Institut National de la Santé et de la Recherche Médicale 1 U-443 and 2 U-394, 33076 Bordeaux Cedex, France
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ABSTRACT |
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Bone development and remodeling depend on
complex interactions between bone-forming osteoblasts and other cells
present within the bone microenvironment, particularly vascular
endothelial cells that may be pivotal members of a complex interactive
communication network in bone. Our aim was to investigate the
interaction between human umbilical vein endothelial cells (HUVEC) and
human bone marrow stromal cells (HBMSC). Cell differentiation analysis
performed with different cell culture models revealed that alkaline
phosphatase activity and type I collagen synthesis were increased only
by the direct contact of HUVEC with HBMSC. This "juxtacrine
signaling" could involve a number of different heterotypic connexions
that require adhesion molecules or gap junctions. A dye
coupling assay with Lucifer yellow demonstrated a functional coupling
between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC
but also in the endothelial cell network and that these two cell types
can communicate via a gap junctional channel constituted at least by
Cx43. Moreover, functional inhibition of the gap junction by
18-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC
cocultures on HBMSC differentiation. This stimulation could be mediated
by the intercellular diffusion of signaling molecules that permeate the
junctional channel.
coupling; intercellular messenger; connexin43; osteoblast; endothelial cells
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INTRODUCTION |
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ANGIOGENESIS is a tightly regulated process involved in growth, repair, and bone remodeling (9, 11-13, 23, 28, 50, 54, 56). Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, in which systemic hormones and paracrine growth factors or cytokines play an active role (4, 24, 53, 55). In a previous study (51), we showed that osteoblast progenitor cells [human bone marrow stromal cells (HBMSC)] behave differently in terms of proliferation and differentiation when cultured in association with endothelial cells [human umbilical vein endothelial cells (HUVEC)] in various coculture models (coculture with or without direct contact, conditioned medium) than when they are cultured alone. An increase in alkaline phosphatase activity (Al-P) was observed only when HBMSC were cocultured in direct contact with HUVEC. The enhancement of this early osteoblastic marker is not provided when HBMSC and HUVEC are cocultured separately with the use of a semipermeable membrane (51) or when HBMSC are seeded onto a matrix obtained from HUVEC cultures (51).
Therefore, the intercommunication between endothelial cells and osteoblast-like cells not only may require diffusible factors but also may involve junctional communications to form a multicellular network (6, 37). Gap junction communications between osteoblastic cells mediate intracellular exchanges of regulatory ions and small molecules (molecular mass <1 kDa), which allows metabolic cooperation between adjacent cells and control cell differentiation and growth (14, 15, 31, 33, 37, 43, 44, 57). Cell-to-cell communication could be critical to the coordinated cell behavior necessary for bone development and remodeling. Lecanda et al. (31) have demonstrated that gap junction communication modulates transcriptional activity of osteoblast-specific promoters, thus pointing to a fundamental physiological role of intercellular communication for the function of specialized tissues such as bone. The major gap junction protein connexin43 (Cx43) (20) has been identified in osteoblastic (14, 15, 36, 37, 57, 61) and endothelial cells (5, 10, 22, 27, 60). Moreover, heterotypic gap channels may be present between endothelial cells and vascular smooth muscle cells in arteriole and sinusoid walls (8, 16, 48, 58), and some ultrastructural and morphological studies have demonstrated the existence of heterotypic gap junction channels between microvascular cells and osteocytes (41) or other cell types (5). Several studies have shown that many factors regulate the number, localization, nature, and function of gap junctional channels. These factors may be physical (10, 22, 27), physicochemical (14), ionic (15), hormonal, and cellular (5, 14, 37, 38).
In the present study, we focused on cell-to-cell interactions between endothelial cells and osteoprogenitor cells to explain the effect of HUVEC on the HBMSC differentiation observed only when these two cell types are cocultured with direct contact (51). Our results provide new evidence pointing to the function of endothelial mesenchymal cell interactions via gap junctional communication for the development of bone-forming cells.
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MATERIALS AND METHODS |
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Cell Culture
Endothelial cell culture. Endothelial cells were extracted from human umbilical veins essentially as described by Bordenave et al. (3), according to the procedure of Jaffe (26). Cell cultures were maintained in Iscove's modified Dulbecco's medium (IMDM) (GIBCO BRL, Gaithersburg, MD) with 20% (vol/vol) FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 90 µg/ml heparin, 20 µg/ml endothelial cell growth supplement (Sigma-Aldrich Diagnostics, St. Louis, MO). Cell characterization was evaluated either by the assessment of von Willebrand factor (vWF) expression, detected by immunocytochemical reaction with the use of DAKO-EPOS anti-vWF coupled with horseradish peroxidase (HRP) (DAKO DAB chromogene tablets) or by uptake of fluorescent acetylated low-density lipoprotein (DiI-Ac-LDL) by the "scavenger cell pathway" of LDL according to the operative conditions of Voyta et al. (52).
HBMSC culture.
Human bone marrow was obtained by aspiration from the femoral diaphysis
or iliac bone from patients (age 20-70 yr) undergoing hip
prosthesis surgery after trauma. Their informed consent was obtained.
Cells were separated into a single suspension by sequential passage
through syringes fitted with 16-, 18-, and 21-gauge needles, cultured
in IMDM supplemented with 10% (vol/vol) FCS, 100 U/ml penicillin, and
100 µg/ml streptomycin, and incubated in a humidified atmosphere of
95% (vol/vol) air-5% (vol/vol) CO2 at 37°C (42, 49). Dexamethasone at 108 M was added to the
culture medium during the first 2 wk of culture to induce alkaline
phosphatase stimulation and mineralization of adherent cells
(1). At the first subculturing, the cells were tested for
their intracellular Al-P activity as described previously
(2) with the Sigma diagnostic kit (85L-2). HBMSC also were
characterized by immunocytochemical detection of intracytoplasmic type
I collagen and osteocalcin (Takara Biomedicals). Moreover, additional
markers besides the latter (osteopontin, calcium-binding proteins,
osteonectin) (49) and their responsiveness to vitamin D3 [1,25(OH)2D3 or
dihydroxycholecalciferol], parathyroid hormone, transforming
growth factor-
(TGF-
), and bone morphogenetic protein 3 (BMP3)
have previously been studied (17, 18).
Coculture of HBMSC and HUVEC with direct contact. HBMSC were plated in 24-multiwell dishes at 104 cells/well in 0.5 ml of IMDM containing 10% (vol/vol) FCS and antibiotics as described above. During the same seeding phase, HUVEC were added to each well at 2 × 104 cells/well in IMDM containing 10% (vol/vol) FCS without growth factors or dexamethasone for direct cocultures.
Quantitative Measurement of Al-P Activity
Intracellular Al-P activity was determined as described by Majeska and Rodan (35) in the different culture conditions. Data are expressed as ratios of nanomoles of inorganic phosphate (Pi) cleaved by the enzyme in 30 min per microgram of protein. The quantitative measurement of cellular proteins was done by using the method of Lowry et al. (34).Quantitative Measurement of Type I Collagen Synthesis
Collagenous protein synthesis was quantified by L-[5-3H]proline (10 mCi/ml; Amersham, Amersham, UK) incorporation in the culture medium for 24 h. After being washed with 0.1 M phosphate-buffered saline (PBS), pH 7.4, labeled materials were extracted from the cells and immunoprecipitated by using a monoclonal antibody against type I collagen incubated overnight at 4°C. Immunocomplex was trapped by adding 100 µl of washed protein A-agarose bead (Upstate Biotechnology) slurry for 2 h at 4°C. Agarose beads were collected by pulsing in the microcentrifuge and washed three times, and the radioactivity retained was counted with a beta counter (Packard).Western Blot Analysis
Cells (HBMSC and HUVEC) were grown to confluence, and crude extract proteins were then obtained in 0.1 M ice-cold PBS, pH 7.4, containing protease inhibitors (5 mM EDTA, 0.3 M benzaminidiumchloride, 0.1 mM iodoacetamide, 4.5 mM phenylmethylsulfonyl fluoride, and 0.1 M 6-amino hexanoic acid) before being subjected to Western blot analysis. Proteins (20 µg/lane) were separated by 10% SDS-PAGE in reducing conditions according to Laemmli (30). Samples were transferred onto nitrocellulose membrane, and the free binding capacity of the membrane was blocked with 3% bovine serum albumin (BSA) in 0.1 M PBS, pH 7.4, for 2 h at 37°C. Cx43 was detected with rabbit polyclonal antibodies (Chemicon, Euromedex) diluted 1:1,000 and revealed with anti-rabbit immunoglobulin G (IgG) linked to HRP (Chemicon, Euromedex) diluted 1:20,000. Loading control was performed by using monoclonal antibodies againstImmunofluorescence Labeling
Cx43 and vWF were localized using indirect immunofluorescence. HBMSC and HUVEC cultured alone and cocultured HBMSC/HUVEC were fixed in 4% (wt/vol) paraformaldehyde for 20 min at 4°C and rinsed in Tris · HCl [50 mM Trisma base, pH 7.4, buffered with HCl (TB)]. Fixed cells were incubated for 30 min in TB containing 1% (wt/vol) BSA and then for 90 min with a monoclonal antibody against Cx43 (Chemicon, Euromedex) diluted 1:200 in TB and/or with a polyclonal antibody against vWF diluted 1:250 in TB, pH 7.4. Subsequently, cells were washed in TB and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch) diluted 1:100 or with rhodamine-conjugated anti-rabbit IgG (Jackson Immunoresearch) diluted 1:100 for 1 h. Cells were rinsed in TB, and cultures were coverslipped with Vectashield (Vector Laboratories) to prevent bleaching. Cultures were examined with an Axiophot microscope (Zeiss) equipped with the appropriate epifluorescence filter sets. Controls were performed without the primary antibody, and cross-reactivity of the secondary antibodies to mouse and rabbit IgG also were investigated.Intercellular Dye Transfer and Staining
Coupling was examined in cells bathed in a control saline solution, pH 7.4, containing 124 mM NaCl, 15 mM KCl, 1.3 mM MgSO4, 1.25 mM KH2PO4, 26 mM NaHCO3, 1.8 mM CaCl2, and 11 mM glucose. For dye coupling experiments, we used patch pipettes with a resistance of 5-10 MRT-PCR Analysis
Total RNA was extracted from HBMSC and HUVEC by using the RNeasy Total RNA kit (Qiagen), and 2 µg were used as template for single-strand cDNA synthesis with the Superscript preamplification system (GIBCO BRL) in a 20-µl final volume containing 20 mM Tris · HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml BSA, 10 mM dithiothreitol (DTT), 0.5 mM each of dATP, dCTP, dGTP, and dTTP, 0.5 µg of oligo(dT)12-18, and 200 units of reverse transcriptase. After incubation for 50 min at 42°C, the reaction was stopped at 70°C for 15 min and kept on ice. Synthesized cDNA were treated with 2 units of RNase H (Escherichia coli) at 37°C for 20 min, and samples were mixed with PCR cocktail containing 20 mM Tris · HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 0.1 µg/µl BSA, 10 mM DTT, 0.5 mM each of dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq polymerase, and 0.5 µM each of forward and reverse primer (Table 1) in a 50-µl final volume. Amplification was performed in a HYBAID thermocycler under the following conditions: denaturation at 94°C for 5 min, 30 cycles of 94°C for 10 s, hybridization at melting temperature (indicated in Table 1) for 30 s, followed by a final extension step at 72°C for 1 min. To control for the integrity of the various RNA preparations, the expression of
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Gap Junction Channel Inhibition Assay
Assay with 18-glycyrrhetinic acid.
HBMSC and HUVEC were plated in 24-multiwell dishes at 104
cells/well and 2 × 104 cells/well, respectively, in
IMDM supplemented with 10% (vol/vol) FCS with antibiotics. Four hours
after seeding, 18
-glycyrrhetinic acid (18
-GA; Sigma-Aldrich
Diagnostics) was added to the culture medium to obtain a concentration
of 100 µM (18
-GA stock solution was at 50 mM in DMSO) (48,
58). Thereafter the medium was changed every day and
supplemented or not with 18
-GA (100 µM) for 1, 3, and 6 days. At
each time indicated, the medium was removed and the cells were washed
twice with 0.1 M PBS, pH 7.4, and then scraped and sonicated for
30 s at 20 kHz and 8% amplitude (Vibra-Cell; Bioblock-Scientific)
in 300 µl of 0.1 M PBS, pH 7.4. They were then frozen at
80°C
until quantitative assays of total protein content and Al-P activity
were performed.
Assay with oligodeoxyribonucleotide antisense Cx43.
HBMSC and HUVEC were plated in 24-multiwell dishes at 104
cells/well and 2 × 104 cells/well, respectively, in
IMDM supplemented with 10% (vol/vol) FCS with antibiotics. Four hours
after seeding, phosphorothioated sense oligodeoxyribonucleotide (ODN)
of Cx43 (TAC CCA CTG ACC TCG CGG) or antisense ODN of Cx43 (GGC GCT CCA
GTC ACC CAT) (ODN-Cx43; Genset, Paris, France) were added to the
culture medium to obtain a concentration of 10 µM. Thereafter, the
medium was changed every day and supplemented or not with sense or
antisense ODN-Cx43 (10 µM) for 3 and 6 days (19). At
each time, the medium was removed and the cells were washed twice with
0.1 M PBS, pH 7.4, and then scraped, sonicated for 30 s at 20 kHz
and 8% amplitude (Vibra-Cell) in 300 µl of 0.1 M PBS, pH 7.4, and
frozen at 80°C until quantitative assays of total protein content
and Al-P activity were performed.
Statistical Analysis
The data were expressed as means ± SD and were analyzed using the nonparametric U-test of Mann and Whitney (used to compare observed means on a few independent samples). ![]() |
RESULTS |
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Effects of HUVEC on HBMSC Differentiation in Coculture With Direct Contact
As shown in Fig. 1 and as previously identified (49), osteoblastic markers were detected in HBMSC obtained from the first subculture. Cytochemical analysis revealed that HBMSC expressed Al-P activity (Fig. 1a), synthesized type I collagen (Fig. 1c), and began to express and synthesize osteocalcin after the first passage, which was then secreted in the matrix (Fig. 1e). The other osteoblastic markers (osteonectin and osteopontin) and their response to 1,25(OH)2D3, BMP3, and TGF-
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When Al-P-positive HBMSC and vWF-positive HUVEC (Fig.
2A) were cultured with direct
contact for 3, 6, and 9 days, the Al-P activity increased greatly (Fig.
2B). Type I collagen synthesis, which is concomitant to Al-P
activity expression in bone cell cultures (45), also was
investigated in these cocultures by using [3H]proline
incorporation followed by an immunoprecipitation with an antibody
against type I collagen. When HBMSC and HUVEC were cultured with direct
contact for 3, 6, and 9 days, type I collagen synthesis increased with
time of coculture (Fig. 2C), as Al-P enzymatic activity did
in the same coculture. In this cell culture model, the endothelial
cells could be considered as being "osteoinductive" mediators
instead of acting at the level of bone cell maturation.
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We next explored the effect of cell seeding density on
osteoblastic phenotype potential (Fig.
3). The optimal effect of HUVEC on HBMSC
Al-P activity was obtained at 3 days, for a HUVEC seeding density of
5-10 × 103 cells/cm2 cocultured with
HBMSC, themselves seeded at 5 × 103
cells/cm2. This effect was maintained at days 6 and 9 for the same HBMSC seeding density (5 × 103 cells/cm2) and also was maintained for
higher HUVEC seeding densities such as 15 × 103
cells/cm2 when Al-P activity was normalized for protein
content.
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Functional Coupling Exists Between HUVEC and HBMSC
Because no effect of HUVEC was noted with a semipermeable membrane (51), we investigated the gap junctional communication between HUVEC and HBMSC. Lucifer yellow dye transfer confirmed that HBMSC were coupled homotypically (Fig. 4, a and b), as were HUVEC (Fig. 4, c and d). Interestingly, in coculture (Fig. 4, e-p), immunostaining of vWF in HUVEC (Fig. 4, g, j, m, and p) performed a few minutes after the dye transfer experiment shows that HBMSC and HUVEC also were coupled heterotypically (Fig. 4, f, i, l, and o).
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In bone cells, the predominant gap junction protein is Cx43,
which plays a fundamental role in osteoblast differentiation and also
has been formally identified in endothelial cells (22). This cellular intercommunication between HUVEC and HBMSC was strongly suggested by the expression of Cx43 mRNA in both cell types (Fig. 5A). Moreover, both Western
blot with anti-Cx43 (Fig. 5B) and fluorescence staining of
Cx43 performed on confluent HBMSC (Fig. 6a) or confluent HUVEC (Fig.
6b) demonstrated that these cells synthesized these
proteins. In both cell types (Fig. 6, a and b)
under indirect immunofluorescence, Cx43 appeared mainly in bright,
punctate regions located where cells were close together, probably
corresponding to gap junctions. Double staining of Cx43 (Fig. 6,
c, f, and i) and vWF (Fig. 6,
d, g, and j) were performed on the
coculture (Fig. 6, e, h, and k), and
immunolabeling of Cx43 was identified in neighboring cells either both
negative to vWF or both positive to vWF. In some areas of the
coculture, a specific labeling of Cx43 between a vWF-positive cell and
a vWF-negative cell was observed, which suggested gap junctional coupling between HBMSC and HUVEC (Fig. 6, c-k).
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HUVEC Effect on Osteoblast Differentiation is Mediated by Gap Junctions
Because Cx43 was able to constitute gap junctional communication between HUVEC and HBMSC, we further investigated the function of these gap junctions in this signaling pathway.Functional gap junction inhibition.
Glycyrrhetinic acid (GA) is a lipophilic aglycone isolated from
licorice root, with a steroidal structure that can occur in - and
-isoforms. Dye transfer studies have shown that 18
-GA reversibly
interrupts gap junctional communication in cultured cells at micromolar
concentrations. This effect remains specific for 18
-GA even
at concentrations up to 100 µM (48, 58). At this
concentration, 18
-GA inhibits communication in HBMSC and HUVEC, as
shown by using Lucifer yellow dye transfer (data not shown), and this
reduced communication significantly decreased the HUVEC effect on HBMSC
Al-P activity by ~76% at day 3 after the coculture and by
~82% at day 6 (Fig. 7).
Concerning HBMSC cultured alone, 18
-GA induced a weak but
significant decrease in enzymatic activity after day 3 of
treatment but not at day 6. As also demonstrated by Schiller
et al. (44), disturbance of gap junctions in osteoblastic
cells by 18
-GA results in decreased Al-P activity.
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Gap junction synthesis inhibition.
To directly inhibit gap junctional channels composed of Cx43, we used
antisense ODN-Cx43. Addition of antisense-ODN-Cx43 at 10 µM inhibited protein synthesis, as demonstrated by
immunocytochemistry performed on HBMSC (data not shown), and decreased
the HUVEC effect on HBMSC-Al-P activity (Fig.
8) by ~82% at day 3 after
the direct contact and by ~79% at day 6 compared with
untreated cells. Both antisense ODN-Cx43 and sense ODN-Cx43 did not
exert any significant effect on HBMSC cultured alone. Moreover, the
effect of sense ODN-Cx43 on HBMSC Al-P activity in coculture is not
significant compared with that in untreated cells. The decrease
observed in HBMSC Al-P activity between sense and antisense treatment
remains significant at day 6 (P < 0.001).
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DISCUSSION |
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Gap junctions provide a fundamental regulatory mechanism controlling cell proliferation and gene expression in tissues whose specialized function requires the synchronization of multicellular activity (31, 33, 63). Coordination of gene expression during osteoblast differentiation and bone remodeling represents an excellent model for the physiological role of gap junctional intercellular communication (31). Palazzini et al. (41) showed that the cells belonging to the osteogenic lineage form a continuous protoplasmic network that extends from the osteocytes to the vascular endothelium, passing through osteoblasts and stromal cells in the perimedullary spaces.
The occurrence of gap junctions in the cytoplasmic network forms a functional syncytium-like structure, suggesting that the activity of the cells pertaining to the osteogenic lineage might be regulated not only by diffusion of systemic or paracrine factors through the intercellular fluids but also by signals transmitted through the cytoplasmic network (31, 41, 59). Therefore, inadequate control of intercellular communication among osteoblasts or a reduction in the number of coupling cells that may occur in pathological conditions could contribute to the impaired synthesis of new bone and reduced trabecular wall thickness, which is typical of osteoporotic bone (31). This is strongly supported by a typical feature of osteoporosis in which a reduction in the number of sinusoids and arterial capillaries in the bone marrow (4) is associated with a bone formation deficiency.
Gap junction channels are aqueous intercellular channels that allow the cell-to-cell diffusion of small molecules and ions. Each gap junction pore is formed by the juxtaposition of two hemichannels in adjacent cells, composed of a hexameric array of transmembrane proteins called connexins. While Cx43, Cx40, and Cx37 have been formally identified in endothelial cells (60), only Cx43 and Cx45 have already been reported in osteoprogenitor cells. These connexins have different molecular permeabilities (46) and differing abilities to interact with each other, e.g., gap junction pores formed by Cx45, which are less expressed in osteoblast-like cells, are less permeable than Cx43 pores to negatively charged dyes such as Lucifer yellow, carboxyfluorescein, or calcein. Lecanda et al. (31) hypothesized that the type of gap junctional communication provided by Cx43 allows the full development of mature osteoblastic phenotypes, because they showed the correlation between permeability to negatively charges, which in osteoblasts is mediated by Cx43 gap junctions, and osteocalcin and bone sialoprotein gene transcription. Li et al. (33) recently demonstrated that Cx45 in rat osteosarcoma cells may contribute to altered gene expression as in osteocalcin or Al-P. These results were confirmed by Furlan et al. (21).
Among the connexins expressed by endothelial cells and cells of osteogenic lineage, Cx43 is most closely associated with the initiation of osteogenesis and bone formation. Minkoff et al. (39) suggested that expression of Cx43 appears to precede the overt expression of the osteogenic phenotype (38, 63).
Our results demonstrate for the first time that human endothelial cells and osteoprogenitor cells arising from human bone marrow stromal cells do couple. Moreover, our data demonstrate 1) that this heterotypic gap junctional coupling contributes to the regulation of gene expression in osteoblastic cells and 2) that lack of coupling, including osteoblast to osteoblast but also endothelial cells to osteoblastic cells through gap junctions composed of the Cx43 expressed in both cells, is sufficient to alter osteoblastic marker expression such as Al-P in HBMSC and its increase occurring in direct contact with HUVEC. Together, our data suggest that Cx43 expression in HUVEC and HBMSC contributes to osteoblastic differentiation.
The nature of the mechanism that links gap junctional communication to gene expression remains elusive, but it certainly depends on the type of signals that permeate the junctional channel. This stimulation could be mediated by the intercellular diffusion of signaling molecules such as inositol phosphates as well as propagation of calcium waves (29, 32), which require gap junctional communication. Other second messengers also could be partly involved in the relationship between our endothelial and osteoblastic cell culture models. Inoue et al. (25) reported that the cyclic nucleotides cAMP and cGMP significantly and reciprocally regulate osteocalcin synthesis and AL-P activity and modulate the formation of mineralized nodules by osteoblast-like cells in culture (7). HUVEC could act as a donor of nitric oxide, which is well known to enhance the level of mRNA osteocalcin in osteoblastic cells (40, 62) and the rate of production of intracellular cGMP by activation of soluble guanylate cyclase. The identification of the signal transduction pathways involved in these cocultures is now underway.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Cooke for help in preparing the manuscript. We thank Dr. L. Jaubert (Bel Air Maternity Hospital, Bordeaux, France) for collecting umbilical cords and Dr. A. Durandeau (Pellegrin Hospital, Bordeaux, France) for the bone marrow samples.
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FOOTNOTES |
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This work was supported by a grant from the Fondation pour la Recherche Medicale and Pole Genie Biologique et Medical Aquitaine.
Address for reprint requests and other correspondence: F. Villars, INSERM U-443, Université Bordeaux 2 Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France (E-mail: joelle.amedee{at}bordeaux.inserm.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00310.2001
Received 6 July 2001; accepted in final form 19 November 2001.
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