molecular weights of prominent tyrosine phosphorylated bands are shown on the right. (B) Comparison of FLG 29.1 and HL-60 cells. Molecular weight markers are indicated on the left. For HL-60 cells, the exposure of the blot was twice as long as for FLG 29.1 to yield a stronger ECL signal. (C) After PMA-induced differentiation, FLG 29.1 cells were treated with indicated concentrations of herbimycin A for 16 h.

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To examine the specificity of the PMA-induced increase in tyrosine phosphorylation in FLG 29.1 cells, HL-60 cells were used for comparison. PMA induced protein tyrosine phosphorylation in HL-60 cells (Fig. 5B, left). However, the pattern of phosphorylated proteins was partly distinct in HL-60 from that of FLG 29.1 cells, particularly at molecular weights above 120 kDa (Fig. 5B). This result indicates that different sets of proteins are induced during differentiation toward osteoclast and macrophage/monocyte phenotypes. The analysis of these proteins may provide insight into mechanisms controlling differentiation along these two related but distinct lineages.

PMA treatment of FLG 29.1 cells enhances both the expression of Src and protein tyrosine phosphorylation with similar kinetics. To assess whether Src activity contributes to increased tyrosine phosphorylation, PMA-treated FLG 29.1 cells were incubated with herbimycin A, a protein kinase inhibitor that shows some selectivity toward Src. Herbimycin A reduced tyrosine phosphorylation in FLG 29.1 cells in a dose-dependent manner (Fig. 5C). The IC50 value for the inhibition of a p160 protein was 0.04 μM, in the range of previously reported values (0.02–0.2 μM) for the inhibition of osteoclastic bone resorption and Src activity in several systems in vitro.(27,30) These data suggest that Src may contribute to tyrosine phosphorylation in differentiated FLG 29.1 cells. Although FLG 29.1 cells cannot efficiently resorb bone, the phosphorylation in FLG 29.1 cells may be representative for biochemical changes relevant for the role of Src in mature osteoclasts with the bone-resorbing capacity.

Pyk2 tyrosine phosphorylation after adhesion of FLG 29.1 cells to fibronectin and to stromal cell line ST2

As demonstrated in Fig. 3B, the expression of Pyk2 strongly increased during differentiation of FLG 29.1 cells. Since Pyk2 has been implicated in the adhesion process and since differentiating FLG 29.1 cells tend to increasingly adhere to plastic,(6) it is possible that Pyk2 plays a role in the adhesion of these cells. To address this question, FLG 29.1 cells were treated for 48 h with PMA and allowed to adhere to fibronectin-coated dishes in the absence of serum. The adherence to fibronectin was almost complete within 30 minutes (data not shown). For estimation of Pyk2 phosphorylation, cellular lysates were subjected to immunoprecipitation with anti-Pyk2 antibodies followed by antiphosphotyrosine Western blotting. Adhesion to fibronectin, but not to plastic, resulted in a significant increase in tyrosine phosphorylation (Fig. 6, upper panel). Enhanced tyrosine phosphorylation of Pyk2 was observed after 15 minutes (data not shown) and usually reached a maximum after 30 minutes of adherence to fibronectin. In exponentially growing cells in suspension, Pyk2 was only weakly phosphorylated (Fig. 6, upper panel; 0 h PMA suspension). This phosphorylation could only be detected by very extended exposure of the blot. Western blotting with Pyk2 antibodies confirmed equivalent amounts of Pyk2 in the immune complexes of PMA-treated cells and a low amount of Pyk2 in nondifferentiated cells (Fig. 6, lower panel). Neither Pyk2 nor tyrosine phosphorylated proteins were detected in immune complexes of an irrelevant control antibody (data not shown). In a preliminary experiment, a similar increase in tyrosine phosphorylation of Fak was detected upon adhesion to fibronectin (data not shown).

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Figure FIG. 6.. Tyrosine phosphorylation of Pyk2 after adhesion of differentiated FLG 29.1 cells to fibronectin or ST2 stromal cells. (A) FLG 29.1 cells were allowed to differentiate in the presence of 0.1 μM PMA for 48 h. Subsequently, cells were transferred to a serum-free medium and placed on fibronectin-coated plates or to plastic plates without coating for the indicated periods of time. For a comparison, nondifferentiated (0 h PMA), growing cells in suspension were analyzed. Cell extracts were prepared, immunoiprecipitated with Pyk2 antibody, and analyzed by 8% SDS-PAGE. The immunoblots were probed with antiphosphotyrosine (PTyr, upper blot) and anti-Pyk2 antibodies (Pyk2, lower blot). The position of Pyk2 is indicated by an arrow. (B) PMA-treated FLG 29.1 cells were plated on ST2 stromal cell cultures for the times indicated, and the proteins were extracted. For immunoprecipitations, equal amount of FLG 29.1 cell protein was used, after normalizing for protein contributed by the ST2 cells. The analysis of Pyk2 phosphorylation was the same as in (A). Note that the ST2 cells did not contain a detectable amount of Pyk2; this, immunoprecipitated Pyk2 comes only from the FLG 29.1 cells.

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We investigated whether Pyk2 phosphorylation is activated in response to the attachment of the FLG 29.1 cells to extracellular matrix proteins other than fibronectin or to stromal cells, which are shown to be relevant for osteoclast differentiation.(31) Since only a small portion of FLG 29.1 cells adhered to collagen type I or vitronectin and the interaction was rather weak, it was not possible to analyze Pyk2 phosphorylation under those conditions. Direct cell-to-cell contacts between osteoclast precursors and stromal cells are considered to be important for the osteoclast formation,(31) while direct interaction with osteoblastic or stromal cells seems to be important for bone resorption by more mature osteoclastic cells.(32,33) The FLG 29.1 cells strongly adhered to the culture of the stromal cell line ST2, and we examined Pyk2 phosphorylation during adhesion. Pyk2 phosphorylation increased above basal levels after 30 and 60 minutes of adhesion to ST2 cells (Fig. 6B). Since no Pyk2 was detected in immunoprecipitates from ST2 cells, the Pyk2 signal comes only from the FLG 29.1 cells (Fig. 6B, bottom panel, right lane). Thus, the adherence of FLG 29.1 cells to either fibronectin or the ST2 stromal cell line leads to increased Pyk2 phosphorylation. Thus, the increase in Pyk2 phosphorylation may be a common signaling event in osteoclastic cells interacting with matrix components or with other cell types and might play a role in osteoclastogenesis and/or bone resorption. Whether Pyk2 expression and phosphorylation by Src are crucial for complex processes such as osteoclastogenesis or bone resorption needs to be addressed in future studies with Src inhibitors more specific than herbimycin, with an antisense oligonucleotide-induced block of Pyk2 expression, and by comparing the biochemical analysis and molecular biological manipulation of FLG 29.1 cells with the functional analysis of primary osteoclastic cells. Studies with FLG 29.1 cells overexpressing Src and with the selective Src inhibitors are underway.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

In this study, we examined the protein expression of the Src family of tyrosine kinases in the human osteoclast-like leukemia cell line FLG 29.1. We detected high levels of p60 Src, p59 Fyn, and p53/p56 Lyn and lower levels of p59 Hck and p62 Yes expression. Upon treatment with PMA, which induces differentiation of FLG 29.1 cells toward an osteoclast-like phenotype, the expression of p60 Src and p59 Fyn increased, the expression of p59 Hck and p53/p56 Lyn decreased, and the levels of p62 Yes were not changed. This pattern of expression is quite different from the expression in another, similar promyelocyte-like leukemia cell line HL-60, which can be induced to differentiate into granulocytes or monocytes/macrophages. In these cells, PMA treatment induces monocytic differentiation, which correlates with an increase in the mRNA expression of p59 Fyn and p53/p56 Lyn, while the expression of p60 Src, p62 Yes, and p59 Fgr did not change.(10) We did not detect p60 Src protein in HL-60 cells treated with PMA. These data confirm a difference in the phenotype of myeloid HL-60 cells and osteoclast-like FLG 29.1 cells. Since both monocytes and, most likely, osteoclasts are derived from hematopoietic cells of the monocyte/macrophage lineage,(5) and since FLG 29.1 cells express some surface markers that are shared with macrophages, this is an important distinction between the two cell lines. Further, FLG 29.1 cells can be regarded as a model for in vitro osteoclastogenesis, and the described changes in the expression of the Src family members point to biochemical differences during two related but distinct differentiation pathways.

It is interesting to compare the expression of Src family kinases in FLG 29.1 osteoclast-like cells to the reported expression in native, terminally differentiated isolated osteoclasts. A study with partially purified chicken osteoclasts reported the expression of p60 Src, small amounts of p59 Fyn, p53/p56 Lyn, and p62 Yes, but no expression of p59 Fgr, p59 Hck, p55 Blk, or p56 Lck.(34) Another study reported expression of Src family kinases in mouse preosteoclasts generated in a coculture system in vitro.(35) In addition to p60 Src, high amounts of p59 Fgr and p59 Hck were detected, the expression of p56 Lyn was low, while p59 Fyn and p56 Lck were not detected. In FLG 29.1 cells, we detected all the Src family members that had been previously detected in either chicken or mouse osteoclasts. Since we used a cell line, the possibility of contamination with another cell type is excluded.

In addition to p60 Src, p56/p59 Hck may be another Src family member with a specific role in osteoclasts.(35) Similar to murine osteoclasts,(35) FLG 29.1 cells apparently express more p60 Src than Hck and more p59 than the p56 Hck form. Moreover, murine bone marrow cells express more p56/p59 Hck than differentiated murine osteoclasts, which is consistent with reduced p59 Hck levels in differentiating FLG 29.1 cells. The apparent lack of Hck expression in avian cells may be species related or due to a low affinity of the antibody, since the signal of the positive control for Hck was rather weak as well.(34) The expression of p53/p56 Lyn was low in all three systems and it declined with differentiation in both murine and in human cells. The expression of p59 Fyn increased in differentiating osteoclastic FLG 29.1 cells and in monocytic HL-60 cells,(10) while low levels of Fyn were detected in avian(34) and murine osteoclasts.(35) Thus, it is possible that p59 Fyn expression in FLG 29.1 cells reflects partly mixed osteoclastic/monocytic phenotype of this cell line. In all three systems, p56 Lck was not detected, and low levels of p62 Yes were detected. Importantly, although performed in a semiquantitative manner with cells at various stages of differentiation originating from different species, our data and the reports of others are consistent in the high expression of Src, which is specific for more differentiated cells of the osteoclast lineage, as compared with undifferentiated bone marrow cells, stromal cells, and the cells of monocytic lineage.

A number of the Src substrates identified in fibroblastic cells are proteins associated with cytoskeleton and focal adhesion plaques.(19) In this study, we analyzed the expression of several such proteins: p120 Cas, p130 Cas, vinculin, and Fak kinase. The expression of p130 Cas, vinculin, and Fak increased after PMA treatment of FLG 29.1 cells. This increase may be related to a change in cell phenotype: differentiation toward osteoclasts involves increased cell–cell as well as cell–substrate contacts. Both these features were detected to various degrees in PMA-treated FLG 29.1 cells. It is interesting to note that it was possible to select a clone of FLG 29.1 cells that grew as an adherent culture, while the original population of FLG 29.1 cells grows mostly as a suspension culture. In this new subclone of FLG 29.1 cells, without PMA treatment the levels of Fak, vinculin, and p130 Cas were already as high as in a PMA-induced “normal” clone of FLG 29.1 cells (data not shown). It appears that the level of expression of these proteins correlates with the ability of FLG 29.1 cells to adhere to substrates and to each other.

An interesting finding is the high expression of Pyk2 kinase in PMA-induced FLG 29.1 cells. This novel tyrosine kinase is related to Fak and was originally cloned from brain.(21) High expression of Pyk2 was originally reported in brain and kidney, and later expression was also found in cells of the hematopoietic lineage.(36,37) The fact that Pyk2 is strongly induced in FLG 29.1 cells treated with PMA suggests that this kinase may play a role in differentiation toward osteoclasts, as well as in mature osteoclasts. This possibility remains to be explored. Pyk2 is also a target for integrin-mediated tyrosine phosphorylation in B cells and megakaryocytes.(37,38) Osteoclasts express an αvβ3 integrin receptor which binds to vitronectin, fibronectin, and osteopontin. Integrin receptor binding was suggested to be important for osteoclast activation since MAb's raised against αvβ3 integrin can inhibit osteoclastic bone resorption.(5) We observed a significant increase in Pyk2 tyrosine phosphorylation upon adherence of FLG 29.1 cells to fibronectin. This result indicates a potential role for Pyk2 in integrin signaling pathways in osteoclastic cells. Since Src is highly expressed in mature osteoclasts and Src has been shown to associate with and phosphorylate Pyk2 in hematopoietic and neuroneal cells, it is possible that Src is the kinase that phosphorylates Pyk2 in osteoclastic cells. Pyk2 is also increasingly phosphorylated upon attachment of FLG 29.1 cells to the stromal cell line ST2. Presently, it is not clear which adhesion molecule mediates this interaction. However, since stromal cells can support osteoclast differentiation and since vascular adhesion molecule 1 (VCAM-1) is thought to play a role in this process,(31,39) it can be suggested that the role of Pyk2 during osteoclast differentiation may be to promote differentiation by mediating signals from stromal cells. It would be interesting to examine whether VCAM-1 is involved in the induction of Pyk2 phosphorylation. In addition, since cell-to-cell contacts between stromal and osteoblastic cells seem to be crucial for bone resorption by purified osteoclasts,(33) it would be interesting to examine Pyk2 phosphorylation in primary osteoclastic cells during the resorption.

The cellular function of Src and its substrates has been so far mostly studied in fibroblasts. Only since the finding that Src-deficient mice exhibited osteopetrosis has there been an interest in examining p60 Src in osteoclasts. These studies were hampered by the lack of appropriate cell lines. Work with isolated cells shows that Cbl may be a relevant p60 Src substrate in osteoclasts.(15) We detected tyrosine-phosphorylated Cbl in PMA-treated FLG 29.1 cells, although the level of its phosphorylation was lower in differentiated than in undifferentiated cells (data not shown). Our data show that the major Src substrates in fibroblasts differ from the major tyrosine-phosphorylated proteins in an osteoclast-like cell line FLG 29.1. This may be due to the different phenotypes of fibroblasts and osteoclasts, which provide expression of different tyrosine kinase substrates in two cell types. In addition, Src may not be the major tyrosine kinase in FLG 29.1 cells.

What other tyrosine kinases except Src could mediate increased tyrosine phosphorylation during differentiation of FLG 29.1 cells? The CSF-1 receptor tyrosine kinase, fms, is implicated in the differentiation of osteoclasts.(5) We could not detect fms by Western blotting, and stimulation of FLG 29.1 cells with CSF-1 produced only a minor increase in tyrosine phosphorylation, suggesting a low expression of its receptor (data not shown). However, the time course of the increase in protein tyrosine phosphorylation closely paralleled that of p60 Src and p59 Fyn expression, and a Src inhibitor herbimycin A reduced tyrosine phosphorylation in FLG 29.1 cells with IC50 values similar to those reported for inhibition of bone resorption and Src activity in vitro,(30) suggesting a contribution from Src and Fyn. The expression of Pyk2 and, to a lesser degree, of Fak also increased, raising the possibility that these tyrosine kinases may also contribute to overall tyrosine phosphorylation in differentiated FLG 29.1 cells and in osteoclasts. These questions can be addressed by the overexpression of Src, Fyn, Fak, or Pyk2 kinases in FLG 29.1 cells and by comparing their tyrosine phosphorylation to that of the PMA-induced parental FLG 29.1 cell line. It will be interesting to analyze the phenotype of Pyk2-deficient mice with respect to osteoclast-mediated bone resorption.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

We thank Prof. Gregory Mundy for the gift of the ST2 stromal cell line and Prof. Anna Teti for lysates of primary murine osteoclasts. We are indebted to Hong-Ngoc Luong-Nguyen and Daisy Rohner for their excellent technical assistance and help in preparation of the figures. We are grateful to Dr. Jonathan Green for critical reading of the manuscript.

Reference

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference
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