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Keywords:

  • cadherin;
  • cell adhesion molecules;
  • bone formation;
  • osteoblast differentiation

Abstract

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

We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCadδC) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCadΔC, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally,45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCadΔC-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and β-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCadΔC cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells.


INTRODUCTION

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

CADHERINS ARE integral membrane proteins that mediate calcium-dependent cell-cell adhesion and provide the adhesive component of adherens junctions.(1) Cell-cell adhesion via cadherins is a key element for many aspects of cell biology, including morphogenesis, acquisition, and maintenance of cell polarity and regulation of cell proliferation and differentiation.(2,3) Cadherins are a family of single chain glycoproteins of approximately 120 kDa, with a long extracellular N-terminal sequence, a single transmembrane domain, and a relatively short cytoplasmic C-terminal tail. The latter interacts with cytoskeletal proteins, including catenins and plakoglobin, and through them cadherins are linked to actin filaments thus providing cell-cell anchorage. The extracellular domain is highly variable among different cadherin isotypes and is constituted by five repeats that define the structure of this class of adhesion molecules and provide calcium-dependent adhesion function. More than a dozen members of this superfamily are currently known, all the products of separate genes, and it is now evident that most tissues express multiple cadherins.(1)

We have previously shown that human osteoblasts express a repertoire of cadherins, which changes during in vitro differentiation.(4) The most abundant osteoblast cadherins are cadherin-11, expressed in most tissues of mesenchymal origin,(5) and N-cadherin, a rather ubiquitous molecule prevalent in neural and mesodermal tissues.(6) Expression and regulation of these molecules also have been reported by other investigators in human and murine osteoblasts and bone marrow stromal cells. (7–9) Although the specific function of each individual molecule remains unknown, accumulated evidence indicates that cadherin-mediated cell-cell adhesion is essential for the commitment of cells to osteoblast differentiation.(4, 9, 10) We have previously reported that interference with cadherin-mediated adhesion by exposure of human osteoblasts to an inhibitory peptide containing the HAV adhesion-recognition motif, thought to be critical for the adhesive function of cadherins, hinders bone morphogenetic protein 2 (BMP-2) induction of alkaline phosphatase activity.(4) Recently, closely similar results have been reported by other investigators using rat osteoblast models,(9) suggesting that direct cell-cell contact among osteoblasts is linked intimately to their function. Although these HAV-containing inhibitory peptides have been useful to probe the potential role of cadherin-mediated adhesion, they are not well suited for a detailed molecular and functional analysis of osteoblast cadherins. In vitro osteoblast differentiation requires long-term cultures (up to 4 weeks), and the exogenous peptides that are metabolized easily do not provide a controlled, sustained inhibition of cell-cell adhesion for such a prolonged time.

To obtain a more reliable inhibition of cadherin function, we have expressed a truncated mutant of N-cadherin (NCadΔC) in the immature mouse calvaria osteoblast cell line, MC3T3-E1. This mutant cadherin, in which most of the extracellular domain has been deleted, previously has been proven to exhibit dominant negative action on cadherin function in cell cultures and in vivo.(11,12) In these studies, we show that interference with cadherin-dependent cell-cell adhesion via expression of NCadΔC alters osteoblast function leading to reduced in vitro matrix mineralization.

MATERIALS AND METHODS

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

Cells and reagents

The mouse calvaria-derived MC3T3-E1 osteoblastic cells were grown in α-modified essential medium (α-MEM) until confluent, with modifications as detailed in each experimental setting. This cell line represents phenotypically immature osteoblasts, derived from spontaneous immortalization of calvaria cells selected by the 3T3 passaging protocol.(13) Rabbit polyclonal antibodies against osteopontin (LF120), bone sialoprotein (LF6), and type I collagen (LF67) were kindly provided by Dr. Larry W. Fisher (National Institutes of Health [NIH], Bethesda, MD, U.S.A.). The polyclonal antibody PEP.1, which recognizes the C-terminal intracellular tail of N-cadherin, was the generous gift of Dr. Barry Gumbiner (Memorial Sloan-Kettering Cancer Center, New York, NY, U.S.A.). Recombinant human BMP-2 (rhBMP-2) was kindly provided by the Genetics Institute (Cambridge, MA, U.S.A.). The complementary DNA (cDNA) probes for mouse osteocalcin, bone sialoprotein, osteopontin, type I collagen, and rat glyceraldehyde-3-phosphate dehydrogenase (GADPH) were kindly supplied by Dr. John Wozney (Genetics Institute), Dr. Marian Young (NIH), and Dr. William A. Parks (Washington University, St. Louis, MO, U.S.A.). cDNA probes that hybridize to mouse N-cadherin and cadherin-11 were prepared as previously described.(14) Unless otherwise indicated, reagents for molecular biology were purchased from Promega (Madison, WI, U.S.A.). Taq polymerase was from Fisher (Pittsburgh, PA, U.S.A.). All the other chemicals, including the tissue culture medium and the membrane permanent dye, PKH-26, were from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Preparation of NCadΔC expression vectors

The NCadΔC construct, cloned in the pSP72 (pSP72NCadΔC) vector was obtained from Dr. Jeffery Gordon (Washington University, St. Louis, MO, U.S.A.), with permission of Dr. Chris Kintner (Salk Institute, San Diego, CA, U.S.A.). This construct, derived from an in-frame deletion of most of the extracellular domain of Xenopus N-cadherin, has been shown to function as a dominant negative cadherin.(11,12) Both the plasmid vector pcDNA3 (Invitrogen Co., Carlsbad, CA, U.S.A.) and the retroviral vector pMV7 (kindly provided by Dr. Nissim Hay, University of Illinois, Chicago, IL, U.S.A.) were employed to generate NCadΔC expression vectors. To clone NCadΔC into pcDNA3 (pcDNA3NCadΔC), a 1249-base pair (bp) insert containing the entire reading frame of NCadΔC (846 bp) plus additional 42 bp at the 5′ end and 361 nontranslated bp at the 3′ end were excised from the pSP72NCadΔC plasmid by Xho I and EcoRV digestion. After end-filling with Klenow polymerase, the construct was inserted into the EcoRV cloning site of pcDNA3. The orientation of the insert was verified by restriction fragment size and by direct DNA sequencing. To clone NCadΔC into the retroviral pMV7 vector (pMV7NCadΔC), an NCadΔC insert was generated by polymerase chain reaction (PCR) using primers encompassing the first 25 bases of the 5′ and 3′ ends of the NCadΔC reading frame. The primers also carried BamHI recognition sequences for cloning the PCR product into pMV7. To generate the retrovirus, the pMV7NCadΔC plasmid was transfected into the ecotropic retroviral packaging cell line BOSC-23 293 via LipofectAMINE (Life Technology, Gaithersburg, MD, U.S.A.) according to manufacturer's instructions. The medium was changed 24 h after transfection and the culture was returned to the incubator. After 24 h, the medium was removed and centrifuged at 500g, and the supernatant containing viral particles was harvested and stored frozen at −80°C.

Generation of control and NCadΔC-expressing MC3T3-E1 cell lines

MC3T3-E1 cells were seeded in 100 mm dishes (3 × 106/dish) in α-MEM with 10% heat-inactivated fetal bovine serum (HIFBS). After overnight recovery, cells were transfected with either pcDNA3NCadΔC or pcDNA3 without insert by using LipofectAMINE Plus reagents according to the manufacturer's protocol. Alternatively, MC3T3-E1 cells were infected with NCadΔC viral supernatant prepared as detailed previously, by 4-h exposure to the viral stock. In either case, cells expressing the exogenous DNA were selected by G418 resistance after a 2- to 3-week incubation in selection medium. Integration of either pcDNA3NCadΔC or pMV7NCadΔC into the genome was verified by PCR using genomic DNA as template and T7 and SP6 as primers or the primers used for construction of the retroviral vector. G418-resistant cells either transfected with pcDNA3NCadΔC or transduced with pMV7NCadΔC exhibited a single product of the expected length (1.25 kilobase [kb] and 0.85 kb, respectively). Two lines of NCadΔC-MC3T3-E1 cells, one generated by plasmid transfection and one by retroviral transduction, were used for the following studies.

Calcium dependent cell-cell adhesion

Cell adhesion assay was performed as previously described.(4) Briefly, cells were released from culture dishes by trypsin/EDTA digestion. Single cell suspension was obtained and labeled with the membrane-permanent fluorescent dye PKH26 (2 μM). After washing with phosphate-buffered saline (PBS) to remove excess dye, the labeled cells were laid on top of a confluent unlabeled monolayer of MC3T3-E1 cells in the presence of 1 mM of calcium chloride. The labeled cells were allowed to settle for 60 minutes, nonadherent cells were gently washed away with PBS, and the number of fluorescent cells adherent to the cell substratum counted as an index of cell-cell adhesion.

Cell proliferation

Cells were seeded in 24-well plates (2 × 104 cells/well) and allowed to recover overnight. The medium was then replaced with fresh medium containing [3H]thymidine (2 μCi/well; Amersham, Arlington Heights, IL, U.S.A.) and 10% HIFBS, and the incubation continued for 18 h. Incorporated [3H]thymidine was measured in trichloroacetic acid precipitated material, as previously described.(15)

Alkaline phosphatase activity

Confluent cells in 24-well plates were washed three times with Tris-buffered saline (TBS; 50 mM Tris, pH 7.4, and 0.15 M NaCl) and stored at −20°C until assayed. Following a previously described method,(15) the cell layer from each well was scraped into 0.5 ml of 10 mM Tris, pH 7.4, containing 0.5 mM MgCl2 and 0.1% TritonX-100, and sonicated with a Fisher Dismembranetor (30-40% of maximum strength; Fisher). Alkaline phosphatase activity was measured using p-nitrophenyl phosphate (3 mM) as substrate in 0.7 M 2-amino-2-methyl-1-propanol, pH 10.3, and 6.7 mM MgCl2, and expressed as p-nitrophenol produced in nmol/minute per milligram of protein. Protein was measured using the Bio-Rad method (Bio-Rad Laboratories, Inc., Richmond, CA, U.S.A.) with bovine serum albumin (BSA) as standard.

Matrix mineralization

As previously described,(16) the mineralization potential of the different cell lines was assessed after incubation for 28 days in mineralizing medium, consisting of α-MEM supplemented with 10% HIFBS, as well as ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mM). At the end of the 4-week culture,45Ca was added to each well (2 μCi/well) and incubation continued for 6 h. After washing,45Ca in the cell layer was extracted with 1N HCl and radioactivity was counted.

Northern blot

PolyA RNA was purified from cell extracts using the Mini RiboSep kit (Collaborative Biochemical Products, Bedford, MA, U.S.A.), as described.(15,16) Samples (5 μg/lane) were separated on 1% formaldehyde agarose gels by electrophoresis, blotted onto nylon membranes, and UV cross-linked. The membranes were hybridized using [32P]-labeled probes made by a random primed oligonucleotide method (Megaprime DNA labeling kit; Amersham) in 40% formamide (Oncor, Inc., Gaithersburg, MD, U.S.A.), 10 mM Tris HCl, pH 7, 5× SSC, 125 mg/ml salmon sperm DNA (5 Prime [RIGHTWARDS ARROW] 3 Prime, Inc., Boulder, CO, U.S.A.), and 1.25× Danhardt's solution, at 42°C, and washed twice in 2× SSC, 0.1% sodium dodecyl sulfate (SDS) at room temperature, followed by one high stringency wash in 0.2× SSC, 0.1% SDS at 52°C for 25 minutes. The abundance of messenger RNA (mRNA) was quantitated in digitized autoradiographic images using SigmaScan (SPSS Science, Chicago, IL, U.S.A.).

Western blot

The methods previously reported were used.(4) Cells were washed three times with PBS and extracted with 0.5 ml of 0.5% Triton X-100 in 10 mM HEPES, pH 7.4, containing 150 mM NaCl, 0.02% sodium azide, 2 mM EDTA, 2 mM ethylene glycol-bis(β-amino ethyl ether)-N,N,N′,N′-tetracetic acid (EGTA), and a cocktail of protease inhibitors (1 mM phenanthrolene, 0.12 trypsin inhibitor unit (TIU)/ml aprotinin, 100 μg/ml N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), 40 μg/ml each of Nα-p-Tosyl-L-lysine chloromethyl ketone (TLCK) and bestatin, 50 μg/ml benzamidine, 10 μg/ml each of leupeptin, pepstatin A, antipain, soybean trypsin inhibitor, chymostatin, and iodoacetamide). Samples containing an equal amount of proteins (assessed by the BioRad method) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted to an Immobilon-P membrane. Membranes were blocked with 25 ml of TBS containing 2% BSA and 3% dry milk, pH 7.4, for one hour. Primary antibody in blocking buffer (1:1000) was added and incubation continued for another hour. After washing in 0.1% Tween 20 in PBS, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibodies (1:5000) for 1 h. Membranes were washed further in 0.3% Tween 20 and 0.1% Tween 20 three times each. Bands were visualized by enhanced chemiluminescence (ECL) using the ECL kit (Amersham).

Statistical analysis

Statistical analysis was performed using Student's t-test. Each experiment was performed at least twice. Unless otherwise indicated, the data are presented as mean ± SEM.

RESULTS

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

Expression of NCadΔC inhibits cell-cell adhesion among MC3T3-E1 osteoblastic cells

Expression of the truncated, mutant cadherin NCadΔC in stable transfected or transduced MC3T3-E1 cells was examined at both mRNA and protein levels. As shown in Fig. 1 (left panel), NCadΔC mRNA (a band of 0.85 kb or 1.25 kb, depending on the plasmid used) was selectively present in MC3T3-E1 cells either transduced with pMV7 virus or transfected with the pcDNA3 plasmid carrying the NCadΔC insert (MC3T3-E1-NCadΔC) but not in control cells transfected with empty vectors. The levels of endogenous cadherin-11 (a band of 4.3 kb) were not different between control and NCadΔC-expressing cells. Endogenous N-cadherin mRNA (a band of 4.5 kb) also was not altered in virus transduced cells, although it appeared to be elevated in NCadΔC MC3T3-E1 cells transfected with the pcDNA3 vector (Fig. 1, left panel). Expression of NCadΔC protein in cell transfectants was confirmed by Western analysis using an antibody that recognizes the intracellular tail of N-cadherin and therefore reacts with both endogenous N-cadherin and the truncated NCadΔC mutant. A band of approximately 45 kDa, corresponding to the truncated NCadΔC, was detected in cells carrying the dominant negative construct but not in control cells (Fig. 1, right panel). Although the abundance of NCadΔC was apparently higher in virus transduced cells, the effects of NCadΔC expression on osteoblast function are similar in these two cell lines. The abundance of endogenous N-cadherin protein (two bands at approximately 125 kDa and 140 kDa) was not altered by the expression of NCadΔC (Fig. 1, right panel).

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Figure FIG. 1. Expression of NCadΔC mRNA and protein in MC3T3-E1 cells. (Left panel) PolyA RNA purified from MC3T3-E1 cell lines either infected with pMV7 or transfected with pcDNA3 expression vectors containing the NCadΔC insert or no insert, as indicated, was blotted on nylon membranes and hybridized with a [32P]-labeled cDNA probes specific for mouse N-cadherin (N-cad), which also hybridizes the truncated mutant NCadΔC. The membrane was washed and sequentially rehybridized with probes for cadherin-11 (cad11) and GAPDH as indicated. (Right panel) Whole cell lysates were separated by SDS-PAGE and blotted with an antibody that recognizes the intracellular tail of N-cad. The solid arrow indicates the truncated NCadΔC; the dotted arrows point to the bands corresponding to endogenous cadherins.

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Having confirmed the expression of NCadΔC in the two cell lines, we next examined whether calcium-dependent cell-cell adhesion was curtailed in these cells, as a proof of the dominant negative action of the cadherin mutant. As shown in the fluorescent micrographs of Fig. 2, the number of PKH26-labeled cells adherent to an unlabeled cell substratum after 1 h of incubation in calcium-containing medium was lower in NCadΔC-expressing cells than in control, vector-transfected cells. Quantitation of adherent cells showed that cell-cell adhesion was approximately 50% lower in NCadΔC transfectants relative to empty vector-transfected cells (Fig. 2, right), confirming the dominant negative action of NCadΔC. Although a change in morphology has been reported in SaOS-2 osteogenic sarcoma cells after introduction of NCadΔC,(9) we did not observe substantial morphological alterations in MC3T3-E1 cells expressing the mutant cadherin (data not shown). Cell proliferation measured by [3H]thymidine incorporation was 17% lower in MC3T3-NCadΔC (43,906 ± 2,284 cpm) relative to vector-transfected cells (52,707 ± 2,269 cpm; p < 0.01). However, this modest antiproliferative effect only marginally delayed the time to reach confluence. At confluence, there was no difference in cell number between MC3T3-NCadΔC and control cells.

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Figure FIG. 2. Expression of NCadΔC inhibits calcium-dependent cell-cell adhesion. NCadΔC-transfected or control MC3T3-E1 cells were labeled with the membrane-permanent fluorescent dye, PKH26, and laid on top of a confluent, unlabeled cell layer of the same cell line. After 1-h incubation in calcium-containing medium, nonadherent cells were removed by gentle washing. (Left panel) Representative fluorescent micrographs of PKH26-labeled adherent cells. (Right panel) Number of adherent cells per microscopic field in the two cell lines, determined in six fields. *p < 0.01 (t-test for unpaired samples).

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NCadΔC expression alters osteoblast differentiation and function

We next examined the consequences of expressing NCadΔC on osteoblast differentiation. First, we analyzed alkaline phosphatase activity by control MC3T3-E1 and MC3T3-NCadΔC cells during maturation in mineralizing medium as well as in response to rhBMP-2, an inducer of osteoblast differentiation. Basal alkaline phosphatase activity was not different between transfected and control cells during the first 2 weeks in culture, but it declined in NCadΔC-expressing cells after 3 weeks and 4 weeks (Fig. 3). Similarly, rhBMP-2 stimulation of alkaline phosphatase activity was 2.0-fold of control in MC3T3-NCadΔC cells compared with the 2.9-fold in control cells after 5-day stimulation, corresponding to approximately 50% reduction in response to rhBMP-2 (Fig. 4).

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Figure FIG. 3. Effect of NCadΔC expression on basal alkaline phosphatase activity. Cells were cultured in 24-well plates for 1-4 weeks. Alkaline phosphatase activity was measured in the cell layer and normalized to cellular protein content. *p < 0.001 versus corresponding empty vector.

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Figure FIG. 4. Effect of NCadΔC expression on rhBMP-2-stimulated alkaline phosphatase activity. Alkaline phosphatase was measured in the two cell lines grown for 8 days in growth medium followed by 5 days in the medium with or without rhBMP-2 (100 ng/ml). *p < 0.01 versus control,@p < 0.05 versus empty vector + rhBMP-2 (t-test for unpaired samples).

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Expression of osteopontin, osteocalcin, and bone sialoprotein also was altered by overexpression of the dominant negative cadherin. After the cells have reached confluence (1 week), steady-state level of osteopontin mRNA was higher in cells expressing NCadΔC, whereas bone sialoprotein mRNA was not different compared with control cells (Fig. 5). In longer-term cultures of MC3T3-E1 cells maintained in mineralizing medium containing ascorbic acid and β-glycerophosphate, down-regulation of osteocalcin mRNA persisted over 4 weeks (Figs. 6 and 7). Inhibition of bone sialoprotein mRNA, which was not evident after the first week, also was observed from week 2 to week 4 in NCadΔC cells. Similarly, type I collagen mRNA was decreased after 2-4 weeks in MC3T3-NCadΔC compared with control cells. By contrast, the initial up-regulation of osteopontin mRNA was not seen after 2 weeks in culture, and its abundance was similar in MC3T3-NCadΔC and control cells up to 4 weeks. Consistent with the mRNA data, Western analysis showed a decrease in bone sialoprotein abundance over a 4-week period in MC3T3-NCadΔC relative to control cells. Variable results were obtained with osteopontin, which was apparently more abundant in MC3T3-NCadΔC cells relative to control cells at 1 week and 4 weeks of culture but not at the intermediate time points (Fig. 8). Expression of NCadΔC also inhibited the induction of bone sialoprotein and osteopontin by rhBMP-2, whereas stimulation of type I collagen was not altered by introduction of the dominant negative cadherin mutant (Fig. 9). The latter experiments also confirmed the lower bone sialoprotein and type I collagen and higher osteopontin protein levels in MC3T3-NCadΔC cells in the absence of rhBMP-2.

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Figure FIG. 5. Bone matrix protein mRNA in NCadΔC-expressing cells. MC3T3-E1 cells transfected with pcDNA3 expression vectors containing either the NCadΔC insert or no insert were cultured in growth medium for 1 week. PolyA RNA was extracted, blotted on nylon membranes, and sequentially hybridized with [32P]-labeled cDNA probes specific for mouse N-cadherin (NCadΔC), bone sialoprotein (BSP), osteopontin (OPN), and GAPDH.

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Figure FIG. 6. Bone matrix protein mRNA accumulation in long-term cultures of MC3T3-E1 cells expressing NCadΔC. After culturing in growth medium for 1 week, confluent cultures were incubated in medium containing β-glycerophosphate and ascorbic acid the rest of the culture period. PolyA RNA was extracted at weekly intervals from equal number of dishes, blotted on nylon membranes, and sequentially hybridized with [32P]-labeled cDNA probes specific for osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN), α1-chain of type I collagen (Col-I), and GAPDH.

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Figure FIG. 7. Changes in mRNA for osteoblast secretory products in MC3T3-E1 cells expressing NCadΔC relative to vector-transfected cells. Cells were cultured for 2, 3, or 4 weeks and processed for mRNA extraction as detailed in Fig. 6. The densities of the hybridizing bands for each matrix protein in three different Northern blots were normalized to that of GAPDH bands and the density ratio between NCadΔC and control vector-transfected cells was calculated. Data are averages ± SD.

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Figure FIG. 8. Abundance of bone sialoprotein (BSP) and osteopontin (OPN) protein in cultures of MC3T3-E1 cells expressing NCadΔC. Cells were cultured for 1-4 weeks in growth medium. Whole cell lysates were separated by SDS-PAGE, transferred to Immobilon-P membranes, and blotted with antibodies against osteopontin or bone sialoprotein. Bands were visualized by chemiluminescence after incubation with peroxidase-conjugated secondary antibodies.

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Figure FIG. 9. Bone matrix protein content in rhBMP-2-stimulated MC3T3-E1 cells expressing NCadΔC. Cells were cultured for 1 week in growth medium followed by a 5-day incubation in the absence (control) or in the presence of rhBMP-2. Whole cell lysates were separated by SDS-PAGE, transferred to Immobilon-P membranes, and blotted with antibodies against osteopontin, bone sialoprotein, and type I collagen α-chain. Bands were visualized by chemiluminescence after incubation with peroxidase-conjugated secondary antibodies.

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As the final assessment on osteoblast differentiation, we studied the effect of overexpressing the dominant negative cadherin on the calcification potential of MC3T3-E1 cells. Incorporation of45Ca into the extracellular matrix after 4 weeks in mineralization medium was reduced by 35% in the NCadΔC-expressing cultures compared with empty vector-transfected cells (Fig. 10). There was no difference in cell number between the two cell types, as measured by cell counting in parallel cultures.

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Figure FIG. 10. Expression of NCadΔC inhibits incorporation of calcium in the extracellular matrix by MC3T3-E1 cells. Cell lines expressing NCadΔC or an empty vector were cultured in the presence of β-glycerophosphate and ascorbic acid for 4 weeks. Six hours before the end of culture, cells were incubated with medium containing45Ca. After washing,45Ca in the cell layer was extracted with acid and radioactivity was counted. *p < 0.05 (t-test for unpaired samples).21

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DISCUSSION

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

In these studies, we show that introduction of a dominant negative N-cadherin mutant alters normal osteoblast gene expression and inhibits matrix mineralization by MC3T3-E1 cells. We also show that NCadΔC limits the ability of these cells to mount a normal response to rhBMP-2 in terms of increased alkaline phosphatase activity and expression of osteopontin and bone sialoprotein. Therefore, cadherin-mediated cell-cell interactions are important for the development of fully functional osteoblasts.

The dominant negative, truncated NCadΔC and other similar N-terminal deleted N-cadherin constructs have been used successfully by other investigators to inhibit cadherin-mediated cell-cell adhesion in tissue cultures,(17,18) embryos,(11,19) and transgenic animals.(12,20) Consistent with these observations, we found that introduction of NCadΔC in the immature MCT3T-E1 cells significantly reduces calcium-dependent cell-cell adhesion and alters the development of fully mature osteoblasts. Although the lower abundance of type I collagen, osteocalcin, and bone sialoprotein in long-term cultures indicates a reduced differentiation potential of MC3T3-NCadΔC cells, the most critical finding of our studies is the significantly decreased incorporation of calcium into the extracellular matrix. The subtle but clear disruption of the finely tuned expression of matrix proteins critical for matrix maturation, such as osteopontin, bone sialoprotein, and osteocalcin, may explain the reduced mineralization by NCadΔC expression. Bone sialoprotein serves as a nucleation center for matrix mineralization,(21) whereas osteocalcin and osteopontin generally inhibit mineral deposition.(22,23) Because the balance among these three matrix proteins appears to be important for normal matrix mineralization to proceed, reduced production of bone sialoprotein is consistent with reduced matrix calcification in NCadΔC-expressing cultures. Although osteocalcin level also is decreased, a condition that would favor mineral deposition, the associated increase of osteopontin, though transient, may contribute to the overall reduced mineral deposition in NCadΔC-expressing cells, along with reduced type I collagen production.

It has been reported that MC3T3-E1cells can secrete BMP-2 and BMP-4 into the matrix and that matrix-associated BMPs are essential for the differentiation of these cells.(24) It is possible that the inhibitory effect of NCadΔC on alkaline phosphatase activity and bone sialoprotein we observed in the absence of exogenous BMP-2 is in part mediated by inhibition of action of matrix-associated BMPs. These and other observations support a mechanistic role of cadherin-mediated cell-cell interactions for osteoblast differentiation and function. Perturbation of cadherin-mediated cell-cell interaction by either inhibitory peptides(4,9) or by expression of a dominant negative cadherin(9) (this study) hinders basal and rhBMP-2-induced expression of osteoblastic genes in human and murine osteoblastic models. Furthermore, Hay and coworkers were able to inhibit BMP-2 induction of alkaline phosphatase activity, osteocalcin, and the osteoblast differentiation transcription factor, Cbfa1, using specific antibodies against N- and E-cadherins.(10)

The exact mechanisms by which cell-cell interactions regulate expression of bone matrix proteins remain elusive. The cytoplasmic domain of cadherins, which mediates interactions with cytoskeletal proteins, is highly conserved and data in other cell systems indicate that truncated cadherin mutants lacking the extracellular domains, including NCadΔC, prevent lateral clustering and dimerization of endogenous cadherins, thus inhibiting the formation of the adhesion complex.(17,25) There also is evidence that NCadΔC competes with endogenous cadherins for binding to β-catenin.(11) Because β-catenin can function as a transcriptional factor, interference with cellular translocation of β-catenin is an obvious potential mechanism of abnormal gene expression regulation observed in NCadΔC-expressing cells. This tantalizing hypothesis will require further work. Based on its mechanism of action, it is very likely that the inhibitory effect on osteoblast function obtained by introduction of the dominant negative construct is the consequence of a global loss of cadherin-mediated adhesion rather than interference with a specific cadherin. In support of this contention is our finding that expression of NCadΔC in MC3T3-E1 cells did not reduce the abundance of endogenous N-cadherin or cadherin-11, as we anticipated from previous reports in other cell systems.(11) Perturbation of expression or function of individual cadherins is necessary to dissect the role of each of these cell adhesion molecules for osteoblast function.

Growing evidence indicates that cadherins are key regulators of bone development and remodeling. N-cadherin is required for mesenchymal condensation and chondrogenesis in the early phases of embryonic limb bud development.(26,27) Cadherin-11, which is highly expressed in mesenchymal cells,(5) also is present in somites and limb buds at sites of embryonic bone formation.(28,29) Moreover, Mbalaviele and coworkers showed that cadherins also mediate heterotypic interactions between the osteoblast and osteoclast cell lineages,(30) and that HAV peptides inhibit osteoclast activity.(31) This effect may result from prevention of either fusion of osteoclast precursors(31) or sealing zone formation.(32) Therefore, cell-cell contact and adhesion via cadherins appears to be important physiologically in both arms of bone remodeling. Identifying the specific roles of different cadherins for the function of bone cells and uncovering the signaling mechanisms linking cell-cell adhesion to gene expression are the challenges for future research.

ACKNOWLEDGMENTS

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

This work is supported by NIH-NIAMS grants AR43470 and AR32087.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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    Takeichi M 1994 The cadherin cell adhesion receptor family: Roles in multicellular organization and neurogenesis. Prog Clin Biol Res 390:145153.
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    Takeichi M 1995 Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7:619627.
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