We have explored the use of adenovirus vector-mediated gene transfer to introduce foreign genes into osteoclasts, terminally differentiated cells responsible for bone resorption. A replication-deficient adenovirus vector that contains a reporter gene encoding β-galactosidase efficiently infected human osteoclast-like cells (OCLs) derived from human giant cell tumors and mouse OCLs formed in vitro. We then constructed an adenovirus vector carrying human epidermal growth factor receptor (EGFR) cDNA (Ax1CAhEGFR) and introduced the EGFR gene into mouse OCLs. Clear induction of EGF receptor was detected in Ax1CAhEGFR-infected OCLs (EGFR-OCLs) by immunocytochemistry and immunoblotting, and EGF stimulation induced rapid tyrosine phosphorylation of several proteins including EGF receptor itself. Large vacuoles appeared in EGFR-OCLs in response to EGF treatment, and pit-forming activity by EGFR-OCLs was dose-dependently suppressed by recombinant human EGF. In addition, survival of EGFR-OCLs was prolonged by EGF. No expression of EGF receptor or effects of EGF were observed in noninfected OCLs or control vector-infected OCLs. These results suggest that adenoviral vectors are useful for modulating osteoclast function by introducing foreign genes into osteoclasts and that they will be a good means of gene therapy of metabolic bone diseases.
OSTEOCLASTS ARE PRIMARY cells for bone resorption, and metabolic bone diseases, such as osteoporosis, Paget's disease, and humoral hypercalcemia of malignancy, result from an abnormal regulation of osteoclastic bone resorption. Rapid progress has been made during the last decade in identifying many therapeutic agents that can potentially regulate osteoclast function, opening a new therapeutic possibility for these bone disorders. One alternative is to modulate osteoclast function through gene therapy, where the gene or cDNA that modulates osteoclast function is transferred to osteoclasts. In recent years, adenovirus-mediated gene transfer has been recognized to provide a means of high-efficiency gene transfer into various kinds of mammalian cells including terminally differentiated cells, such as nerve cells and liver cells.1–5 Here, we report that a replication-deficient adenovirus vector has the ability to infect osteoclasts in vitro. Using an adenovirus vector– mediated gene transduction system, we successfully introduced the human epidermal growth factor (EGF) receptor gene as well as a reporter enzyme gene (lacZ gene) in human and mouse osteoclast-like cells (OCLs). OCLs expressing human EGF receptor showed a dramatic morphological change and prolonged survival rate in response to recombinant human EGF, and the pit-forming activity of these cells was suppressed by EGF.
MATERIALS AND METHODS
Animals and chemicals
Newborn ddY mice and 8-week-old male ddY mice were purchased from Shizuoka Laboratories Animal Center (Shizuoka, Japan). Alpha modified-minimum essential medium (α-MEM) and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO BRL, Life Technologies, Inc. (Rockville, MD, U.S.A.), and fetal bovine serum (FBS) was from Cell Culture Laboratory (Cleveland, OH, U.S.A.). Bacterial collagenase and 1α,25-dihydroxyvitamin D3 were purchased from Wako Pure Chemical Co. (Osaka, Japan), and dispase was from Godo Shusei Co. (Tokyo, Japan). Collagen gel solutions (cell matrix, type I-A) were purchased from Nitta Gelatin Co. (Osaka, Japan). Dentine (ivory) slices were kindly provided by Dr. N. Takahashi (Showa University). X-Gal was obtained from Takara (Otsu, Japan). Antiphosphotyrosine antibody, clone 4G10 was obtained from UBI (Lake Placid, NY, U.S.A.). Antihuman EGF receptor antibody was purchased from Transduction Laboratories (Lexington, KY, U.S.A.). Other chemicals and reagents used in this study were of analytical grade.
Human OCLs were obtained from giant cell tumors after surgical procedures at the hospital of the University of Tokyo following the method reported by Nesbitt et al.,6 and were cultured on 24-well culture plates (Corning Glass Works, Corning, NY, U.S.A.) in DMEM with 10% FBS. Mouse OCLs were prepared as described by Akatsu et al.7 Briefly, osteoblastic cells were obtained from calvaria of newborn ddY mice.8 Osteoblastic cells (5 × 105 cells) and bone marrow cells (5 × 106 cells) were cocultured in the presence of 10 nM 1α,25-dihydroxyvitamin D3 on culture dishes (ϕ 6 cm) coated with 0.2% collagen gel matrix. OCLs were recovered by digesting the collagen gel with 0.2% collagenase. This preparation was used in the conventional pit assay as previously reported.9 For biochemical studies, OCLs were further purified as described,10,11 and tartrate-resistant acid phosphatase (TRAP) staining was used for their identification.12
Constructs and gene transduction
The recombinant adenovirus carrying an Escherichia coli β-galactosidase (β-gal) expression cassette with the CAG (cytomegalovirus IE enhancer + chicken β-actin promoter + rabbit β-globin poly(A) signal) promoter (AxCASLacZ) and control vector (Ax1w1) were kindly provided by Dr. Izumu Saito (The University of Tokyo). The recombinant adenovirus carrying wild-type human EGF receptor with the CAG promoter (Ax1CAhEGFR) was constructed by homologous recombination between the expression cosmid cassette and the parental virus genome as described previously.13,14 Isolated human OCLs were incubated with DMEM containing the recombinant adenoviruses at an indicated multiplicity of infection (MOI) for 2 h at 37°C, and then 10 times more medium with 10% FBS was added. Mouse cocultures on day 5, when OCLs began to appear, were incubated with α-MEM containing the adenoviruses for 2 h at 37°C. Experiments were performed 2 days after the infection. EGF stimulation was carried out following the method previously described.15 For cell survival assay, Ax1CAhEGFR-infected OCLs (EGFR-OCLs) were further cultured for 3 days in the presence or absence of EGF. Surviving OCLs were detected by TRAP staining.
All extraction procedures were performed at 4°C. Cells were lysed with RIPA buffer (10 mM Tris-HCl, pH 7.4, 1% [v/v] Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 0.1% sodium deoxycholate, 1 mM EDTA, 2 mM sodium orthovanadate, 10 mM sodium fluoride, and aprotinin at 10 μg/ml). RIPA extracts were prepared by centrifugation at 12,000 g for 10 minutes. Samples containing an equal amount of proteins (10 μg) were electrophoresed on SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to Immobilon-P (Millipore Co., Bedford, MA, U.S.A.). Immunostaining with antibodies was performed using ECL Western blotting reagents (Amersham Co., Arlington Heights, IL, U.S.A.) according to the conditions recommended by the supplier.
X-gal histochemistry and immunocytochemistry
Detection of β-gal activity was performed with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) as described.16 In short, cells were fixed at room temperature in 3.7% formaldehyde prepared in phosphate-buffered saline for 10 minutes and thoroughly washed in phosphate-buffered saline three times. β-gal activity was detected by immersing the cells into staining solution (5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2, and 1 mg/ml of X-gal) for 1 h at 37°C. Expression of EGF receptors was examined by Western blot analysis and immunocytochemistry with anti-EGF receptor antibody as previously described.15
Pit formation assay
The pit-forming activity of OCLs was assayed as reported.9 The OCL preparation obtained from cocultures performed on collagen gel-coated dishes was resuspended in α-MEM containing 10% FBS, and plated on dentine slices (ϕ 4 mm) in 96-well culture plates. After 90 minutes of incubation, the slices were transferred to 24-well plates and incubated for another 48 h in the presence or absence of indicated concentrations of recombinant human EGF. Dentine slices were then treated with 1 N NH4OH to remove adherent cells. Resorption pits were stained with 0.5% toluidine blue, and the resorbed area was measured using an image analysis system (SYSTEM SUPPLY, Nagano, Japan).
Each series of experiments was repeated at least three times. The results were expressed as the means ± SD of four determinants. Significant differences were determined using Student's t-test.
Adenovirus-mediated gene expression in human and mouse osteoclast-like cells
Cultured human OCLs obtained from giant cell tumors and mouse OCLs formed in cocultures were infected with recombinant adenovirus encoding lacZ gene (AxCASLacZ) in order to investigate the efficiency of adenoviral gene transduction in OCLs. Cultures were infected with AxCASLacZ at different MOIs, and β-gal activity of OCLs was examined cytochemically using X-gal substrate after 2 days of infection. As shown in Fig. 1C, strong staining was observed in OCLs and some mononuclear cells within 1 h. The labeling was not limited to nuclei despite the nuclear localization signal sequence added to β-gal, but diffuse cytoplasmic staining was observed in many of the OCLs. The proportion of β-gal-positive OCLs increased in accordance with the number of recombinant adenoviruses inoculated in the culture, and more than 85% of human OCLs became strongly positive for β-gal activity at an MOI of 100, with no obvious morphological changes or toxic effects (Figs. 1A and 1C). As shown in Fig. 1C, some mononuclear cells were also positive for β-gal activity. The gene transduction seemed less efficient in mouse OCLs, but 300 MOI was enough to transduce the lacZ gene in almost 100% of OCLs (data not shown). It should be noted that even noninfected OCLs were positively stained for β-gal when they were stained for longer than 8 h, probably because of their intrinsic β-gal activity, but the activity was much weaker than that in the AxCASLacZ-infected OCLs, and they did not show any blue staining within 1 h (Fig. 1B).
Adenovirus-mediated EGF receptor expression in OCLs
It has been reported that signaling mediated by nonreceptor and receptor-type protein-tyrosine kinases, such as c-Src and c-Fms, modulates osteoclast function as well as osteoclast differentiation.17 We therefore attempted to modulate osteoclast function by transducing EGF receptor gene into osteoclasts. Ax1CAhEGFR was constructed following the method previously reported.13 Adenoviral infection at an MOI of 100 was carried out for 2 h in mouse OCLs formed in cocultures of mouse osteoblastic cells and bone marrow cells performed on collagen gel on day 5 of culture. After 2 days of infection, OCLs were recovered by digesting the collagen gel with 0.2% collagenase, resuspended in α-MEM containing 10% FBS, and replaced on culture dishes. For protein analysis, OCLs were further purified following the method previously described.11 As shown in Fig. 2A, clear expression of EGF receptor was observed in EGFR-OCLs by Western blot analysis. Immunostaining also demonstrated an induction of EGF receptor in EGFR-OCLs as well as in mononuclear cells (Fig. 3B). In contrast, no expression of hEGFR was observed in noninfected OCLs or Ax1w1-infected OCLs by either Western blotting or immunostaining (Figs. 2A and 3A).
EGF induces tyrosine phosphorylation and suppresses the function of EGFR-OCLs
Tyrosine phosphorylation of several proteins including EGF receptor itself was observed in response to EGF stimulation in EGFR-OCLs, while no apparent induction of tyrosine phosphorylation was observed in noninfected or Ax1w1-infected OCLs even in the presence of EGF (Fig. 2B). Interestingly, obvious morphological changes were observed in EGFR-OCLs in response to EGF treatment. Within 6 h of EGF treatment, huge vacuoles began to appear in the cytoplasm, and cellular retraction was observed (Figs. 4B and 4C). Such morphological changes disappeared after 48 h. No similar morphological changes were observed in macrophage colony-stimulating factor (M-CSF)–treated OCLs (data not shown). We next examined the effect of EGF on bone-resorbing activity of EGFR-OCLs. EGFR-OCLs were cultured on dentine slices in the presence or absence of recombinant human EGF. As shown in Fig. 5, pit-forming activity of EGFR-OCLs was inhibited by EGF in a dose-dependent manner. This inhibitory effect was mild, and even the highest concentration of EGF tested was not enough to suppress the pit formation completely (Fig. 5A). Such inhibitory effect was not observed when noninfected OCLs were mixed with Ax1CAhEGFR-infected osteoblastic cells (data not shown), indicating that EGF directly acts on EGFR-OCLs.
EGF prolongs the survival of EGFR-OCLs
Because it has recently been reported that M-CSF, whose receptor also has tyrosine kinase activity, supports the viability of osteoclasts,18,19 we next examined if EGF can prolong the viability of EGFR-OCLs. After replating on culture dishes, crude preparations of OCLs were treated with 0.1% collagenase and 0.2% dispase to remove osteoblastic cells and hematopoietic cells and then incubated in α-MEM containing 10% FBS with or without EGF. Most of the EGFR-OCLs disappeared within 72 h in the absence of EGF, while EGF treatment obviously promoted their survival (Fig. 6). No such effect of EGF was observed in Ax1w1-infected OCLs (Fig. 6).
The transduction of foreign genes into osteoclasts has been extremely difficult, mainly because osteoclasts are terminally differentiated cells without proliferating activity, and only limited success has been reported so far.11,20–24 In this report, we demonstrate that adenovirus vectors can be efficiently used as gene transfer agents for postmitotic osteoclasts at least in vitro. Recombinant adenovirus carrying the lacZ gene can infect more than 85% of human OCLs at an MOI of 100 with no apparent morphological changes or cellular toxicity (Figs. 1A and 1B). Adenovirus vectors have several advantages in introducing foreign genes into osteoclasts. First, these vectors are capable of infecting a variety of terminally differentiated cells, such as neurons and hepatocytes.1–5 Second, recombinant adenovirus can be easily amplified to a very high titer in vitro. Third, adenovirus infection to the cells has been reported to require the interaction of the RGD sequence in the penton base of the virus with the cell surface vitronectin receptors (αvβ3 or αvβ5 integrins), which are expressed at high levels on the cell surface of osteoclasts.25 In fact, as shown in Fig. 1B, when infection of AxCASLacZ was performed at an MOI of 30, OCLs were almost exclusively positive for β-gal staining within 1 h, indicating high β-gal activity in the infected OCLs. Some mononuclear cells were also positive for β-gal activity (Fig. 1B), most of which are also positive for TRAP activity, indicating that they are osteoclast precursors. Some osteoblastic (or fibroblastic) cells were also positive for β-gal staining, but only 5% of the cells were β-gal positive at an MOI of 30, while about 60% of OCLs were positively stained. This suggests that osteoclasts and their precursors are more easily infectable for adenovirus vectors because they express high levels of vitronectin receptors. Our results and previous observations show that the adenovirus vector is a suitable gene transfer agent to osteoclasts. Whether adenovirus vectors are also available for in vivo gene transfer into osteoclasts is now under investigation in our laboratory.
Previous reports have demonstrated that both osteoclast differentiation and function are regulated by the signaling pathways mediated by receptor- and non-receptor-type tyrosine kinases such as M-CSF receptors and c-Src.17,26 Not only is M-CSF necessary for osteoclast differentiation,27 but it also regulates osteoclast function and survival.18,19 The signaling of M-CSF is mediated by its specific receptor encoded by c-fms proto-oncogene. The M-CSF receptor belongs to subclass III of receptor tyrosine kinase with five immunoglobulin-like repeats in the extracellular domains, while EGF receptor (EGFR) belongs to subclass I with disulfide-linked heterotetrameric α2β2 structures and cystein-rich sequences (reviewed in Ref. 28). Although these two receptors utilize several common second messengers in transducing their signals, they also have different signaling pathways which account for the difference in their effects on cells.29 Therefore, it is interesting to compare the effect of EGF on EGFR-OCLs with the effect of M-CSF on osteoclasts. Western blotting and immunostaining demonstrated the lack of expression of EGFR in noninfected OCLs and a clear induction of the receptor in OCLs following the infection of the recombinant adenovirus. EGF treatment stimulated a rapid tyrosine phosphorylation of several proteins, including EGFR itself in EGFR-OCLs, especially proteins with molecular weight ∼100–120 kDa (Fig. 2B), which we have not identified yet. Similar to M-CSF, the dentin-resorbing activity of EGFR-OCLs was moderately suppressed by recombinant human EGF in a dose-dependent manner, and their survival was prolonged by EGF treatment. Interestingly, EGF treatment induced a dramatic morphological change in EGFR-OCLs, i.e., the appearance of large vacuoles and cellular contraction, a phenomenon that cannot be seen in OCLs treated with M-CSF (data not shown). This may suggest that the signaling pathways of EGFR in EGFR-OCLs are different from those of M-CSF receptors. The molecular mechanism that leads to the morphological changes and suppression of bone-resorbing activity of OCLs requires further investigation. In summary, adenoviral vectors are useful for modulating osteoclast function by transducing foreign genes into osteoclasts and are a promising means of gene therapy of metabolic bone diseases.
We thank I. Saito and J. Miyazaki for generous gifts of the recombinant adenovirus vector system and the CAG promoter, respectively. We also thank Y. Fukui for technical assistance. This work was supported by a grant from the Japan Orthopaedic and Traumatology Foundation, Inc., no. 0089 (Alcare Award) and Grants-in-Aids from the Ministry of Education, Science and Culture of Japan to S.T.