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J. Biol. Chem., Vol. 277, Issue 24, 21446-21452, June 14, 2002

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H-Ras/Mitogen-activated Protein Kinase Pathway Inhibits Integrin-mediated Adhesion and Induces Apoptosis in Osteoblasts^*

Yoshiya Tanaka^§, Shingo Nakayamada, Hiroko Fujimoto^¶, Yosuke Okada, Hisanori Umehara, Tohru Kataoka^**, and Yasuhiro Minami^¶

From the  First Department of Internal Medicine, University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan, the ^¶ Department of Biomedical Regulation, Kobe University School of Medicine, Kobe 650-0017, Japan, the  Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School of Medicine, Kyoto 605-8507, Japan, and the ^** Second Department of Physiology, Kobe University School of Medicine, Kobe 650-0017, Japan

Received for publication, March 7, 2002, and in revised form, March 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have studied the relevance of H-Ras and its downstream effectors to osteoblast functions. 1) Purified human osteoblasts highly expressed integrins ₁, ₄, ₅, ₆ and the activation epitope of ₁. However, these molecules were markedly down-regulated on osteoblasts transfected with expression vector encoding fully activated H-Ras^V12, H-Ras^V12T35S, activating Raf-1/mitogen-activated protein kinase (MAPK), or an active Raf-1 but not on cells having H-Ras^V12Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant. 2) Although osteoblasts spontaneously adhered to fibronectin and laminin in ₁-dependent manner, the expression of H-Ras^V12 or H-Ras^V12T35S, but not H-Ras^V12Y40C, in osteoblasts reduced their adhesion. 3) Osteoblasts bearing H-Ras^V12, H-Ras^V12T35S, or Raf-1 failed to proliferate, whereas those with H-Ras^V12Y40C proliferated well. (4) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-Ras^V12, H-Ras^V12T35S, or Raf-1. (5) Most of the cells having H-Ras^V12, H-Ras^V12T35S, or Raf-1 became annexin-V^high/propidium iodide (PI)^{high or low} and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL)^high/PI^low after 24 and 72 h incubation, respectively. Thus, we propose that H-Ras signals followed by Raf-1/MAPK pathway but not PI3K not only reduces ₁-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Various biological processes such as proliferation, apoptosis, adhesion, cytokine production, and chemotaxis are tightly regulated by intracellular signaling. Recent evidence indicates that small G proteins (guanine nucleotide-binding regulatory proteins) control signaling pathways critical for such diverse cellular functions by activating multiple effector molecules. Bone metabolism in health and disease is based on a self-regulating cellular event. Osteoblasts play a central role in bone formation by synthesizing multiple bone matrix proteins and differentiating into bone cells and also regulate osteoclast maturation by producing bone-resorbing cytokines and by direct cell attachment, resulting in bone resorption (1-4). Such diverse functions of osteoblasts are induced or regulated by multiple soluble factors including growth factors, hormones, and prostaglandins. Recent findings imply that many critical factors such as estrogen, parathyroid hormone, glucocorticoid, prostaglandins, tumor necrosis factor-, and chemokines control osteoblast functions by binding to their cognate G-protein-coupled receptors and/or subsequent small G-proteins-mediated signaling (5-9). However, little is known regarding the mechanisms of the integration of G-proteins in controlling osteoblast functions.

Among several small G-proteins, Ras has been characterized as a central molecule for the regulation of signal transduction pathways in various types of cells (10-14). It is noteworthy that Ras proteins both physically and functionally couple with multiple effectors including Raf-1/mitogen-activated protein kinase (MAPK),¹ Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase (PI3K), protein tyrosine kinases, and small GTPases. Namely, Ras proteins are molecular switches similar to a "hub," which radiates multiple signaling pathways critical for diverse cellular functions. This is a dynamic phenomenon involving an array of protein-protein interactions modulated by chemical modifications, structural rearrangements, and intracellular relocalizations. Thus, Ras proteins are activated by multiple extracellular stimuli and are involved in regulatory biological processes from the outside of the cell to its interior through a complex array of downstream effectors, thereby controlling a variety of cellular responses such as proliferation, apoptosis, adhesion, and cytokine/matrix production. However, the relevance of Ras to signaling and functions in osteoblasts remains unclear.

We have previously reported that H-Ras plays a pivotal role in integrin-mediated adhesion and proliferation of lymphocytes (15, 16). Here we have studied the relevance of H-Ras and its downstream effectors to functions of osteoblasts by shedding light upon the difference of Raf-1/MAPK and PI3K. The current report demonstrates that H-Ras/Raf-1/MAPK pathways might be involved in down-regulation of integrins and integrin-mediated adhesion to matrix proteins as well as induction of apoptosis presumably via Fas/Bcl-2 systems in human-purified osteoblasts.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of Human Osteoblastic Cells-- Osteoblast-like cells were purified from metaphyseal trabecular bone in the proximal femur of five osteoarthritis patients during total hip arthroplasty by the established procedures of Russell and colleagues (17-19). All five patients were female (mean age: 57.3 ± 8.6 years). After removing pieces of cortical bone, articular cartilage, and soft connective tissue, the fragments were cut into small pieces and washed extensively. The bone explants were cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Grand Island, NY) containing 10% fetal calf serum (FCS) (GIBCO) in 25-cm² culture flasks (Falcon, Lincoln Park, NJ) at a humidified 5% CO₂ atmosphere. When cell monolayers were confluent after the 6-8-week culture, the explants were removed and the cells were replated and incubated, which resulted in new cellular outgrowth and eventually a confluent monolayer of cells. At confluence, the cells were trypsinized, passaged at a 1:3 split ratio, and recultured. The medium was changed twice each week, and the cells were used after 3-7 passages. The obtained cells showed a flattened polygonal shape with multiple spindlelegs and possessed characteristics of osteoblast-like phenotype including osteocalcin (OC), bone sialoprotein, type I collagen (COLL-I), and bone alkaline phosphatase (ALP) as described previously (18).

Antibodies and Other Reagents-- The following monoclonal antibodies (mAbs) were used as purified immunoglobulin (Ig) in the preparation of staining and analysis of cell surface or cytoplasmic molecules and adhesion assays as follows: control mAb thy-1.2 (ATCC, Manassas, VA); human integrin ₁ (CD29) mAb MAB13; human ₅ (CD49e) mAb MAB16 (provided by K. Yamada, National Institutes of Health, Bethesda, MD); human ₄ (CD49d) mAb NIH49d-1; human ₄ (CD49f) mAb NIH49f-1 (provided by S. Shaw, National Institutes of Health); anti-ligand binding sites of human integrin ₁ mAb HUTS-21 (provided by F. Sanchez-Madrid, The Princess Hospital, Madrid, Spain) (20); human integrin ₁ (CD29) mAb Lia1/2 (Immunotech, Marseille, France); human ₃ (CD49c) mAb P1B5 (Fujisawa, Tokyo, Japan); anti-human bone ALP mAb ALP-mAb (provided by M. Miura, Mitsubishi Chemical BCL, Tokyo, Japan); anti-human OC mAb 10B (provided by K. Hosoda, Teijin, Tokyo, Japan); anti-human IL-6 mAb MQ2-13A5 (Fujisawa); anti-human Fas (CD95) mAb DX2 (Fujisawa); and Bcl-2 mAb Ab-1 (Cosmo-Bio, Tokyo, Japan).

The obtained oligonucleotides, a human active form of H-Ras expression plasmid pEF-BOS-HA-Ras^V12, a human Raf-1-binding/extracellular signal-regulated kinase (ERK) subfamily of MAPK-activating form of H-Ras expression plasmid pEF-BOS-HA-Ras^V12T35S, a human Raf-1-non-binding form of H-Ras expression plasmid pEF-BOS-HA-Ras^V12E37G, a human PI3K-binding/activating form of H-Ras expression plasmid pEF-BOS-HA-Ras^V12Y40C, and a human active form of Raf-1 expression plasmid pEF-BOS-HA-Raf-1 were introduced into osteoblasts using a cationic liposome-mediated transfection method (21-23). Oligonucleotides and plasmids were mixed with 5 µl of Lipofectin reagent (LipofectAMINE 2000, Invitrogen) and incubated for 10 min at room temperature. The oligonucleotide and liposome complex was added to osteoblasts plated in a 6-well culture dish (3 × 10⁵ cells/well, Falcon) and incubated in a 10% FCS containing DMEM for 24-72 h. The concentration of oligonucleotides in the conditioned medium was 2.2 µM, and the expression of each H-Ras was confirmed by staining with anti-HA Ab using flow cytometer. The transfection efficiency of pEF-BOS-HA-Ras^V12 into osteoblasts was 50-80% detected by anti-HA Ab and differed among donors. Marked difference of the transfection efficiency among all of the used mutants of H-Ras was not observed in COS cells (data not shown).

Flow Microfluorometry-- Staining and flow cytometric analyses of osteoblasts with or without transfection of several plasmid as mentioned above were performed using a FACScan (BD PharMingen) and standard procedures as described previously (26). 2 × 10⁵ cells were incubated with negative control mAb thy-1.2, integrin ₁ mAb, anti-ligand binding sites of ₁ mAb, ₃ mAb, ₄ mAb, ₅ mAb, ₆ mAb, or anti-Fas mAb in FACS medium consisting of Hanks' balanced salt solution (Nissui, Tokyo, Japan), 0.5% human serum albumin (HAS) (Yoshitomi, Osaka, Japan), and 0.2% NaN₃ (Sigma) for 30 min at 4 °C. The cytoplasmic antigens of osteoblasts, which were pretreated with cell permeabilization kit (Caltag, Burlingame, CA), were stained by anti-Bcl-2 mAb, anti-OC mAb, anti-ALP mAb, or anti-IL-6 mAb in FACS medium for 30 min at 4 °C. After washing the cells three times with FACS medium, they were further incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG Ab, goat anti-rabbit IgG Ab, or rabbit anti-goat IgG Ab for 30 min at 4 °C. The staining of cells with mAbs was detected using FACScan. The quantification of cell surface antigens on single cells was calculated using standard beads QIFKIT (DAKO Japan, Kyoto, Japan) as already described (15, 27). The data were used for the construction of the calibration curve (mean fluorescence intensity) against antibody-binding capacity (ABC). The cell specimen was analyzed on the FACScan and ABC calculated by interpolation on the calibration curve. When green fluorescence laser detector was set at 450 level in the FACScan used, ABC = 414.45 × exponential (0.0092 × mean fluorescence intensity) (R² = 0.9999). Subsequently, specific antibody-binding capacity was obtained after corrections for background, an apparent ABC of the negative control mAb thy-1.2. Specific antibody-binding capacity corresponds to the mean number of accessible antigenic sites per cell referred to as antigen density and expressed in sites per cell.

Adhesion Assay-- Adhesion assay of osteoblasts to extracellular matrix glycoproteins was performed as described previously (15, 16, 24, 25). 48-well culture plates (Costar, Cambridge, MA) were coated with 10 µg/ml fibronectin (FN), laminin (LM), vitronectin (VN), or COLL-I (all from Cosmo-Bio) for 24 h at 4 °C. After washing by phosphate-buffered saline three times, nonspecific proteins were blocked by 2% human serum albumin for 2 h at 37 °C. The plates were washed three times with phosphate-buffered saline before the addition of osteoblasts. 2 × 10⁵ osteoblasts were labeled with ⁵¹Cr (PerkinElmer Life Sciences) in DMEM with 1% human serum albumin and were added to the plates in the absence or presence of adhesion-blocking anti-₁ mAb Lia1/2 (10 µg/ml). After a settling phase of 30 min at 4 °C, the plates were rapidly warmed to 37 °C for 30 min and then gently washed twice with DMEM at room temperature to completely remove non-adherent monocytes. The contents of each well containing adherent osteoblasts were lysed with 250 µl of 1% Triton X-100 (Sigma), and the emission of the contents of each well was measured using a -counter.

Proliferation Assay-- Osteoblasts (1 × 10⁴) were seeded and incubated on 96-well flat-bottomed microfilter plates (Costar) in DMEM and 10% FCS for 24-72 h at 37 °C. After cells were stained with TetraColor One kit including tetrazolium and electron carrier mixture (Seikagaku, Tokyo, Japan) for 1 h at 37 °C, the optical density value of each well was measured by a enzyme-linked immunosorbent assay plate reader at 450 nm.

Detection of Apoptosis-- The quantitation of early apoptotic osteoblasts by the annexin-V and propidium iodide (PI) (Kamiya Biomedical, Seattle, WA) on osteoblasts, which were precultured for 24 h at 4 °C, were conducted by dual-color flow cytometry. After the cells were treated with RNase A (2 ng/ml, Sigma) for 15 min at 37 °C, PI and fluorescein isothiocyanate-conjugated annexin-V were added to the cells and incubated for 2 min. The staining of cells was detected using FACScan. The quantitation of apoptotic cells by the TUNEL technique using ApopTag Direct (Intergen, Purchase, NY) and PI on cultured synovial cell subpopulations was conducted by dual-color flow cytometry. After the cells were fixed with 200 µl of 1% paraformaldehyde for 15 min at 4 °C and washed twice, the cells were resuspended in 70% ethanol and were kept at 20 °C. After washing twice, the cells were resuspended in 75 µl of equilibration buffer, were washed again, were resuspended in 25 µl of working strength Tdt enzyme, and were incubated for 30 min at 37 °C. After adding 250 µl of working strength stop/wash buffer, the cells were washed and resuspended in 250 µl of working strength stop/wash buffer, and 50 µg/ml of PI was added to the cells. The obtained cells were assessed using FACScan.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of H-Ras^V12, H-Ras^V12T35S, or Active Raf-1 Reduced Expression of Integrins ₁, ₄, ₅, ₆, and Activated Epitope of ₁ on Osteoblasts-- Initially, we assessed the ability of H-Ras and its mutants to regulate integrins on osteoblasts when expressed ectopically in the cells. Purified human osteoblastic cells (osteoblasts) spontaneously and highly expressed an integrin ₁ as recognized by anti-conventional ₁ (CD29) mAb MAB13 (Fig. 1). However, the expression of ₁ was decreased on osteoblasts expressing H-Ras^V12 but not on those expressing mock plasmid. ₁ requires an active configuration to bind to its ligand, a process that can be induced by a variety of stimuli, and can be assessed by HUTS-21 mAb, which reacts with a ligand-induced binding site located on the ₁ (20). Osteoblasts spontaneously expressed the ligand binding sites of ₁ as recognized by HUTS-21 mAb, whereas osteoblasts bearing H-Ras^V12 but not mock plasmid expressed significantly lesser amounts of the sites on the cell surface. The expression of  subunits was also screened on osteoblasts. Osteoblasts expressed ₃ (CD49c), a receptor for LM; ₄ (CD49d), a receptor for FN and vascular cell adhesion molecule-1; ₅ (CD49e), a receptor for FN; and ₆ (CD49f), a receptor for LM. The expression of ₄, ₅, and ₆ but not ₃ was reduced on osteoblasts expressing H-Ras^V12, whereas these  chains on osteoblasts expressing mock plasmid was comparable with  chains on control osteoblasts.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Integrin 1, 4, 5, and 6 were inhibited by expressing H-RasV12 on osteoblasts. Osteoblasts transfected with or without the expression vectors encoding H-RasV12 or a mock plasmid were analyzed for the expression of 1 (CD29) as recognized by MAB13 mAb (A), an activated form of 1 as recognized by HUTS-21 mAb (B), 4 (CD49d) by NIH49d-1 mAb (C), 5 (CD49e) by MAB16 mAb (D), 3 (CD49c) by P1B5 mAb (E), and 6 (CD49f) by NIH49f-1 mAb (F) using flow cytometry. The data are expressed as the mean percentage and mean ± S.E. of the number of molecules expressed per one cell calculated by standard QIFKIT beads from five different experiments using five different donors.

Because H-Ras exhibits its actions through binding to a set of effector proteins involved in Ras/Raf-1/MAPK and Ras/PI3K pathways (10-14), it important to determine which Ras effector is required to induce ₁. It is noteworthy that osteoblasts expressing H-Ras^V12T35S mutant, which selectively binds to Raf-1 and activates Raf-1, reduce the expression of not only ₁ but also an activated form of ₁ (Fig. 2). In contrast, the expression of both ₁ and an activated form of ₁ on the cells expressing a H-Ras^V12Y40C mutant that selectively binds to PI3K and a H-Ras^V12E37G mutant that does not bind to Raf-1 was comparable with their spontaneous expression on osteoblasts expressing a mock plasmid. Furthermore, osteoblasts expressing an active Raf-1 reduced the expression of ₁ and an activated form of ₁. These results imply that H-Ras signals, especially those mediated by Raf-1/ERK pathway, reduce the expression of ₁ and an activated form of ₁ on osteoblasts.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   1 and an activated form of 1 were inhibited by expressing H-RasV12T35S on osteoblasts. Osteoblasts transfected with or without the expression vectors encoding H-RasV12, H-RasV12Y40C, H-RasV12T35S, H-RasV12E37G, or active Raf-1, respectively, were analyzed for the expression of 1 (A) and an activated form of 1 using flow cytometry (B). The data are expressed as the mean percentage and the mean ± S.E. of the number of molecules expressed per one cell calculated by standard QIFKIT beads from three different experiments using three different donors.

Expression of H-Ras^V12 or H-Ras^V12T35S Reduced Integrin ₁-mediated Adhesion of Osteoblasts to FN, LM, and VN-- We next assessed the ability of H-Ras and its mutants to regulate adhesion of osteoblasts to matrix proteins when expressed ectopically in the cells. Purified human osteoblasts spontaneously adhered to FN, LM, VN, and COLL-I (Fig. 3). mAb-blocking studies indicated that osteoblast adhesion to FN, LM, and VN was integrin ₁-dependent, whereas the adhesion to COLL-I was ₁-independent. However, the adhesion of osteoblasts, transfected with the expression vector encoding H-Ras^V12T35S mutant, to FN, LM, and VN, but not COLL-I was markedly reduced to the levels that ₁-mAb blocking studies showed. In contrast, the adhesion of the cells expressing H-Ras^V12Y40C was comparable with spontaneous adhesion of osteoblasts. These results imply that H-Ras signals, especially those mediated by Raf-1/ERK pathway, reduces ₁-mediated adhesion of osteoblasts to matrix proteins such as FN, LM, and VN.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Expression of H-RasV12T35S suppressed 1-mediated adhesion of osteoblasts to FN, LM, and VN. 51Cr-labeled control human osteoblasts transfected with or without expression vectors encoding H-RasV12Y40C or H-RasV12T35S were incubated on plastic wells precoated with FN, LM, VN, or COLL-I (10 µg/ml) in the presence (hatched bar) or absence (open bar) of 1 mAb (10 µg/ml) at 37 °C for 30 min. After washing out non-adherent osteoblasts, -emissions of the lysates of only adherent cells were determined. Data are expressed as the mean percentage and the mean ± S.E. of the binding of added osteoblasts from triplicate wells of a representative result among five different donors.

Intracellular Expression of ALP, OC, and IL-6 Was Not Changed in Osteoblasts and Osteoblasts Expressing H-Ras Mutants-- The expression of intracellular ALP, OC, and IL-6 in osteoblasts was assessed. To this end, osteoblasts expressing H-Ras^V12, H-Ras^V12Y40C, or H-Ras^V12T35S were fixed by formaldehyde and permeabilized by saponin and subsequent staining, and flow cytometric analyses of the cells were performed with the indicated mAbs using FACScan. Control osteoblasts and osteoblasts transfected with the expression vectors encoding H-Ras^V12, H-Ras^V12Y40C, or H-Ras^V12T35S, respectively, exhibited similar levels of intracellular ALP, OC, and IL-6 (Fig. 4). The secretion of IL-6 from osteoblasts expressing H-Ras^V12, H-Ras^V12Y40C, or H-Ras^V12T35S did not change (data not shown). These results suggest that not only the differentiation of osteoblasts but also the production of one bone-resorbing cytokine IL-6 is not affected by the expression of H-Ras or its mutants.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Intracellular expression of ALP, OC, and IL-6 in osteoblasts. After treating the osteoblasts or osteoblasts transfected with the expression vectors encoding H-RasV12, H-RasV12Y40C, or H-RasV12T35S, respectively, with cell permeabilization kit, staining and flow cytometric analyses of the cells were performed with anti-human bone-type ALP mAb ALP-mAb, anti-OC mAb 10B and anti-IL-6 mAb, and fluorescein isothiocyanate-conjugated second Ig using FACScan. The data are expressed as the mean percentage and mean ± S.E. of the number of molecules expressed per one cell calculated by standard QIFKIT beads from three different experiments using three different donors.

Expression of H-Ras^V12 or H-Ras^V12T35S Suppressed Proliferation of Osteoblasts-- The proliferation assay of control osteoblasts or osteoblasts transfected with the expression vectors encoding H-Ras^V12, H-Ras^V12Y40C, H-Ras^V12T35S, H-Ras^V12E37G, or active Raf-1 was performed next using TetraColor One including tetrazolium and electron carrier mixture for evaluating cell proliferation. Osteoblasts proliferated well at least up to 72 h (Fig. 5). However, the proliferation rate of osteoblasts transfected with H-Ras^V12 was markedly reduced, and the proliferation rate of osteoblasts expressing H-Ras^V12T35S mutant was completely inhibited. The proliferation of osteoblasts expressing an active Raf-1 was also decreased within 24 h. The cell counts of osteoblasts expressing H-Ras^V12 and H-Ras^V12T35S were also down-regulated at 24 and 72 h compared with control osteoblasts (data not shown). In contrast, the proliferation of osteoblasts expressing H-Ras^V12Y40C or H-Ras^V12E37G was comparable with spontaneous proliferation of osteoblasts. These results imply that H-Ras signals, especially those followed by a Raf-1/ERK pathway, reduce the proliferation of osteoblasts.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Proliferation of osteoblasts was inhibited by expression of H-RasV12T35S. Proliferation assay of control osteoblasts or osteoblasts transfected with the expression vectors encoding H-RasV12, H-RasV12Y40C, H-RasV12T35S, H-RasV12E37G, or active Raf-1 was performed. After these osteoblasts were incubated in DMEM containing 10% FCS for 24 h (open bar) and 72 h (hatched bar), cells were stained with TetraColor One including tetrazolium and electron carrier mixture for detecting cell proliferation. The optical density value was measured by enzyme-linked immunosorbent assay plate reader at 450 nm. The data are expressed as the mean optical density value and mean ± S.E. in osteoblasts from triplicate wells of a representative result among five different donors. OD, optical density.

Expression of H-Ras^V12 or H-Ras^V12T35S Reciprocally Regulated of Expression of Fas and Bcl-2-- Fas is known to be involved in apoptosis, whereas Bcl-2 is essential to proliferative responses (28). These molecules on osteoblasts expressing H-Ras^V12, H-Ras^V12Y40C, H-Ras^V12T35S, or active Raf-1 were observed with anti-Fas mAb DX2 or anti-Bcl-2 mAb Bcl-2/100 using FACScan. Control osteoblasts expressed both cell surface Fas and intracellular Bcl-2 (Fig. 6). Of note, the expression of H-Ras^V12, H-Ras^V12T35S, or an active Raf-1 further augmented Fas expression on osteoblasts, whereas it completely inhibited intracellular Bcl-2. In contrast, the expression of H-Ras^V12Y40C did not change Fas and Bcl-2 levels in osteoblasts. Taken together, our observation of increased expression of Fas and reduced expression of Bcl-2 in osteoblasts expressing H-Ras^V12, H-Ras^V12T35S, or an active Raf-1 suggests that the cells might be apoptotic at least partially mediated by Fas.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Reciprocal regulation of Fas and Bcl-2 on osteoblasts expressing H-RasV12T35S. Staining and flow cytometric analyses of osteoblasts expressing H-RasV12, H-RasV12Y40C, H-RasV12T35S, or active Raf-1 were carried out with control mAb thy-1.2, CD95 (Fas) mAb DX2 (A) and Bcl-2 mAb Bcl-2/100 (B) using FACScan. The data are expressed as the mean percentage and mean ± S.E. of the number of molecules expressed per one cell calculated by standard QIFKIT beads from three different experiments using three different donors.

Expression of H-Ras^V12T35S Induced Apoptosis of Osteoblasts-- Accordingly, we assessed apoptotic features of control osteoblasts and osteoblasts expressing H-Ras^V12T35S. Annexin-V/PI-staining indicates that the majority of control osteoblasts were annexin-V^low/PI^low by Dot-blot analysis using flow cytometer, whereas most of the osteoblasts expressing H-Ras^V12T35S were annexin-V^high/PI^low or annexin-V^high/PI^high, namely early apoptotic after 24 h incubation (Fig. 7). The percentage of annexin-V^high/PI^low osteoblasts expressing an active Raf-1 was also significantly increased within 24 h (Table I). Furthermore, PI/TUNEL staining of the osteoblasts indicates that half of the osteoblasts expressing H-Ras^V12 and most of the osteoblasts expressing H-Ras^V12T35S were TUNEL^high/PI^low by Dot-blot analysis using ApopTag Direct kit and subsequent flow cytometer after 72-h incubation, whereas all of the control osteoblasts cells were TUNEL^low (Fig. 8). These results imply that H-Ras signals, especially those followed by Raf-1/ERK pathway, reduce proliferation and induce apoptosis of osteoblasts.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Early apoptosis of osteoblasts expressing H-RasV12T35S.After control osteoblasts (A) or osteoblasts expressing H-RasV12T35S (B) were incubated in DMEM with 10% FCS for 24 h, the quantitation of early apoptotic cells by the annexin-V and PI in the cells were conducted by dual-color flow cytometry. Shown is the Dot-blot analysis stained with annexin-V (x axis, logarithmic scale) and PI (y axis, linear scale). The data of osteoblasts were obtained from one representative result among five donors. The solid line represents the gate set to discriminate negative from of positive-stained cells as determined by control thy1.2 mAb.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Apoptosis of osteoblasts expressing H-RasV12T35S. The quantitation of apoptotic cells by the TUNEL and PI on control osteoblasts (A), osteoblasts expressing H-RasV12 (B), or H-RasV12T35S (C), which were cultured for 72 h, were conducted by dual-color flow cytometry. Shown is the histogram stained with TUNEL, which was gated in PIlow population. The data of osteoblasts were obtained from one representative result among three donors. The solid line represents the gate set to discriminate negative from positive stained cells as determined by control thy1.2 mAb.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The main findings obtained in this study are as follows. 1) Osteoblasts adhere to matrix protein such as FN, LM, and VN in a ₁ integrin-dependent manner. However, the osteoblasts transfected with the expression vector encoding fully activated H-Ras^V12 mutant or H-Ras^V12T35S, which selectively binds to Raf-1 and activates Raf-1/MAPK, failed to adhere to them, whereas the expression of H-Ras^V12Y40C mutant, which selectively binds to PI3K, or H-Ras^V12E37G mutant, which does not bind to Raf-1, did not affect the adhesion. 2) The expression levels of cell surface ₁, ₄, ₅, and ₆ and of ligand-binding activation epitope of ₁ were decreased on osteoblasts expressing H-Ras^V12, H-Ras^V12T35S, or an active Raf-1. 3) The expression of any of the H-Ras mutants did not affect on the intracellular expression of ALP, OC, and IL-6 in osteoblasts. 4) The osteoblasts expressing H-Ras^V12, H-Ras^V12T35S, or active Raf-1 failed to proliferate, whereas control osteoblasts and osteoblasts expressing H-Ras^V12Y40C or H-Ras^V12E37G proliferated well. 5) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-Ras^V12 or H-Ras^V12T35S compared with control cells. 6) The osteoblasts expressing H-Ras^V12, H-Ras^V12T35S, or active Raf-1 were apoptotic, because most of them were annexin-V^high/PI^{high to low} after 24-h incubation and TUNEL^high/PI^low after 72-h incubation. Thus, we propose that H-Ras signals, especially those followed by Raf-1/MAPK pathway but not by PI-3K, not only reduces ₁-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis via the Fas up-regulation and Bcl-2 down-regulation.

Regeneration is a process common in keeping homeostasis of several tissues and is also essential to bone metabolism designated bone remodeling. During bone remodeling cycle, osteoblasts play a central role not only in bone formation by synthesizing multiple bone matrix proteins but also in bone resorption by regulating osteoclast maturation and activation (1-4). Integrins are a superfamily of cell surface receptors involved in cell-cell and cell-matrix adhesion. Functional osteoblasts, which adhere to matrix via integrins in the "formation" phase, are achieved by combining the ability to create mechanically functional adhesion to matrices or opposing cells and signal-transducing capabilities. Signals from matrices transduced by integrins play critical roles in regulating gene expression, tissue-specific differentiation, and survival of primary osteoblasts and fibroblasts (2, 29, 30). Such functions of osteoblasts during the remodeling cycle are determined by hormones, cytokines, prostaglandins, and growth factors, most of which transduce signals by binding to their cognate G-protein-coupled receptors and/or subsequent small G-proteins-mediated signaling (5-9). Among several small G-proteins, Ras has been characterized as a central molecule for the regulation of signal transduction pathways in various types of cells (10-14). We here observed that H-Ras signals, especially those followed by Raf-1/MAPK pathway but not by PI3K, reduces integrins ₁, ₄, ₅, and ₆ and also inhibits ligand-binding activation epitope of ₁ on the surface of osteoblasts and subsequent ₁-mediated adhesion of osteoblasts to matrix proteins without changing the synthesis of matrix proteins and IL-6. H-Ras/Raf-1 pathway appeared to be involved in osteoblast adhesion to FN, VN, and LM, although it did not mediate the adhesion to COLL-I, a major bone matrix compartment. Although further evidence is required, we suppose that the pathway could function well when osteoblasts encounter with the circumstance in which the ratio of FN, VN, or LM is increased in bone matrix rather than usual COLL-I-enriched matrix.

It was also reported that the expression of an active form of H-Ras and its effector kinase, Raf-1, in CHO cells stably expressing an active chimeric integrin suppressed the function of the chimeric ₆A, ₁, and ₃. The suppression of integrin function correlated with the activation of the Ras/Raf/MAPK kinase pathway (31). In contrast, we reported that H-Ras^V12Y40C mutant, which binds to PI3K in T cells, induce the activated form of leukocyte function-associated antigen (LFA)-1 (_L2) and LFA-1-dependent adhesion to ICAM-1 (intercellular adhesion molecule 1) and that activation of LFA-1 is inhibited by PI3K inhibitors (15). We also found that the expression of active form of H-Ras induces the activation of the ₁ in B cells (16). Accumulating evidence demonstrates that PI3K appears to play a central role in integrin triggering (27, 32, 33). One plausible explanation for such discrepant and complex nature of H-Ras functions can be considered to be that second signals induced by H-Ras may be differently involved in "on and off switch" for integrin triggering. Ras is known to be a hub that radiates multiple signaling pathway including Raf-1/MAPK and PI3K (10). From our findings and others, we propose that H-Ras-sensitive PI3K activation is involved in "on switch" for integrin functions, whereas the H-Ras/Raf-1/MAPK may function as an "off switch" for integrin functions.

We also observed that H-Ras signals, especially those followed by Raf-1/MAPK pathway but not PI3K, inhibits proliferation and induces apoptosis of osteoblasts presumably via the reciprocal regulation of Fas/Bcl-2 expression. Such a regulation of cell survival/apoptosis is an important determinant of the life span of cells in regenerating tissues including bone in which continuous bone remodeling keeps its homeostasis. More than half of the osteoblasts, which initially present at the remodeling sites and complete their bone-forming function, undergo apoptosis, and that the process can be modulated by growth factors and/or cytokines produced in the bone microenvironment and by exogenous administration of glucocorticoids (34, 35). Thus, apoptosis of osteoblasts is a fundamental regulatory event during bone tissue differentiation (36). Fas gene is known to be a target gene of p53 during apoptosis, and the p53 mediates down-regulation of Bcl-2 protein presumably by binding to a cis-acting p53-negative response element located in the 5'-untranslated region of the bcl-2 gene (37). In addition, anti-Fas antibody stimulates apoptosis of human osteoblastic MG-63 cells, and Bcl-2 prevents this change (34). Our observation of increased expression of Fas and reduced expression of Bcl-2 in osteoblasts expressing H-Ras^V12 or H-Ras^V12T35S implies that the cells might be apoptotic at least partially mediated by Fas. Furthermore, the adhesion of osteoblasts to FN is required for the survival of osteoblasts and subsequent bone formation (38). The reduced ₁-mediated adhesion of osteoblasts to matrix proteins such as FN, which is induced by H-Ras/Raf-1/MAPK signals, might further augment apoptotic features of osteoblasts.

The potential importance of the balance between survival and apoptosis of osteoblasts during bone remodeling is well accepted. Taken together, we propose that H-Ras signals, especially those followed by Raf-1/MAPK pathway but not PI3K, not only reduces the expression of functionally active ₁ and ₁-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation, and that such a regulation of cell cycle arrest is an important determinant of the life span of cells in regenerating bone in which continuous remodeling keeps its homeostasis. As described, the functions of osteoblasts during the remodeling cycle are tightly regulated by hormones, cytokines, prostaglandins, and growth factors, most of which transduce small G-proteins-mediated signaling. Thus, the regulation of Ras-mediated signaling might also lead to novel pharmacotherapeutic strategies for osteoporosis and other pathologic conditions in which tissue mass diminution has compromised functional integrity.

	FOOTNOTES

^* This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a grant from the Ministry of Health and Welfare.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 81-93-603-1611, Ext. 2426; Fax: 81-93-691-9334; E-mail: tanaka@med.uoeh-u.ac.jp.

Published, JBC Papers in Press, April 4, 2002, DOI 10.1074/jbc.M202238200

	ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; OC, osteocalcin; COLL-I, type I collagen; ALP, alkaline phosphatase; mAb, monoclonal antibody; IL, interleukin; ERK, extracellular signal-regulated kinase; Ab, antibody; HA, hemagglutinin; FACS, fluorescence-activated cell sorter; ABC, antibody-binding capacity; FN, fibronectin; LM, laminin; VN, vitronectin; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling; LFA, leukocyte function-associated antigen.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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