Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication

Henry J. Donahue, Zhongyong Li, Zhiyi Zhou, and Clare E. Yellowley

Musculoskeletal Research Laboratory, Departments of Orthopedics and Rehabilitation and of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gap junctional channels facilitate intercellular communication and in doing so may contribute to cellular differentiation. To test this hypothesis, we examined gap junction expression and function in a temperature-sensitive human fetal osteoblastic cell line (hFOB 1.19) that when cultured at 37°C proliferates rapidly but when cultured at 39.5°C proliferates slowly and displays increased alkaline phosphatase activity and osteocalcin synthesis. We found that hFOB 1.19 cells express abundant connexin 43 (Cx43) protein and mRNA. In contrast, Cx45 mRNA was expressed to a lesser degree, and Cx26 and Cx32 mRNA were not detected. Culturing hFOB 1.19 cells at 39.5°C, relative to 37°C, inhibited proliferation, increased Cx43 mRNA and protein expression, and increased gap junctional intercellular communication (GJIC). Blocking GJIC with 18alpha -glycyrrhetinic acid prevented the increase in alkaline phosphatase activity resulting from culture at 39.5°C but did not affect osteocalcin levels. These results suggest that gap junction function and expression parallel osteoblastic differentiation and contribute to the expression of alkaline phosphatase activity, a marker for fully differentiated osteoblastic cells.

connexin; alkaline phosphatase; osteocalcin; proliferation; bone


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GAP JUNCTIONS ARE membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules to pass from cell to cell. Studies of gap junctional intercellular communication (GJIC) in carcinogenesis suggest that GJIC is critically important in cell proliferation and differentiation. For instance, neoplastic oncogenes decrease GJIC and expression of connexins, the protein subunits of gap junctions (2). Furthermore, transforming growth factor-beta and retinoids, which inhibit cell proliferation, also can increase GJIC (20, 28), and transfection of gap junction-deficient cells with connexins inhibits proliferation and restores differentiation potential (30, 39). Although data on the role of gap junctions in transformed cells suggest a role for GJIC in cell proliferation and differentiation, the data on gap junctions in normal cell differentiation and proliferation are limited and somewhat inconsistent. For instance, GJIC is decreased in differentiating keratinocytes relative to proliferating keratinocytes (15), whereas GJIC is increased in highly differentiated smooth muscle cells relative to poorly differentiated skin fibroblasts (1). Furthermore, increasing GJIC in cultured hepatocytes maintains cell differentiation (37).

To further address this issue, we examined the role of gap junctions in bone cell differentiation. There are several reasons why bone-forming osteoblasts are useful in examining the role of gap junctions in cell differentiation. Osteoblasts communicate with one another via gap junctions in organ culture (21) and in vitro (3, 4, 10, 33, 34, 36), and recent data suggest that cell-to-cell communication is critical for the coordinated cell behavior necessary in bone tissue development (5, 11, 41) and function (8, 35). Additionally, the particular connexins expressed by osteoblasts have been identified and partially characterized (4, 10, 35, 36). Furthermore, the expression of connexin 43 (Cx43) is increased in more highly differentiated osteoblasts (32) and in osteocytes (22). Finally, we (26) and others (23) have demonstrated that the expression of phenotypic markers characteristic of fully differentiated osteoblasts is decreased in ROS 17/2.8 cells (osteoblast-like rat osteosarcoma cell line) rendered coupling deficient. Therefore, to examine the hypothesis that GJIC contributes to osteoblastic cell differentiation, we characterized gap junction expression and function in differentiating human fetal osteoblastic cells (hFOB 1.19).

hFOB 1.19 are human fetal osteoblastic cells conditionally immortalized with a gene encoding for a temperature-sensitive mutant (tsA58) of the simian virus 40 (SV40) large T antigen (19). At permissive temperatures (37°C) the temperature-sensitive gene is expressed and this cell line proliferates rapidly, whereas at restrictive temperatures (39.5°C) the gene is not expressed and the cells proliferate less rapidly and express increased alkaline phosphatase activity and osteocalcin levels relative to cells cultured at the permissive temperature. Thus to some degree hFOB 1.19 proliferation and differentiation in vitro can be controlled by temperature, providing a useful model to examine the role of gap junctions in osteoblastic differentiation. We found that GJIC and expression of Cx43, the predominant connexin in bone, parallel hFOB 1.19 differentiation. Furthermore, inhibition of GJIC inhibited alkaline phosphatase activity, a phenotypic marker of fully differentiated osteoblasts.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. DMEM and Ham's F-12 were purchased from GIBCO (Life Technologies, Gaithersburg, MD). Menadione, ascorbic acid, 18alpha -glycyrrhetinic acid (GA), DMSO, and alkaline phosphatase enzyme assay kits were purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT), and 1,25-dihydroxy vitamin D3 was purchased from Biomol (Plymouth Meeting, PA). Calcein, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), and Lucifer yellow were from Molecular Probes (Eugene, OR). Horseradish-linked goat anti-mouse IgG was purchased from Bio-Rad (Hercules, CA), and enhanced chemiluminescence (ECL) detection reagents were purchased from Amersham (Amersham, United Kingdom). Osteocalcin immunoradiometric assay (IRMA) kits were purchased from Nichols Institute (San Juan Capistrano, CA, no. 40-2245). Monoclonal antibody against Cx43 was purchased from Zymed (South San Francisco, CA), and cDNA probes for Cx43, Cx32, and Cx26 were prepared previously (25). The cDNA probes for Cx45 were provided by Eric Beyer (University of Chicago). All other reagents were purchased from Sigma and were tissue culture grade.

Cell culture and cell counting. For initial characterization of connexin protein and mRNA expression, hFOB 1.19 cells, provided by Dr. Steven Harris (Bayer, West Haven, CT), in DMEM-Ham's F-12 (1:1) with 10% charcoal-stripped FBS, 1% penicillin-streptomycin, 10-8 M menadione, 100 µg/ml ascorbic acid, and 10-8 M 1,25-dihydroxy vitamin D3 were plated at 8 × 103 cells/mm in 100-mm round tissue culture dishes and cultured to confluency (6 days). For quantification of Lucifer yellow dye spread, hFOB 1.19 cells were plated onto 25-mm-diameter round coverslips at 2 × 105 cells/slide and cultured to confluency.

To examine changes in cell behavior associated with differentiation, hFOB 1.19 cells were plated at 1 × 103 cells/mm into 24-well plates for osteocalcin assays, 24-well plates or 100-mm round tissue culture dishes for alkaline phosphatase assays, 100-mm round tissue culture dishes for Northern and Western blot analysis, or 25-mm-diameter round glass coverslips for dual-label parachute assays and were cultured at 37°C in culture medium. Medium was changed and cells were collected for assay on days 3, 6, 9, and 12. On day 6, one-half of the tissue culture plates were placed in a separate but identical incubator at 39.5°C. These cells were also collected for assay on days 9 and 12. In some experiments, cells were exposed to GA, a gap junction uncoupler (6, 7), or vehicle control (DMSO), with the addition of fresh medium, on days 3, 6, 9, and 12. Cells were trypsinized and counted using a hemocytometer and trypan blue dye exclusion.

Quantification of dye coupling. We used two techniques to quantify GJIC. Lucifer yellow dye spread was utilized as previously described (9, 10) to initially quantify GJIC and the effect of GA on GJIC. Although this technique was useful for assessing GJIC in cells that had just reached confluency, it proved difficult in cells cultured for extended periods of time and beyond confluency. To overcome this problem and thus examine GJIC in cells as they differentiated in culture, we utilized a dual-label parachute technique. This technique is similar to the parachute technique described by Ziambaras et al. (40), but it was modified to better visualize the parachuted cell (17). Cells were simultaneously labeled with 10 µM calcein-AM and 10 µM DiI. Although calcein is able to permeate gap junctions, DiI is impermeant. Labeled cells were then trypsinized, centrifuged at 200 g for 5 min, resuspended in the appropriate growth medium, and counted. Labeled cells were then dropped (parachuted) onto confluent plates of unlabeled cells at a 1:500 ratio of labeled to unlabeled cells. After gap junctions are established between cells in the monolayer and the labeled parachuted cells, calcein transfers through these channels from labeled to unlabeled cells, which then fluoresce green. DiI, which fluoresces red and does not transfer from cell to cell, can thus be used to visualize the original labeled cell. Cells were visualized 45 min after parachuting, using both fluorescein filters (excitation 450-490 nm; emission 520 nm) and rhodamine filters (excitation 546 nm; emission 590 nm) to locate calcein- and DiI-loaded cells, respectively. The number of neighboring cells that calcein had transferred to from one double-labeled parachuted cell was recorded. In some experiments, GA was used to assess the role of gap junctions in calcein transfer. Unlabeled cells were treated with 30 µM GA in medium for 10 min before addition of labeled cells.

RNA isolation and Northern blot analysis. hFOB cells were collected on days 3, 6, 9, and 12, and total RNA was isolated as previously described (25). Briefly, 20 µg of total RNA, as determined by absorption at 260 nm, were subjected to electrophoresis on a 1% agarose-formaldehyde gel. The gel was capillary blotted with 0.1 M sodium phosphate onto membranes (GeneScreen hybridization transfer membrane, DuPont NEN Research Products) and prehybridized for 15 min at 55°C in 1% BSA, 0.35 M sodium phosphate, 7% SDS, and 30% (vol/vol) deionized formamide. The gel was then hybridized overnight in the same solution with [alpha -32P]dCTP-labeled probes for a 1.1-kb Hinc II-Bst fragment of Cx26 cDNA (38), a full-length 1.5-kb cDNA for Cx32 (25), a 0.89-kb Ava II fragment of Cx43 cDNA (13), a full-length 1.2-kb Cx45 or a 1.4-kb Pst I fragment of rat glyceraldehyde-3-phosphate dehydrogenase cDNA (14). The blots were washed once in 150 mM sodium phosphate and 0.1% SDS at room temperature and two more times at 55°C. The abundance of specific mRNAs was determined with a PhosphorImage system. For graphical representation, the membranes were exposed to Kodak X-OMAT AR film for various times.

Quantitative real-time RT-PCR. hFOB cells were cultured for 12 days at either 37 or 39.5°C. RNA was then isolated using a commercially available kit (RNeasy, Qiagen). Real-time RT-PCR was accomplished with a Perkin-Elmer ABI Prism 7700 sequence detection system. This technology is based on the TaqMan PCR core reagent kit (Perkin-Elmer), which allows for the detection of PCR product concentration in real time, using a custom-designed fluorogenic oligonucleotide probe for 5' nuclease assay (16, 27). Human Cx43 cDNA primers and probes were designed using sequence data from Fishman et al. (12) (GenBank no. M65188) and the real-time RT-PCR probe/primer design software Primer Express (version 1.0, Perkin-Elmer), which optimized the sequences for use in real-time RT-PCR. The sequences designed were 5'-TCT CAC CTA TGT CTC CTC CTG GGT ACA A-3' for the probe, 5'-GCT CCT CAC CAA CCG CT-3' for the forward primer, and 5'-TTG CGG CAG GAG GAA TTG-3' for the reverse primer. These sequences were synthesized, and PCR conditions were optimized with respect to concentrations of Mg2+, probe, and both primers to maximize signal.

For real-time RT-PCR analysis, 2.5 µl of mRNA (20 ng/ml) sample were added to a reverse transcription reaction mix consisting of 0.5 µl RNase inhibitor (40 U/µl), 2.0 µl 10× TaqMan universal master mix buffer, 3.6 µl MgCl2 (25 mM), 2.0 µl reverse Cx43 primer (10 µM), 1.0 µl 18S reverse primer, 1.0 µl each of ATP, CTP, GTP, and UTP (10 mM), 0.44 µl (50 U/µl) murine leukemia virus RT, and 5.5 µl diethyl pyrocarbonate-treated deionized H2O. This mixture was placed in the thermocycler and subjected to 1 h at 42°C, 5 min at 72°C, and 2 min at 25°C. Eight microliters of this mixture were then added to the PCR reaction mix consisting of 5 µl 10× TaqMan universal master mix buffer, 4.0 µl MgCl2 (25 mM), 2.0 µl each forward and reverse Cx43 primer (10 µM), 10.0 µl fluorogenic probe (1.0 µM), 1.0 µl forward and reverse 18S primer (0.25 µM), 1.0 µl 18S probe (1.0 µM), 1.0 µl each of ATP, CTP, GTP, and UTP (10 mM), 0.25 µl TAQ Gold (5 U/µl), and 18.35 µl deionized H2O and subjected to 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C in the Perkin-Elmer ABI Prism 7700. The fluorescent time history was recorded, and the number of amplification steps required to reach an arbitrary intensity threshold (Ct) was computed. The ratio of Cx43 mRNA to 18S mRNA was calculated from the formula
A<SUB>Cx43</SUB>/A<SUB>18S</SUB> = 2C<SUB>t,18S</SUB>/2C<SUB>t,Cx43</SUB> = 2(C<SUB>t,18S</SUB> − C<SUB>t,Cx43</SUB>)
where ACx43 and A18S are the initial concentrations of Cx43 and 18S rRNA, respectively, and Ct,Cx43 and Ct,18S are the number of cycles necessary to reach threshold intensity for Cx43 and 18S rRNA, respectively.

Western blot analysis. hFOB cells were collected on days 3, 6, 9, and 12, and the crude membrane fraction protein was isolated from cells and subjected to Western blot analysis as previously described (25). Briefly, 10-µg protein samples were resolved by 12% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes that were blocked by Blotto (10% nonfat milk, 10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) at pH 8.0. The membranes were then incubated for 3 h at room temperature with antibodies against amino acids 252-272 of Cx43 diluted 1:1,000 in Blotto. The membranes were washed three times and incubated for 1 h with goat anti-mouse IgG linked to horseradish peroxidase diluted 1:5,000. After three additional washes with PBS, the membrane was soaked in ECL detection reagents according to the manufacturer's protocol. The sheet was then air dried and exposed to Kodak X-Omat AR film. The abundance of Cx43 protein signals was quantified by densitometry.

Alkaline phosphatase activity. Cells were cultured as described above. Medium was removed and saved for osteocalcin immunoradiometric assays. Cellular alkaline phosphatase activity was determined by the conversion of p-nitrophenyl phosphate to p-nitrophenol (29). Two slightly different protocols were used. In earlier experiments (see Fig. 2B for results), cells were cultured in 100-mm tissue culture dishes. After reaching confluency, the cells were washed twice with PBS and incubated for 30 min in AP reaction buffer (0.5 ml of 0.75 M 2-amino-2-methyl-1-propanol with 0.5 ml of 2 mg/ml p-nitrophenol phosphate); 50 µl of the reaction mixture were mixed with 200 µl of 0.1 N NaOH in the wells of a 96-well microplate. Absorption was measured at 410 nm, and conversion to enzyme activity was made using a p-nitrophenol standard absorption curve. In these experiments, protein concentration was determined from cells in 100-mm dishes run in parallel. In later experiments (see Fig. 7B for results), a protocol was utilized that allows protein and alkaline phosphatase to be determined from the same well. Cells were cultured in 24-well plates and washed twice with PBS, and 200 µl of Triton X-100 were added to each well. The cells were frozen and thawed twice, 5 µl were removed for determination of protein concentration, 300 µl of AP reaction buffer were added, and the solution was incubated for 30 min. The remainder of the protocol was the same as that for 100-mm dishes. Because of these differences in the two protocols, the alkaline phosphatase activity was lower in cells isolated from 24-well plates, but relative changes as a function of incubator temperature and duration of culture were similar.

Osteocalcin immunoradiometric assay. Medium was removed from cells cultured as previously described, and osteocalcin levels were quantified using an IRMA kit according to the manufacturer's protocol. Briefly, 400 µl of medium or of medium plus known osteocalcin standards were mixed in a tube with 200 µl of an I125-labeled monoclonal antibody to amino acids 37-49 of human osteocalcin. A single avidin-coated bead, to which a biotinylated monoclonal antibody to amino acids 7-19 of human osteocalcin had been immobilized, was then added to each tube, and the mixture was incubated at 25°C for 3 h on a horizontal rotor. The contents of each tube were aspirated, and the beads were washed and then counted in a gamma counter. The radioactivity of the antibody-bead complex is directly proportional to the amount of osteocalcin in the sample. The concentration of osteocalcin in the samples was determined by use of a standard curve constructed by counting samples of known osteocalcin concentration.

Statistical analysis. All data are reported as means ± SE except as stated otherwise. One-way ANOVA followed by a Student-Newman-Keuls test was used to analyze all data. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Expression of mRNA for three prevalent connexins (Cx26, Cx32, and Cx43) was examined by Northern blot analysis of hFOB cells cultured for 72 h at 37°C until confluency. Cx26 and Cx32 mRNA were not detected in the cell lines examined (data not shown), but Cx43 mRNA and protein were detected as described below.

To examine gap junction function in cells that had just reached confluency, Lucifer yellow dye spread from single dye-loaded cells to neighboring cells was recorded in cells cultured for 48 h at 37°C. Typically, after dye loading, if the cell was coupled, the dye rapidly (within 15 s) diffused into neighboring cells. Diffusion continued for up to 5 min, depending on the degree to which the particular cell was coupled. After 10 min, if the cell was highly coupled, the dye had diffused or faded to such a degree that quantification of the number of cells containing dye was difficult. Therefore, we routinely counted the number of cells containing dye at 5 min. Figure 1 shows quantitative analyses of cell coupling in hFOB cells in the presence of vehicle control or the gap junction uncoupler GA. In the absence of GA, 100% of the cells examined were coupled and >40% of the cells were coupled to five or more adjacent neighbors. However, in the presence of GA, >50% of the cells were uncoupled and no cells were coupled to more than four neighboring cells. The mean (±SD) number of cells coupled (i.e., those to which dye transferred) in the absence of GA was significantly greater than the number coupled in the presence of GA (9.3 ± 2.5 vs. 1.1 ± 1.4, P < 0.02).


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Fig. 1.   Quantitative analysis of gap junctional intercellular communication (GJIC) in hFOB 1.19 cells. Individual cells were loaded with Lucifer yellow in presence of vehicle control (A) or the gap junction uncoupler 18alpha -glycyrrhetinic acid (GA; 2 µM; B). Number of neighboring cells containing dye was recorded 5 min after loading. Histograms depict frequency with which individual cells were coupled to a given number of neighboring cells. Data are normalized to 100% to allow comparison among control (32 cells total) and GA (40 cells total) groups. Zero neighboring cells coupled means that loaded cell was not coupled. Mean (±SD) numbers of cells coupled per loaded cell are shown and were significantly different from one another (P < 0.05).

We next examined proliferation and differentiation of hFOB cells cultured at 37 and 39.5°C. hFOB cells cultured at the permissive temperature (37°C) proliferated rapidly with time in culture, whereas cells stimulated to differentiate by growth at the restrictive temperature (39.5°C) displayed a sharp reduction in proliferation (Fig. 2A). This is consistent with a reciprocal relationship between proliferation and differentiation. Alkaline phosphatase activity peaked at day 6 in cells cultured at 37°C and declined thereafter (Fig. 2B). Alkaline phosphatase activity was increased on days 9 and 12 in cells cultured at 39.5°C; the increase relative to 37°C reached statistical significance on day 12. Osteocalcin protein synthesis followed a similar pattern (Fig. 2C).


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Fig. 2.   Proliferation and differentiation of hFOB cells. Cells cultured at 37°C were plated at 1 × 103 cells/mm and assayed on days 3, 6, 9, and 12. On day 6, one-half of cells were cultured at 39.5°C (dashed lines) while other cells remained at 37°C (solid lines). Cells cultured at 37°C continued to proliferate throughout 12-day experiment, whereas cells cultured at 39.5°C did not (A). Additionally, cells at 39.5°C displayed greater alkaline phosphatase (AP) activity (B) and osteocalcin concentration (C) than cells cultured at 37°C. For each experimental condition, 4 experiments were completed in triplicate for proliferation and 3 experiments were completed in duplicate for alkaline phosphatase and osteocalcin. * Significantly greater than cells at 39.5°C, P < 0.05.

Expression of Cx43 and Cx45 was examined as hFOB cells differentiated in culture. Steady-state Cx45 mRNA expression was rather modest on day 3 of culture and did not change appreciably thereafter (Fig. 3). In contrast, steady-state Cx43 mRNA expression increased with time in culture and to a greater degree when cells were stimulated to differentiate by culture at 39.5°C (Fig. 4). To confirm the increase in Cx43 mRNA in cells stimulated to differentiate, we utilized real-time RT-PCR to quantify steady-state levels of Cx43 mRNA. Cx43 mRNA normalized to 18S was approximately threefold greater in cells cultured at 39.5°C than in those cultured at 37°C (Fig. 5). These results are similar to those obtained by Northern blot analysis. Cx43 protein displayed a pattern of expression similar to that of Cx43 mRNA (Fig. 6).


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Fig. 3.   Connexin 45 (Cx45) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in hFOB cells. hFOB cells were plated at 1 × 103 cells/mm and cultured at 37°C for 3, 6, 9, and 12 days; 20 µg of total RNA were subjected to Northern blot analysis using either a 1200-bp Cx45 cDNA or a 1400-bp radiolabeled GAPDH probe. hFOB Cx45 signal did not change appreciably with time in culture.



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Fig. 4.   Cx43 and GAPDH mRNA expression in differentiating hFOB cells. Cells were cultured for 6 days at 37°C, and then one-half were cultured at 39.5°C and one-half at 37°C for another 6 days, as described in MATERIALS AND METHODS. On days 3, 6, 9, and 12, cells were collected, total RNA was extracted, and 20 µg were subjected to Northern blot analysis using either a 890-bp radiolabeled rat Cx43 cDNA or a 1400-bp radiolabeled GAPDH probe. A: a single experimental result. B: summarized results of 7 experiments. Data are means ± SD of abundance, determined by phosphorimaging, of Cx43 mRNA normalized to GAPDH mRNA abundance and relative to day 3. Steady-state Cx43 mRNA abundance in cells cultured at 37°C (solid line) increased from day 3 to day 6 but not thereafter. In cells cultured at 39.5°C (dashed line), Cx43 mRNA followed a similar trend but continued to increase from day 9 to day 12. * Level at 39.5°C was significantly greater than that at 37°C on day 12.



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Fig. 5.   Cx43 mRNA in hFOB cells. hFOB cells were cultured for 12 days at either 37 or 39.5°C, and steady-state Cx43 mRNA levels relative to 18S mRNA (means ± SD) were quantified by real-time RT-PCR. Ratios were determined from Ct values, which are number of PCR cycles necessary to reach an arbitrarily selected fluorescence intensity threshold, as described in MATERIALS AND METHODS. Differentiation of hFOB by culture at 39.5°C resulted in a >3-fold increase in steady-state Cx43 mRNA levels. Three experiments were completed in duplicate at 37°C and 39.5°C. * Significantly different from 37°C, P < 0.05.



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Fig. 6.   Western blot analysis of Cx43 protein in differentiating hFOB cells. Samples (10 µg) of crude membrane fraction isolated from cells cultured as described for Fig. 4 were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed using a polyclonal antibody to Cx43. A: result of single experiment. B: summarized results of 5 experiments. Data are means ± SD of protein abundance, determined by densitometry, relative to day 3 abundance. Cx43 protein abundance did not change appreciably with time in culture at 37°C (solid line). However, cells cultured at 39.5°C displayed a dramatic increase in Cx43 protein abundance on days 9 and 12 (dashed line). * Significantly greater than 37°C, P < 0.05.

To determine whether gap junction coupling also increased in cells stimulated to differentiate, we examined coupling with the dual-label parachute assay. Cells were cultured at 37 or 39.5°C for 6, 9, or 12 days, as described above for differentiation experiments, in the presence of vehicle control or 30 µM GA, the gap junction uncoupler. GJIC was assessed by calcein dye transfer on days 6, 9, and 12 but not on day 3, since at this point the cells are still very sparse and lacked the cell contacts necessary for GJIC. GJIC modestly increased with time in cells cultured at 37°C (Fig. 7A). However, in cells stimulated to differentiate by culture at 39.5°C there was a dramatic increase in GJIC to a level three times greater, by day 12, than that of cells cultured at 37°C. Cells cultured in the presence of GA did not display an increase in GJIC when cultured at 39.5°C. Calcein dye transfer was generally less overall than Lucifer yellow dye transfer (Fig. 1). This is likely because Lucifer yellow is negatively charged and smaller (mol wt 457) than the more polar and larger (mol wt 995) calcein. Thus Lucifer yellow would be expected to transfer more easily through Cx43 gap junction channels.


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Fig. 7.   GJIC, alkaline phosphatase activity, and osteocalcin concentration in differentiating hFOB cells. Cells were plated on 25-mm-diameter round glass coverslips for GJIC assay and 35-mm culture dishes for other assays, and cultured for 12 days in presence of vehicle control or 30 µM GA. On day 6, one-half of coverslips were placed in an incubator at 39.5°C while other one-half were maintained at 37°C. A: GJIC was determined by parachute dual-labeling technique, as described in MATERIALS AND METHODS, on day 6, before any cells had been cultured at 39.5°C, and on days 9 and 12 after one-half of coverslips were moved to 39.5°C incubator. Cells were not assayed on day 3 because there was not sufficient cell-cell contact to initiate GJIC. In control cells cultured at 37°C (thick solid line), GJIC increased gradually with time in culture, whereas GJIC decreased in presence of GA (thin solid line). Control cells stimulated to differentiate by culture at 39.5°C (thick dashed line) displayed a dramatic increase in GJIC, which was inhibited by GA (thin dashed line). Each day, n = 38-56 cells were imaged for each experimental group. * Significantly different from other groups on same day, P < 0.05. B: in cells cultured at 37°C, alkaline phosphatase activity increased gradually with time in culture in absence or presence of GA. At 39.5°C, alkaline phosphatase activity dramatically increased in absence of GA but only gradually increased in cells cultured in presence of GA. Three experiments were completed in duplicate for each experimental condition. * Significantly greater than cells cultured in presence of GA, P < 0.05. C: osteocalcin concentration in cells cultured at 37°C increased modestly with time in culture in absence or presence of GA. Osteocalcin concentration increased more dramatically in cells cultured at 39.5°C, whether in absence or presence of GA. Five experiments were completed in duplicate for each experimental condition. * Significantly greater than cells cultured at 37°C, P < 0.05.

To examine the role of GJIC in hFOB differentiation, we cultured cells for 12 days in the presence of 30 µM GA or vehicle control. As shown in Fig. 7B, cells cultured at 37°C displayed increased alkaline phosphatase activity with time in culture. When cells were switched to 39.5°C, in the presence of vehicle control, alkaline phosphatase activity increased, as demonstrated in our previous experiment. Interestingly, alkaline phosphatase activity did not increase in cells grown at 39.5°C in the presence of the gap junction uncoupler GA. We repeated these experiments and examined osteocalcin concentration in cell culture medium. As was the case with alkaline phosphatase, osteocalcin levels increased with time in culture at 37°C (Fig. 7C). Additionally, cells switched to 39.5°C displayed an increase in osteocalcin concentration in the presence of vehicle control. However, unlike alkaline phosphatase activity, osteocalcin levels increased at 39.5°C even in the presence of GA. Thus, whereas GJIC was necessary for maximal alkaline phosphatase activity, this was not the case for osteocalcin secretion.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we examined the expression of markers of differentiation as well as gap junction expression and function in a human fetal osteoblastic cell line, hFOB 1.19, that was conditionally immortalized with a gene coding for a temperature-sensitive mutant (tsA58) of the SV40 large T antigen (19). We found that hFOB cells expressed Cx43 and, to a lesser degree, Cx45. However, Cx26 and Cx32 were not expressed. These results are similar to what has been reported for other human osteoblastic cells.

Our results suggest that hFOB 1.19 cells are quite sensitive to subtle changes in temperature. When hFOB 1.19 cells are cultured at 37°C, the restrictive temperature at which the large T antigen is not expressed, the cells proliferate rapidly. However, when the cells are switched to the permissive temperature of 39.5°C, cell proliferation slows and cell number does not increase further. Harris et al. (19) found similar results with hFOB 1.19 cells cultured at 33.5 and 39.5°C. Considering the reciprocal relationship between cell proliferation and differentiation displayed by many cell types, including osteoblasts (31), these results suggest that hFOB 1.19 cells cultured at 39.5°C were stimulated to differentiate.

To further address this issue, we examined alkaline phosphatase activity and osteocalcin synthesis in hFOB 1.19 cells grown at the restrictive and permissive temperatures. Both alkaline phosphatase activity and osteocalcin synthesis increased more rapidly and to a greater extent when cells were cultured at 39.5°C, relative to 37°C, suggesting that culture at this temperature stimulated differentiation. These findings emphasize the usefulness of this particular cell line in studying osteoblastic differentiation.

We next examined gap junction function and expression as hFOB 1.19 cells differentiated with time in culture. We found that both Cx43 expression and function increased in hFOB 1.19 cells stimulated to differentiate by culture at 39.5°C. We also found that GA, which rapidly and reversibly inhibits gap junctional coupling, possibly through a mechanism involving disruption of connexon particles (18), prevented the increase in alkaline phosphatase activity resulting from culture at 39.5°C. These results suggest a role of GJIC in activation of alkaline phosphatase and possibly in differentiation.

Although GA decreased differentiation-associated changes in alkaline phosphatase activity, it had no effect on osteocalcin synthesis, suggesting that GJIC is not critical for osteocalcin synthesis. However, it has previously been demonstrated that ROS 17/2.8 cells that are genetically rendered gap junction deficient, either by expression of Cx45 (a gap junction protein not normally expressed by ROS) or by expression of antisense Cx43, display decreased osteocalcin synthesis (23, 26), suggesting that GJIC does contribute to osteocalcin synthesis.

One explanation for these contradictory findings is that the dependence of osteocalcin synthesis on GJIC is cell type dependent, existing in the rat osteosarcoma cell line ROS17/2.8 but not in the human osteoblastic cell line hFOB 1.19. However, a more likely explanation is that GA does not completely block GJIC in hFOB 1.19 cells, whereas Cx43 antisense or overexpression of Cx45 does completely block GJIC in ROS17/2.8 cells. Indeed, Li et al. (24) demonstrated that GA completely blocks Lucifer yellow dye transfer but only partially blocks electrical conductance in Cx43-expressing C6 glioma cells. If this is also the case in hFOB 1.19 cells, it would suggest that alkaline phosphatase activity is regulated by signals passing through gap junctions of a size or charge that can be blocked even when the gap junctional channel is only partially inhibited. On the other hand, osteocalcin is regulated by signals of a charge or size that can be blocked only when the gap junction channel is completely closed. Thus when the channels are completely closed, as in Cx43 antisense- or Cx45-expressing ROS 17/2.8 cells, osteocalcin synthesis is inhibited, but when the channels are only partially blocked, as in GA treatment of hFOB 1.19 cells, osteocalcin is not inhibited. The lack of effect of GA on osteocalcin synthesis also suggests that its effect on alkaline phosphatase activity is not the result of toxic or nonspecific effects on overall cell metabolism.

In summary, our studies suggest that Cx43 expression and GJIC parallels osteoblastic cell differentiation and that partially inhibiting GJIC by incubation with GA inhibits expression of the fully differentiated phenotype in human fetal osteoblastic cells. These findings suggest that GJIC at least partially contributes to expression of the fully differentiated osteoblast phenotype.


    ACKNOWLEDGEMENTS

We thank Debbie Firestine for help in preparing the manuscript and Dr. Deborah Grove for designing primers and completing RT-PCR protocols.


    FOOTNOTES

This work was supported by National Institute on Aging Grant AG-13087.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. J. Donahue, Musculoskeletal Research Laboratory, Dept. of Orthopedics and Rehabilitation, Milton S. Hershey Medical Center, Pennsylvania State University, PO Box 850, 500 University Dr., Hershey, PA 17033-0850 (E-mail: hdonahue{at}psu.edu).

Received 25 May 1999; accepted in final form 17 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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