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βig-h3 mediates osteoblast adhesion and inhibits differentiation
Bone 36 (2005) 232 – 242 www.elsevier.com/locate/bone
hig-h3 mediates osteoblast adhesion and inhibits differentiation Narendra Thapa, Kae-Bok Kang, In-San Kim* Institute of Cell and Matrix Biology Research, Department of Biochemistry, School of Medicine, Kyungpook National University, Daegu 700-422, South Korea Received 12 May 2004; revised 2 August 2004; accepted 12 August 2004
Abstract hig-h3 is an extracellular matrix (ECM) protein induced by TGF-h, and it has motifs interacting with the a3h1, avh5, and avh3 integrins. Our previous study shows the role of hig-h3 in osteoblast differentiation and its involvement in melorheostosis, a rare bone disease. Here we demonstrate that hig-h3 expression is down-regulated during the early stage of differentiation of the murine preosteoblastic cell line, KS483. The recombinant hig-h3 and its FAS1 domain significantly inhibited in vitro osteoblast differentiation as evaluated by matrix mineralization/bone nodule formation. Furthermore, inhibition of expression of osteoblast differentiation marker genes [such as type I collagen, alkaline phosphatase, and osteocalcin (OC)] was accompanied by suppression of osteoblast-specific transcription factors, Cbfa1/ Runx2 and osterix. Flow cytometric analyses, cell adhesion, and inhibition assays disclosed avh3 and avh5 as the principal integrins mediating the adhesion of osteoblastic cells to hig-h3. The disruption of interactions between hig-h3 and osteoblasts by a function-blocking antibody specific for avh3 but not for avh5 abolished the inhibitory effect of hig-h3 on osteoblast differentiation. We suggest that these interacting integrins may play an important role in hig-h3-mediated inhibition of osteoblast differentiation. D 2004 Elsevier Inc. All rights reserved. Keywords: hig-h3; Osteoblast differentiation; avh3/h5 integrin; Bone formation; TGF-h
Introduction The differentiation of osteoblasts in vivo and in vitro during bone formation is primarily characterized by the expression of extracellular matrix (ECM) proteins, principally, type I collagen (Col I) and other noncollagenous proteins such as fibronectin (FN), vitronectin (VN), osteocalcin (OC), osteopontin, and bone sialoprotein [1– 3]. Earlier reports reveal alterations in proliferation and differentiation profiles of osteoblasts cultured on collagen, bone sialoprotein, and osteopontin [4–6]. The interaction of osteoblasts with the surrounding extracellular matrix proteins and the biological signals generated represent essential environmental signals necessary for regulating differentiation [7]. This interaction is mediated primarily through the integrin family [8]. Osteoblasts express a wide array of * Corresponding author. Department of Biochemistry, Kyungpook National University, School of Medicine, 101 Dongin-dong, Jung-gu, Daegu, 700-422, South Korea. Fax: +82 53 422 1466. E-mail address: [email protected] (I.-S. Kim). 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.08.007
integrin heterodimers, both in vivo and in vitro, particularly those interacting with the extracellular matrix proteins of bone [9–13]. A number of investigators report the expression of a broad range of integrin subunits in bone cells, including a1h1, a2h1, a3h1, a5h1, avh3, and avh5 [9,10,14]. Furthermore, osteoblasts represent a good model for the study of interactions between extracellular matrix proteins and cells due to their involvement in secretion and organization of the extracellular matrix [15]. The hig-h3 protein was initially identified by differential screening of a cDNA library produced from A549 human lung adenocarcinoma cells treated with TGF-h1 [16]. The protein consists of 683 amino acids, an amino terminal signal peptide, and 4 internal FAS1 domain repeats [16]. The FAS1 domains are homologous to fasciclin-1 in Drosophila and are characterized by presence of two highly conserved sequences. Many secreted and membrane proteins of several species, including mammals, insects, sea urchins, plants, yeast, and bacteria, contain FAS1 domains [17]. hig-h3 is involved in cell growth, tumorigenesis, wound healing, apoptosis, and migration [16,18–20]. In situ
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hybridization studies in developing bone using a full-length hig-h3 cDNA probe revealed specific hybridization to osteoblasts of primary spongiosa and a significant amount of specific hybridization to newly recruited preosteoblasts in both the periosteum and perichondrium during the early stages of bone formation, but not in mature bone [21]. Previous analyses demonstrated an inhibitory effect of this protein on osteoblast differentiation and its involvement in melorheostosis, a rare bone disease [22]. The melorheostosis is characterized by hyperostosis along the cortex, resembling wax dripping down one side of candle, and periosseous fibrosis of soft tissues. Histologic studies show the endosteal thickening during infancy and childhood. Periosteal new bone formation occurs at adulthood. The present report provides additional information on the molecular mechanism of inhibition, the role of interacting integrins, and the specific binding motif in hig-h3 for osteoblasts. We employed the recombinant hig-h3 protein as a cell adhesion/integrin ligand by coating it onto a culture plate and analyzed its effects on matrix mineralization/bone nodule formation and regulation of expression of osteoblast differentiation marker genes, Col I, ALP, and OC. The expression of osteoblast-specific transcription factors, Cbfa1/Runx2 and osterix, was also analyzed in hig-h3mediated inhibition of osteoblast differentiation. Furthermore, the functional integrin receptors mediating adhesion of osteoblasts to hig-h3 were determined.
Materials and methods Materials The generation of recombinant hig-h3 proteins is described in previous reports [22,23]. The YH synthetic peptide was purchased from AnyGen Co. Ltd. (Kwangju, Korea). Function-blocking monoclonal antibodies specific for integrin subunits a1 (FB12), a2 (P1E6), a3 (P1B5), a4 (P1H4), a5 (P1D6), a6 (GoH3), h1 (6S6), h2 (P4H9), avh3 (LM609), and avh5 (P1F6) were purchased from Chemicon International Inc. (Temecula, CA). The [a-35P]-deoxycytosine triphosphate (dCTP), Megaprime DNA labeling systems, and Hybond-N+ nylon membrane were purchased from Amersham Pharmacia Biotech (UK). Type I collagen and vitronectin were obtained from Chemicon International and Promega, respectively. Fibronectin was prepared from plasma by affinity chromatography. Cell culture The mouse calvarial preosteoblastic cell line, KS483, was cultured at 378C in 8% CO2 in alpha-minimal essential medium (a-MEM) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin G (100 U/ml) and streptomycin (100
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Ag/ml). The mouse preosteoblastic cell line, MC3T3-E1 cells, and mouse osteoblastic cell line, KUSA/A1, cells were also cultured in same medium in 5% CO2. The human osteosarcoma cell line (HOS) was maintained in Dulbecco’s modified Eagle medium (DMEM-high) (Life Technologies, Inc.) supplemented with 10% FBS in the presence of penicillin G (100 U/ml) and streptomycin (100 Ag/ml). Primary human osteoblasts were derived from bone fragments as a by-product of surgery (Kyungpook National University Hospital, Daegu, Republic of Korea) on IRB approval. Bone fragments were thoroughly rinsed three times in phosphate-buffered saline (PBS) and minced into small pieces. Next, fragments were placed into cell culture dishes (Corning, Corning, NY, USA) containing DMEMhigh supplemented with 10% FBS and 50 Ag/ml ascorbic acid (Sigma) in the presence of penicillin G (100 U/ml) and streptomycin (100 Ag/ml). Cultures were maintained in a sterile incubator at 378C and 5% CO2, with medium replacement every 3 days. After 4 weeks, growth of osteoblast-like cells from bone tissues was observed. Tissues were removed, and the medium was replaced with a-MEM supplemented with 10% FBS. Following confluence, cells were trypsinized and replated in fresh medium. Matrix mineralization/bone nodule formation assay ECM proteins, such as Col I, VN, FN, hig-h3, and the fourth FAS1 domain (Do-IV) (50 Ag/ml) were coated onto 24-well tissue culture plates (Corning Glass Works) and incubated overnight at 48C [6,22]. Plates were washed twice with PBS, and nonspecific binding sites were blocked with heat-inactivated 2% BSA for 1 h at room temperature. Similarly, 100-mm culture plates were coated with 50 Ag/ml hig-h3 protein. A trypsinized suspension of KS483 cells was seeded at concentration of 2 104 cells per plate after washing twice with PBS. Cells reached confluence after 2 days. Osteogenic medium containing a-MEM supplemented with 10% FBS, 50 Ag/ml ascorbic acid, 5 mM hglycerophosphate, and 10 nM dexamethasone was supplied and used to replace the medium every 3 days for 21 days. Matrix mineralization/bone nodule formation was assessed using an alizarin red-S histochemical stain (Sigma) as described previously [22]. Similarly, matrix mineralization/bone nodule formation of MC3T3-E1 cells and KUSA/A1 cells were evaluated after 21 and 12 days of culture, respectively, in osteogenic medium containing a-MEM supplemented with 10% FBS, 50 Ag/ml ascorbic acid, and 5 mM h-glycerophosphate. MTT test Proliferation and viability of KS483 cells in hig-h3 protein-coated culture plate were assessed by MTT test [24]. Briefly, cells were seeded in the concentration of 1.5 103
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cells per well in 100 Al of serum-free medium onto BSA, FN, hig-h3 protein, and Do-IV-coated 96-well culture plates (20 Ag/ml). Then, 50 Al of thiazolyl blue (MTT) (Sigma) solution in final concentration of 0.5 mg/ml was added in the culture plate and incubated for 4 h at 378C. The colored product was solubilized in 150 Al of dimethyl sulfoxide (DMSO), and absorbance was quantified at 570 nm in a Bio-Rad model 550 microplate reader. The test was performed in the interval of 24 h for 5 days. Cell adhesion assay The cell adhesion assay was performed as described previously [23]. Briefly, flat-bottomed 96-well enzymelinked immunosorbent assay (ELISA) plates (Costar, Corning Inc.) were incubated overnight at 48C with 10 Ag/ml recombinant hig-h3 protein or other extracellular matrix (ECM) proteins in PBS and blocked for 1 h at room temperature with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). Cells were suspended in medium at a density of 3 105 cells/ml, and 0.1 ml of the cell suspension was added to each well of the coated plates. Following incubation for 30 min at 378C, unattached cells were removed by rinsing once with PBS. Attached cells were incubated for 1 h at 378C in 50 mM citrate buffer, pH 5.0, containing 3.75 mM pnitrophenyl-N-acetyl h-d-glycosaminide and 0.25% Triton X-100. Enzyme activity was blocked by adding 50 mM glycine buffer (pH 10.4) containing 5 mM EDTA, and absorbance was measured at 405 nm in a Bio-Rad model 550 microplate reader. Inhibition assay To identify the receptor for hig-h3, monoclonal antibodies specific for different integrin types (5 Ag/ml) (Chemicon, International Inc.) were preincubated at 378C for 30 min with primary human osteoblasts in a 0.1 ml cell suspension (3 105 cells/ml). Cells were transferred onto plates precoated with recombinant hig-h3 proteins and incubated further for 30 min at 378C. Attached cells were quantified as described above. Function-blocking monoclonal antibodies specific for the following integrin subunits were employed: a1 (FB12), a2 (P1E6), a3 (P1B5), a4 (P1H4), a5 (P1D6), a6 (GoH3), av (P3G8), h1 (6S6), h2 (P4H9), avh3 (LM609), and avh5 (P1F6). Flow cytometric analyses To confirm the expression of avh3 and avh5 integrins on osteoblast cell surface, fluorescence-activated cell sorter analysis was performed. Human primary osteoblasts and osteosarcoma cells grown to confluency were detached from plates by treatment with 0.25% trypsin-0.05% EDTA. After washing twice in PBS, cells were suspended in PBS and incubated for 1 h at 48C with antibodies specific for a3
(ASC-1), av (P3G8), avh3 (LM609), and avh5 (P1F6). Cells were incubated for 1 h at 48C with 10 Ag/ml secondary goat-antimouse IgG conjugated to FITC (Santa Cruz Biotechnology, Inc., CA) and analyzed at 488 nm on the flow cytometer FACSCalibur system (Becton Dickinson, San Jose, CA) equipped with a 5-W argon laser. Total RNA isolation and Northern blot analysis Total RNA was isolated from cells using guanidinium isothiocyanate solution [25], resolved on a 1% formaldehyde-agarose gel (10 Ag) and transferred to a Nylon membrane (Sigma) by the capillary method using 20 SSC buffer (sodium chloride sodium citrate, pH 7.0). The RNA was cross-linked to membranes by UV irradiation using UV StratalinkerTM 1800 (Stratagene). The DNA probes used included mouse hig-h3, Col I, OC, ALP, Cbfa1/ Runx2, and osterix. The cDNA probes for OC, ALP, Cbfa1/ Runx2, and osterix were generous gifts of Dr. Ryoo (Department of Biochemistry, School of Dentistry, KNU, Taegu, South Korea). All probes were labeled with 30 ACi [a-32P] dCTP using the random priming Megaprime DNA labeling Kit (Amersham Pharmacia Biotech, UK Ltd.). Hybridization was performed at 688C for 1 h to overnight using ExpressHybTM (Clontech). Blots were washed in high salt solution containing 0.05% SDS and 2 SSC at 308C, followed by low salt solution containing 0.1% SDS in 0.1 SSC for different time periods, depending on the type of probe used. Blots were exposed to X-ray film (AGFA) overnight at 808C. To study the effect of interrupting the interaction of high3 protein with its integrin receptor in HOS cells, we treated the trypsinized cell suspension with function-blocking monoclonal antibody for avh3 and avh5 (Chemicon) at concentration of 1.4 Ag/ml before seeding into the hig-h3 protein-coated culture plate in complete growth medium. The RNA was isolated after 3 days for analysis of type I collagen gene expression. Binding assay of big-h3 A binding assay was performed as described previously [26]. Briefly, primary human osteoblasts were suspended in medium at a density of 1 105 cells/ml, and 1 ml of the cell suspension was preincubated with anti-avh3 (LM609) or avh5 (P1F6) for 30 min at 378C. The cells were incubated with biotinylated hig-h3 in serum-free medium for 5 h at 48C. The cells were washed three times with PBS before lysis. Equal amounts of proteins were separated on 10% SDS–PAGE gel, transferred to membrane, and incubated with streptavidin conjugated to horseradish peroxidase (Amersham Biosciences). The membrane was developed by using enhanced chemiluminescence (ECL; Amersham Biosciences). For internal control, membrane was stripped and reprobed with beta-tubulin (Santa Cruz Biotechnology).
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Cell transfection KS483 cells were transfected with human h3 integrin subunit in pcDNA 3.1(+) (kind gift of Dr. Jaffrey Smith, Burnham Institute, San Diego, USA) by using lipofecAMINE plusk reagent (Life Technologies Inc.) according to manufacturer’s protocol. The transfected cells were selected in a-MEM medium with 10% FBS in presence of 500 Ag/ml G418 (Life Technologies Inc.) for 2 weeks. The stably transfected clones overexpressing h3 were verified by Western blot analysis by using antihuman h3 antibody (Chemicon International). Statistical analysis The values were expressed as the mean F SD. A oneway analysis of variance and Dunnett t test were used to determine the statistical significance. Differences were considered significant at P b 0.05.
Results big-h3 gene expression is down-regulated during osteoblast differentiation To determine the expression level of hig-h3 during in vitro differentiation of osteoblasts, total RNA was extracted at indicated time points from KS483 cells cultured in osteogenic medium (Fig. 1A). Northern blot analyses revealed down-regulation of hig-h3 expression during differentiation of KS483 cells. Gene expression of hig-h3 was the highest at an early stage of proliferation and dramatically decreased in subsequent days in this study. This finding indicates that the down-regulation of hig-h3 gene expression is important for osteoblast differentiation. big-h3 protein inhibits matrix mineralization/bone nodule formation Next, we determined the role of hig-h3 in matrix mineralization/bone nodule formation during osteoblast differentiation, using alizarin red-S histochemical staining. Cells were cultured for 21 days on 24-well plates coated with hig-h3, the fourth FAS1 domain of hig-h3 (Do-IV), or various ECM proteins, such as FN, VN, and Col I. Decreased mineralization/bone nodule formation by high3 protein and Do-IV (Fig. 1B) was observed, compared to the control (noncoated). The inhibitory effect of hig-h3 protein was specific, in view of the finding that the other ECM proteins examined (except FN) displayed a similar or higher extent of bone nodule formation/matrix mineralization, compared to the control (Fig. 1B). Similarly, inhibitory effect of hig-h3 in matrix mineralization/bone nodule formation was also observed in MC3T3-E1 and KUSA/A1 cells (Fig. 1C). In our MTT assay, hig-h3 and Do-IV
Fig. 1. hig-h3 expression in KS483 cells and its effect on matrix mineralization/bone nodule formation. (A) KS483 cells were seeded in 100 mm culture plates at a concentration of 6 105 cells per plate and incubated in osteogenic media for indicated time periods (in days). Total RNA was extracted, and Northern blotting was performed using a specific probe for mouse hig-h3 mRNA. The consistency of RNA loading was assessed by reprobing the membrane with 18S rRNA. (B) KS483 cells were cultured in 24-well plates coated with recombinant hig-h3 protein, Do-IV, or other ECM proteins, such as plasma FN, VN, and Col I, for 21 days in osteogenic medium. Matrix mineralization/bone nodule formation was evaluated by alizarin red-S staining (macroscopic and microscopic view, 100). (C) Alizarin red-S staining of MC3T3-E1 and KUSA/A1 cells cultured in hig-h3-coated plates for 21 and 12 days, respectively, in osteogenic medium. (D) KS483 cells were seeded in 96-well plates coated with BSA, FN, hig-h3, and Do-IV in serum-free medium. The absorbance was taken after addition of MTT solution as described in Materials and methods. The values are the means F SD of triplicate determination. bP b 0.01 vs. BSA.
supported the proliferation and viability of KS483 cells indicating that inhibitory effect of hig-h3 is not due to negative effect on proliferation and viability of KS483 cells (Fig. 1D).
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big-h3 inhibits the expression of osteoblast differentiation marker genes and osteoblast-specific transcription factors The hig-h3 protein inhibited the expression of osteoblast differentiation marker genes, ALP and OC. Inhibition of ALP expression was evident at an early time point (Fig. 2A) and did recover with time of culture in our study. The OC gene, a late-stage marker of osteoblast differentiation, was also distinctly inhibited in hig-h3-coated plates (Fig. 2A). Interactions between collagen and integrin are indispensable for osteoblastic differentiation, and type I collagen is a very
early marker of osteoblast differentiation. Northern blot analyses revealed significant suppression of Col I expression by hig-h3 (Fig. 2A). We examined whether hig-h3 protein affects the expression of osteoblast-specific transcription factors. Coated recombinant hig-h3 protein down-regulated expression of the runt domain-containing transcription factor, Cbfa1/ Runx2 (Fig. 2B), and the recently identified transcription factor, osterix (Fig. 2C). The mRNA levels of both Cbfa1/ Runx2 and osterix in KS483 cells cultured on hig-h3-coated culture plates were clearly down-regulated over different time points studied, compared to the control. Additionally, we found an inhibited expression level of Col I and OC in MC3T3-E1 cells and KUSA/A1 cells (Fig. 2D) cultured in hig-h3-coated plates. big-h3 protein and its FAS1 domains support the adhesion of primary human osteoblasts and KS483 cells The hig-h3 protein and all four FAS1 domains (I–IV) support cell adhesion of both KS483 cells (Fig. 3B) and primary human osteoblasts (Fig. 3C). Each domain displayed comparable cell adhesion activity to FN or hig-h3 protein in both cell types, indicating that the FAS1 domain contributes to cell adhesion to osteoblasts. Cell adhesion activity of KS483 cells was increased to some extent at increasing concentrations of hig-h3 and all four FAS1 domains (Fig. 3D). Integrins avb3 and avb5 are involved in mediating the adhesion of osteoblasts to big-h3 protein
Fig. 2. Inhibition of expression of osteoblast differentiation marker genes and osteoblast-specific transcription factors by hig-h3 protein. KS483 cells were seeded at a concentration of 6 105 cells per plate in 100 mm plates either coated with hig-h3 protein (+) or left uncoated ( ), and cultured for the indicated time periods (days) in osteogenic medium. Total RNA was isolated, and Northern blotting was performed using specific probes. (A) Col I, ALP, and OC; (B) Cbfa1/Runx2; (C) osterix. (D) Total RNA was extracted from MC3T3-E1 and KUSA/A1 cells cultured in osteogenic medium for 14 and 7 days, respectively, in plates either coated with hig-h3 protein (+) or left uncoated ( ). Northern blotting was performed for Col I and OC expression. The consistency of RNA loading was assessed by reprobing the membrane with 18S rRNA.
To identify the specific integrin(s) mediating adhesion of osteoblasts to hig-h3, we performed an inhibition assay using function-blocking monoclonal antibodies (Chemicon). The adhesion of primary human osteoblasts to hig-h3 was specifically inhibited with antibodies against avh3 and avh5 integrins (Fig. 4A). In contrast, no significant effects were observed with other anti-integrin antibodies, including a1, a2, a3, a4, and h1. This finding suggests that both avh3 and avh5 are functional receptors in mediating the adhesion of osteoblasts to hig-h3 protein. Additionally, adhesion of osteoblasts to vitronectin is mediated by avh5 (Fig. 4B). To confirm the expression of these integrin subunits in osteoblasts, FACS analyses were performed in primary human osteoblasts. The similar level of avh3 and avh5 integrins expression was observed on osteoblasts. The expression of av and a3 integrins was also prominent. However, we did not perform the FACS analysis for other integrins examined in inhibition study. To investigate the interaction between hig-h3 and integrins found in osteoblasts, we studied the binding of biotinylated hig-h3 to osteoblasts cell surface. The hig-h3 binds in a dose-dependent manner (Fig. 5A), and this binding was inhibited by antibodies to either avh3 or avh5 integrins, but not by control IgG (Fig. 5B). This result
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reveals the interaction of hig-h3 and avh3/avh5 integrins found on cell surface of osteoblasts. The synthetic YH peptide inhibits the adhesion of osteoblasts to big-h3 protein Our group has recently identified a YH motif in the FAS1 domain of hig-h3 protein that mediates adhesion of endothelial cells via the avh3 integrin [26] and a
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fibroblastic cell line, MRC-5, via the avh5 integrin [27]. Since the YH motif interacts with both the avh3 and avh5, we hypothesize that this sequence in hig-h3 protein provides a potential interacting site for avh3 and avh5 integrins expressed in osteoblasts. A YH18 synthetic peptide corresponding to amino acids 563–580 of hig-h3 significantly inhibited cell adhesion of both KS483 cells and primary human osteoblasts to hig-h3 protein, as shown in Figs. 6A and B, respectively. A YH18 control sequence containing all 18 amino acids of the YH18 peptide but randomly ordered did not affect cell adhesion activity. avb3 integrin plays a role in big-h3-mediated inhibition of osteoblast differentiation The inhibitory effect of integrin avh3 in osteoblast differentiation has been reported [28]. The bone nodule formation and matrix mineralization of KS483 cells overepressing human h3 integrin subunit were dramatically reduced in compared with mock-transfected cells (Fig. 7A). To elucidate the role of interacting integrins in hig-h3mediated inhibition of osteoblast differentiation, we employed a function-blocking monoclonal antibody specific for avh3 and avh5 integrins to disrupt the interactions between osteoblasts and hig-h3 protein. We employed HOS cells for this study. Our FACS analyses additionally reveal expression of these integrins in HOS cells (Fig. 7 B). The adhesion of HOS cells to hig-h3 was found more dependent on avh3 integrin than on avh5 integrin (Fig. 7 C). Treatment with a function-blocking antibody specific for avh3 abolished the inhibitory effect of hig-h3 protein, as evaluated by the expression of type I collagen (Fig. 7D). The function-blocking monoclonal antibody specific for avh5 did not significantly affect the inhibitory effect of high3 protein.
Discussion We previously demonstrated an inhibitory effect of hig-h3 protein on osteoblast differentiation [22]. In the present study, Fig. 3. The hig-h3 protein and all its FAS1 domains support the adhesion of KS483 cells and primary human osteoblasts. (A) Diagram of hig-h3 protein and position of four FAS1 domains (Do-I, residues 134–236; Do-II, residues 242–372; Do-III, residues 373–501; Do-IV, residues 502–632). The hatched and cross-hatched boxes indicate the highly conserved sequences in each domain. For adhesion assay, a 96well plate was coated with protein overnight at 48C. After blocking with 2% BSA and PBS washing, 2 104 cells per well in 100 Al complete media were seeded and incubated at 378C for 40 min. After incubation, unattached cells were washed with PBS, and attached cells were quantified using a hexosaminidase assay, as described in Materials and methods. Plasma FN (10 Ag/ml) and 2% BSA were employed as the positive and negative controls, respectively. (B) KS483 cells; (C) primary human osteoblasts; (D) dose-dependent cell adhesion of KS483 cells to high3 protein and its FAS1 domains. The values are the means F SD of triplicate determinations. aP b 0.001 versus BSA.
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we explain the molecular mechanism of action and identify the interacting integrin receptor in hig-h3-mediated inhibition of osteoblast differentiation. Osteoblast differentiation is mediated by a complex series of cell–cell and cell–matrix interactions. The process represents a typical model for analyzing interactions between cells and ECM proteins, since osteoblasts are specifically involved in secretion and organization of the ECM during bone formation. The studies have identified hig-h3 as a bone matrix protein that plays an important role in osteogenesis [21,22]. The expression of
hig-h3 in vivo is abundant in preosteoblasts during early bone formation [21]. Our study additionally revealed high expression of hig-h3 at the early stage of in vitro differentiation of KS483 cells, which was followed by a significant decrease. The regulated expression level of hig-h3 was noted during in vitro differentiation of pluripotent human bone marrow stromal cells [21]. We have also found a dramatic change in expression level of hig-h3 gene in chondrocyte differentiation of ATDC5 cell line (data not shown). We employed recombinant hig-h3 as a cell adhesion substrate/integrin ligand with a view to elucidating the function of the protein. Coated ECM proteins provide natural ligands for adhesion and activation of specific integrin receptors present on the cell surface of osteoblasts [24]. Proliferation and differentiation profiles of osteoblasts are influenced by ECM proteins coated onto culture plates, such as collagen, osteopontin, and bone sialoprotein [4–6]. Since KS483 cells form a hydroxyapatitecontaining mineralized matrix in vitro when grown in osteogenic conditions [29], we assessed the matrix mineralization/bone nodule formation of these cells cultured on high3, Do-IV, and other ECM proteins, including vitronectin, type I collagen, and fibronectin. hig-h3 and Do-IV significantly inhibited bone nodule formation, analogous to our previous report [22]. The extent of matrix mineralization/ bone nodule formation of KS483 cells on other ECM matrix protein-coated wells was similar or increased, compared to the control (noncoated). Indeed, matrix mineralization was more enhanced in the well coated with type I collagen, consistent with earlier results [30]. Fibronectin, which is expressed only at early stages of osteogenesis [2], also inhibited matrix mineralization/bone nodule formation to a small extent, in agreement with our previous data [22]. Furthermore, inhibition of matrix mineralization/bone nodule formation by hig-h3 was accompanied by suppression of expression of osteoblast differentiation marker genes, Col I, ALP, and OC. Interactions between osteoblasts and type I collagen are critical for differentiation [31], suggesting that inhibition of osteoblast differentiation by hig-h3 is mediated to some extent by down-regulating the expression of other
Fig. 4. avh3 and avh5 are functional receptors mediating the adhesion of primary human osteoblasts to hig-h3 protein. (A) Primary human osteoblasts were preincubated with different function-blocking monoclonal antibodies specific for integrins (final concentration of 5 Ag/ml) for 30 min at 378C. A Cell adhesion assay was performed using 10 Am/ml hig-h3-WT coated in 96-well culture plates, as described in Materials and methods. (B) Identification of integrins mediating the adhesion of osteoblasts to vitronectin. Primary human osteoblasts were preincubated with function-blocking monoclonal antibodies to integrin avh3 (LM609), avh5 (P1F6), and av (P3G8). Cell adhesion to vitronectincoated plates (5 Ag/ml) was determined. (C) Flow cytometric analysis of avh3 and avh5 integrins on the cell surface of primary human osteoblasts. Cells were stained with saturating concentrations of antibodies specific for a3 (ASC-1), av (P3G8), avh3 (LM609), and avh5 (P1F6). Data are expressed as cell number (y-axis) plotted as a function of fluorescence intensity (x-axis) and are representative of three separate experiments. Negative control cells were incubated with the secondary antibody alone. c P b 0.05, bP b 0.01, aP b 0.001.
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Fig. 5. Binding of biotinylated hig-h3 to osteoblasts. (A) Dose-dependent binding of biotinylated hig-h3 to osteoblasts cell surface. The primary human osteoblasts were incubated with biotinylated hig-h3, and amount of biotinylated hig-h3 associated with osteoblasts was determined by Western immunoblotting with the streptavidin-conjugated horseradish peroxidase. (B) Function-blocking antibody to either avh3 or avh5 integrins inhibits the binding of biotinylated hig-h3 to osteoblasts. The primary human osteoblasts were preincubated with 5 Ag of anti-avh3 (LM609) or antiavh5 (P1F6) or control IgG followed by incubation with biotinylated high3 in serum-free medium for 5 h at 48C. The amount of biotinylated hig-h3 associated with osteoblasts was determined by Western immunoblotting with the streptavidin-conjugated horseradish peroxidase as described in Materials and methods.
ECM proteins and subsequently altering integrin-ECM protein interactions and cell signaling. The result of MTT assay suggests that hig-h3 does not have negative effect on cell proliferation and viability, and thus the inhibitory effect of hig-h3 on matrix mineralization/bone nodule formation is not likely due to inhibition of cell growth. The inhibitory effect of Do-IV was found more pronounced than that of high3, and it may be due to efficient coating and stability of DoIV compared with hig-h3 in long-term culture condition. Interactions between a particular ECM ligands and specific integrin heterodimers influence osteoblast function via regulation of transcription factors [32]. The expression of osteoblast-specific transcription factors, core binding factor/runt-related gene 2 (Cbfa1/Runx2), and zinc fingercontaining novel transcription factor, osterix, was evaluated in KS483 cells cultured on a hig-h3 protein-coated plate. These transcription factors are known to play an important role in osteoblast differentiation [33,34]. Interestingly, high3 suppressed the expression of both Cbfa1/Runx2 and osterix, indicating a role of these osteoblast-specific transcription factors in hig-h3-mediated inhibition of osteoblast differentiation. We propose that the interaction of osteoblasts with high3 through integrins triggers signals that inhibit osteoblast differentiation. The hig-h3 protein and all four FAS1 domains displayed comparable cell adhesion activity, indicating the existence of an integrin-interacting motif in each domain. The antibody inhibition assay revealed that avh3 and avh5 are the principal integrin heterodimers that mediate adhesion of osteoblasts to hig-h3. Additionally,
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binding of hig-h3 to osteoblasts is mediated by these two integrins. Our previous studies have shown that avh3 and avh5 integrins mediate the interaction of human umbilical vein endothelial cells (HUVEC) and fibroblastic cell line, MRC-5 cells, to hig-h3, respectively [26,27]. Data from our FACS analyses of the expression of avh3 and avh5 integrins on the cell surface of primary human osteoblasts are consistent with other studies [10,11,13,14]. We have identified highly conserved amino acids (aspartic acid and isoleucine) in the second and fourth FAS1 domains of high3 as the binding motif for a3h1 integrin in human corneal epithelial cells [23], and a YH motif for avh3 integrin in endothelial cells [26] and avh5 in the fibroblastic MRC-5 cell line [27]. We suggest that YH is also the binding motif in the case of osteoblasts, since it interacts with both avh5 and avh3 integrins. The synthetic YH18 peptide containing
Fig. 6. The YH motif is responsible for cell adhesion of hig-h3 to osteoblasts. The amino acid sequence of the synthetic YH18 peptide corresponding to hig-h3 is specified. Before the cell adhesion assay, trypsinized suspensions of KS483 cells (A) and primary human osteoblasts (B) were preincubated with 100 AM synthetic and control YH peptides for 15 min, and added to plates. The number of adhering cells was quantified, as explained in Materials and methods. The values are the means F SD of triplicate determinations. bP b 0.01.
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the 18 amino acids of hig-h3 (positions 563 to 580) almost completely inhibited both primary human osteoblasts and KS483 cells adhesion to hig-h3. In contrast, the scrambled form of this peptide had no effect. This finding implies that the YH motif is a potential binding site for osteoblasts in hig-h3 protein.
Integrin-mediated interactions of cells with ECM proteins and the cell signals generated have been implicated in a diverse range of biological activities, including cell differentiation [35]. The avh3 integrin negatively regulates osteoblast differentiation [28] and is expressed in the inactive form in human osteoblasts [11]. Studies on oligodendrocytes and skeletal muscle cells additionally confirm the inhibitory role of this integrin in cell differentiation [36,37]. The avh3 and avh5 integrins are involved in mediating adhesion of osteoblasts to hig-h3. Accordingly, we hypothesize that avh3 plays a role in the inhibition of osteoblast differentiation by hig-h3. We could not use KS483 cells due to lack of function-blocking monoclonal antibody specific for mouse avh3 and avh5 integrins. The poor osteogenic potentiality of our primary human osteoblasts optioned us to use HOS cells. The FACS analyses reveal expression of these integrins in HOS cells. We analyzed the expression of type I collagen in HOS cells cultured on hig-h3 protein-coated plates in the presence of function-blocking antibodies for avh3 and avh5 integrins, with the aim of disrupting the interactions between HOS cells and hig-h3 protein. Treatment with a function-blocking anti-integrin antibody specific for avh3 abolished the inhibitory effect of hig-h3, supporting the theory that activation of avh3 and subsequent cell signaling in high3 protein mediates the inhibition of osteoblast differentiation. Col I is one of the differentiation marker genes of osteoblasts [34]. We could not examine the effect on matrix mineralization and other marker genes of osteoblasts differentiation because the HOS cells used in this study could not differentiate faithfully and were detached from culture plate once cells became confluent. We found avh5, rather than the avh3, integrin-mediated interactions of osteoblasts with coated vitronectin, and this result is
Fig. 7. avh3 integrin plays a role in hig-h3-mediated inhibition of osteoblast differentiation. (A) Western blot analysis and alizarin red-S staining of KS483 cells stably overexpressing human h3 integrin subunit. The cell lysate was prepared and subjected to 10% SDS–PAGE gel. The membrane was incubated with anti-h3 antibody for overnight at 48C. The binding of antibody was detected by ECLk Western Blotting Reagents (Amersham Biosciences). The human h3 chain of approximately 95 kDa is shown by an arrow. The alizarin red-S staining was performed after culturing the mock and h3-overexpressing KS483 cells in osteogenic medium for 21 days. (B) Flow cytometric analysis of HOS cells. All antibodies used and procedures were the same as described above for primary human osteoblasts. (C) Identification of integrins mediating the adhesion of HOS cells to hig-h3. HOS cells were preincubated with different function-blocking monoclonal antibodies specific for integrins (final concentration of 5 Ag/ml) for 30 min at 378C. A cell adhesion assay was performed using 10 Ag/ml hig-h3-WT coated in 96-well culture plates, as described in Materials and methods. The values are the means F SD of triplicate determinations. aP b 0.001. (D) The trypsinized cell suspensions of HOS cells either treated with 1.4 Ag/ml function-blocking antibody specific for avh3 (anti-avh3) and avh5 (anti-avh5) integrins or left untreated were seeded into hig-h3 protein-coated (+) and uncoated ( ) culture plates. RNA was isolated after 3 days, and Northern blotting was performed for human Col I. The consistency of RNA loading was evaluated by reprobing the membrane with 18S rRNA.
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consistent with that of Cheng et al. [38]. This may provide indirect evidence why coated vitronectin does not have an inhibitory effect on osteoblast differentiation. Accordingly, we suggest that avh3 integrin plays a role in inhibition of osteoblast differentiation by hig-h3. In conclusion, hig-h3 expression is down-regulated during differentiation of osteoblasts. Moreover, the protein inhibits osteoblast differentiation, and avh3/avh5 integrins mediate the interaction of osteoblasts to hig-h3. Further studies are required to clarify the roles of this newly identified bone matrix protein and the relevant integrin heterodimers involved in early bone formation, osteoblast differentiation, and pathogenesis of bone diseases.
[13]
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[17]
Acknowledgments We thank Dr. Ryoo (Department of Biochemistry, School of Dentistry, KNU, Daegu, South Korea) for providing cDNA probes for Northern blotting. This work was supported by a program of the National Research Laboratory (M1010400036-01J0000-01610).
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hig-h3 mediates osteoblast adhesion and inhibits differentiation Narendra Thapa, Kae-Bok Kang, In-San Kim* Institute of Cell and Matrix Biology Research, Department of Biochemistry, School of Medicine, Kyungpook National University, Daegu 700-422, South Korea Received 12 May 2004; revised 2 August 2004; accepted 12 August 2004
Abstract hig-h3 is an extracellular matrix (ECM) protein induced by TGF-h, and it has motifs interacting with the a3h1, avh5, and avh3 integrins. Our previous study shows the role of hig-h3 in osteoblast differentiation and its involvement in melorheostosis, a rare bone disease. Here we demonstrate that hig-h3 expression is down-regulated during the early stage of differentiation of the murine preosteoblastic cell line, KS483. The recombinant hig-h3 and its FAS1 domain significantly inhibited in vitro osteoblast differentiation as evaluated by matrix mineralization/bone nodule formation. Furthermore, inhibition of expression of osteoblast differentiation marker genes [such as type I collagen, alkaline phosphatase, and osteocalcin (OC)] was accompanied by suppression of osteoblast-specific transcription factors, Cbfa1/ Runx2 and osterix. Flow cytometric analyses, cell adhesion, and inhibition assays disclosed avh3 and avh5 as the principal integrins mediating the adhesion of osteoblastic cells to hig-h3. The disruption of interactions between hig-h3 and osteoblasts by a function-blocking antibody specific for avh3 but not for avh5 abolished the inhibitory effect of hig-h3 on osteoblast differentiation. We suggest that these interacting integrins may play an important role in hig-h3-mediated inhibition of osteoblast differentiation. D 2004 Elsevier Inc. All rights reserved. Keywords: hig-h3; Osteoblast differentiation; avh3/h5 integrin; Bone formation; TGF-h
Introduction The differentiation of osteoblasts in vivo and in vitro during bone formation is primarily characterized by the expression of extracellular matrix (ECM) proteins, principally, type I collagen (Col I) and other noncollagenous proteins such as fibronectin (FN), vitronectin (VN), osteocalcin (OC), osteopontin, and bone sialoprotein [1– 3]. Earlier reports reveal alterations in proliferation and differentiation profiles of osteoblasts cultured on collagen, bone sialoprotein, and osteopontin [4–6]. The interaction of osteoblasts with the surrounding extracellular matrix proteins and the biological signals generated represent essential environmental signals necessary for regulating differentiation [7]. This interaction is mediated primarily through the integrin family [8]. Osteoblasts express a wide array of * Corresponding author. Department of Biochemistry, Kyungpook National University, School of Medicine, 101 Dongin-dong, Jung-gu, Daegu, 700-422, South Korea. Fax: +82 53 422 1466. E-mail address: [email protected] (I.-S. Kim). 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.08.007
integrin heterodimers, both in vivo and in vitro, particularly those interacting with the extracellular matrix proteins of bone [9–13]. A number of investigators report the expression of a broad range of integrin subunits in bone cells, including a1h1, a2h1, a3h1, a5h1, avh3, and avh5 [9,10,14]. Furthermore, osteoblasts represent a good model for the study of interactions between extracellular matrix proteins and cells due to their involvement in secretion and organization of the extracellular matrix [15]. The hig-h3 protein was initially identified by differential screening of a cDNA library produced from A549 human lung adenocarcinoma cells treated with TGF-h1 [16]. The protein consists of 683 amino acids, an amino terminal signal peptide, and 4 internal FAS1 domain repeats [16]. The FAS1 domains are homologous to fasciclin-1 in Drosophila and are characterized by presence of two highly conserved sequences. Many secreted and membrane proteins of several species, including mammals, insects, sea urchins, plants, yeast, and bacteria, contain FAS1 domains [17]. hig-h3 is involved in cell growth, tumorigenesis, wound healing, apoptosis, and migration [16,18–20]. In situ
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hybridization studies in developing bone using a full-length hig-h3 cDNA probe revealed specific hybridization to osteoblasts of primary spongiosa and a significant amount of specific hybridization to newly recruited preosteoblasts in both the periosteum and perichondrium during the early stages of bone formation, but not in mature bone [21]. Previous analyses demonstrated an inhibitory effect of this protein on osteoblast differentiation and its involvement in melorheostosis, a rare bone disease [22]. The melorheostosis is characterized by hyperostosis along the cortex, resembling wax dripping down one side of candle, and periosseous fibrosis of soft tissues. Histologic studies show the endosteal thickening during infancy and childhood. Periosteal new bone formation occurs at adulthood. The present report provides additional information on the molecular mechanism of inhibition, the role of interacting integrins, and the specific binding motif in hig-h3 for osteoblasts. We employed the recombinant hig-h3 protein as a cell adhesion/integrin ligand by coating it onto a culture plate and analyzed its effects on matrix mineralization/bone nodule formation and regulation of expression of osteoblast differentiation marker genes, Col I, ALP, and OC. The expression of osteoblast-specific transcription factors, Cbfa1/Runx2 and osterix, was also analyzed in hig-h3mediated inhibition of osteoblast differentiation. Furthermore, the functional integrin receptors mediating adhesion of osteoblasts to hig-h3 were determined.
Materials and methods Materials The generation of recombinant hig-h3 proteins is described in previous reports [22,23]. The YH synthetic peptide was purchased from AnyGen Co. Ltd. (Kwangju, Korea). Function-blocking monoclonal antibodies specific for integrin subunits a1 (FB12), a2 (P1E6), a3 (P1B5), a4 (P1H4), a5 (P1D6), a6 (GoH3), h1 (6S6), h2 (P4H9), avh3 (LM609), and avh5 (P1F6) were purchased from Chemicon International Inc. (Temecula, CA). The [a-35P]-deoxycytosine triphosphate (dCTP), Megaprime DNA labeling systems, and Hybond-N+ nylon membrane were purchased from Amersham Pharmacia Biotech (UK). Type I collagen and vitronectin were obtained from Chemicon International and Promega, respectively. Fibronectin was prepared from plasma by affinity chromatography. Cell culture The mouse calvarial preosteoblastic cell line, KS483, was cultured at 378C in 8% CO2 in alpha-minimal essential medium (a-MEM) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin G (100 U/ml) and streptomycin (100
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Ag/ml). The mouse preosteoblastic cell line, MC3T3-E1 cells, and mouse osteoblastic cell line, KUSA/A1, cells were also cultured in same medium in 5% CO2. The human osteosarcoma cell line (HOS) was maintained in Dulbecco’s modified Eagle medium (DMEM-high) (Life Technologies, Inc.) supplemented with 10% FBS in the presence of penicillin G (100 U/ml) and streptomycin (100 Ag/ml). Primary human osteoblasts were derived from bone fragments as a by-product of surgery (Kyungpook National University Hospital, Daegu, Republic of Korea) on IRB approval. Bone fragments were thoroughly rinsed three times in phosphate-buffered saline (PBS) and minced into small pieces. Next, fragments were placed into cell culture dishes (Corning, Corning, NY, USA) containing DMEMhigh supplemented with 10% FBS and 50 Ag/ml ascorbic acid (Sigma) in the presence of penicillin G (100 U/ml) and streptomycin (100 Ag/ml). Cultures were maintained in a sterile incubator at 378C and 5% CO2, with medium replacement every 3 days. After 4 weeks, growth of osteoblast-like cells from bone tissues was observed. Tissues were removed, and the medium was replaced with a-MEM supplemented with 10% FBS. Following confluence, cells were trypsinized and replated in fresh medium. Matrix mineralization/bone nodule formation assay ECM proteins, such as Col I, VN, FN, hig-h3, and the fourth FAS1 domain (Do-IV) (50 Ag/ml) were coated onto 24-well tissue culture plates (Corning Glass Works) and incubated overnight at 48C [6,22]. Plates were washed twice with PBS, and nonspecific binding sites were blocked with heat-inactivated 2% BSA for 1 h at room temperature. Similarly, 100-mm culture plates were coated with 50 Ag/ml hig-h3 protein. A trypsinized suspension of KS483 cells was seeded at concentration of 2 104 cells per plate after washing twice with PBS. Cells reached confluence after 2 days. Osteogenic medium containing a-MEM supplemented with 10% FBS, 50 Ag/ml ascorbic acid, 5 mM hglycerophosphate, and 10 nM dexamethasone was supplied and used to replace the medium every 3 days for 21 days. Matrix mineralization/bone nodule formation was assessed using an alizarin red-S histochemical stain (Sigma) as described previously [22]. Similarly, matrix mineralization/bone nodule formation of MC3T3-E1 cells and KUSA/A1 cells were evaluated after 21 and 12 days of culture, respectively, in osteogenic medium containing a-MEM supplemented with 10% FBS, 50 Ag/ml ascorbic acid, and 5 mM h-glycerophosphate. MTT test Proliferation and viability of KS483 cells in hig-h3 protein-coated culture plate were assessed by MTT test [24]. Briefly, cells were seeded in the concentration of 1.5 103
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cells per well in 100 Al of serum-free medium onto BSA, FN, hig-h3 protein, and Do-IV-coated 96-well culture plates (20 Ag/ml). Then, 50 Al of thiazolyl blue (MTT) (Sigma) solution in final concentration of 0.5 mg/ml was added in the culture plate and incubated for 4 h at 378C. The colored product was solubilized in 150 Al of dimethyl sulfoxide (DMSO), and absorbance was quantified at 570 nm in a Bio-Rad model 550 microplate reader. The test was performed in the interval of 24 h for 5 days. Cell adhesion assay The cell adhesion assay was performed as described previously [23]. Briefly, flat-bottomed 96-well enzymelinked immunosorbent assay (ELISA) plates (Costar, Corning Inc.) were incubated overnight at 48C with 10 Ag/ml recombinant hig-h3 protein or other extracellular matrix (ECM) proteins in PBS and blocked for 1 h at room temperature with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). Cells were suspended in medium at a density of 3 105 cells/ml, and 0.1 ml of the cell suspension was added to each well of the coated plates. Following incubation for 30 min at 378C, unattached cells were removed by rinsing once with PBS. Attached cells were incubated for 1 h at 378C in 50 mM citrate buffer, pH 5.0, containing 3.75 mM pnitrophenyl-N-acetyl h-d-glycosaminide and 0.25% Triton X-100. Enzyme activity was blocked by adding 50 mM glycine buffer (pH 10.4) containing 5 mM EDTA, and absorbance was measured at 405 nm in a Bio-Rad model 550 microplate reader. Inhibition assay To identify the receptor for hig-h3, monoclonal antibodies specific for different integrin types (5 Ag/ml) (Chemicon, International Inc.) were preincubated at 378C for 30 min with primary human osteoblasts in a 0.1 ml cell suspension (3 105 cells/ml). Cells were transferred onto plates precoated with recombinant hig-h3 proteins and incubated further for 30 min at 378C. Attached cells were quantified as described above. Function-blocking monoclonal antibodies specific for the following integrin subunits were employed: a1 (FB12), a2 (P1E6), a3 (P1B5), a4 (P1H4), a5 (P1D6), a6 (GoH3), av (P3G8), h1 (6S6), h2 (P4H9), avh3 (LM609), and avh5 (P1F6). Flow cytometric analyses To confirm the expression of avh3 and avh5 integrins on osteoblast cell surface, fluorescence-activated cell sorter analysis was performed. Human primary osteoblasts and osteosarcoma cells grown to confluency were detached from plates by treatment with 0.25% trypsin-0.05% EDTA. After washing twice in PBS, cells were suspended in PBS and incubated for 1 h at 48C with antibodies specific for a3
(ASC-1), av (P3G8), avh3 (LM609), and avh5 (P1F6). Cells were incubated for 1 h at 48C with 10 Ag/ml secondary goat-antimouse IgG conjugated to FITC (Santa Cruz Biotechnology, Inc., CA) and analyzed at 488 nm on the flow cytometer FACSCalibur system (Becton Dickinson, San Jose, CA) equipped with a 5-W argon laser. Total RNA isolation and Northern blot analysis Total RNA was isolated from cells using guanidinium isothiocyanate solution [25], resolved on a 1% formaldehyde-agarose gel (10 Ag) and transferred to a Nylon membrane (Sigma) by the capillary method using 20 SSC buffer (sodium chloride sodium citrate, pH 7.0). The RNA was cross-linked to membranes by UV irradiation using UV StratalinkerTM 1800 (Stratagene). The DNA probes used included mouse hig-h3, Col I, OC, ALP, Cbfa1/ Runx2, and osterix. The cDNA probes for OC, ALP, Cbfa1/ Runx2, and osterix were generous gifts of Dr. Ryoo (Department of Biochemistry, School of Dentistry, KNU, Taegu, South Korea). All probes were labeled with 30 ACi [a-32P] dCTP using the random priming Megaprime DNA labeling Kit (Amersham Pharmacia Biotech, UK Ltd.). Hybridization was performed at 688C for 1 h to overnight using ExpressHybTM (Clontech). Blots were washed in high salt solution containing 0.05% SDS and 2 SSC at 308C, followed by low salt solution containing 0.1% SDS in 0.1 SSC for different time periods, depending on the type of probe used. Blots were exposed to X-ray film (AGFA) overnight at 808C. To study the effect of interrupting the interaction of high3 protein with its integrin receptor in HOS cells, we treated the trypsinized cell suspension with function-blocking monoclonal antibody for avh3 and avh5 (Chemicon) at concentration of 1.4 Ag/ml before seeding into the hig-h3 protein-coated culture plate in complete growth medium. The RNA was isolated after 3 days for analysis of type I collagen gene expression. Binding assay of big-h3 A binding assay was performed as described previously [26]. Briefly, primary human osteoblasts were suspended in medium at a density of 1 105 cells/ml, and 1 ml of the cell suspension was preincubated with anti-avh3 (LM609) or avh5 (P1F6) for 30 min at 378C. The cells were incubated with biotinylated hig-h3 in serum-free medium for 5 h at 48C. The cells were washed three times with PBS before lysis. Equal amounts of proteins were separated on 10% SDS–PAGE gel, transferred to membrane, and incubated with streptavidin conjugated to horseradish peroxidase (Amersham Biosciences). The membrane was developed by using enhanced chemiluminescence (ECL; Amersham Biosciences). For internal control, membrane was stripped and reprobed with beta-tubulin (Santa Cruz Biotechnology).
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Cell transfection KS483 cells were transfected with human h3 integrin subunit in pcDNA 3.1(+) (kind gift of Dr. Jaffrey Smith, Burnham Institute, San Diego, USA) by using lipofecAMINE plusk reagent (Life Technologies Inc.) according to manufacturer’s protocol. The transfected cells were selected in a-MEM medium with 10% FBS in presence of 500 Ag/ml G418 (Life Technologies Inc.) for 2 weeks. The stably transfected clones overexpressing h3 were verified by Western blot analysis by using antihuman h3 antibody (Chemicon International). Statistical analysis The values were expressed as the mean F SD. A oneway analysis of variance and Dunnett t test were used to determine the statistical significance. Differences were considered significant at P b 0.05.
Results big-h3 gene expression is down-regulated during osteoblast differentiation To determine the expression level of hig-h3 during in vitro differentiation of osteoblasts, total RNA was extracted at indicated time points from KS483 cells cultured in osteogenic medium (Fig. 1A). Northern blot analyses revealed down-regulation of hig-h3 expression during differentiation of KS483 cells. Gene expression of hig-h3 was the highest at an early stage of proliferation and dramatically decreased in subsequent days in this study. This finding indicates that the down-regulation of hig-h3 gene expression is important for osteoblast differentiation. big-h3 protein inhibits matrix mineralization/bone nodule formation Next, we determined the role of hig-h3 in matrix mineralization/bone nodule formation during osteoblast differentiation, using alizarin red-S histochemical staining. Cells were cultured for 21 days on 24-well plates coated with hig-h3, the fourth FAS1 domain of hig-h3 (Do-IV), or various ECM proteins, such as FN, VN, and Col I. Decreased mineralization/bone nodule formation by high3 protein and Do-IV (Fig. 1B) was observed, compared to the control (noncoated). The inhibitory effect of hig-h3 protein was specific, in view of the finding that the other ECM proteins examined (except FN) displayed a similar or higher extent of bone nodule formation/matrix mineralization, compared to the control (Fig. 1B). Similarly, inhibitory effect of hig-h3 in matrix mineralization/bone nodule formation was also observed in MC3T3-E1 and KUSA/A1 cells (Fig. 1C). In our MTT assay, hig-h3 and Do-IV
Fig. 1. hig-h3 expression in KS483 cells and its effect on matrix mineralization/bone nodule formation. (A) KS483 cells were seeded in 100 mm culture plates at a concentration of 6 105 cells per plate and incubated in osteogenic media for indicated time periods (in days). Total RNA was extracted, and Northern blotting was performed using a specific probe for mouse hig-h3 mRNA. The consistency of RNA loading was assessed by reprobing the membrane with 18S rRNA. (B) KS483 cells were cultured in 24-well plates coated with recombinant hig-h3 protein, Do-IV, or other ECM proteins, such as plasma FN, VN, and Col I, for 21 days in osteogenic medium. Matrix mineralization/bone nodule formation was evaluated by alizarin red-S staining (macroscopic and microscopic view, 100). (C) Alizarin red-S staining of MC3T3-E1 and KUSA/A1 cells cultured in hig-h3-coated plates for 21 and 12 days, respectively, in osteogenic medium. (D) KS483 cells were seeded in 96-well plates coated with BSA, FN, hig-h3, and Do-IV in serum-free medium. The absorbance was taken after addition of MTT solution as described in Materials and methods. The values are the means F SD of triplicate determination. bP b 0.01 vs. BSA.
supported the proliferation and viability of KS483 cells indicating that inhibitory effect of hig-h3 is not due to negative effect on proliferation and viability of KS483 cells (Fig. 1D).
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big-h3 inhibits the expression of osteoblast differentiation marker genes and osteoblast-specific transcription factors The hig-h3 protein inhibited the expression of osteoblast differentiation marker genes, ALP and OC. Inhibition of ALP expression was evident at an early time point (Fig. 2A) and did recover with time of culture in our study. The OC gene, a late-stage marker of osteoblast differentiation, was also distinctly inhibited in hig-h3-coated plates (Fig. 2A). Interactions between collagen and integrin are indispensable for osteoblastic differentiation, and type I collagen is a very
early marker of osteoblast differentiation. Northern blot analyses revealed significant suppression of Col I expression by hig-h3 (Fig. 2A). We examined whether hig-h3 protein affects the expression of osteoblast-specific transcription factors. Coated recombinant hig-h3 protein down-regulated expression of the runt domain-containing transcription factor, Cbfa1/ Runx2 (Fig. 2B), and the recently identified transcription factor, osterix (Fig. 2C). The mRNA levels of both Cbfa1/ Runx2 and osterix in KS483 cells cultured on hig-h3-coated culture plates were clearly down-regulated over different time points studied, compared to the control. Additionally, we found an inhibited expression level of Col I and OC in MC3T3-E1 cells and KUSA/A1 cells (Fig. 2D) cultured in hig-h3-coated plates. big-h3 protein and its FAS1 domains support the adhesion of primary human osteoblasts and KS483 cells The hig-h3 protein and all four FAS1 domains (I–IV) support cell adhesion of both KS483 cells (Fig. 3B) and primary human osteoblasts (Fig. 3C). Each domain displayed comparable cell adhesion activity to FN or hig-h3 protein in both cell types, indicating that the FAS1 domain contributes to cell adhesion to osteoblasts. Cell adhesion activity of KS483 cells was increased to some extent at increasing concentrations of hig-h3 and all four FAS1 domains (Fig. 3D). Integrins avb3 and avb5 are involved in mediating the adhesion of osteoblasts to big-h3 protein
Fig. 2. Inhibition of expression of osteoblast differentiation marker genes and osteoblast-specific transcription factors by hig-h3 protein. KS483 cells were seeded at a concentration of 6 105 cells per plate in 100 mm plates either coated with hig-h3 protein (+) or left uncoated ( ), and cultured for the indicated time periods (days) in osteogenic medium. Total RNA was isolated, and Northern blotting was performed using specific probes. (A) Col I, ALP, and OC; (B) Cbfa1/Runx2; (C) osterix. (D) Total RNA was extracted from MC3T3-E1 and KUSA/A1 cells cultured in osteogenic medium for 14 and 7 days, respectively, in plates either coated with hig-h3 protein (+) or left uncoated ( ). Northern blotting was performed for Col I and OC expression. The consistency of RNA loading was assessed by reprobing the membrane with 18S rRNA.
To identify the specific integrin(s) mediating adhesion of osteoblasts to hig-h3, we performed an inhibition assay using function-blocking monoclonal antibodies (Chemicon). The adhesion of primary human osteoblasts to hig-h3 was specifically inhibited with antibodies against avh3 and avh5 integrins (Fig. 4A). In contrast, no significant effects were observed with other anti-integrin antibodies, including a1, a2, a3, a4, and h1. This finding suggests that both avh3 and avh5 are functional receptors in mediating the adhesion of osteoblasts to hig-h3 protein. Additionally, adhesion of osteoblasts to vitronectin is mediated by avh5 (Fig. 4B). To confirm the expression of these integrin subunits in osteoblasts, FACS analyses were performed in primary human osteoblasts. The similar level of avh3 and avh5 integrins expression was observed on osteoblasts. The expression of av and a3 integrins was also prominent. However, we did not perform the FACS analysis for other integrins examined in inhibition study. To investigate the interaction between hig-h3 and integrins found in osteoblasts, we studied the binding of biotinylated hig-h3 to osteoblasts cell surface. The hig-h3 binds in a dose-dependent manner (Fig. 5A), and this binding was inhibited by antibodies to either avh3 or avh5 integrins, but not by control IgG (Fig. 5B). This result
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reveals the interaction of hig-h3 and avh3/avh5 integrins found on cell surface of osteoblasts. The synthetic YH peptide inhibits the adhesion of osteoblasts to big-h3 protein Our group has recently identified a YH motif in the FAS1 domain of hig-h3 protein that mediates adhesion of endothelial cells via the avh3 integrin [26] and a
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fibroblastic cell line, MRC-5, via the avh5 integrin [27]. Since the YH motif interacts with both the avh3 and avh5, we hypothesize that this sequence in hig-h3 protein provides a potential interacting site for avh3 and avh5 integrins expressed in osteoblasts. A YH18 synthetic peptide corresponding to amino acids 563–580 of hig-h3 significantly inhibited cell adhesion of both KS483 cells and primary human osteoblasts to hig-h3 protein, as shown in Figs. 6A and B, respectively. A YH18 control sequence containing all 18 amino acids of the YH18 peptide but randomly ordered did not affect cell adhesion activity. avb3 integrin plays a role in big-h3-mediated inhibition of osteoblast differentiation The inhibitory effect of integrin avh3 in osteoblast differentiation has been reported [28]. The bone nodule formation and matrix mineralization of KS483 cells overepressing human h3 integrin subunit were dramatically reduced in compared with mock-transfected cells (Fig. 7A). To elucidate the role of interacting integrins in hig-h3mediated inhibition of osteoblast differentiation, we employed a function-blocking monoclonal antibody specific for avh3 and avh5 integrins to disrupt the interactions between osteoblasts and hig-h3 protein. We employed HOS cells for this study. Our FACS analyses additionally reveal expression of these integrins in HOS cells (Fig. 7 B). The adhesion of HOS cells to hig-h3 was found more dependent on avh3 integrin than on avh5 integrin (Fig. 7 C). Treatment with a function-blocking antibody specific for avh3 abolished the inhibitory effect of hig-h3 protein, as evaluated by the expression of type I collagen (Fig. 7D). The function-blocking monoclonal antibody specific for avh5 did not significantly affect the inhibitory effect of high3 protein.
Discussion We previously demonstrated an inhibitory effect of hig-h3 protein on osteoblast differentiation [22]. In the present study, Fig. 3. The hig-h3 protein and all its FAS1 domains support the adhesion of KS483 cells and primary human osteoblasts. (A) Diagram of hig-h3 protein and position of four FAS1 domains (Do-I, residues 134–236; Do-II, residues 242–372; Do-III, residues 373–501; Do-IV, residues 502–632). The hatched and cross-hatched boxes indicate the highly conserved sequences in each domain. For adhesion assay, a 96well plate was coated with protein overnight at 48C. After blocking with 2% BSA and PBS washing, 2 104 cells per well in 100 Al complete media were seeded and incubated at 378C for 40 min. After incubation, unattached cells were washed with PBS, and attached cells were quantified using a hexosaminidase assay, as described in Materials and methods. Plasma FN (10 Ag/ml) and 2% BSA were employed as the positive and negative controls, respectively. (B) KS483 cells; (C) primary human osteoblasts; (D) dose-dependent cell adhesion of KS483 cells to high3 protein and its FAS1 domains. The values are the means F SD of triplicate determinations. aP b 0.001 versus BSA.
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we explain the molecular mechanism of action and identify the interacting integrin receptor in hig-h3-mediated inhibition of osteoblast differentiation. Osteoblast differentiation is mediated by a complex series of cell–cell and cell–matrix interactions. The process represents a typical model for analyzing interactions between cells and ECM proteins, since osteoblasts are specifically involved in secretion and organization of the ECM during bone formation. The studies have identified hig-h3 as a bone matrix protein that plays an important role in osteogenesis [21,22]. The expression of
hig-h3 in vivo is abundant in preosteoblasts during early bone formation [21]. Our study additionally revealed high expression of hig-h3 at the early stage of in vitro differentiation of KS483 cells, which was followed by a significant decrease. The regulated expression level of hig-h3 was noted during in vitro differentiation of pluripotent human bone marrow stromal cells [21]. We have also found a dramatic change in expression level of hig-h3 gene in chondrocyte differentiation of ATDC5 cell line (data not shown). We employed recombinant hig-h3 as a cell adhesion substrate/integrin ligand with a view to elucidating the function of the protein. Coated ECM proteins provide natural ligands for adhesion and activation of specific integrin receptors present on the cell surface of osteoblasts [24]. Proliferation and differentiation profiles of osteoblasts are influenced by ECM proteins coated onto culture plates, such as collagen, osteopontin, and bone sialoprotein [4–6]. Since KS483 cells form a hydroxyapatitecontaining mineralized matrix in vitro when grown in osteogenic conditions [29], we assessed the matrix mineralization/bone nodule formation of these cells cultured on high3, Do-IV, and other ECM proteins, including vitronectin, type I collagen, and fibronectin. hig-h3 and Do-IV significantly inhibited bone nodule formation, analogous to our previous report [22]. The extent of matrix mineralization/ bone nodule formation of KS483 cells on other ECM matrix protein-coated wells was similar or increased, compared to the control (noncoated). Indeed, matrix mineralization was more enhanced in the well coated with type I collagen, consistent with earlier results [30]. Fibronectin, which is expressed only at early stages of osteogenesis [2], also inhibited matrix mineralization/bone nodule formation to a small extent, in agreement with our previous data [22]. Furthermore, inhibition of matrix mineralization/bone nodule formation by hig-h3 was accompanied by suppression of expression of osteoblast differentiation marker genes, Col I, ALP, and OC. Interactions between osteoblasts and type I collagen are critical for differentiation [31], suggesting that inhibition of osteoblast differentiation by hig-h3 is mediated to some extent by down-regulating the expression of other
Fig. 4. avh3 and avh5 are functional receptors mediating the adhesion of primary human osteoblasts to hig-h3 protein. (A) Primary human osteoblasts were preincubated with different function-blocking monoclonal antibodies specific for integrins (final concentration of 5 Ag/ml) for 30 min at 378C. A Cell adhesion assay was performed using 10 Am/ml hig-h3-WT coated in 96-well culture plates, as described in Materials and methods. (B) Identification of integrins mediating the adhesion of osteoblasts to vitronectin. Primary human osteoblasts were preincubated with function-blocking monoclonal antibodies to integrin avh3 (LM609), avh5 (P1F6), and av (P3G8). Cell adhesion to vitronectincoated plates (5 Ag/ml) was determined. (C) Flow cytometric analysis of avh3 and avh5 integrins on the cell surface of primary human osteoblasts. Cells were stained with saturating concentrations of antibodies specific for a3 (ASC-1), av (P3G8), avh3 (LM609), and avh5 (P1F6). Data are expressed as cell number (y-axis) plotted as a function of fluorescence intensity (x-axis) and are representative of three separate experiments. Negative control cells were incubated with the secondary antibody alone. c P b 0.05, bP b 0.01, aP b 0.001.
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Fig. 5. Binding of biotinylated hig-h3 to osteoblasts. (A) Dose-dependent binding of biotinylated hig-h3 to osteoblasts cell surface. The primary human osteoblasts were incubated with biotinylated hig-h3, and amount of biotinylated hig-h3 associated with osteoblasts was determined by Western immunoblotting with the streptavidin-conjugated horseradish peroxidase. (B) Function-blocking antibody to either avh3 or avh5 integrins inhibits the binding of biotinylated hig-h3 to osteoblasts. The primary human osteoblasts were preincubated with 5 Ag of anti-avh3 (LM609) or antiavh5 (P1F6) or control IgG followed by incubation with biotinylated high3 in serum-free medium for 5 h at 48C. The amount of biotinylated hig-h3 associated with osteoblasts was determined by Western immunoblotting with the streptavidin-conjugated horseradish peroxidase as described in Materials and methods.
ECM proteins and subsequently altering integrin-ECM protein interactions and cell signaling. The result of MTT assay suggests that hig-h3 does not have negative effect on cell proliferation and viability, and thus the inhibitory effect of hig-h3 on matrix mineralization/bone nodule formation is not likely due to inhibition of cell growth. The inhibitory effect of Do-IV was found more pronounced than that of high3, and it may be due to efficient coating and stability of DoIV compared with hig-h3 in long-term culture condition. Interactions between a particular ECM ligands and specific integrin heterodimers influence osteoblast function via regulation of transcription factors [32]. The expression of osteoblast-specific transcription factors, core binding factor/runt-related gene 2 (Cbfa1/Runx2), and zinc fingercontaining novel transcription factor, osterix, was evaluated in KS483 cells cultured on a hig-h3 protein-coated plate. These transcription factors are known to play an important role in osteoblast differentiation [33,34]. Interestingly, high3 suppressed the expression of both Cbfa1/Runx2 and osterix, indicating a role of these osteoblast-specific transcription factors in hig-h3-mediated inhibition of osteoblast differentiation. We propose that the interaction of osteoblasts with high3 through integrins triggers signals that inhibit osteoblast differentiation. The hig-h3 protein and all four FAS1 domains displayed comparable cell adhesion activity, indicating the existence of an integrin-interacting motif in each domain. The antibody inhibition assay revealed that avh3 and avh5 are the principal integrin heterodimers that mediate adhesion of osteoblasts to hig-h3. Additionally,
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binding of hig-h3 to osteoblasts is mediated by these two integrins. Our previous studies have shown that avh3 and avh5 integrins mediate the interaction of human umbilical vein endothelial cells (HUVEC) and fibroblastic cell line, MRC-5 cells, to hig-h3, respectively [26,27]. Data from our FACS analyses of the expression of avh3 and avh5 integrins on the cell surface of primary human osteoblasts are consistent with other studies [10,11,13,14]. We have identified highly conserved amino acids (aspartic acid and isoleucine) in the second and fourth FAS1 domains of high3 as the binding motif for a3h1 integrin in human corneal epithelial cells [23], and a YH motif for avh3 integrin in endothelial cells [26] and avh5 in the fibroblastic MRC-5 cell line [27]. We suggest that YH is also the binding motif in the case of osteoblasts, since it interacts with both avh5 and avh3 integrins. The synthetic YH18 peptide containing
Fig. 6. The YH motif is responsible for cell adhesion of hig-h3 to osteoblasts. The amino acid sequence of the synthetic YH18 peptide corresponding to hig-h3 is specified. Before the cell adhesion assay, trypsinized suspensions of KS483 cells (A) and primary human osteoblasts (B) were preincubated with 100 AM synthetic and control YH peptides for 15 min, and added to plates. The number of adhering cells was quantified, as explained in Materials and methods. The values are the means F SD of triplicate determinations. bP b 0.01.
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the 18 amino acids of hig-h3 (positions 563 to 580) almost completely inhibited both primary human osteoblasts and KS483 cells adhesion to hig-h3. In contrast, the scrambled form of this peptide had no effect. This finding implies that the YH motif is a potential binding site for osteoblasts in hig-h3 protein.
Integrin-mediated interactions of cells with ECM proteins and the cell signals generated have been implicated in a diverse range of biological activities, including cell differentiation [35]. The avh3 integrin negatively regulates osteoblast differentiation [28] and is expressed in the inactive form in human osteoblasts [11]. Studies on oligodendrocytes and skeletal muscle cells additionally confirm the inhibitory role of this integrin in cell differentiation [36,37]. The avh3 and avh5 integrins are involved in mediating adhesion of osteoblasts to hig-h3. Accordingly, we hypothesize that avh3 plays a role in the inhibition of osteoblast differentiation by hig-h3. We could not use KS483 cells due to lack of function-blocking monoclonal antibody specific for mouse avh3 and avh5 integrins. The poor osteogenic potentiality of our primary human osteoblasts optioned us to use HOS cells. The FACS analyses reveal expression of these integrins in HOS cells. We analyzed the expression of type I collagen in HOS cells cultured on hig-h3 protein-coated plates in the presence of function-blocking antibodies for avh3 and avh5 integrins, with the aim of disrupting the interactions between HOS cells and hig-h3 protein. Treatment with a function-blocking anti-integrin antibody specific for avh3 abolished the inhibitory effect of hig-h3, supporting the theory that activation of avh3 and subsequent cell signaling in high3 protein mediates the inhibition of osteoblast differentiation. Col I is one of the differentiation marker genes of osteoblasts [34]. We could not examine the effect on matrix mineralization and other marker genes of osteoblasts differentiation because the HOS cells used in this study could not differentiate faithfully and were detached from culture plate once cells became confluent. We found avh5, rather than the avh3, integrin-mediated interactions of osteoblasts with coated vitronectin, and this result is
Fig. 7. avh3 integrin plays a role in hig-h3-mediated inhibition of osteoblast differentiation. (A) Western blot analysis and alizarin red-S staining of KS483 cells stably overexpressing human h3 integrin subunit. The cell lysate was prepared and subjected to 10% SDS–PAGE gel. The membrane was incubated with anti-h3 antibody for overnight at 48C. The binding of antibody was detected by ECLk Western Blotting Reagents (Amersham Biosciences). The human h3 chain of approximately 95 kDa is shown by an arrow. The alizarin red-S staining was performed after culturing the mock and h3-overexpressing KS483 cells in osteogenic medium for 21 days. (B) Flow cytometric analysis of HOS cells. All antibodies used and procedures were the same as described above for primary human osteoblasts. (C) Identification of integrins mediating the adhesion of HOS cells to hig-h3. HOS cells were preincubated with different function-blocking monoclonal antibodies specific for integrins (final concentration of 5 Ag/ml) for 30 min at 378C. A cell adhesion assay was performed using 10 Ag/ml hig-h3-WT coated in 96-well culture plates, as described in Materials and methods. The values are the means F SD of triplicate determinations. aP b 0.001. (D) The trypsinized cell suspensions of HOS cells either treated with 1.4 Ag/ml function-blocking antibody specific for avh3 (anti-avh3) and avh5 (anti-avh5) integrins or left untreated were seeded into hig-h3 protein-coated (+) and uncoated ( ) culture plates. RNA was isolated after 3 days, and Northern blotting was performed for human Col I. The consistency of RNA loading was evaluated by reprobing the membrane with 18S rRNA.
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consistent with that of Cheng et al. [38]. This may provide indirect evidence why coated vitronectin does not have an inhibitory effect on osteoblast differentiation. Accordingly, we suggest that avh3 integrin plays a role in inhibition of osteoblast differentiation by hig-h3. In conclusion, hig-h3 expression is down-regulated during differentiation of osteoblasts. Moreover, the protein inhibits osteoblast differentiation, and avh3/avh5 integrins mediate the interaction of osteoblasts to hig-h3. Further studies are required to clarify the roles of this newly identified bone matrix protein and the relevant integrin heterodimers involved in early bone formation, osteoblast differentiation, and pathogenesis of bone diseases.
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Acknowledgments We thank Dr. Ryoo (Department of Biochemistry, School of Dentistry, KNU, Daegu, South Korea) for providing cDNA probes for Northern blotting. This work was supported by a program of the National Research Laboratory (M1010400036-01J0000-01610).
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