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Stem Cells, Vol. 16, No. 3, 229-238, May 1998
© 1998 AlphaMed Press

Tyro 3 Receptor Tyrosine Kinase and its Ligand, Gas6, Stimulate the Function of Osteoclasts

Yuka S. Nakamuraa, Yoshiyuki Hakedab, Nobuyuki Takakuraa, Takashi Kamedab, Isao Hamaguchia, Takeshi Miyamotoa, Shinji Kakudoc, Toru Nakanoc, Masayoshi Kumegawab, Toshio Sudaa

a Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan;
b Department of Anatomy, Meikai University School of Dentistry, Saitama, Japan;
c Shionogi Research Laboratories, Osaka, Japan

Key Words. Osteoclasts • Tyro 3 • Gas6 • Bone resorption

Dr. Toshio Suda, Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto 860-0811 Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone is continuously being formed and resorbed. This process is accomplished by the precise coordination of two cell types: osteoblasts and osteoclasts. Osteoclasts are large, multinucleated cells that are derived from the same hematopoietic precursors as macrophages. However, these bone-resorbing cells are difficult to study directly because of their relative inaccessibility. The purification of primary osteoclasts from rabbit bones by their adherent nature provides an opportunity for investigating the molecules in osteoclasts. We have examined the expression of receptor tyrosine kinase by polymerase chain reaction (PCR) and found that Tyro 3 was frequently identified from primary osteoclasts in PCR cloning. Immunohistochemistry revealed that Tyro 3 was expressed on the multinucleated osteoclasts which were positive for tartrate-resistant acid phosphatase (TRAP), but not on mononuclear TRAP-positive cells. The Tyro 3 ligand, Gas6, induced the phosphorylation of Tyro 3 receptors in osteoclasts in two to five min. Gas6 and protein S directly enhanced the bone resorbing activity of mature osteoclasts. This effect of Gas6 was inhibited by the addition of a tyrosine kinase inhibitor, herbimycin A. However, Gas6 did not affect the differentiation of osteoclasts from bone marrow cells. Gas6 and protein S are dependent on vitamin K, a cofactor for the enzyme responsible for carboxylation of glutamic acid residues. The findings in this study are the first to indicate a new biological activity of Gas6 and protein S as a direct regulator of osteoclastic function; they give an insight into the role of these vitamin K-dependent ligands in bone resorption in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone formation and resorption are accomplished by the precise coordination of osteoblasts and osteoclasts. Osteoclasts are giant, multinucleated cells that mediate the process of the bone resorption. They are of hematopoietic stem cell (HSC) origin [1-4]. Many receptor tyrosine kinases (RTK) phosphorylate themselves and other proteins in response to ligands. Macrophage-colony stimulating factor (M-CSF), M-CSF receptor (c-fms), macrophage-stimulating protein (MSP), and MSP-receptor (Ron/Stk) signal transduction systems play an important role in the development and function of osteoclasts [5-7].

It has been our aim to characterize RTK in signaling events in bone resorption. We have isolated Tyro 3 from purified rabbit osteoclasts by reverse transcriptase- polymerase chain reaction (RT-PCR). This receptor has been cloned from other tissues and is also known as Sky [8], Rse [9], Brt [10], and Tif [11]. Tyro 3 has structural homology with Tyro 7/Axl/Ufo/Ark (hereafter, Axl) [12-14] and with Tyro 12/Eyk/Mer (hereafter, Mer) [15, 16], and together these receptors define a new family. All three of these related receptor-like tyrosine kinases have similar ectodomains, sharing about 35% amino acid identity and consisting of two immunoglobulin-like domains and two fibronectin type III repeats [17]. Tyro 3 is most prominently expressed in the adult nervous system, although it is expressed in some nonneural tissues and a number of hematopoietic cell lines [8, 18, 19]. The Axl receptor was originally isolated as a protein encoded by a transforming gene from primary human chronic myeloid leukemia cells by DNA-mediated transformation of 3T3 cells [12], and further studies in normal and malignant hematopoiesis have provided supportive evidence for a role in hematopoiesis. Axl has been shown to be expressed in CD34+ hematopoietic progenitors, bone marrow (BM) stromal cells, and peripheral blood monocytes [20]. Mer is also expressed in the nervous system. Although the ligand for Mer remains to be determined, the ligands for Tyro 3 and Axl have been reported [21, 22]. The growth arrest-specific protein, Gas6, and the anticoagulant cofactor, protein S, have been identified as their ligand. Gas6 and protein S are related vitamin K-dependent proteins that share overall domain organization and 43% amino acid identity [23]. We investigated the expression of Tyro 3 in isolated rabbit osteoclasts and murine bones and the role of Tyro 3 and its ligands in their development and function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of Osteoclasts on Plastic Dishes
Rabbit osteoclasts were isolated on plastic dishes as described previously [24]. Tibiae, femora, humeri, ulnae, and radii were obtained from 10-day-old rabbits (Japan White, Saitama Experimental Animals; Saitama, Japan). After connective tissues attached to bones were removed, these bones were minced into pieces in {alpha}-minimal essential medium ({alpha}-MEM). Cells were dissociated from bone fragments by vigorous vortexing. After removal of bone fragments by sedimentation under normal gravity, cells were collected from the supernatant by centrifugation and used as unfractionated bone cells. The unfractionated bone cells were plated into 100-mm tissue culture dishes at 1 x 108 living cells per dish. After overnight culture, the nonadherent cells were removed by several washes, and the adherent cells were incubated in phosphate-buffered saline (PBS) containing 0.001% pronase E (Kaken Chemical; Tokyo, Japan) and 0.02% EDTA for 10 min at room temperature. By this incubation, cells other than osteoclasts became detached from the dishes and were washed off. More than 95% of the cells prepared were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated. We used these cells as isolated osteoclasts for preparation of rabbit osteoclast cDNA library and analysis for tyrosine phosphorylation of proteins in osteoclasts.

RT-PCR Amplification of Protein Tyrosine Kinase (PTK)-Related Sequences
Total RNA was obtained from rabbit osteoclasts prepared as mentioned above by acid guanidinium thiocyanate-phenol-chloroform extraction. The first-strand cDNA was synthesized from 2 ug of total RNA using a superscript preamplification system (GIBCO BRL; Gaithersburg, MD). Two sets of degenerate oligonucleotide primers corresponding to the consensus sequences of kinase domains (sense primer; subdomain VI, antisense primer; subdomain IX) were synthesized. One set was directed to the sequence IHRDL (PTK I) and DVWSFG (PTK II), and the other was directed to the sequence HRDLA (TK I), HRDLAAR (TK II), and DVWSY/FG (TK III). TK I-III primers were designed to be more degenerate than PTK I and II, as previously described [25]. Flanking sequences for Sal I and EcoRI sites were added to the TK I-III primers for subsequent subcloning. The sequences are as follows: PTK I, 5'-CGGATCCAC(A/C)GNGA(C/T)(C/T)-3'; PTK II, 3'-CT(A/G)CA(C/G)ACCAGGA(A/T)ACCTTAAGG-5'; TK I, 5'-TAGTCGACA(C/T)(A/C)G(A/G)GA (C/T)(C/T)T(A/C/G)CG-3'; TKII, 5'-TTGTCGACAC(A/C) G(A/G)GA(C/T)(C/T)T(C/G)GCNGCN(A/C) G-3'; TK III, 3'-CT(A/G)CA(C/G)ACC(A/T)(C/G)(A/G)A(A/T)ACCTTAAGGT-5'.

All of the cDNA was used as the template for amplification. The cycling parameters were one min at 94°C, two min at 37°C, and three min at 72°C. The cDNA was primed with oligo(dT), and the first amplification proceeded for 30 cycles with 1 mg of PTK I and PTK II primers. After the PCR products were purified using a CHROMA SPIN-100 column (CLONTECH; Palo Alto, CA), an additional 20 cycles of amplification were performed with 1 ug of TK I and TK III or TK II and TK III primers. The amplified cDNA of around 210 bp was subcloned into the Bluescript vector (Stratagene; La Jolla, CA) after digestion with Sal I and EcoRI. Inserts of randomly selected clones were sequenced by dideoxynucleotide chain termination, using an AutoRead sequence kit (Pharmacia; Uppsala, Sweden) and the Automated Laser Fluorescent ALF DNA Sequencer (Pharmacia).

Factors
Rat Gas6 was kindly provided by Shionogi Research Laboratory (Osaka, Japan). Human protein S was purchased from SERBIO (Celsus Laboratory; Cincinnati, OH). The standard concentrations of factors used in our culture were as follows: rat Gas6, 0.3 to 30 nM; human protein S, 0.4 to 12.0 nM. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was purchased from BIOMOL (Plymouth Meeting, PA).

In Vitro Formation of Osteoclast by Adult BM Cells
BM cells were obtained from femurs of six-week-old C57BL/6 mice and suspended in {alpha}-MEM. BM cells were flushed out with a syringe containing {alpha}-MEM, and a cell suspension was prepared by pipetting. Unfractionated BM cells were plated in plastic dishes (Becton-Dickinson; Lincoln Park, NJ) or chamber slides (Nunc; Naperville, IL) at 5 x 105 cells/ml, and cultured at 37°C in a humidified 5% CO2 air incubator. The medium consisted of {alpha}-MEM containing 10% fetal calf serum (FCS), and 100 U/ml mouse recombinant interleukin 3 (IL-3). After seven days of culture, various concentrations of Gas6 and/or 1,25(OH)2D3 (10–8 M) were added to the medium. After 14 days of culture, cells were stained for TRAP activity using a commercially available kit (Sigma; St. Louis, MO) at 37°C for one h. Then TRAP-positive multinucleated cells were counted in several microscopic fields at a final magnification of 100.

Immunohistochemistry for Tyro 3
After washing off nonadherent cells from the culture of rabbit unfractionated bone cells on collagen gel, adherent cells including mature osteoclasts and stromal cells were released by incubation in 0.1% collagenase in PBS. The cells were plated in type-1 collagen-coated dishes and cultured overnight. Then, the cells were fixed with a cold fixation solution consisting of 25 mM sodium phosphate (pH 7.5), 45% acetone and 9% formaldehyde for one min. After blocking with 5% skim milk in PBS, the fixed cells were immunoreacted with goat anti-Tyro 3 IgG polyclonal antibody, RSE (2 µg/ml anti-Tyro 3 PoAb, Santa Cruz Biotechnology; Santa Cruz, CA) or nonimmune goat IgG (2 µg/ml) in the absence or presence of synthetic peptide (20 µg/ml, Santa Cruz Biotechnology) containing an epitopic sequence for anti-Tyro 3, and subsequently with biotin-conjugated anti-goat IgG (1 µg/ml). Prior to these immunoreactions, the anti-Tyro 3 antibody was preincubated with or without the synthetic epitopic peptide for one h. The anti-Tyro 3 immunoreactive cells were visualized using avidin-biotin-peroxidase complex immunostaining reagents (Vector Technology Corp., Houston, TX) following the manufacturer's instructions.

Murine fetal bones were prepared as follows: C57BL/6 mice were purchased from Japan SLC Inc. (Shizuoka, Japan). Females were paired with males overnight and if a vaginal plug was detected the next morning, that day was designated day 0.5 day post coitum (0.5 dpc). Fetal (16.5 dpc) and adult murine bones were also immunostained by anti-Tyro 3 PoAb. Fetal bones were immersed in 4% paraformaldehyde. For prefixation, microwave treatment was performed for 20 seconds at 600 w in a microwave oven (ER-11, Toshiba; Tokyo, Japan), then specimens were post-fixed overnight at 4°C in the same solution. Fetal bone was embedded in OCT compound (Miles Laboratories Inc.; Elkhart, IN), and frozen sections were made using a cryomicrotome (MICRON; Heidelberg, Germany). Adult bone was decalcified in 0.5 M EDTA solution (WAKO; Osaka, Japan), embedded in polyester wax, and sectioned at 5 mm using a sliding microtome (Yamato; Tokyo, Japan). Immunostaining procedures were as described [26], except that biotin-conjugated anti-goat IgG antibody (1 µg/ml in blocking solution, Southern Biotechnology; Birmingham, AL) as the second antibody and complex of biotinylated peroxidase and streptavidin (streptABComplex/HRP, DAKO; Glostrup, Denmark) for developing biotin were used. The procedure for TRAP staining on tissue sections is described above.

Mouse BM cells were cultured with 1,25(OH)2D3 as described above. After 14 days of culture, cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C. After washing with PBS, cells were treated with 0.3% H2O2 in PBS for 15 min to block endogenous peroxidase activity. After blocking with PBST (0.1% triton X100 in PBS) containing 1% normal rabbit serum, 0.2% BSA and 2% skim milk for 15 min at 4°C, they were stained with 1 µg/ml anti-Tyro 3 PoAb by the indirect immunoperoxidase method using HRP-conjugated anti-goat IgG. Peroxidase activity was visualized using diaminobenzidine as a substrate.

Isolation of Mature Osteoclasts in Suspension from Collagen Gel
Osteoclasts were isolated from collagen gel by the method of Kakudo et al. [27]. Briefly, rabbit unfractionated bone cells prepared as above were plated onto 0.24% collagen gel (type-IA, Nitta Gelatin; Osaka, Japan) in 100-mm tissue culture dishes (Falcon, Becton-Dickinson) and incubated for two h at 37°C in a 5% CO2 incubator. The culture was treated with 0.001% pronase E and 0.02% EDTA in PBS for 10 min, washed three times with PBS, and subsequently treated with 0.01% bacterial collagenase for five min at room temperature to completely remove cells other than osteoclasts. After several washings, the culture was finally digested with 0.1% collagenase for 10 min to free the remaining cells from the gel. The released cells were collected by centrifugation and suspended in {alpha}-MEM with 5% fetal calf serum (FCS). More than 95% of the cells prepared were TRAP-positive and multinucleated, like the cells isolated on plastic dishes. We used these cells for determination of osteoclastic bone resorbing activity.

Bone Resorption Assay
One hundred isolated osteoclasts from collagen gel, prepared as mentioned above, were plated and cultured on dentine slices (6 mm in diameter and 0.1 mm in thickness) with {alpha}-MEM supplemented with 5% FCS. After one h incubation, the medium was replaced with {alpha}-MEM containing 0.1% bovine serum albumin (BSA) with or without Gas6. After incubation for the period indicated, the cells were scraped off the dentine slices, and the slices were stained with acid hematoxylin (Sigma) to visualize the pits formed. The stained pit areas were determined under a microscope by counting the number of mesh squares covering the pit to evaluate the possible osteoclastic bone resorption.

Assay for Tyrosine Phosphorylation of Cellular Proteins
The isolated osteoclasts on plastic dishes were cultured in {alpha}-MEM supplemented with 0.1% BSA for eight h. Then, the osteoclasts were treated with 3 nM Gas6 for various periods. The cells were quickly washed with PBS containing 5 mM EDTA and 0.1 mM Na3VO4, and lysed with a lysis buffer consisting of 10 mM sodium phosphate (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 1 mM aminoethyl-benzenesulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The protein concentration in the cell lysate was measured using a bicinchoninic acid protein assay kit (Pierce Chemical Co.; Rockford, IL). Each cell lysate containing equal amounts of protein was subjected to 8% SDS-polyacrylamide gel electrophoresis; proteins separated in the gel were subsequently electrotransferred onto a polyvinylidene difluoride membrane. After being blocked with 5% skim milk, the membrane was incubated with monoclonal antibody against phosphotyrosine (Upstate Biotechnology; Lake Placid, NY) and subsequently with peroxidase-conjugated anti-mouse IgG antibody. Phosphotyrosine-containing proteins were visualized using Western blot chemiluminescence reagents (Dupont New England Nuclear Products; Boston, MA) following the manufacturer's instructions. After this visualizing, the antibodies on the membrane were stripped by incubating in a buffer consisting of 62.5 mM Tris-HCl (pH 6.8), 2% SDS and 100 mM 2-ME at 50°C for 30 min. The stripped membrane was then immunoreacted with anti-Tyro-3 PoAb and subsequently with peroxidase-conjugated anti-goat IgG, and the anti-Tyro 3-immunoreactive proteins were visualized under the same conditions as above.

Statistical Analyses
Intergroup differences in mean values were statistically analyzed using Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PCR Amplification of Tyrosine Kinase-Related cDNA
To identify the RTKs expressed in osteoclasts, we used the strategy of PCR amplification previously described [25]. In addition to the primers of PTK I and PTK II, we prepared more degenerate oligonucleotide primers (TK I-III) corresponding to the amino acid sequences HRDLAAR (subdomain VI) and DVWS/YFG (subdomain IX). Sense primers TK I and TK II were designed to preferentially clone RTK genes (HRDLAAR), and not to amplify Src-related genes (HRDLRAA). As a source of RT-PCR amplification, we isolated rabbit osteoclasts from unfractionated bone cells on plastic dishes. The purity of the isolated rabbit osteoclasts was more than 95% in cell number.

Random sequence analysis of PCR-amplified cDNA clones identified 14 distinct kinase-related genes ( Table 1). Six of them were nonreceptor PTK, and the remaining eight genes were RTK: Tie, c-kit, Fms, Met, Axl, Tyro 3, FGF-R1, and INS-R. The profile of the identified cDNAs differed according to the primers, and the more degenerate primers (sense primer; HRDLAAR) amplified an extended family of kinase-related genes, but not Src-related genes. Tyro 3, FGF-R1, Fms, and Met were frequently cloned. The roles of Fms and Met in osteoclasts have been investigated, and FGF-R is expressed in many kinds of cell lineage. So far, little evidence on the roles of Tyro 3 has been demonstrated in primary cells. Here, we have investigated the expression and function of Tyro 3 in osteoclasts. Since the ligands for Tyro 3 were recently identified as Gas6 and protein S, we also examined the role of Tyro 3 and its ligands in osteoclasts.


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  		Table 1. Profile of PTKs isolated from osteoclasts
 
Immunohistochemistry of Tyro 3 on Osteoclasts
To detect Tyro 3 expression on osteoclasts at the protein level, we performed immunohistochemical staining of osteoclasts using an anti-Tyro-3 antibody. As shown in Figure 1, rabbit osteoclasts were stained well with an anti-Tyro-3 antibody. The specific reactivity of antibody with Tyro 3 was confirmed by the finding that positive staining was completely blocked by an excess molar ratio of antigen peptides ( Fig. 1). In mouse bone specimens, TRAP staining demonstrated the existence of osteoclasts in a demarcation between metaphysis and cartilage. Tyro 3-positive staining of osteoclasts was confirmed by comparing with TRAP staining in serial sections ( Fig. 2). To assess when Tyro 3 is expressed in the differentiation pathway to osteoclasts, murine BM cells were cultured in the presence of IL-3 and 1,25(OH)2D3 to develop osteoclast-like multinucleated cells. Immunohistochemical staining of osteoclast-like cells showed that only multinucleated TRAP-positive cells were positive for Tyro 3 ( Fig. 3). The positive staining was completely blocked by an excess molar ratio of antigen peptides in murine system (data not shown). These findings suggest that Tyro 3 expression is restricted to mature osteoclasts.



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  		Figure 1. Immunohistochemical analysis of Tyro 3 on rabbit osteoclasts and stromal cells. (A) Non-immune IgG did not react with rabbit osteoclasts and stromal cells. (B) Rabbit osteoclasts showed positive staining with anti-Tyro 3 PoAb. (C) The positive staining was completely blocked in the presence of an excess of antigen peptide. Bar indicates 25 µm. Arrowheads indicate multinucleated osteoclasts.

 


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  		Figure 2. Localization of TRAP-positive osteoclasts and expression of Tyro 3 in the embryonic and adult mouse bone. Femurs of embryo at 16.5 dpc (A, C) and adult mouse (B, D) were stained by TRAP (A, B) and anti-Tyro 3 PoAb (C, D). Arrows indicate TRAP-positive (A) and Tyro 3-positive (C) cells, respectively. Note that TRAP-positive (A, B) and Tyro 3-positive (C,D) cells adhere to the bone . Abbreviations: b = bone; bc = bone marrow cavity. Bar in (D) indicates 50 µm (A, C) and 10 µm (B, D), respectively.

 


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  		Figure 3. Formation of TRAP-positive and Tyro 3-positive multinucleated cells in the culture of mouse BM cells with IL-3 and 1.25(OH)2D3. (A) IHC analysis of Tyro 3. Multinucleated giant cells were strongly positive. (B) TRAP staining was performed to delineate osteoclasts. Multinucleated giant cells were TRAP-positive. Bar indicates 25 µm.

 
Activation of Tyro 3 Receptor in Rabbit Osteoclasts by Gas6
To investigate Gas6-induced signals in osteoclasts, we examined whether Gas6 induces autophosphorylation of Tyro 3 in osteoclasts. Gas6 (0.08-10 nM)-stimulation induced a significant autophospholylation of 170-kD Tyro 3 in osteoclasts after two min of stimulation and continued at least for 30 min ( Fig. 4), suggesting that Tyro 3 mediates the signals for Gas6 in osteoclasts.



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  		Figure 4. Time-dependent effect of Gas6 on tyrosine phosphorylation in rabbit osteoclasts. Rabbit osteoclasts isolated on plastic dishes were stimulated with 3 nM Gas6 for the periods indicated. Proteins in cell lysates were electrophoresed and then Western blotted with antiphosphotyrosine (left) and anti-Tyro 3 (right). The bands above the 170-kDa protein which were blotted with anti-Tyro 3 were phosphorylated.

 
Evaluation of Bone-Resorbing Activity by Pit Formation
To investigate whether Gas6 plays a functional role in osteoclasts, we examined the bone-resorbing activity of rabbit osteoclasts in the presence of Gas6 ( Fig. 5 ). The increase of pit area (number of mesh/dentine slice) was observed at a concentration as low as 0.3 nM as compared with control, while the number of pits was not significantly changed (data not shown). The greatest difference in bone resorption between Gas6-treated and -nontreated culture was observed at a concentration of 3.0 nM. At that concentration, the level of pit formation was approximately 2.5 times higher than the control. The stimulatory effect of Gas6 on osteoclastic bone-resorbing activity appeared as early as six h after Gas6 addition and continued up to 24 h. The stimulation of bone-resorbing activity was also observed when protein S was added to cultures ( Fig. 5). Stimulatory effects of Gas6 on rabbit osteoclastic bone resorption were inhibited by 1 mM herbimycin A ( Fig. 6).



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  		Figure 5. Dose-dependent effect of Gas6 and protein S (A, C) and time-dependent effect of Gas6 (B; closed circles) compared with control (B; open circles) on bone resorption by rabbit mature osteoclasts. Mature osteoclasts were purified and isolated from collagen gel, and plated on dentine slices. After culturing in various concentrations or with stimulation for various durations, the cells were brushed off the dentine slices and the slices stained with acid hematoxylin. The pit area on the dentine was measured microscopically. Gas6 and protein S exceeded the control in pit formation. Values are means ± SD for three cultures. *p < 0.01 versus Gas6-untreated culture.

 


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  		Figure 6. Inhibition of the stimulatory effect of Gas6 on rabbit osteoclastic bone resorption by herbimycin A. Rabbit osteoclasts purified from collagen gels were pretreated with (HMA and HMA + Gas6) or without (control and Gas6) herbimycin A (HMA, 1 mM) for one h. Then, the cells were treated with or without Gas6 (3 nM) in the presence or absence of HMA (1 mM) for 12 h. At the end of the treatment, pit number (closed bar) and pit area (open bar) on the dentine slice were counted as described in Materials and Methods. Values are means ± SD for four cultures. *p < 0.01 versus control.

 
Effect of Gas6 on the Formation of TRAP-Positive Cells
To investigate the effect of Gas6 on formation of TRAP-positive multinucleated cells in BM cells cultured with 1,25(OH)2D3, Gas6 was added to the culture system of BM cells. The number of TRAP-positive multinucleated cells was not significant between the cultures with and without Gas6 ( Fig. 7).



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  		Figure 7. The effect of Gas6 on the development of TRAP-positive multinucleated cells (MNCs) during BM culture with IL-3 and 1,25(OH)2D3. Gas6 did not affect the formation of TRAP-positive cells. Values are means ± SD for four cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Osteoclasts are derived from monocyte/macrophage precursors. Mononuclear osteoclast precursor cells fuse with each other and become mature multinucleated cells which can resorb bone. Mutations generating the osteopetrotic phenotype provide insights into the molecular mechanisms of bone resorption. The transcription factors PU.1 and Fos are involved in osteoclast determination and differentiation [28-30]. M-CSF stimulates the generation of osteoclasts [31, 32]. Carbonic anhydrase II and H+-ATPase are essential enzymes for the function of polarization and bone-resorbing activity [33, 34]. Moreover, integrins and nonreceptor tyrosine kinases such as c-Src, FAK, c-Cbl, and Cas have been demonstrated to play important roles in regulating the osteoclastic activity [35-38]. Most recently, M-CSF has also been demonstrated to induce cytoskeletal reorganization of mature osteoclasts via intracellular interaction between c-Fms, M-CSF receptor, and c-Src [39].

The purification of primary osteoclasts from rabbit bones by their adherent nature [27, 40] provides us with an opportunity to investigate the molecules in osteoclasts. The purity of osteoclasts was based on more than 95% TRAP-positive multinucleated cells. We have examined the expression of receptor tyrosine kinases by PCR and found that Tyro 3, Fms, and Met were frequently cloned from primary osteoclasts in PCR cloning.

Although the cloning frequency does not always correspond to the degree of expression, the profile of RTK cloned from osteoclasts is indicative ( Table 1). Among them, it is surprising to see the frequent cloning of Tyro 3, which has rarely been cloned from HSC [25]. Tyro 3 is most prominently expressed in the adult nervous system [17], but it is also expressed in some nonneuronal tissues such as kidney, ovary, and testis [8]. Tyro 3 was reported to be abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, liver cells, and fetal thymus from day 14 until birth [18]. One of the ligands for these three related receptors has been identified as Gas6, which was cloned as a growth arrest-specific gene [21]. Gas6 is expressed in the heart, lung, stomach, and kidney at high levels [23]. Mitogenic activity of Gas6 for fibroblasts, vascular smooth muscles and Schwann cells has been reported [41-43], however, the function of Gas6 on primary cells is not known. While Axl was expressed at moderate levels in adult BM, expression of Tyro 3 in BM was barely detectable [44]. We confirmed that Tyro 3 was expressed on HSC (lineage-negative c-kit-positive) but not lineage-specific mature cells by RT-PCR including macrophages (data not shown).

In this paper, we have shown that Tyro 3 was expressed in rabbit and murine multinucleated osteoclasts and cultured osteoclast-like cells by immunohistochemistry ( Figs. 1 -3GoGoGo) . We have examined the effect of Gas6 on rabbit osteoclasts and shown that it stimulates the bone resorption of osteoclasts dose-dependently ( Fig. 5). The optimum dose was 3 nM, and an excess amount of ligand suppressed the pit formation. This dome-shaped dose response is seen in the activity of chemokines. It is noted that primary BM stromal cells and a stromal cell line, ST-2, both of which have the ability to support osteoclastic bone resorption, express Gas6, and that the expression is regulated by osteotrophic hormones such as 1,25(OH)2D3 and parathyroid hormone (unpublished data, Y. Hakeda et al.). We have shown that the phosphorylation of 170 kD Tyro 3 protein was induced two min after the addition of Gas 6 ( Fig. 4). Moreover, herbimycin A inhibited the Gas6 effect on osteoclastic bone resorption ( Fig. 6). These data may support the theory that Tyro 3 mediates the effect of Gas6 through tyrosine phosphorylation. Taken together, Gas6/Tyro 3 signaling is a novel mechanism for regulating osteoclastic function. However, Gas6 did not affect the differentiation of osteoclasts from BM progenitor cells. This is consistent with the restricted expression of Tyro 3 in multinucleated osteoclasts but not in osteoclast progenitors. However, since the serum contains Tyro 3 ligand, it is possible that Tyro 3 on osteoclast progenitors is already stimulated. Protein S, the putative ligand of Tyro 3 [22, 45] affected the function of osteoclasts. When cultured osteoclasts were placed on dentine slices in the presence of protein S, the area of pits increased substantially over those in the absence of ligands ( Fig. 5). Findings that protein S is produced by bone cells and that its deficiency exhibits osteopenia suggests a role for protein S in bone turnover and metabolism [46, 47]. Interestingly, human protein S can bind to murine Tyro 3 [22, 44]. However, Ohashi et al. reported that Gas6, but not human protein S, specifically bound to the soluble form of human Sky-Fc chimeric protein [48]. Thus, it remains to be determined whether protein S is a ligand of Tyro 3. Since we used rat Gas6 and human protein S for the activation of mouse or rabbit Tyro 3, it will be necessary to determine the function of Tyro 3 using the ligand from the same species.

Gas6, protein S, and an osteocalcin, bone-specific protein are dependent on vitamin K, a cofactor for the enzyme responsible for carboxylation of glutamic acid residues. It remains to be clarified how these vitamin K-dependent proteins are involved in bone resorption and formation in vivo. To date, M-CSF, HGF, and MSP have been reported as osteotropic factors whose receptors such as c-fms, Met, and Stk/Ron have a tyrosine kinase activity. Although all these factors are expressed in osteoclasts, their biological effects differ. M-CSF stimulates survival and chemotactic behavior in mature osteoclasts. M-CSF is known to play an important role in osteoclast formation, but paradoxically, it inhibits bone resorption by mature osteoclasts [6]. HGF was reported to increase the number of osteoclasts, induce changes in osteoclast shape, and stimulate chemotactic migration and DNA replication in the osteoclast precursors; however, it did not induce bone resorption [49, 50]. Although MSP did not stimulate the formation of osteoclasts, it induced the bone-resorbing activity of osteoclasts [7]. Therefore, the function of Gas6-Tyro 3 is similar to that of the MSP-Stk/Ron system.

In conclusion, a novel receptor tyrosine kinase is expressed in mature osteoclasts, and its ligand, Gas6, stimulates the bone-resorbing activity of osteoclasts.


   Acknowledgments
 
Y.S.N. and Y.H. contributed equally to this study and are considered the top authors.

This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication December 19, 1997.



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