The PPARgamma-selective ligand BRL-49653 differentially regulates the fate choices of rat calvaria versus rat bone marrow stromal cell populations
- Takuro Hasegawa†1,
- Kiyoshi Oizumi†2, 4,
- Yuji Yoshiko†2, 3Email author,
- Kazuo Tanne1,
- Norihiko Maeda3 and
- Jane E Aubin2Email author
© Hasegawa et al; licensee BioMed Central Ltd. 2008
Received: 30 October 2007
Accepted: 14 July 2008
Published: 14 July 2008
Osteoblasts and adipocytes are derived from a common mesenchymal progenitor and an inverse relationship between expression of the two lineages is seen with certain experimental manipulations and in certain diseases, i.e., osteoporosis, but the cellular pathway(s) and developmental stages underlying the inverse relationship is still under active investigation. To determine which precursor mesenchymal cell types can differentiate into adipocytes, we compared the effects of BRL-49653 (BRL), a selective ligand for peroxisome proliferators-activated receptor (PPAR)γ, a master transcription factor of adipogenesis, on osteo/adipogeneis in two different osteoblast culture models: the rat bone marrow (RBM) versus the fetal rat calvaria (RC) cell system.
BRL increased the number of adipocytes and corresponding marker expression, such as lipoprotein lipase, fatty acid-binding protein (aP2), and adipsin, in both culture models, but affected osteoblastogenesis only in RBM cultures, where a reciprocal decrease in bone nodule formation and osteoblast markers, e.g., osteopontin, alkaline phosphatase (ALP), bone sialoprotein, and osteocalcin was seen, and not in RC cell cultures. Even though adipocytes were histologically undetectable in RC cultures not treated with BRL, RC cells expressed PPAR and CCAAT/enhancer binding protein (C/EBP) mRNAs throughout osteoblast development and their expression was increased by BRL. Some single cell-derived BRL-treated osteogenic RC colonies were stained not only with ALP/von Kossa but also with oil red O and co-expressed the mature adipocyte marker adipsin and the mature osteoblast marker OCN, as well as PPAR and C/EBP mRNAs.
The data show that there are clear differences in the capacity of BRL to alter the fate choices of precursor cells in stromal (RBM) versus calvarial (RC) cell populations and that recruitment of adipocytes can occur from multiple precursor cell pools (committed preadipocyte pool, multi-/bipotential osteo-adipoprogenitor pool and conversion of osteoprogenitor cells or osteoblasts into adipocytes (transdifferentiation or plasticity)). They also show that mechanisms beyond activation of PPARγ by its ligand are required for changing the fate of committed osteoprogenitor cells and/or osteoblasts into adipocytes.
Osteoblasts and adipocytes derive from a common mesenchymal progenitor and appear to display plasticity between the phenotypes and an inverse relationship between expression of the two lineages under certain pathological and several experimental conditions . For example, a reciprocal relationship between marrow adipocyte content and bone mass has been reported in osteoporosis . Studies on the senescence-accelerated (SAMP6)  and aging mouse  models also suggest that osteoblastogenesis is decreased concomitant with an increase in the number of marrow adipocytes.
As summarized previously , however, it can often be difficult to interpret the developmental and cellular basis of the inverse relationship between osteoblasts and adipocytes, when both bi-/multipotential progenitors reside in the same populations as restricted monopotential progenitors that may display plasticity or transdifferentiation capacity. Data from different approaches confirm multiple possible cellular events underlying transitions between the two lineages (see, e.g., [6, 7]). Thus, the number of adipocytes in bone marrow stromal or bone-derived populations may reflect the frequency of committed adipocyte precursors (pathway 2), the conversion of osteoprogenitor cells and/or osteoblasts into adipocytes (pathway 3), and changes to the balance in commitment choices of mesenchymal stem cells (pathway 1). Specific treatments and different culture conditions including different cell densities may dependently or independently affect any or all of these lineage choices.
Adipocyte differentiation is under the control of peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor superfamily, in concert with members of the CCAAT/enhancer-binding protein (C/EBP) family of basic leucine zipper nuclear transcription factors . The analyses of homo- and heterozygous PPARγ-deficient mice and ES cells have suggested that PPARγ may positively and negatively determine the fate of osteo-adipocyte precursors respectively at least during early differentiation events . The thiazolidinedione antidiabetic agents, which are PPARγ-selective ligands, induce adipogenesis in a variety of culture models including mesenchymal stem/stromal cells and cell lines (see, e.g., [9–12]). However, different ligands of the thiazolidinedione class with different capacities for PPARγ activation appear to differentially modulate adipogenesis versus osteoblastogenesis in the mouse model . Taken together, these reports suggest that PPARγ-selective ligands may induce adipogenesis not only in mesenchymal stem or multipotential progenitor cells (pathway 1), but also in osteoprogenitors and/or osteoblasts (pathway 3).
Three members of the C/EBP family (C/EBPα, β, and δ) have also been implicated in adipocyte differentiation . Analyses of the differentiation program in adipocytic cell lines and genetically altered mice have shown that C/EBP and PPAR work sequentially and cooperatively to stimulate the molecular events required for adipogenesis. C/EBPβ and δ also activate osteocalcin gene transcription and synergize with runt-related transcription factor 2 (Runx2), a master regulator for osteoblastogenesis, at the C/EBP element to regulate bone-specific expression . To elucidate the contribution of recruitment from a committed osteoblast precursor pool (pathway 3) versus multipotential progenitor pool (pathway 1) to adipogenesis induced by the PPARγ-selective ligand BRL-49653 (BRL), we compared primary cultures of fetal rat calvaria (RC) cells (in which osteoblasts derive mainly from committed osteoprogenitors, a model of pathway 3, and in which committed preadipocytes also reside, a model of pathway 2) with rat bone marrow (RBM) cultures (representing a model of pathway 1) .
The effect of BRL on osteo/adipogenic differentiation outcomes in RC single cell-derived colonies
Oil red O+
ALP+/Oil red O+
ALP-/Oil red O-
It is worth considering that because RBM (from mesoderm) and RC cells (from a mixture of neuroectoderm and mesoderm) are embryologically distinct, the regulation of fate selection by progenitors in the stromal versus calvarial populations may be different. For example, some hormones and growth factors regulate intramembranous or periosteal bone formation (calvaria or periosteal growth of other bones) differently than endosteal bone formation (stromal cell-derived trabeculae and endosteal growth) (e.g., parathyroid hormone effects; ). Thus, RC and RBM cells may be subject to differential regulation by activation of PPARγ (BRL), while responding similarly to other factors, e.g., dexamethasone [1, 5, 14] and endogenous glucocorticoids as evidenced by 11β-hydroxysteroid dehydrogenase type 2-overexpressing mice driven by α1(I)-collagen promoter .
Bone marrow stromal cell populations, often referred to as mesenchymal stem cells, are capable of undergoing differentiation along multiple mesenchymal lineages, but are heterogeneous in the capacity of individual colonies (CFU-F) to express multilineage versus more restricted capacity (see, e.g., , and discussion in ). However, the frequency of multipotential progenitors in stromal populations appears quite high, e.g., ~30% of single cell-derived colonies in human stroma  and >90% in a recently-described alternative isolation/enrichment method . As described above, RC cell populations also contain a mixture of cell lineages and types, e.g., osteoprogenitors and osteoblasts at different differentiation stages, fibroblastic cells, adipocyte precursors , as well as a multipotential side population . However, the frequency of functionally multipotential precursor or stem cells, e.g., the RCJ3.1, clonally-derived multipotential cell line , bipotential adipo-osteoprogenitors  or SP cells  appears to be very low in RC populations. Additionally, RC populations contain preadipocytes [25, 26] (pathway 2; Fig. 6) and possibly circulating progenitor cells from bone marrow . Our data support the view that BRL induces differentiation/maturation of adipocytes mainly from a committed preadipocyte pool in RC populations. However, our data on single cell colonies suggest that a subpopulation of committed osteoprogenitors or relatively mature osteoblasts is also induced to switch on the adipogenic pathway (pathway 3) when PPARγ is activated, as we also recently proposed with leukemia inhibitory factor treatments . The expression of PPARγ and/or C/EBPs in some osteogenic cells in our models and MC3T3-E1 cells  may also predispose them to the pathway 3. It is also possible that at least some of the osteo-adipogenic cells in BRL-treated RC populations represent recruitment from multipotential or bipotential progenitor pools equivalent to those in stromal cell populations; the low frequency of such cells in RC populations would not be expected to markedly alter overall osteoblast or adipocyte colony numbers (Fig. 4, 5A, and see ), although we cannot discount the possibility that activation of PPARγ dramatically changes their frequency.
In BRL-treated single cell colonies, mineralized colonies with and without adipsin expression (Fig. 5B) correlated with high and low levels of PPARγ respectively, which is consistent with the hypothesis that RC cell populations comprise two kinds or differentiation stages of progenitor or osteoblastic cells as we described previously with respect to glucocorticoid regulation [29, 30], i.e., a subpopulation responsive to the PPARγ selective ligand and a non-responsive subpopulation, with the former capable of expressing both the adipocyte and osteoblast programs and the latter expressing only the osteoblast program. Our data are also consistent with the view that activation of PPARγ alone by its ligand is not sufficient to induce complete conversion of osteoprogenitor cells into adipocytes. This view is consistent with an earlier report in which the PPARγ selective ligand was unable to increase PPARγ expression and cause adipocyte differentiation in human adult trabecular-derived bone cells, although these cells were able to undergo adipogenesis in the presence of isobutylmethylxanthine (IBMX) plus dexamethasone with concomitant increase in expression of PPARγ . Treatment of MC3T3-E1 cells retrovirally overexpressing PPARγ with insulin, dexamethasone and IBMX increases the adipogenic capacity, which is further enhanced when C/EBPα is co-overexpressed . Together these data suggest that mechanisms beyond activation of PPARγ by its ligand (BRL) are required for changing the fate of committed osteoprogenitor cells and/or osteoblasts into adipocytes.
BRL is a potent PPARγ-selective ligand but it is also known to increase PPARγ expression , as it did in RC cells, along with C/EBPα. These two transcription factors display interactive regulatory roles and cooperate to promote adipocyte differentiation . The role of these two factors, or other members of the families in osteoblasts has been investigated recently (see e.g., ). Wnt/β-catenin signaling suppresses C/EBPα and PPARγ, which shifts mesenchymal cell fate toward osteoblastogenesis at the expense of adipogenesis [35, 36]. The lack of coordinate expression of PPAPγ and C/EBPα during osteoblast differentiation in our models suggests that they may not cooperate in osteoblast differentiation as they do in adipocyte differentiation.
The present study showed clear differences in the capacity of the PPARγ-selective ligand BRL-49653 to alter the fate choices of precursor cells in stromal versus calvarial cell populations and that recruitment of adipocytes can occur from multiple precursor cell pools (committed preadipocyte pool, multi-/bipotential osteo-adipoprogenitor pool and conversion of osteoprogenitor cells or osteoblasts into adipocytes (transdifferentiation or plasticity)). They also show that mechanisms beyond activation of PPARγ by its ligand are required for changing the fate of committed osteoprogenitor cells and/or osteoblasts into adipocytes.
Animal use and procedures were approved by the University of Toronto Animal Care Committee and Research Facilities for Laboratory Animal Science, Natural Science Center for Basic Research and Development, Hiroshima University.
RBM stromal cell cultures
Bone marrow stromal cells from the femora of young adult male Wistar rats (110–130 g) were cultured essentially as described . Briefly, femora were dissected and immersed α-MEM with antibiotics. After removal of the epiphysis, the marrow was collected by flushing MEM, supplemented with antibiotics and 10% fetal calf serum (FCS), through the shafts with a syringe. The resulting cell suspension was plated into a T75-tissue culture flask and incubated in the same medium supplemented additionally with ascorbic acid (50 μg/ml) and dex (10 nM) (differentiation medium) for a week at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were then harvested with trypsin and collagenase and subcultured at 0.2 × 104 cells/cm2 in differentiation medium; medium was changed every 2–3 days until bone nodules were observed. Cells were treated chronically with or without BRL (10–100 nM). To promote mineralization, 10 mM β-glycerophosphate was added for the last 5 days.
RC Cell Cultures
Cells were enzymatically isolated from calvariae of 21-d Wistar rat fetuses by sequential digestion with collagenase as described . Cells obtained from the last four of five digestion steps were grown in α-MEM containing 10% FCS and antibiotics. After 24 h, cells were collected by trypsinization, and cultured at the same cell density as RBM in the presence or absence of BRL (0.1–1 μM) in differentiation medium as above but without dex. To obtain single cell colonies, RC cells were also cultured at very low density (500 cells/100 mm dish) in differentiation medium .
Total RNA was harvested at appropriate culture time points with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Twenty micrograms of total RNA were electrophoresed on 1% agarose-17% formaldehyde gels and transferred onto positively charged nylon membranes (Hybond-N+, GE Healthcare, Buckinghamshire, UK). The membranes were cross-linked, prehybridized and hybridized with specific probes as described (see below, and ). After washing, the membranes were exposed to X-ray film at -80°C for various times. cDNAs for rat BSP (pBSP1), OCN (pOC9), ALP and OPN were described previously . The aP2 and adipsin cDNAs were cloned from BRL-treated mouse bone marrow cells, using specific primers designed with Primer Picking (Primer 3). The primer sequences were as follows: aP2, 5'-ATA GCA CCC TCC TGT GCT G-3' and 5'-CCA GCC TCT TCC TTT GCT C-3'; adipsin, 5'-TGT ACT TCG TGG CTC TGG TG-3' and 5'-ATC CGG TAG GAT GAC ACT CG-3'. A mouse LPL cDNA was purchased from the American Type Culture Collection (63117; Rockville, MD). All probes were labeled with [α32P]dCTP using a Multiprime DNA labelling system (GE Healthcare). L32 was used as internal control.
Real-time and semiquantitative RT-PCR
cDNA was synthesized from 2 μg or less (100–400 ng from single cell colonies) of total RNA isolated from cells and tissues as above, using Superscript II (Invitrogen) or RevatraAce (Toyobo, Osaka, Japan). The sequence of PCR primers were designed using Primer 3; PPARα, 5'-CGA CAA GTG TGA TCG AAG CTG CAA G-3' and 5'-GTT GAA GTT CTT CAG GTA GGC TTC-3'; PPARδ/β, 5'-GGG CTG ACG GCC AGC GAG GGA-3' and 5'-TGG GGA GAA CCG GGT GCC GA-3'; PPARγ, 5'-GCG GAG ATC TCC AGT GAT ATC-3' and 5'-TCA GCG ACT GGG ACT TTT CT-3'; BSP, 5'-CGC CTA CTT TTA TCC TCC TCT G-3' and 5'-CTG ACC CTC GTA GCC TTC ATA G-3'; OCN, 5'-AGG ACC CTC TCT CTG CTC AC-3'and 5'-AAC GGT GGT GCC ATA GAT GC-3'; Runx2, 5'-CTT CAT TCG CCT CAC AAA C-3' and 5'-CAC GTC GCT CAT CTT GCC GG-3'; L32, 5'-CAT GGC TGC CCT TCG GCC TC-3' and 5'-CAT TCT CTT CGC TGC GTA GCC-3'. The primers for C/EBPα,β,δ, and adipsin were as follows: C/EBPα, 5'-GAA TCT CCT AGT CCT GGC TC-3' and 5'-GAT GAG AAC AGC AAC GAG TAC-3 ; C/EBPβ, 5'-GCC ACG GAC ACC TTC GAG G-3' and 5'-CGG CTC CGC CTT GAG CTG-3' ; C/EBPδ, 5'-GCG GAT CCG AGG TGA CAG CCC AAC TTG-3' and 5'-GGA ATT CGG TCG TTC GGA GTC TCT AAG-3' ; adipsin, 5'-TGT ACT TCG TGG CTC TGG TG-3' and 5'-ATC CGG TAG GAT GAC ACT CG-3' . Real-time PCR was carried out by using the LightCycler system (SYBR Green 1; Roche Diagnostics, Indianapolis, IN) according to manufacturer's instructions. L32 was used as internal control. For semiquantitative assessment of expression levels, each PCR reaction was done over an increasing series of cycles from 17 to 45 cycles and PCR products were size fractionated on 1% ethidium bromide/agarose gels. A representative gel is shown in which the bands are visualized from cycle number within the exponential phase of amplification as determined by densitometric analysis of amplimers (Image Quant software, MD Apps) (see for example, ).
For lipid-containing adipocyte detection, cells were fixed in 10% neutral buffered formalin and stained with oil red O solution . To detect osteoblasts, cells were incubated with either single or double-stained for ALP and mineral (2.5% silver nitrate (von Kossa)) as described . For single colony analyses, colonies were double-stained with ALP and oil red O.
Experiments were repeated on a minimum of three independent cell isolates. In some cases, as specified in the figure legends, a representative experiment is shown in which data points are the mean ± SD of triplicate samples. Statistical significance was computed by ANOVA and Dunnet's t-test and set at the level of p < 0.05 for high density cultures. For low density cultures, at least three independent experiments were done; data were analyzed by Fisher's exact test and significance was set at p < 0.05.
This work was supported by a grant from the Canadian Institutes of Health Research (MOP-83704) to JEA and Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (13771074) to YY.
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