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PPAR Res. 2006; 2006: 67297. Published online 2006 September 20. doi: 10.1155/PPAR/2006/67297. | PMCID: PMC1779577 |
Peroxisome Proliferator-Activated Receptor-γ Promotes Adipogenic Changes in Growth Plate Chondrocytes In Vitro Lai Wang, Yvonne Y. Shao, and R. Tracy Ballock* Orthopaedic Research Center, Department of Orthopaedic Surgery and Biomedical Engineering, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA Received April 9, 2006; Revised July 8, 2006; Accepted July 10, 2006. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited. Chondrocytes and adipocytes are two differentiated cell types which are both derived from mesenchymal cells. The purpose of this study was to investigate whether peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor involved in lineage determination during adipogenesis, is able to induce adipogenic differentiation in growth plate chondrocytes. Isolated epiphyseal chondrocytes were infected with a PPARγ adenovirus or treated with the PPARγ agonist ciglitazone. Both of these treatments resulted in lipid droplet accumulation and expression of the adipogenic markers aP2, lipoprotein lipase, and adipsin in chondrocytes. Proteoglycan matrix synthesis was decreased in the PPARγ-infected cells, as was the expression of the chondrogenic genes Col2a1 and aggrecan. Growth plate cells transfected with a PPARγ expression plasmid under the control of the collagen α1(II) promoter also demonstrated a similar adipogenic changes. Terminal differentiation of growth plate chondrocytes induced by thyroid hormone was also inhibited by overexpression of PPARγ and ciglitazone treatment, with decreased expression of alkaline phosphatase and Runx2/Cbfa1 genes. These in vitro data suggest that PPARγ is able to promote adipogenic differentiation in growth plate chondrocytes, while negatively regulating chondrogenic differentiation and terminal differentiation. Longitudinal growth of the skeleton is a result of endochondral ossification that occurs at the growth
plate [ 1]. Endochondral ossification is a multistep process
that includes differentiation of mesenchymal cells into
chondrocytes, cell proliferation, hypertrophic differentiation,
matrix mineralization, apoptosis, vascular invasion, and
eventually the replacement of the cartilage by bone. The first step of growth plate development is the commitment of
mesenchymal stem cells to the chondrogenic lineage. Mesenchymal
stem cells exhibit a high differentiation plasticity. They are
capable of differentiating into chondrocytes, osteoblasts,
adipocytes, and other tissues of mesenchymal origin [ 2].
Interconversion between mesenchymal phenotypes is thought to be
under control of specific transcription factors, including the Sox
family in chondrogenesis [ 3], Runx2/Cbfa1 in osteogenesis
[ 4], and PPAR γ (peroxisome proliferator-activated
receptor- γ), and C/EBP (CCAAT/enhancer-binding protein) in
adipogenesis [ 5]. PPAR γ is a key transcriptional regulator of adipogenesis
[ 5]. PPAR γ is also expressed in preosteoblastic cells
and is thought to play a role in regulation of bone metabolism.
PPAR γ and PPAR γ activators inhibit the maturation
of preosteoblastic cells to osteoblasts [ 6– 8]. Free
fatty acids activate PPARs and induce adipocyte-like
differentiation of osteosarcoma cell lines [ 6]. Lecka-Czernik
et al observed that PPAR γ2 negatively regulates stromal
cell plasticity by suppressing expression Osf2/Cbfa1 and
osteoblast-like biosynthetic activity, while promoting
differentiation to adipocytes [ 7]. Conversely, PPAR γ
insufficiency enhances osteogenesis through increased osteoblast
formation from bone marrow progenitors. Homozygous PPAR γ-deficient ES cells fail to differentiate into adipocytes, but
increase bone mass by stimulating osteoblastogenesis from bone
marrow progenitors [ 8]. Transdifferentiation of chondrocytes to adipocytes has been
previously reported by Heermeier et al, who observed that
chondrocytes of the mouse xiphoid process undergo
transdifferentiation into adipocytes in the presence of 10% fetal
calf serum [ 9]. Based on the finding that PPAR γ is expressed in growth
plate chondrocytes [ 10], as well as the evidence that
PPAR γ is able to compete with the thyroid hormone receptor
(TR) for binding to retinoic acid receptor X to inhibit growth
plate cell hypertrophy [ 11], the purpose of this study was to
investigate whether PPAR γ and its ligands are able to
promote adipogenic differentiation and suppress chondrogenic
differentiation in growth plate chondrocytes. Cell culture Chondrocytes were isolated from the resting zone of the distal
femoral growth plate of 2-day old neonatal Sprague-Dawley rats by
sequential digestion in trypsin/EDTA (Invitrogen, Carlsbad, Calif)
for 1 hour at 37°C, followed by 0.3% collagenase type I
(Worthington, Lakewood, NJ) for 4 hours at 37°C [ 12].
Cells were resuspended in DMEM/F12 medium (Invitrogen)
supplemented with a defined media supplement (ITS+1, Sigma, St
Louis, Mo) and plated in monolayer at a density of
5×10 5 cells/cm 2, or in a pellet culture of
1×10 5 cells/mL as indicated [ 12].
Tri-iodothyronine (T3, Sigma) at a concentration of
100 ng/mL and ciglitazone (BioMol, Plymouth Meeting, Pa) at a
concentration of 5 μM were added to the medium as
indicated. Immunoblotting Total cellular protein was extracted from chondrocytes treated
with 5 μM of ciglitazone using RIPA buffer [ 9]. An
equal amount of protein was subjected to SDS-PAGE, and transferred
onto nitrocellulose membranes. The blots were incubated with
anti-PPAR γ and anti-actin (Santa Cruz Biotechnology, Santa
Cruz, Calif) followed by a HRP-conjugated secondary antibody.
Immunoreactive proteins were visualized by Western Blotting
Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology).
Immunoblot bands were quantitated with Kodak 1D Image Analysis
Software (Eastman Kodak Company, Rochester, NY). Adenovirus infection Recombinant adenovirus carrying PPAR γ1 (Ad-PPAR γ)
was kindly provided by Dr J. L. Jameson (Northwestern University
Medical School, Chicago, Ill). Ad-PPAR γ contains mouse
PPAR γ1 cDNA driven by the CMV promoter/enhancer with an
SV40 polyadenylation sequence [ 13]. Ad-Gal, which contains
β-galactosidase driven by CMV promoter, was used to
evaluate the efficiency of gene transduction. Eighteen hours after
plating in monolayer, growth plate chondrocytes were infected with
adenoviral vectors at a multiplicity of infection (MOI) of 100.
Fresh media were added 24 hours after infection and
incubated for 72 hours to collect the cell
protein extracts. β-galactosidase expression was detected
in 80% of cells after 24 hours of infection with Ad-Gal.
Expression of introduced PPAR γ genes was confirmed by
immunoblot. Plasmid construction and transient transfection The full-length cDNA of mouse PPAR γ was excised by
Asp718/NheI digestion from pCMX-PPAR γ (kindly
provided by Dr R. Evans, Salk Institute, La Jolla, Calif). The
ends of this fragment were blunted with Klenow polymerase and
ligated to a blunt-ended BamHI site in the p1757 plasmid
containing the rat α1(II) collagen promoter (kindly
provided by Dr Y. Yamada, NIDR, Bethesda, Md) [ 14]. The cDNA
encoding the mouse PPAR γ was thus located downstream of
the rat α1 (II) collagen promoter element (−977 to
+110). Nucleotide sequence analysis confirmed the correct
orientation of the PPAR γ cDNA. Growth plate cells were transfected with 10 μg of
p1757-PPAR γ or p1757 as a negative control by lipofection
(Fugene 6, Roche, Indianapolis, Ind) in the presence of
4 units/mL of hyaluronidase. Sixteen hours later, the cells
were trypsinized and centrifuged to pellets cultured in DMEM/F12
plus ITS+ supplements [ 11]. Histochemical staining For the analysis of adipogenic differentiation, adipogenesis and
lipid accumulation in the growth plate cells were examined by
staining with Oil Red-O. After 10 days of culture, cells were
washed gently with PBS followed by staining with a filtered
solution of 0.5% Oil Red-O (Sigma) in 60% isopropanol for 20
minutes. After washing cells with PBS three times, cells were kept
in 75% glycerol solution and observed under a phase-contrast
microscope. Alcian blue staining was used to detect chondrocyte-specific
proteoglycans at 10 days of culture. Cells were stained with a
4 : 1 ratio of 0.1 M HCl/0.5% Alcian blue stock
(0.5% Alcian blue in 95% ethanol) overnight at 37°C in a
humidified atmosphere. Cells were then washed twice with PBS to
stop reaction and once with 70% ethanol to reduce background. For alkaline phosphatase (ALP) staining, cultured plates were
rinsed with PBS at 10 days of culture, fixed in 3.7% formaldehyde
at room temperature for 10 minutes, and stained in the dark for 30
minutes in a 0.1 M Tris-HCl
solution (pH 8.5) containing 0.2 mg/mL of Napthol AS-MX
phosphate and 0.6 mg/mL of Fast Blue BB salt (Sigma). Quantitative real-time RT-PCR The expression of chondrocyte or adipocyte-specific RNA markers
was analyzed using quantitative real-time RT-PCR. Total RNA was
isolated from cultured growth plate chondrocytes using the RNeasy
Kit (Qiagen, Valencia, Calif) 4 days after adenovirus infection or
plasmid transfection. Reverse transcription was performed using
random primers and Superscript III (Invitrogen). Real-time PCR
reactions were conducted in an ABI Prism 7700 Sequence Detection
System using SYBR Green PCR core reagents (Applied Biosystems,
Foster City, Calif). The comparative C T method
(ΔΔC T method) was utilized for relative
quantitation of gene levels of expression. 18S rRNA was used as an
internal control for normalization of target gene expression. The
forward and reverse primers for the amplifications are listed in
Table 1. | Table 1Primer sequences used for real-time PCR. |
Statistical analysis The data for real-time PCR are represented as mean ± standard
deviation. Values are assessed by one-way ANOVA with the
Bonferroni post-hoc test and Student t test at a significance
level of P < .05. Ciglitazone upregulates PPARγ
expression in growth plate chondrocytes Treatment of growth plate cells with ciglitazone resulted in
increases of both PPARγ mRNA and protein. PPARγ
mRNA was increased 9-fold after addition of ciglitazone
(5 μM) for 4 days (), while
PPARγ protein levels were increased approximately 5-fold
(). PPARγ induces adipogenic
differentiation in growth plate chondrocytes Phase-contrast microscopy demonstrated that the Ad-PPARγ-infected growth plate chondrocytes acquired the morphology
characteristic of adipocytes after culture in monolayer for 10
days. Approximately 50% of the cells had accumulated vacuoles,
which were positive for Oil Red-O staining of lipid accumulation
(). The control cells infected with Ad-Gal
demonstrated few Oil Red-O positive vacuoles. Cells treated with
5 μM of ciglitazone alone for 10 days also showed enhanced
Oil Red-O staining (20% of the total cells), while the
combination of ciglitazone treatment and Ad-PPARγ
infection further enhanced lipid accumulation (over 70% of cells
staining positively with Oil Red-O). To characterize the phenotype of the transformed cells in more
detail, the cells were cultured in three-dimensional cell pellets
and the expression of adipocyte differentiation marker genes
examined by real-time RT-PCR at day 4 of the culture period.
Compared with the control samples, the levels of expression of the
adipogenic marker genes aP2, LPL,and adipsin increased 6.6-, 4.4-
and 4.6-folds, respectively, on day 4 in the 5 μM
ciglitazone-treated cells (). Expression of
aP2, LPL, and adipsin genes increased in the
Ad-PPARγ-infected cells by 75.1-, 40.2-, and 76.6-folds,
respectively in the absence of ciglitazone, and 101.1-, 44.9-, and
96.3-folds, respectively, in the presence of ciglitazone
(5 μM). In order to address the possibility that PPARγ was acting
on an undifferentiated mesenchymal stem cell as opposed to a
differentiated chondrocyte, p1757-PPARγ expression
plasmid was generated in which a PPARγ cDNA was
placed under the transcriptional control of the rat COL2A1 gene
promoter and enhancer sequences. Oil Red-O staining of the
p1757-PPARγ-transfected cells maintained in
three-dimensional pellet culture for 10 days showed markedly
increased lipid accumulation (). Real-time
RT-PCR demonstrated that aP2, LPL, and adipsin mRNA expressions
were upregulated 21.1-, 12.9-, and 17.9-folds, respectively,
compared with the cells transfected with the empty p1757 plasmid
at day 4 after transfection (). PPARγ induces loss of chondrocytic phenotype in growth plate cells Alcian blue staining was used to detect the accumulation of
cartilage-specific proteoglycan. At day 10, the control cultures
of growth plate cells still accumulated abundant proteoglycan
(). No significant difference in proteoglycan
accumulation was observed in the growth plate chondrocytes that
were treated with 5 μM of ciglitazone alone. Compared to
the Ad-Gal-infected cells, the Ad-PPARγ-infected cultures
were stained less intensely with Alcian blue, especially the surrounding
of the cells that contained vacuoles. Addition of 5 μM of
ciglitazone to the Ad-PPARγ-infected cultures resulted in
a further decrease in proteoglycan matrix. Quantitative RT-PCR demonstrated that the chondro-cyte-specific
genes COL2A1 and aggrecan were downregulated by both PPARγ
and ciglitazone (). Treatment with
5 μM of ciglitazone for 4 days resulted in a 33% decrease
of COL2A1 mRNA and a 17% decrease in aggrecan mRNA expression.
Combination of both PPARγ adenovirus and 5 μM of
ciglitazone resulted in a 50% decrease of COL2A1 mRNA and a 22%
decrease in aggrecan mRNA expression. PPARγ inhibits T3-induced hypertrophy
and mineralization in growth plate chondrocytes Thyroid hormone is a crucial regulator in growth plate chondrocyte
hypertrophic differentiation and matrix mineralization
[ 15– 17]. Growth plate cells treated with thyroid hormone and 5 μM of ciglitazone demonstrated decreased alkaline
phosphatase histochemical staining compared to cells treated with
T3 alone (). Quantitative RT-PCR analysis of
growth plate cells in pellet cultures treated with T3 and
5 μM of ciglitazone for 4 days confirmed a 64% decrease
in ALP mRNA compared to cells treated with T3 alone
(). Infection with PPAR γ adenovirus
in cells treated with T3 also decreased expression of ALP mRNA
approximately 71% in the absence of ciglitazone, and 76% in the
presence of 5 μM of ciglitazone. Runx2/Cbfa1 is expressed
in chondrocytes as they initiate chondrocyte hypertrophy and
maturation. Ciglitazone at a concentration of 5 μM
decreased the T3-induced expression of Runx2 by 36%.
Ad-PPAR γ infection decreased the expression of Runx2 mRNA
by 54% in the absence of ciglitazone and by 66% in the
presence of 5 μM of ciglitazone. Growth plate chondrocytes originate from multipotential
mesenchymal stem cells that can differentiate into other cell
types including adipocytes. We present evidence in this study that
growth plate cells continue to display differentiation plasticity
and are able to undergo adipogenic changes and a reciprocal
decrease of chondrocytic markers when PPARγ is overexpressed. It has been previously reported that chondrocytes are able to
transdifferentiate into adipocytes in vitro [ 9]. The fatty
acid content of the serum added to the culture media has been
implicated as a potential cause of this trans-differentiation
process [ 6]. We used a serum-free culture system in these
experiments to avoid the possibility that fatty acids in the serum
might induce the adipogenic changes observed. Ciglitazone is one of the thiazolidinedione classes of
antidiabetic compounds which can activate PPAR γ [ 18].
Ciglitazone not only increases endogenous PPAR γ
transcriptional activity [ 11], but also upregulates
PPAR γ mRNA and protein expression in growth plate
chondrocytes, as observed in this study. Activation of endogenous PPARγ by ciglitazone or
adenoviral overexpression of PPARγ in growth plate
chondrocytes resulted in acquisition of adipogenic features in
both high-density monolayer cultures and three-dimensional pellet
cultures of growth plate chondrocytes, as evidenced by cell
morphology, lipid accumulation, and expression of adipocyte marker
genes aP2, LPL, and adipsin. Growth plate cells maintained in
monolayer cultures seemed to acquire features of the adipocytic
phenotype and lose features of the chondrocytic phenotype more
readily than those in the pellet cultures (data not shown). To confirm that the adipocyte-like cells were differentiated
directly from chondrocytes and not from other cell types such as
undifferentiated mesenchymal stem cells, growth plate cells were
transfected with a PPARγ plasmid under the control of a
collagen α1(II) promoter. Acquisition of the adipogenic
phenotype in these transfected cells was similar to the cells
infected with an adenovirus encoding PPARγ and driven by
the CMV promoter/enhancer. While PPARγ and ciglitazone converted cells of the
chondrocyte lineage to an adipocytic phenotype, features of the
chondrocyte phenotype were simultaneously suppressed. PPARγ inhibited the ability of chondrocytes to terminally
differentiate into hypertrophic cells, and suppressed the
expression of genes encoding chondrocyte-specific extracellular
matrix proteins. Slipped capital femoral epiphysis (SCFE) is an obesity-related hip
disease in children characterized by weakness in the growth plate
of the proximal femur, delayed skeletal maturation, and eventual
mechanical failure of the physis [ 19– 21]. We speculate
that obesity may induce the expression of PPAR γ isoforms
in growth plate chondrocytes, resulting in phenotypic changes that
interrupt normal skeletal maturation at the growth plate through
interference with thyroid hormone signaling. This interference
with thyroid hormone-mediated terminal differentiation of growth
plate cells and resulting decreased mineralization of the
cartilage matrix would be expected to reduce the resistance of the
growth plate to shear stresses. Therefore this delay in maturation
at the growth plate, combined with both the increased mechanical
stress resulting from increased body weight and the decreased
shear stress resulting from delayed maturation, may combine to
cause the proximal femoral epiphysis to slip. We thank Dr J. L. Jameson (Northwestern University Medical School,
Chicago, Ill) for providing the recombinant adenovirus carrying
PPARγ1, Dr. R. Evans (Salk Institute, La Jolla, Calif) for
providing the PPARγ expression plasmids, and Dr Y. Yamada
(National Institutes of Health, Bethesda, Md) for
providing p1757 plasmid. This work was supported by a grant from
the National Institutes of Health to R. Tracy Ballock (1
RO1 AR47955). 1. Ballock RT, O'Keefe RJ. Physiology and pathophysiology of the growth plate. Birth Defects Research Part C: Embryo Today: Reviews. 2003;69(2):123–143. 2. Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Research and Therapy. 2003;5(1):32–45. [PubMed] 3. de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W. Transcriptional mechanisms of chondrocyte differentiation. Matrix Biology. 2000;19(5):389–394. [PubMed] 4. Komori T. Runx2, a multifunctional transcription factor in skeletal development. Journal of Cellular Biochemistry. 2002;87(1):1–8. [PubMed] 5. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM. Transcriptional regulation of adipogenesis. Genes & Development. 2000;14(11):1293–1307. [PubMed] 6. Diascro DD, Jr, Vogel RL, Johnson TE, et al. High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells. Journal of Bone and Mineral Research. 1998;13(1):96–106. [PubMed] 7. Lecka-Czernik B, Gubrij I, Moerman EJ, et al. Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARγ2. Journal of Cellular Biochemistry. 1999;74(3):357–371. [PubMed] 8. Akune T, Ohba S, Kamekura S, et al. PPARγ insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. The Journal of Clinical Investigation. 2004;113(6):846–855. [PubMed] 9. Heermeier K, Strauss PG, Erfle V, Schmidt J. Adipose differentiation of cartilage in vitro. Differentiation. 1994;56(1-2):45–53. [PubMed] 10. Shao YY, Wang L, Hicks DG, Tarr S, Ballock RT. Expression and activation of peroxisome proliferator-activated receptors in growth plate chondrocytes. Journal of Orthopaedic Research. 2005;23(5):1139–1145. [PubMed] 11. Wang L, Shao YY, Ballock RT. Peroxisome proliferator activated receptor-γ (PPARγ) represses thyroid hormone signaling in growth plate chondrocytes. Bone. 2005;37(3):305–312. [PubMed] 12. Ballock RT, Reddi AH. Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated
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