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1Department of Craniofacial Sciences, School of Dental Medicine, University of Connecticut Health Center and 2Department of Genetics and Developmental Biology, School of Medicine, University of Connecticut Health Center, Farmington, Connecticut
Submitted 5 May 2006 ; accepted in final form 5 October 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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parathyroid hormone; cell proliferation; apoptosis; osteoblast differentiation; flow cytometry
At the cellular level, the increased bone formation is primarily the result of an increase in the number of active osteoblasts. Depending on the culture systems, animal models, and experimental conditions, different studies have shown variable results with respect to the anabolic effect of PTH as it relates to osteoblast proliferation. For instance, in vitro organ cultures of embryonic mouse radii and rat calvarie showed that PTH increases osteoblast number (15) and stimulates osteoblast replication (5, 8) but decreases the collagen synthesis (10, 40). PTH stimulated cell proliferation in cultures of the human osteosarcoma cell line TE-85 (12, 30); however, it inhibited cell proliferation in UMR-106 osteosarcoma cell cultures (33). Whereas intermittent PTH stimulated proliferation and differentiation of osteoprogenitor cells in bone marrow and primary spongiosa (19, 28), other in vivo studies showed a lack of cell proliferation in response to PTH treatment (11, 27, 31). DNA labeling in histomorphometry studies has shown no evidence of increased osteoprogenitor proliferation stimulated by PTH (11, 27). Various studies indicate that the anabolic effect of PTH may result from mechanisms other than proliferation in the osteoblast lineage. Instead, an increase in the number of osteoblasts may be achieved either by the inhibition of apoptosis, which prolongs the longevity of mature osteoblasts (16, 23), or by the activation of quiescent bone-lining cells into active osteoblasts (11).
Part of the reasons why the cellular mechanisms underlying enhanced bone formation are still not fully understood may relate to the complex and heterogeneous cellular nature of bone. In a tissue containing osteogenic cells that range from multipotential osteoprogenitors to terminal osteocytes, the unique response of each population to the hormone presents difficulties in the interpretation of cellular events in experiments when heterogeneous bone cells are present. Literature has indicated the importance of understanding how much this proliferative response may contribute to the anabolic effect of PTH and clarifying the effect of PTH on proliferation in different subpopulations of osteogenic cells. Toward this, it is necessary to have an experimental model that has the ability to identify the cellular targets of PTH and distinguish among the effects of PTH in the heterogeneous osteoblast lineage.
Previously, we have shown that primary calvarial osteoblast cultures derived from mice transgenic for promoter-driven stage-specific green fluorescent protein (GFP) marker genes pOBCol3.6GFP and pOBCol2.3GFP (24) respond to an early transient PTH exposure (from day 1 to 7) and, consequently, after the removal of PTH, acquire intensified expression of fluorescent markers and increased formation of mineralized bone nodules by day 21 (43). However, at day 21, the final DNA content in PTH-treated cultures was never higher than that of control cultures, making it unlikely that the early transient PTH exposure stimulates progenitor cell proliferation. Based on these observations, the purpose of the present study was to analyze the effect of PTH on early proliferation and apoptosis in the osteoprogenitor cell population (before the removal of PTH) and to assess the effect of PTH on subsequent osteoblast differentiation (after the removal of PTH). In pOBCol3.6GFP cultures, the initiation of GFP expression marks the transition of the osteoblast lineage from multipotential osteoprogenitors to preosteoblasts that are highly committed to osteoblast differentiation. With the use of fluorescence-activated cell sorting (FACS), it is now possible to examine the effects of PTH on cell proliferation and apoptosis in two subpopulations of osteoprogenitor cells [i.e., the GFP(+) and GFP(–) cells] well before they undergo full osteoblast differentiation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Calvarial osteoblast cultures derived from mice transgenic for pOBCol3.6GFP (24), in which the GFP was driven by a rat 3.6-kb type I collagen promoter, were used to examine osteoblast development. Calvarial cells were isolated from 6- to 8-day-old neonatal mice. After removal of sutures and adherent mesenchymal tissues, calvaria were subjected to four sequential 15-min enzyme digestions at 37°C in solution containing 0.05% trypsin-EDTA and 0.1% collagenase P (Roche Diagnostics, Indianapolis, IN). Cells released from the second to fourth digestions were pooled, centrifuged, resuspended, and plated at 1.5 x 104 cells/cm2 (1.5 x 105 cells/well) in 35-mm six-well culture plates in DMEM (Invitrogen, Carlsbad, CA) containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µM nonessential amino acids. The day of plating was counted as day 0. At day 1, medium was refreshed to remove cells that were not attached to culture plate. Plated cells became confluent around days 5–6; and then at day 7 the culture medium was changed to differentiation medium, which consisted of 
-minimal essential medium (Invitrogen) containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml ascorbic acid, and 4 mM 
-glycerophosphate. Medium was changed every other day for the entire duration of culture. For harvest, cells were dissociated from the dish with 0.25% trypsin/1 mM EDTA. The experimental protocol was approved by the University of Connecticut Health Center Animal Care Committee (Protocol 2004-037).
PTH Administration
Human PTH-(1–34) powder (Bachem Bioscience, King of Prussia, PA) was dissolved in 4 mM HCl/0.1% BSA to make reconstituted stock solution. Based on the previous results of Isogai et al. (22) and Wang et al. (43) that PTH produces an anabolic effect on early calvarial osteoblast culture, in the present study the duration of PTH treatment from day 1 to 7 was chosen to examine the effect of PTH on cell proliferation, apoptosis, and differentiation. PTH was present in culture from day 1 to 7 at a final concentration of 25 nM (or 103 ng/ml; added at day 1, 3, and 5 when medium was refreshed). No PTH was present after day 7 when medium was changed to differentiation medium. In the control cultures, only vehicle (no PTH) was added to medium.
Observation of Fluorescent Expression
Cell cultures were examined and photographed with a Zeiss Axiovert 200 (Carl Zeiss, Thornwood, NY) microscope using Openlab software (Improvision, Lexington, MA). The Zeiss Axiovert 200 microscope is equipped with the motorized X-Y-Z platform, motorized fluorescent cube, and AxioCam color digital camera that are controlled by a user-defined computation program. The microscope workstation allows the user to reproducibly record images of cultures at the same location at defined time points (43). Expression of green fluorescence [i.e., GFP] and red fluorescence [i.e., ethidium homodimer-1 (EthD-1), rhodamine, and xylenol orange (XO)] was examined using yellow fluorescent protein (YFP) and tetramethyl rhodamine isothiocyanate (TRITC) filters, respectively (Chroma Technology, Rockingham, VT).
Flow Cytometry
Isolated cells labeled with bromodeoxyuridine (BrdU; for cell proliferation) or annexin V (for apoptosis) were filtered through a 35-µm cell strainer to avoid clogging during flow cytometric analysis. Flow cytometry was carried out on FACScan Calibur (BD Biosciences, San Jose, CA), which was equipped with a 488-nm air-cooled argon ion laser and a 635-nm diode laser. Forward/side scattering and detection of three ranges of fluorescence (FL1, FL2, and FL3) were performed with the 488-nm laser. In addition, a fourth range of fluorescence (FL4) was available using the 635-nm diode laser. Emission was detected using a 500-nm longpass filter. Cells from non-GFP cultures were used as control to determine the baseline for the intensity of GFP expression. Analysis of cell proliferation and apoptosis used FL1 for GFP, FL3 for 7-amino-actinomycin D (7-AAD), and FL4 for BrdU-allophycocyanin (APC) and annexin V-APC (see sections below for details). To sort cells into GFP(+) and GFP(–) populations, harvested cultured cells were adjusted to a density of 10 x 106 cells/ml. FACS for GFP was carried out on a FACS-Vantage SE (Becton-Dickinson) with the speed of 5,000 cells/s using 488-nm excitation wavelength generated by a 200-mW argon ion laser. Emission was detected using a FITC 525/30-nm bandpass filter. The fractionated GFP(+) and GFP(–) populations were collected in DMEM before further usage.
Examination of Cell Proliferation
BrdU was added to medium (10 µM) to label cells in the S (DNA synthesis) phase. A BrdU Flow Kit (BD Bioscience) was used to prepare the BrdU-incorporated cells for flow cytometric analysis. In brief, after incubation with BrdU at 37°C for 2 h, cells were dissociated from culture dishes with 0.25% trypsin/1 mM EDTA. After being washed and centrifuged, cells were resuspended, fixed, permeabilized, and DNase treated to expose incorporated BrdU. BrdU-labeled cells were detected with an anti-BrdU antibody, which was conjugated with APC. In flow cytometry, to avoid the interference from GFP expression that is detected using the FL1 channel, APC-conjugated anti-BrdU is detected using the FL4 channel. In addition, cells were stained with 7-AAD, which intercalates into the DNA fragments digested by DNase, to measure total DNA content. Fluorescence of 7-AAD was detected using the FL3 channel. In cell cycle analysis, whole cells were first divided into GFP(+) and GFP(–) populations, and then each population was separately analyzed using the fluorescence of APC (for BrdU incorporation) and 7-AAD (for DNA content) as shown in Fig. 1A to obtain three clusters of cells that represented the G0/G1 phase (i.e., R4), S phase (i.e., R6), and G2+M phase (i.e., R5) phase (Fig. 1A).
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Cell viability. To avoid interference from GFP expression in living cultures, a red fluorescent dead-cell indicator (EthD-1; Molecular Probes, Eugene, OR) was chosen to determine cell viability. EthD-1, with high affinity for DNA and low membrane permeability, permits the use of very low concentrations to stain dead cells. In the present study, cultures were incubated with EthD-1 at a final concentration of 2 µM for 30 min and examined under the fluorescent microscope with a TRITC filter to measure cell viability. A fluorescent image of EthD-1 staining (red color) was photographed and then quantitated by counting the total number of red dots using a computation program developed from Openlab software.
Apoptosis. Fluorochrome-conjugated annexin V has been used in flow cytometry to detect apoptosis (42). In the present study, harvested cultured cells were resuspended in annexin-binding buffer, which consisted of 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at pH 7.4. APC-conjugated annexin V (Molecular Probes) was added to the cell suspension at 1:20 dilution to label the apoptotic cells for 15 min. In flow cytometry, cells were analyzed using channel FL4 to detect the apoptotic cells labeled with APC-conjugated annexin V and FL1 to detect cells with GFP expression. Results for GFP(+)/APC(+) (top right), GFP(+)/APC(–) (bottom right), GFP(–)/APC(+) (top left), and GFP(–)/APC(–) (bottom left) are shown in Fig. 1B.
Secondary Cultures of FACS-fractionated Cells
In the present study, replicas of 7-day-old primary calvarial osteoblast cultures incubated with or without PTH (days 1–7) were dissociated from dishes and subjected to sorting for GFP. FACS-isolated GFP(+) and GFP(–) cells were centrifuged, resuspended, and replated in six-well culture dishes at the same density as primary cultures (1.5 x 104 cells/cm2). These secondary cultures were grown for another 21 days using the same protocol as that for primary calvarial osteoblast cultures except no PTH was added to the cultures. To examine the mineralization in living cultures, XO was added to the culture medium (final concentration: 20 µM) overnight. Our previous result has shown that XO staining correlates exactly with the standard von Kossa staining for mineralized nodules (43).
Quantitative PCR Analysis of Receptor Activator of Nuclear Factor-
B Ligand and Osteoprotegerin Expression
Total RNA was extracted from FACS-isolated GFP(+) and GFP(–) cells from 7-day-old primary calvarial osteoblast cultures incubated with or without PTH from day 1 to day 7. The extraction of total RNA and reverse transcription was performed using TRIzol Reagent (Invitrogen) and a SuperScript First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions, respectively. The PCR mixtures were prepared using reagents from a SYBR Green PCR kit (Qiagen, Valencia, CA). PCR amplification primer pairs for receptor activator of nuclear factor-B ligand (RANKL) were 5'-CAGAAGACAGCACTCACTGCT and 3'-CATTGATGGTGAGGTGTGCAA and for osteoprotegerin (OPG) were 5'-GCAGAGACGCACCTAGCACTG and 3'-GCCAGCTGTCCGTATAAGAGT. Gene expression of RANKL and OPG was normalized with hypoxanthine guanine phosphoribosyltransferase, which was amplified using primer pairs 5'-CACAGGACTAGAACACCTGC and 3'-GCTGGTGAAAAGGACCTCT. Real-time PCR was performed in an iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The PCR mixtures were preheated to 95°C for 15 min followed by 40 cycles of amplification (95°C for 30 s; 60°C for 30 s; 72°C for 40 s). Quantitative PCR results were analyzed using the iCycler Optical System Software, version 3.1 (Bio-Rad).
Statistical Analysis
Statistical analysis was carried out using SPSS-11.0 software (SPSS, Chicago, IL). All experiments were repeated three times, and data were analyzed with one-way ANOVA followed by a Scheffé post hoc test. All values are means ± SE, and a P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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PTH Reduced Cell Number and Inhibited Cell Proliferation in the Preconfluent Cultures
PTH was added to primary calvarial osteoblast cultures from day 1 to day 7. Culture plates from different time points were harvested for total cell counts. Compared with the controls, PTH significantly reduced the total cell number at day 4 and day 6 (Fig. 2A). After the discontinuation of PTH (1 day), the PTH-pretreated cultures continued to have fewer cells than the controls at day 8 (Fig. 2A). This observation is consistent with cell counts based on DNA quantitation that showed PTH decreased the DNA content of early calvarial osteoblast cultures (22, 43). To understand the reduction of cell number in the presence of PTH, BrdU incorporation was used to examine the effect of PTH on cell proliferation in cultures from day 3 (preconfluent) to day 9 (postconfluent). In the present study, cultures became confluent around day 5 or day 6. The incorporation of BrdU during DNA synthesis was detected with an APC-conjugated anti-BrdU antibody, and the percentage of cells in the S phase was analyzed using flow cytometry. In both PTH-treated and control cultures, the rate of cell proliferation decreased as the cultures moved from the preconfluent to the postconfluent stage (Fig. 2B). Once the cultures became confluent, the rate of proliferation stabilized. In the presence of PTH, there was a significant decrease of the proliferation rate in the preconfluent cultures at day 3 and day 4. However, once the cultures reached confluence after day 5, there was no difference between the controls and the PTH-treated cultures (Fig. 2B).
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Two populations of cells can be distinguished based on the expression of the pOBCol3.6GFP, which we believe marks a transition within the osteoblast lineage from multipotential osteoprogenitors [i.e., GFP(–) cells] to differentiating preosteoblasts [i.e., GFP(+) cells; see Refs. 24 and 43]. We observed that both GFP(+) and GFP(–) cells were able to proliferate in cultures detected by BrdU incorporation (revealed by rhodamine-conjugated anti-BrdU). In the GFP(+) population, while nonproliferating cells showed only green fluorescence (Fig. 3A), those cells in proliferation displayed a yellow/orange nucleus [overlapping of green (GFP) and red (rhodamine); Fig. 3A]. In the GFP(–) population, cells in proliferation showed a red nucleus (Fig. 3A), and nonproliferating cells were identified by the background staining (Fig. 3A').
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38–32%) with a corresponding increase in G0/G1 and G2+M phases in both GFP(+) and GFP(–) populations (Table 1). The decrease of percentage in S phase by PTH treatment apparently resulted from an increase in both G0/G1 and G2+M phases.
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38–9% in control and 32–10% in PTH treated; Table 1). In day 6 control cultures, the GFP(–) population still showed a pattern that had a higher percentage of cells in G0/G1 and a lower percentage in G2+M relative to the GFP(+) population (Table 1). In contrast to its inhibitory effect in day 3 cultures, the PTH treatment did not affect the percentage of cells in S phase in day 6 cultures. However, within the GFP(+) population, PTH treatment did significantly increase the proportion of cells in the G0/G1 phase (74.6 vs. 71.9%) and decrease the proportion in the G2+M phase (15.1 vs. 18.3%; Table 1). In summary, PTH treatment significantly altered the cell cycle, in particular reducing the S phase, in both GFP(+) and GFP(–) populations in the preconfluent day 3 cultures. However, this effect was lost by the time confluence was reached.
PTH Increased Cell Viability and Inhibited Apoptosis in the Preconfluent Cultures
To test whether PTH affects cell viability, EthD-1 (a red-fluorescent dead-cell indicator) was added to cultures. At day 6, quantitation of EthD-1 staining showed that there was 50% less EthD-1 staining in the PTH-treated culture (Fig. 3C') when compared with the control (Fig. 3B'). This observation indicates an increase of cell survival induced by PTH treatment but contradicts the fact that PTH reduced the total cell number in cultures. It is possible that the decrease of total cell number in the PTH-treated cultures (Fig. 2A) resulted from the reduction of cell proliferation (Table 1). However, in addition to the change in the rate of cell proliferation, the total cell number in the culture could also be affected by the change in the rate of cell death. Therefore, we examined the effect of apoptosis on the total cell number in cultures.
Annexin V was used to detect apoptosis in living cultures. Annexin V has a high affinity for cytoplasmic phosphatidylserine, which is translocated to the outer surface of the cell membrane during apoptosis. We validated the efficacy of annexin V to detect apoptosis in etoposide-treated calvarial osteoblast cultures (data not shown). Table 2 summarizes the results of annexin V labeling in the total cell population as well as in the GFP(+) and GFP(–) populations. In the control cultures at day 3, 13.2% of the total cells were labeled with annexin V. These annexin V-labeled cells consisted of 3.1% of the GFP(+) cells and 22.6% of the GFP(–) cells, indicating that apoptosis occurs more frequently in the GFP(–) population (Table 2). PTH treatment significantly reduced the incidence of apoptosis in day 3 cultures (10.8 vs. 13.2%). This reduction resulted entirely from the decrease of apoptosis in the GFP(–) population (17.5 vs. 22.6%). Thus PTH treatment increased the survival of GFP(–) cells in preconfluent cultures. At day 6, when cultures reached confluence, there was a reduction in the proportion of cells labeled with annexin V mainly because of a decrease in the GFP(–) population (Table 2). In contrast to its inhibitory effect in preconfluent cultures, PTH treatment did not significantly alter the occurrence of apoptosis in postconfluent cultures.
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Our previous results (43) demonstrate that the early transient PTH treatment (days 1–7) enhanced osteoblast differentiation and mineralized nodule formation in day 21 cultures. It is possible that the present result of decreased cell proliferation associated with increased cell survival appears as an early manifestation of an enhanced commitment effect by PTH on progenitor populations. Thus we were interested in examining which population of cells [i.e., GFP(+) or GFP(–)] within the cultures was being influenced toward increased osteoblast commitment by the 7-day exposure to PTH.
Two sets of 7-day-old pOBCol3.6GFP cultures that were treated with or without 25 nM PTH (days 1–7) were subjected to FACS to sort the GFP(+) and GFP(–) populations. Initial flow cytometric analysis showed that the composition of the controls and PTH-treated cultures was similar, each containing nearly 50% GFP(+) and 50% GFP(–) cells (Fig. 4A). After sorting, the isolated GFP(+) and GFP(–) populations were 99% homogeneous in terms of GFP expression (Fig. 4, B and C). Sorted GFP(+) and GFP(–) populations were separately replated and cultured for 21 days to examine their subsequent differentiation. Both fractionated populations showed the ability to continue to proliferate, differentiate, and express GFP after replating (Fig. 4D).
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strong expression mineralization) that was previously reported in the primary calvarial osteoblast cultures (43). On the other hand, in the GFP(–) cultures, the expression of pOBCol3.6GFP and differentiation of osteoblast nodules in both control and PTH-pretreated cells were not seen until the end of the 2nd wk (Fig. 4D, rows 3 and 4). Compared with the control GFP(+) culture, the PTH-pretreated GFP(+) culture had greater pOBCol3.6GFP expression and more mineralized nodules, as shown by XO staining at day 21 (Fig. 4D). Similarly, the prior PTH treatment also significantly increased the areas of GFP expression and mineralized nodule formation in the GFP(–) culture at day 21 (Fig. 4D). These observations were also substantiated by the results of quantitation on GFP expression and mineralized nodule formation as shown in Fig. 4E. To examine whether the PTH pretreatment affected cell proliferation, apoptosis, and DNA content in these secondary cultures, flow cytometry analysis and DNA content quantitation were performed. Results showed that there was no significant difference between the PTH-pretreated and control cells as well as between GFP(+) and GFP(–) cells with respect to cell proliferation (Fig. 4F), apoptosis (Fig. 4F), and DNA content (Fig. 4G) in day 3 or day 14 cultures.
In primary calvarial osteoblast cultures, we have observed that strong expression of pOBCol3.6GFP is an indication of osteoblast differentiation (43). We were interested to see if early PTH treatment affects the efficiency of osteoblast differentiation and mineralization in GFP(+) and GFP(–) cultures. Observation showed that there was a similar proportion (4044%) of GFP-expressing areas that underwent mineralization in the control GFP(+) and PTH-pretreated GFP(+) cultures (Fig. 4E). Although they had fewer areas of GFP-expressing and mineralization, GFP(–) cultures had a higher proportion (6087%) of GFP-expressing areas that underwent mineralization, especially in the PTH-pretreated GFP(–) cultures (87%; Fig. 4E). In summary, the PTH pretreatment not only intensified GFP expression but also enhanced mineralized nodule formation in the secondary cultures established from sorted GFP(+) and GFP(–) cells.
PTH Increased RANKL Expression in GFP(+) and GFP(–) Cells
A recent microarray study (25) in the GFP subpopulations sorted from 7-day-old pOBCol3.6GFP calvarial osteoblast cultures demonstrated that a number of genes associated with dendritic cells or cells within the macrophage/osteoclast lineage are expressed in the GFP(–) cells, indicating that cells from the macrophage/osteoclast lineage are present within these cultures. It is known that the osteoblast lineage is a cell source of cytokines for osteoclast differentiation and the formation of osteoclasts is differentially regulated by osteoblast-derived RANKL and its decoy receptor OPG. It would be interesting to learn whether PTH affects the expression of these two molecules in the present culture model. Thus we examined the effect of PTH on RANKL and OPG mRNA expression in the GFP(+) and GFP(–) cells sorted from the 7-day-old pOBCol3.6GFP calvarial osteoblast cultures that were treated with 25 nM PTH from day 1 to day 7. Results from quantitative PCR showed that PTH greatly increased (15-fold) the expression of RANKL in both GFP(+) and GFP(–) cells when compared with the control non-PTH-treated cells (Fig. 5). However, there was no detectable expression of OPG in either control or PTH-treated cells. The strong induction of RANKL, but not OPG, by PTH treatment indicates a possible induction of osteoclastogenesis in these cultures.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Various studies have shown a conflicting stimulatory and inhibitory effect of PTH on cell proliferation. This inconsistency may have resulted from the different PTH targets or differentiation stages of osteoprogenitor cells that are present in various model systems. Although PTH inhibits cell proliferation in cultures derived from the proliferative and undifferentiated osteoprogenitor cells of primary spongiosa (32), another in vivo BrdU-labeling study reveals an increase of cell proliferation in the same primary spongiosa (19). Our results showed that PTH initially inhibited cell proliferation and decreased total DNA content, which is consistent with the result of Isogai et al. (22), who showed that PTH treatment significantly reduces DNA content in preconfluent osteoblast cultures. In addition, cell proliferation was decreased from day 3 to day 7 cultures, which was highly correlated with the degree of confluence of the cultures. The preconfluent osteoblast cultures consist of highly proliferative and undifferentiated osteoprogenitor cells. To take the advantage of promoter-driven GFP expression in our cultures, flow cytometry was used to further examine the difference of cell proliferation between GFP(+) and GFP(–) subpopulations.
Multiparameter flow cytometry has been widely used to identify various subpopulations of cells in the heterogeneous hematopoietic system (20, 39). Using FACS to measure the expression of GFP, the DNA content, and the incorporation of BrdU, we were able to examine the effects of PTH on the cell cycle in two different osteoprogenitor populations [i.e., GFP(+) and GFP(–) cells]. In control cultures, although no difference was observed in S phase, there was a significant increase in the G0/G1 phase and a significant decrease in the G2+M phase in GFP(–) cells relative to GFP(+) cells, reflecting the heterogeneity of these progenitor populations. In day 3 preconfluent cultures, PTH decreased the proportion of cells in S phase and concurrently increased the proportion of cells in the G0/G1 and G2+M phases in both GFP(+) and GFP(–) subpopulations. Although the reduction of apoptosis may rescue cells primarily in the GFP(–) subpopulation, the reduction of proliferation in both GFP(+) and GFP(–) subpopulations may contribute to the decrease of total cell number in culture. Further study is needed to determine whether PTH slows down the whole cell cycle or retards the cell cycle in a particular phase.
The anti-proliferative effect of PTH on UMR-106 cells results from a block of cellular entry into S phase and an accumulation of cells in the G1 phase (29). PTH activates different signal pathways that lead to an inhibitory and stimulatory effect on cell proliferation. In UMR-106 cell cultures, PTH exerted inhibitory and stimulatory effects that are dependent, respectively, on the activated cAMP/protein kinase A (PKA) and Ca2+/protein kinase C (PKC) pathway, through PTH1 receptors (PTH1R; see Refs. 33, 37, 41). Both pathways have been closely coupled with mitogen-activated protein kinase (MAPK) activity, which is associated with cell proliferation. It has been demonstrated that PTH inhibits MAPK activity and cell proliferation via the PKA pathway (41). On the other hand, the stimulation of MAPK activity and cell proliferation by PTH is dependent on the PKC pathway (37). Thus, in future studies, with the ability of FACS to isolate different subpopulations, it will be interesting to determine whether the distribution of PTH1R, the activity of MAPK, and the activation of PKA/PKC pathways are altered in GFP(+) and GFP(–) subpopulations during different phases of the cell cycle.
The confluence status of cultures influences the action of PTH not only on cell proliferation, but also apoptosis. The anti-apoptotic effect of PTH has been reported previously in calvarial osteoblast cells (23), chick embryonic hypertrophic chondrocytes (44), and MC3T3-E1 and C3H/10T1/2 cells (6). Similar to these reports, our results showed that PTH inhibited apoptosis in the preconfluent, but not the postconfluent, calvarial osteoblast cultures. In particular, the anti-apoptotic effect of PTH occurred solely in the GFP(–) subpopulation. In MC3T3-E1 and C3H/10T1/2 cell cultures overexpressing normal or mutant PTH1R, Chen et al. (6) demonstrated that PTH is anti-apoptotic in preconfluent cultures and pro-apoptotic in more differentiated postconfluent cultures. Recently, a study showed that the anti-apoptotic effect requires Runx2-mediated transcription of survival genes (such as Bcl-2) and the duration of this anti-apoptotic effect is regulated by the PTH-induced proteasomal degradation of Runx2 (3). Thus intermittent PTH exerts its anti-apoptotic effect through survival genes to increase the number of functional osteoblasts in each PTH exposure; in contrast, continuous PTH exposure induces the degradation of Runx2 to abolish the anti-apoptotic effect (3).
Based on the observations that the preconfluent cultures are less differentiated relative to the postconfluent cultures and the anti-apoptotic effect of PTH on the preconfluent cultures was restricted solely to the GFP(–) cells, we propose that the early PTH treatment may selectively inhibit apoptosis in a group of early undifferentiated cells to increase their survival and thereby promote osteoblastic commitment and differentiation.
To better understand the anabolic effect of transient PTH treatment in the present GFP-marked culture model, control and PTH-treated cells were fractionated in GFP(+) and GFP(–) cells using FACS based on GFP expression. Fractionated subpopulations were independently cultured to assess their subsequent osteoblast differentiation. The use of flow cytometry to enrich osteogenic populations has advanced our understanding of the diversity and the differentiation capacity of different osteoprogenitor subpopulations in bone formation (7, 14). Our result showed that transient PTH treatment enhances the ability of both GFP(+) and GFP(–) subpopulations to subsequently express GFP and form mineralized bone nodules. Specifically, PTH treatment greatly increased osteoblastic differentiation and bone nodule formation in the sorted GFP(–) cultures. The GFP fractionation selects for cells with enhanced [i.e., GFP(+)] or reduced [i.e., GFP(–)] osteogenic properties presumably because the GFP(+) population is already committed to osteogenesis, whereas the GFP(–) population has been depleted of that cell pool. Still, early progenitors in the GFP(–) population have the property for osteogenic differentiation, and it is this potential that is enhanced by the limited exposure to PTH.
Previous studies have indicated the necessity of heterotypic cell-cell interactions during osteoprogenitor differentiation in cultures (1, 34). In the present study, when compared with the primary cultures, the secondary cultures of fractionated GFP(+) and GFP(–) cells showed less osteoblast differentiation and fewer bone nodule formations (data not shown). These results are consistent with the need for cell-cell interactions between the GFP(+) and GFP(–) population to promote the full differentiation of osteoblasts.
Gene expression analysis in the GFP subpopulations sorted from day 7 calvarial osteoblast cultures using Northern blot showed that there is no difference between GFP(+) and GFP(–) cells regarding bone-associated molecular markers (25). On the other hand, with the use of microarray analysis, it was surprising to see the elevated gene expression associated with dendritic/macrophage/osteoclast cell lineage in the GFP(–) cells (25). Recent clinical and animal studies have suggested that active osteoclasts are required for the anabolic actions of PTH. Clinical studies have demonstrated that the combination of anti-resorptive reagents such as bisphosphonates together with intermittent PTH does not give rise to greater bone formation (4, 13). On the contrary, this combination negates the anabolic effect of PTH. Animal studies using genetically manipulated mice have provided evidence that the anabolic effect of PTH may be mediated by cells of the osteoclast lineage (26). It was demonstrated that osteopetrotic c-fos-deficient mice, which have a profound osteoclast defect, fail to show an anabolic response to PTH (9). These data strongly indicate the necessity of osteoclasts and their derived activity for the anabolic actions of PTH. Our quantitative PCR suggests that PTH acting on early progenitor cells strikingly upregulates their expression of RANKL, which in turn induces osteoclast differentiation from resident myeloid progenitors. Taking advantage of stage-specific expression of GFP combined with the ability of FACS to isolate immunolabeled cells in the macrophage/osteoclast lineage, it should now be possible to directly test this relationship in vitro and analyze the effect of PTH on the coupling between osteoclasts and different stages of osteoprogenitors.
In summary, our results showed that 1) the PTH treatment inhibited cell proliferation of both GFP(+) and GFP(–) cells in preconfluent cultures, 2) the reduction of apoptosis by PTH treatment in preconfluent cultures occurred primarily in the GFP(–) population, and 3) the fractionated cells pretreated with PTH, particularly the GFP(–) population, exhibited a greater degree of osteoblast differentiation. Based on the present results and our previous observation that a higher proportion of progenitor cells acquire the ability to produce a mineralized matrix following early transient exposure to PTH, it appears that PTH increases the survival of the progenitor population and enhances the commitment of the progenitor population to an osteogenic fate, resulting in a higher proportion of cells that achieve full osteoblast differentiation. We suggest that the anabolic effect of intermittent PTH in vivo may be a result of consecutive waves of committed osteoblast differentiation accumulated from each PTH exposure, in which PTH only acts on the early osteoprogenitor cells that are newly derived from the bone marrow.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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