Oxysterols regulate differentiation of mesenchymal stem cells: pro-bone and anti-fat.
ABSTRACT Pluripotent mesenchymal stem cells can undergo lineage-specific differentiation in adult organisms. However, understanding of the factors and mechanisms that drive this differentiation is limited. We show the novel ability of specific oxysterols to regulate lineage-specific differentiation of mesenchymal stem cells into osteogenic cells while inhibiting their adipogenic differentiation. Such effects may have important implications for intervention with osteoporosis.
Oxysterols are products of cholesterol oxidation and are formed in vivo by a variety of cells including osteoblasts. Novel pro-osteogenic and anti-adipogenic effects of specific oxysterols on pluripotent mesenchymal cells are demonstrated in this report. Aging and osteoporosis are associated with a decrease in the number and activity of osteoblastic cells and a parallel increase in the number of adipocytic cells.
The M2-10B4 pluripotent marrow stromal cell line, as well as several other mesenchymal cell lines and primary marrow stromal cells, was used to assess the effects of oxysterols. All results were analyzed for statistical significance using ANOVA.
Pro-osteogenic and anti-adipogenic effects of specific oxysterols were assessed by the increase in early and late markers of osteogenic differentiation, including alkaline phosphatase activity, osteocalcin mRNA expression and mineralization, and the decrease in markers of adipogenic differentiation including lipoprotein lipase and adipocyte P2 mRNA expression and adipocyte formation. Complete osteogenic differentiation of M2 cells into cells expressing early and late markers of differentiation was achieved only when using combinations of specific oxysterols, whereas inhibition of adipogenesis could be achieved with individual oxysterols. Oxysterol effects were in part mediated by extracellular signal-regulated kinase and enzymes in the arachidonic acid metabolic pathway, i.e., cyclo-oxygenase and phospholipase A(2). Furthermore, we show that these specific oxysterols act in synergy with bone morphogenetic protein 2 in inducing osteogenic differentiation. These findings suggest that oxysterols may play an important role in the differentiation of mesenchymal stem cells and may have significant, previously unrecognized, importance in stem cell biology and potential therapeutic interventions.
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ABSTRACT: Localized induction of bone formation is essential during orthopedic procedures that involve skeletal repair, such as surgical treatment of non-union bone fractures and degenerative disk disease. Herein we disclose the synthesis and biological evaluation of novel oxysterol derivatives designed as anabolic bone growth agents. Structure-activity relationship studies of oxysterol 4 have identified analogues such as 18, 21 and 30. These new analogues are characterized by higher potency in an osteoblast differentiation assay and/or by increased metabolic stability in human liver microsomes. Oxysterols 4, 18 and 21 were evaluated in vivo in a rat spinal fusion model.Bioorganic & medicinal chemistry letters 07/2012; 22(18):5893-7. · 2.65 Impact Factor
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ABSTRACT: The Hedgehog (Hh) pathway is a developmental signaling pathway that plays multiple roles during embryonic development and in adult tissues. Constitutive Hh signaling has been linked to the development and progression of several forms of cancer, and the application of small-molecule pathway inhibitors as anticancer chemotherapeutics is well studied and clearly defined. Activation of the Hh pathway as a therapeutic strategy for a variety of degenerative or ischemic disorders has also been proposed; however, the development of small-molecule Hh agonists has received less attention. The goal of this review is to highlight the recent evidence supporting the therapeutic potential of Hh pathway activators and to provide a comprehensive overview of small-molecule pathway agonists.ChemMedChem 11/2013; · 2.84 Impact Factor
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ABSTRACT: Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Among them, 7-ketocholesterol (7-KC) is one of the most important. It has potent effects in cell death processes, including cytoxicity and apoptosis induction. Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Very little is known about the effects of oxysterols in MSCs. Here, we describe the short-term cytotoxic effect of 7-ketocholesterol on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from two young, healthy women. After 24 h incubation with 7-KC, mitochondrial hyperpolarization was observed, followed by a slight increase in the level of apoptosis and changes in actin organization. Finally, the IC50 of 7-KC was higher in these cells than has been observed or described in other normal or cancer cell lines.Biochemical and Biophysical Research Communications 01/2014; · 2.28 Impact Factor
Oxysterols Regulate Differentiation of Mesenchymal Stem Cells:
Pro-Bone and Anti-Fat
Hoa Ton Kha,1Benjamin Basseri,1Daniel Shouhed,1Jennifer Richardson,1Sotirios Tetradis,2Theodore J Hahn,1,3
and Farhad Parhami1
ABSTRACT: Pluripotent mesenchymal stem cells can undergo lineage-specific differentiation in adult organ-
isms. However, understanding of the factors and mechanisms that drive this differentiation is limited. We
show the novel ability of specific oxysterols to regulate lineage-specific differentiation of mesenchymal stem
cells into osteogenic cells while inhibiting their adipogenic differentiation. Such effects may have important
implications for intervention with osteoporosis.
Introduction: Oxysterols are products of cholesterol oxidation and are formed in vivo by a variety of cells including
osteoblasts. Novel pro-osteogenic and anti-adipogenic effects of specific oxysterols on pluripotent mesenchymal cells
are demonstrated in this report. Aging and osteoporosis are associated with a decrease in the number and activity of
osteoblastic cells and a parallel increase in the number of adipocytic cells.
Materials and Methods: The M2–10B4 pluripotent marrow stromal cell line, as well as several other mesenchymal
cell lines and primary marrow stromal cells, was used to assess the effects of oxysterols. All results were analyzed
for statistical significance using ANOVA.
Results and Conclusion: Pro-osteogenic and anti-adipogenic effects of specific oxysterols were assessed by the
increase in early and late markers of osteogenic differentiation, including alkaline phosphatase activity, osteocalcin
mRNA expression and mineralization, and the decrease in markers of adipogenic differentiation including lipoprotein
lipase and adipocyte P2 mRNA expression and adipocyte formation. Complete osteogenic differentiation of M2 cells
into cells expressing early and late markers of differentiation was achieved only when using combinations of specific
oxysterols, whereas inhibition of adipogenesis could be achieved with individual oxysterols. Oxysterol effects were
in part mediated by extracellular signal-regulated kinase and enzymes in the arachidonic acid metabolic pathway, i.e.,
cyclo-oxygenase and phospholipase A2. Furthermore, we show that these specific oxysterols act in synergy with bone
morphogenetic protein 2 in inducing osteogenic differentiation. These findings suggest that oxysterols may play an
important role in the differentiation of mesenchymal stem cells and may have significant, previously unrecognized,
importance in stem cell biology and potential therapeutic interventions.
J Bone Miner Res 2004;19:830–840. Published online on January 12, 2004; doi: 10.1359/JBMR.040115
Key words:oxysterols, stem cells, osteogenesis, adipogenesis, bone morphogenetic protein
cells commonly known as mesenchymal stem cells or marrow
stromal cells (MSCs).(1,2)These cells are present in a variety of
tissues and are prevalent in bone marrow stroma.(3,4)They can
be readily isolated and expanded in culture, and on treatment
with a variety of agonists, they can differentiate into lineage-
specific cells including osteoblasts, chondrocytes, myocytes,
adipocytes, and fibroblasts.(1–4)MSCs are thought to be an
excellent potential tool for interventions in many diseases that
result from reduced or impaired functioning of these cells.(5,6)
ESENCHYMAL TISSUE REGENERATION during adult life is
highly dependent on a population of pluripotent stem
Such interventions may include regeneration of defective ex-
tracellular matrix by systemic infusion of MSCs in individuals
with generalized defects such as in osteogenesis imper-
fecta,(7,8)repair of damaged tissues such as bone and cartilage
by localized injection into the affected areas,(9,10)and the
therapeutic delivery of specific genes to diseased tissues.(11)
All this, however, requires an understanding of the factors and
molecular mechanisms involved in determining lineage-
specific differentiation of MSCs and the identification of new
strategies for driving lineage-specific differentiation in vitro
and in vivo.
One situation in which MSCs might be used in human
disease is in age-related and postmenopausal osteoporosis,
where the decreased number and osteogenic activity of
osteoprogenitor MSCs, in part, leads to decreased bone
The authors have no conflict of interest.
1Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA;2University of California at Los
Angeles Dental School, Los Angeles, California, USA;3West Los Angeles VA Medical Center, Los Angeles, California, USA.
JOURNAL OF BONE AND MINERAL RESEARCH
Volume 19, Number 5, 2004
Published online on January 12, 2004; doi: 10.1359/JBMR.040115
© 2004 American Society for Bone and Mineral Research
formation.(6,12–14)A reduction in bone formation by osteo-
blasts in the face of increased bone resorption by osteoclasts
in these disorders results in decreased bone mass, increased
susceptibility to fractures, and impaired fracture healing.(14)
Therefore, the systemic and/or local application of osteo-
progenitor MSCs, or factors that enhance their osteogenic
differentiation, could be of great potential benefit in boost-
ing bone forming capacity in osteoporosis. Future improved
treatment of osteoporosis will likely require the use of bone
anabolic agents that can enhance the osteogenic differenti-
ation and bone-forming capacity of osteoblastic precursor
cells, and hence increase bone mass and reduce fracture
risk.(15–17)Several growth factors and hormones have been
tested in this regard, including bone morphogenetic protein
(PTH).(15,16)BMPs play critical roles in the differentiation
of MSCs into osteoblasts both in vitro and in vivo.(18,19)
BMP2 is the most potent known inducer of bone formation
in vivo, and it enhances the differentiation of osteoprogeni-
tor and non-osteoprogenitor precursors into cells with os-
teoblastic phenotype.(20)The use of BMPs in fracture heal-
ing is currently hampered by the large concentrations of the
recombinant protein necessary to induce adequate bone
formation.(20)Currently, only PTH, which on intermittent
injection increases bone mass and reduces bone fracture
incidence, has been approved by the Food and Drug Ad-
ministration (FDA). However, because of its potential ad-
verse side effects and cost, it is currently only used for
severely osteoporotic patients. Hence identification of novel
factors and strategies for enhancing bone formation is cur-
rently an area of intense investigation.
Because MSCs are the common progenitors for both
osteoblasts and adipocytes,(1–4)reductions in the number of
osteoblastic cells in aging and osteoporosis have been at-
tributed, in part, to an increased differentiation of these
common progenitor cells into adipocytes rather than osteo-
blasts.(14)It has been observed that the numbers of adipo-
cytes in the bone marrow increases in parallel with a de-
crease in the number of osteoblasts in a variety of types of
osteoporosis.(21)Moreover, the volume of adipose tissue in
bone increases with age in normal subjects and is substan-
tially elevated in age-related osteoporosis,(22)with the num-
ber of adipocytes adjacent to bone trabeculae increasing in
parallel to the degree of trabecular bone loss.(23)Based on
this and similar observations, it has been suggested that
bone loss in age-related osteoporosis is at least in part
caused by a shift in MSC differentiation from the osteoblas-
tic to the adipocytic pathway.(14,21)If this is true, it is
possible that intervening in this apparent shift in lineage-
specific differentiation of MSCs could increase the number
of cells capable of undergoing differentiation into function-
Oxysterols form a large family of oxygenated derivatives
of cholesterol that are present in the circulation and in
human and animal tissues.(24–26)These compounds may be
formed either by auto-oxidation, as a secondary byproduct
of lipid peroxidation, or by the action of specific mono-
oxygenases, most of which are members of the cytochrome
P450 family of enzymes.(27)In addition, oxysterols may be
derived from dietary intake.(28)Oxysterols have potent ef-
fects in physiologic and pathological processes including
cholesterol metabolism, inflammation, apoptosis, steroid
production, and atherosclerosis.(24–26,29)Moreover, because
of the abundance of cholesterol in living organisms, the
pro-oxidant nature of our cellular metabolic processes, and
the multitude of enzymatic and non-enzymatic pathways for
oxysterol production, we speculate that oxysterols may play
additional, as yet unidentified, roles in biological systems.
Recently, several reports have noted the possible role of
oxysterols in cellular differentiation.(30–32)For example, the
oxysterols 22(R)- and 25-hydroxycholesterol induce the dif-
ferentiation of human keratinocytes in vitro,(30,31)whereas
monocyte differentiation is induced by the oxysterol
We previously reported that products of the cholesterol
biosynthetic pathway are important for the proper osteo-
genic differentiation and activity of MSCs.(33)Because ox-
ysterols may be a derivative of the endogenous cellular
cholesterol biosynthetic pathway,(25–27)we hypothesized
that the oxysterols generated by osteoprogenitor cells, as
well as those derived from exogenous sources, may be
involved in their osteogenic differentiation. In this report,
we present the first line of evidence for osteogenic activity
of specific oxysterols and their ability to inhibit adipogenic
differentiation. In addition, we show the synergistic inter-
action of these oxysterols with BMP2.
MATERIALS AND METHODS
Oxysterols, ?-glycerophosphate (?GP), silver nitrate, and
Oil red O were obtained from Sigma (St Louis, MO, USA);
RPMI 1640, ?-MEM, and DMEM were from Irvine Scien-
tific (Santa Ana, CA, USA); and FBS was from Hyclone
(Logan, UT, USA). PD98059 was purchased from BI-
OMOL Research Labs (Plymouth Meeting, PA, USA); TO-
901317, SC-560, NS-398, Ibuprofen, and Flurbiprofen were
from Cayman Chemical (Ann Arbor, MI, USA); ACA and
AACOCF3 were from Calbiochem (La Jolla, CA, USA);
and recombinant human BMP2 was from R&D Systems
(Minneapolis, MN, USA). Antibodies to phosphorylated
and native extracellular signal-regulated kinases (ERKs)
were obtained from New England Biolabs (Beverly, MA,
USA) and troglitazone was from Sankyo (Tokyo, Japan).
The M2–10B4 mouse marrow stromal cell line obtained
from American Type Culture Collection (Rockville, MD,
USA) was derived from bone marrow stromal cells of a
(C57BL/6J ? C3H/HeJ) F1 mouse and supports human and
murine myelopoiesis in long-term cultures (as per ATCC).
These cells were cultured in RPMI 1640 containing 10%
heat-inactivated FBS and supplemented with 1 mM sodium
pyruvate, 100 U/ml penicillin, and 100 U/ml streptomycin
(all from Irvine Scientific). The MC3T3-E1 mouse preos-
teoblastic cell line was purchased from ATCC and cultured
in ?-MEM containing 10% heat-inactivated FBS and sup-
plements as described above. C3H-10T1/2 mouse pluripo-
tent embryonic fibroblast cells were kindly provided by Dr
Kristina Bostrom (UCLA) and were cultured in DMEM
831 OXYSTEROLS INDUCE OSTEOGENIC DIFFERENTIATION
containing 10% heat-inactivated FBS and supplements as
described above. Primary mouse marrow stromal cells were
isolated from male 4- to 6-month-old C57BL/6J mice and
cultured and propagated as previously reported.(34)
Alkaline phosphatase activity assay
Colorimetric alkaline phosphatase (ALP) activity assay
on whole cell extracts was performed as previously de-
von Kossa and Oil red O staining
Matrix mineralization in cell monolayers was detected by
silver nitrate staining as previously described.(34)Oil red O
staining for detection of adipocytes was performed as pre-
45Ca incorporation assay
Matrix mineralization in cell monolayers was quantified
45Ca incorporation assay as previously de-
Western blot analysis
After treatments, cells were lysed in lysis buffer, protein
concentrations were determined using the Bio-Rad protein
assay (Hercules, CA, USA), and SDS-PAGE was performed
as previously described.(34)Probing for native and phos-
phorylated ERKs was performed as previously reported.(34)
RNA isolation and Northern blot analysis
After treatment of cells under appropriate experimental
conditions, total RNA was isolated using the RNA isolation
kit from Stratagene (La Jolla, CA, USA). Total RNA (10
mg) was run on a 1% agarose/formaldehyde gel and trans-
ferred to Duralon-UV membranes (Strategene) and cross-
linked with ultraviolet light. The membranes were hybrid-
ized overnight at 60°C with a32P-labeled mouse osteocalcin
cDNA probe,(36)mouse lipoprotein lipase (LPL), mouse
adipocyte protein 2 (aP2) PCR-generated probes, and hu-
man 28S and 18S rRNA probes obtained from Geneka
Biotechnology (Montreal, Quebec, Canada) and Maxim
Biotech (San Francisco, CA, USA), respectively. The PCR
products were generated using primer sets synthesized by
Invitrogen (Carlsbad, CA, USA) with the following speci-
fications: mouse aP2 gene (accession no. M13261); sense
(75–95) 5?-CCAGGGAGAACCAAAGTTGA-3?, antisense
(362–383) 5?-CAGCACTCACCCACTTCTTTC-3?, gener-
ating a PCR product of 309 bp. Mouse LPL (accession no.
XM_134193); sense (1038–1058) 5?-GAATGAAGAAAA-
CCCCAGCA-3?, antisense (1816–1836) 5?-TGGGCCA-
TTAGATTCCTCAC-3?, generating a PCR product of 799
bp. The PCR products were gel-purified and sequenced by
the UCLA sequencing core, showing the highest similarity
to their respective GenBank entries. After hybridization, the
blots were washed twice at room temperature with 2? SSC
and 0.1%SDS and twice at 60°C with 0.5? SSC and 0.1%
SDS and exposed to X-ray film. The extent of gene induc-
tion was determined by densitometry.
Conditioned medium from M2 cells cultured in 24-well
plates was prepared after treatment with oxysterols or con-
trol buffer for 24 or 48 h, followed by rinsing the cells to
remove the treatment media and replacing it with 0.5 ml
RPMI containing 0.1% bovine serum albumin (BSA)/well.
After 24 h, the conditioned medium was collected, centri-
fuged to remove debris, and used in the BMP2 ELISA
immunoassay according to the manufacturer’s instructions
Computer-assisted statistical analyses were performed us-
ing the StatView 4.5 program. All p values were calculated
using ANOVA and Fisher’s projected least significant dif-
ference (PLSD) significance test. A value of p ? 0.05 was
Novel osteogenic activities of oxysterols
To begin testing our hypothesis, we examined the effects
of oxysterols on indices of osteoblastic differentiation in in
vitro cultures of MSCs. In cultures of MSCs in vitro, stim-
ulation of ALP activity, osteocalcin gene expression, and
mineralization of cell colonies are indices of increased
differentiation into osteoblast phenotype.(37,38)We found
that specific oxysterols, namely 22(R)-hydroxycholesterol
hydroxycholesterol (22S), induced ALP activity, an early
marker of osteogenic differentiation, in pluripotent M2–
10B4 murine MSCs (M2; Fig. 1A). This effect was specific,
because other oxysterols, including 7-ketocholesterol (7K),
did not induce ALP activity in these cells (Fig. 1A). The
induction of ALP activity was both dose- and time-
dependent at concentrations between 0.5 and 10 ?M, and
showed a relative potency of 20S ? 22S ? 22R. A 4-h
exposure to these oxysterols followed by replacement with
osteogenic medium without oxysterols was sufficient to
induce ALP activity in M2 cells, measured after 4 days in
Although induction of ALP activity is an early marker
of osteogenic differentiation and plays an important role
in both the differentiation and eventual mineralization pro-
cesses,(39)this response was not sufficient for the induction
of mineralization in M2 cells by individual oxysterols. In-
dividual oxysterols (22R, 20S, and 22S) at concentrations
between 0.5 and 10 ?M were unable to induce mineraliza-
tion after as many as 14 days of exposure, despite their
ability to cause large increases in ALP activity measured 4
days after treatment (data not shown). The individual oxys-
terols also had minimal to no effect on osteocalcin gene
expression after as many as 14 days of treatment (data not
shown). However, ALP activity (Fig. 1B), robust mineral-
ization (Figs. 1C and 1D), and osteocalcin gene expression
(Figs. 1E and 1F) were all induced in M2 cultures by a
combination of the 22R ? 20S or 22S ? 20S oxysterols.
Other combinations of oxysterols including 22R ? 22S, or
combinations of 22R or 22S with 7K, did not induce min-
832 KHA ET AL.
eralization in M2 cell cultures (data not shown). The com-
bination of 20S with either 22R or 22S also produced
osteogenic effects in the mouse pluripotent embryonic fi-
broblast C3H10T1/2 cells (Fig. 1G), in murine calvarial
pre-osteoblastic MC3T3-E1 cells, and in primary mouse
MSCs (Fig. 1H), as assessed by stimulation of ALP activity
and mineralization. Thus, we concluded that the combina-
tion of 20S with either S or R stereoisomers of 22-
MSCs. (A) M2 cells at confluence were treated
with control vehicle (C) or 10 ?M individual
oxysterols as indicated, in an osteogenic medium
consisting of RPMI 1640, to which 10% FBS, 50
?g/ml ascorbate, and 3 mM ?GP were added.
After 3 days of incubation, ALP activity was
determined in cell homogenates by a colorimet-
ric assay as previously described. Results from a
representative of five experiments are shown,
reported as the mean ? SD of quadruplicate
determinations, normalized to protein concentra-
tion (*p ? 0.01 for C vs. oxysterol-treated cells).
(B) M2 cells at confluence were treated in os-
teogenic medium with control vehicle (C) or a
combination of 22R and 20S oxysterols, at the
indicated concentrations. ALP activity was mea-
sured after 3 days as described above. Results
from a representative of four experiments are
shown, reported as the mean ? SD of quadru-
plicate determinations, normalized to protein
concentration (*p ? 0.01 for C vs. oxysterols).
(C) M2 cells at confluence were treated in os-
teogenic medium with control vehicle or 5 ?M
oxysterols, alone or in combination as indicated.
After 14 days, mineralization was identified by
von Kossa staining, which appears black, as pre-
viously described. (D) M2 cells were treated
with control vehicle (C) or a combination of 22R
and 20S oxysterols at increasing concentrations
as indicated. After 14 days, matrix mineraliza-
tion in cultures was quantified using a45Ca in-
corporation assay as previously described. Re-
sults from a representative of four experiments
are shown, reported as the mean ? SD of qua-
druplicate determinations, normalized to protein
concentration (*p ? 0.01 for C vs. oxysterol-
treated cultures). (E) M2 cells at confluence were
treated with control vehicle (C) or a combination
of 22R and 20S oxysterols (Ox; 5 ?M each) in
osteogenic medium. After 4 and 8 days, total
RNA from duplicate samples was isolated and
analyzed for osteocalcin (Osc) and 28S rRNA
expression by Northern blotting as described.
Data from densitometric analysis of the Northern
blot is shown in F as the average of duplicate
C3H10T1/2 cells at confluence were treated in
osteogenic medium with control vehicle (C) or a
combination of 22R and 20S oxysterols, at the
indicated concentrations. ALP activity was mea-
sured after 3 days as described above. Results
from a representative of three experiments are
shown, reported as the mean ? SD of quadru-
plicate determinations, normalized to protein
concentration (*p ? 0.01 for C vs. oxysterols).
(H) Primary MSCs at confluence were treated in
osteogenic medium with control vehicle (C) or a
combination of 22R and 20S oxysterols, at the
indicated concentrations. After 10 days, matrix
mineralization in cultures was quantified using a
45Ca incorporation assay as described earlier.
Results from a representative of three experi-
ments are reported as the mean ? SD of qua-
druplicate determinations, normalized to protein
concentration (*p ? 0.05 for C vs. oxysterols).
Osteogenic effects of oxysterols in
to 28SrRNA. (G)
833OXYSTEROLS INDUCE OSTEOGENIC DIFFERENTIATION
hydroxycholesterol has osteogenic effects on osteoblast pre-
cursor cells. Although stimulation of MSCs by BMP2 can
enhance their osteogenic differentiation,(37)the osteogenic
effects of the oxysterols were not caused by the induction of
BMP2 expression in M2 cells. RT-PCR analysis of BMP2
mRNA expression in M2 cells treated for 4 or 8 days with
22R ? 20S oxysterols (5 ?M) showed no induction by
oxysterol treatment (data not shown). In addition, ELISA
assay using conditioned media from M2 cells treated with
22R ? 20S (5 ?M) for 24 and 48 h also did not show any
induction of BMP2 protein expression (data not shown).
Interestingly, the BMP inhibitor noggin (Ng) at 200 ng/ml
caused a 40% inhibition in 22R ? 20S (RS, 2.5 ?M)-
induced ALP activity and a 90% inhibition of that induced
by rhBMP2 (100 ng/ml; Control ? 4 ? 2; RS ? 75 ? 7; RS
? Ng ? 42 ? 5; BMP2 ? 120 ? 11; BMP2 ? Ng ? 10 ?
2 activity units/mg protein; p ? 0.05 for Control versus RS
and BMP2 and for RS and BMP2 in the presence and
absence of Ng). We speculate that in light of our RT-PCR
and ELISA data that did not show any induction of BMP2
by oxysterols, the inhibition of response to oxysterols by
noggin might be caused by inhibition of synergism between
oxysterols and BMP2 present in FBS. Alternatively, oxys-
terols may induce the expression of other members of the
BMP family that can interact with noggin, such as BMP4
and BMP7, the expression of which would not be detected
by our BMP2-specific ELISA assay.
Synergistic osteogenic effects of oxysterols with BMP2
As with other osteoprogenitor cells, BMP2 is able to
induce osteoblastic differentiation of M2 cells in vitro.(40)
Interestingly, we found that osteogenic combination of
22R ? 20S oxysterols acted synergistically with BMP2
in inducing ALP activity (Fig. 2A), osteocalcin mRNA
expression (Figs. 2C and 2D), and mineralization by M2
cells (Fig. 2B). Although synergism in stimulating ALP
activity was found when individual oxysterols 20S, 22S,
and 22R were added with BMP2, synergy in induction of
mineralization was only produced when oxysterols were
added in combinations of 22R ? 20S or 22S ? 20S
oxysterols with BMP2.
Novel anti-adipogenic activities of oxysterols
Adipogenesis of adipocyte progenitors including MSC is
regulated by the transcription factor peroxisome proliferator
activated receptor ? (PPAR?), which on activation by
ligand-binding, regulates transcription of adipocyte-specific
genes.(41)We previously reported that, as expected with
MSCs, M2 cells have the ability to undergo adipogenic
differentiation in response to the PPAR? activator, Trogli-
tazone (Tro).(34)In M2 cells treated with Tro to induce
adipogenesis, 20S, 22S, and 22R, alone or in combination,
inhibited adipogenesis (Figs. 3A and 3B). The relative anti-
adipogenic potency of these oxysterols was similar to their
relative potency in stimulating ALP activity in M2 cells,
with 20S ? 22S ? 22R. Similar to its lack of osteogenic
effect, 7K was also unable to inhibit adipogenesis in M2
cells (data not shown). Inhibition of adipogenesis was also
assessed by an inhibition of the expression of the adipogenic
genes LPL and aP2 by 20S (Figs. 3C and 3D). However,
addition of the oxysterols to already formed adipocytes in
M2 cell cultures did not reduce the number of adipocytes
after 8 days of treatment (data not shown), suggesting that
the oxysterols were active only at the early stages of adi-
pogenesis. Inhibitory effects of these three oxysterols on
adipogenesis were also demonstrated using C3H10T1/2 and
primary mouse MSC, in which adipogenesis was induced
either by Tro or a standard adipogenic cocktail consisting of
Mechanism of osteogenic activity of oxysterols
Mesenchymal cell differentiation into osteoblasts is reg-
ulated by cyclo-oxygenase (COX) activity.(42–44)We exam-
ined the possible role of COX in mediating the osteogenic
effects of oxysterols on M2 cells. In the presence of FBS,
which corresponds to our experimental conditions, M2 cells
in culture express both COX-1 and COX-2 mRNA at all
stages of osteogenic differentiation (data not shown). Con-
sistent with the role of COX in osteogenesis, our studies
showed that the COX-1 selective inhibitor SC-560, at 1–20
MSCs. (A) M2 cells at confluence were treated with control vehicle
(C), 50 ng/ml recombinant human BMP2, or a combination of 22R and
20S oxysterols (RS, 2.5 ?M each), alone or in combination in osteo-
genic medium as described in Fig. 1. ALP activity was measured after
2 days as previously described. Results from a representative of four
experiments are shown, reported as the mean ? SD of quadruplicate
determinations, normalized to protein concentration (*p ? 0.001 for
BMP ? RS vs. BMP and RS alone). (B) M2 cells were treated as
described in A. After 10 days, matrix mineralization in cultures was
quantified using a45Ca incorporation assay as previously described.
Results from a representative of four experiments are shown, reported
as the mean ? SD of quadruplicate determinations, normalized to
protein concentration (p ? 0.01 for BMP ? RS vs. BMP and RS
alone). (C) M2 cells were treated under similar conditions as those
described above. After 8 days, total RNA was isolated and analyzed for
osteocalcin (Osc) and 18S rRNA expression by Northern blotting as
previously described. Data from densitometric analysis of the Northern
blot is shown in D as the average of duplicate samples, normalized to
Synergistic osteogenic effects of oxysterols and BMP2 in
834 KHA ET AL.
?M, significantly inhibited the osteogenic effects of the 22R
? 20S and 22S ? 20S oxysterol combinations. SC-560
inhibited oxysterol-induced ALP activity (Fig. 4A), miner-
alization (Fig. 4B), and osteocalcin gene expression (Figs.
4C and 4D). Although less effective than SC-560, the non-
selective COX inhibitors, ibuprofen and flurbiprofen, at
nontoxic doses of 1–10 ?M, also significantly inhibited the
osteogenic effects of 22R ? 20S oxysterol combination by
25–30%. In contrast, the selective COX-2 inhibitor, NS-
398, at the highest nontoxic dose of 20 ?M, had only
negligible inhibitory effects. SC-560 (10 ?M) also inhibited
the synergistic induction of ALP activity by oxysterols and
BMP2 (Fig. 4G). Furthermore, the osteogenic effects of the
oxysterol combination on ALP activity (Fig. 4E) and min-
eralization (Fig. 4F) were also inhibited by the general
phospholipase A2(PLA2) inhibitor ACA and by the selec-
tive cytosolic PLA2inhibitor, AACOCF3 (AAC). More-
over, rescue experiments showed that the effects of the
COX-1 and PLA2inhibitors on oxysterol-induced ALP ac-
tivity were reversed by the addition of 1 ?M PGE2(Fig. 4H)
and 25 ?M arachidonic acid (Fig. 4I), respectively.
The ERK pathway is another major signal transduction
pathway previously associated with osteoblastic differenti-
ation of osteoprogenitor cells.(45,46)Interestingly, the 20S
oxysterol used alone or in combination with 22R oxysterol
caused a sustained activation of ERK1 and ERK2 in M2
cells (Figs. 5A and 5B). Inhibition of the ERK pathway by
the inhibitor PD98059 inhibited oxysterol-induced mineral-
ization (Fig. 5C) but not ALP activity or osteocalcin mRNA
expression in M2 cell cultures (data not shown). These
results suggest that sustained activation of ERK is important
in regulating certain specific, but not all, osteogenic effects
Liver X receptors (LXR) are nuclear hormone recep-
tors that, in part, mediate certain cellular responses to
oxysterols, including 22R and 20S, but not 22S.(47,48)
LXR? is expressed in a tissue-specific manner, whereas
LXR? is ubiquitously expressed.(47,48)By Northern blot
analysis, we demonstrated the expression of LXR?, but
not LXR?, in confluent cultures of M2 cells (data not
shown). To assess the possible role of LXR in mediating
the effects of osteogenic oxysterols, we examined
whether activation of LXR? by the pharmacologic LXR
ligand TO-901317 (TO) had effects similar to those ex-
erted by 22R and 20S in M2 cells. Interestingly, in
contrast to 22R and 20S, TO at 1–10 ?M caused a
dose-dependent inhibition of ALP activity in M2 cells
(control [C]: 18 ? 2; ligands used at 10 ?M: 22R ? 45 ?
5; 20S ? 140 ? 12; and TO ? 3 ? 0.5 activity units/mg
protein; p ? 0.01 for C versus all ligands). Furthermore,
TO treatment did not induce osteocalcin gene expression
or mineralization after 10 days (data not shown). There-
fore, the osteogenic effects of the oxysterols on M2 cells
seem to be independent of the LXR receptor, as sug-
gested by the potent osteogenic activity of the non-LXR
oxysterol ligand 22S and the lack of osteogenic effects in
response to the LXR ligand TO.
oxysterols. (A and B) M2 cells at confluence
were treated in RPMI containing 10% FBS with
control vehicle or 10 ?M Tro in the absence or
presence of 10 ?M 20S or 22S oxysterols. After
10 days, adipocytes were visualized by Oil red O
staining and quantified by light microscopy,
shown in B, and as previously described. Data
from a representative of four experiments are
shown, reported as the mean ? SD of quadru-
plicate determinations (*p ? 0.001 for Tro vs.
Tro ? 20S and Tro ? 22S). (C and D) M2 cells
were treated at confluence with 10 ?M Tro alone
or in combination with 10 ?M 20S oxysterol.
After 10 days, total RNA was isolated and ana-
lyzed for LPL or aP2 gene expression or 18S
rRNA expression by Northern blotting as de-
scribed. Data from densitometric analysis of the
Northern blot is shown in D as the average of
duplicate samples, normalized to 18S rRNA. (E)
M2 cells were treated with control vehicle (C) or
osteogenic medium containing ascorbate and
?-glycerophosphate (Ost) alone or in combina-
tion with troglitazone (Tro, 20 ?M) and 20S
oxysterol (5 ?M). ALP activity was measured
after 4 days. Results from a representative of
three experiments are reported as the mean ?
SD, normalized to protein concentration (*p ?
0.01 for C vs. Ost, Ost vs. Ost ? Tro, and Ost ?
Tro vs. Ost ? Tro ? 20S).
Inhibition of adipogenesis in MSCs by
835OXYSTEROLS INDUCE OSTEOGENIC DIFFERENTIATION
or 10 ?M COX-1 inhibitor SC-560 (SC) in osteogenic medium as described earlier. Next, a combination of 22R and 20S oxysterols (RS, 2.5 ?M
each) was added in the presence or absence of SC as indicated. After 3 days, ALP activity was measured as described earlier. Data from a
representative of three experiments are shown, reported as the mean ? SD of quadruplicate determinations, normalized to protein concentration
(*p ? 0.001 for RS vs. RS ? SC). (B) M2 cells were treated as described in A, and after 10 days, matrix mineralization in cultures was quantified
by a45Ca incorporation assay as described earlier. Results from a representative of three experiments are shown, reported as the mean ? SD of
quadruplicate determinations, normalized to protein concentration (*p ? 0.01 for RS vs. RS ? SC). (C) M2 cells were pretreated with 20 ?M
SC for 4 h, followed by the addition of RS in the presence or absence of SC as described above. After 8 days, total RNA was isolated and analyzed
for osteocalcin (Osc) and 18S rRNA expression by Northern blotting as previously described. Data from densitometric analysis of the Northern
blot is shown in D as the average of duplicate samples, normalized to 18S rRNA. (E) M2 cells at confluence were pretreated for 2 h with control
vehicle (C) or PLA2inhibitors ACA (25 ?M) and AACOCF3 (AAC, 20 ?M) in osteogenic medium. Next, a combination of 22R and 20S
oxysterols (RS, 2.5 ?M) was added in the presence or absence of the inhibitors as indicated. After 3 days, ALP activity was measured as previously
described. Data from a representative of three experiments are shown, reported as the mean ? SD of quadruplicate determinations, normalized
to protein concentration (*p ? 0.01 for RS vs. RS ? ACA and RS ? AAC). (F) M2 cells were treated as described in E. After 10 days, matrix
mineralization in cultures was quantified using a45Ca incorporation assay as previously described. Results from a representative of three
experiments are shown, reported as the mean ? SD of quadruplicate determinations, normalized to protein concentration (*p ? 0.01 for RS vs.
RS ? ACA and RS ? AAC). (G) M2 cells at confluence were treated in osteogenic medium with control vehicle (C), rhBMP2 (BMP, 50 ng/ml),
and oxysterol combination 22R ? 20S (RS, 2.5 ?M) alone or in combination. Pretreatment of some cells with SC-560 (SC, 10 ?M) was done
for 2 h before the addition of BMP and RS. ALP activity was measured after 4 days. Results from a representative of three experiments are reported
as mean ? SD of quadruplicate determinations, normalized to protein concentrations (*p ? 0.005 for BMP ? RS vs. BMP ? RS ? SC). (H)
M2 cells were treated in osteogenic medium with RS oxysterol combination (2.5 ?M) alone or in combination with COX-1 inhibitor SC-560 (SC,
10 ?M) and PGE2(1 ?M). ALP activity was measured after 4 days and reported as described above. Results from a representative of three
experiments are reported as the mean ? SD of quadruplicate determinations, normalized to protein concentrations (*p ? 0.01 for RS vs. RS ?
SC and RS ? SC and RS ? SC ? PGE2). (I) M2 cells were treated in osteogenic medium with RS oxysterol combination (2.5 ?M) alone or in
combination with PLA2inhibitor ACA (25 ?M) and arachidonic acid (AA, 25 ?M). ALP activity was measured as described above. Results from
a representative of three separate experiments are reported as the mean ? SD of quadruplicate determinations, normalized to protein concentration
(*p ? 0.01 for RS vs. RS ? ACA and for RS ? ACA and RS ? ACA ? AA).
Mechanism of osteogenic activity of oxysterols in MSC. (A) M2 cells at confluence were pretreated for 4 h with control vehicle (C)
836KHA ET AL.
Mechanism of anti-adipogenic activity of oxysterols
As noted above, ERKs are important in the proliferation
and osteogenic differentiation of MSCs. In addition to pos-
itively regulating the osteogenic activity of osteoblast pro-
genitor cells, ERK activation also negatively regulates
the adipogenic differentiation of adipocyte progenitor cells,
and inhibition of ERK enhances adipogenic different-
iation.(49–51)Consistent with this effect of ERK, inhibition
of oxysterol-induced ERK activation by ERK pathway in-
hibitor PD98059 completely abolished the anti-adipogenic
effects of 20S and 22S on Tro-induced adipogenesis in M2
cells (Fig. 5D). Interestingly, PD98059 potentiated the adi-
pogenic effects of Tro (Fig. 5D), suggesting that, similar to
the situation in pre-adipocytes,(51)spontaneous activity of
ERK is inhibitory to the adipogenic differentiation of MSC.
In contrast, the selective inhibitors for COX-1 and COX-2,
SC-560, and NS-398, respectively, were not able to abolish
the anti-adipogenic effects of the oxysterols (data not
shown). These results suggested that the anti-adipogenic
activity of oxysterols is mediated through activation of the
This is the first demonstration of the ability of oxysterols
to regulate lineage-specific differentiation of MSCs in favor
of osteoblastic and against adipogenic differentiation. This
effect of the oxysterols is in part mediated through COX/
PLA2- and ERK-dependent mechanisms (Fig. 6). COX-1
and COX-2 are both present in osteoblastic cells and seem
to be primarily involved in bone homeostasis and repair,
respectively.(42,52)Metabolism of arachidonic acid into
prostaglandins, including prostaglandin E2(PGE2), by the
COXs mediates the osteogenic effects of these enzymes.(53)
COX products and BMP2 have complementary and additive
osteogenic effects.(44)Activation of PLA2releases arachi-
donic acid from cellular phospholipids and makes it avail-
able for further metabolism by COX enzymes into prosta-
oxysterol-stimulated metabolism of arachidonic acid,(56,57)
the present results suggest that the osteogenic activity of the
oxysterols in MSC are in part mediated by the activation of
PLA2-induced arachidonic acid release and its metabolism
into osteogenic prostanoids by the COX pathway. Further-
more, COX activity also seems to be important in regulating
the synergism between the osteogenic oxysterols and
BMP2, but not in the anti-adipogenic effects of the oxys-
terols, suggesting the specific role of COX/PLA2pathway in
mediating the oxysterol-induced lineage specific differenti-
ation of M2 cells into osteogenic cells.
These results also show another signaling pathway medi-
ating M2 responses to oxysterols is the ERK pathway.
Sustained activation of ERKs mediates the osteogenic dif-
ferentiation of human MSCs,(45)and activation of ERKs in
withprevious reports of
1% FBS, followed by treatment with control vehicle or 5 ?M 20S oxysterol for 1, 4, or 8 h. Next, total cell extracts were prepared and analyzed
for levels of native or phosphorylated ERK (pERK) using specific antibodies as previously described. Data from a representative of four
experiments are shown; each treatment is shown in duplicate samples. (B) M2 cells were treated as described in A, with 5 ?M 20S and 22R alone
or in combination. After 4 h, cell extracts were prepared and analyzed for levels of native and pERK. Data from a representative of two
experiments are shown; each treatment is shown in duplicate samples. (C) M2 cells at confluence were pretreated for 2 h with control vehicle (C)
or 20 ?M PD98059 (PD) in osteogenic medium as previously described. Next, a combination of 22R and 20S oxysterols (RS, 5 ?M each) were
added to appropriate wells as indicated. After 10 days of incubation, matrix mineralization was quantified by the45Ca incorporation assay as
previously described. Data from a representative of three experiments are reported as the mean ? SD of quadruplicate determinations, normalized
to protein concentration (*p ? 0.01 for RS vs. RS ? PD). (D) M2 cells at confluence were pretreated for 2 h with 20 ?M PD98059 (PD) in RPMI
containing 5% FBS. Next, the cells were treated with control vehicle (C), 10 ?M Tro, or 10 ?M of 20S or 22S oxysterols alone or in combination
as indicated. After 10 days, adipocytes were visualized by Oil red O staining and quantified by light microscopy as previously described. Data
from a representative of three experiments are reported as the mean ? SD of quadruplicate determinations.
Role of ERK in mediating the responses of MSCs to oxysterols. (A) M2 cells at confluence were pretreated for 4 h with RPMI containing
837OXYSTEROLS INDUCE OSTEOGENIC DIFFERENTIATION
human osteoblastic cells results in upregulation of expres-
sion and DNA binding activity of Cbfa1, the master regu-
lator of osteogenic differentiation.(46)Furthermore, ERK
activation seems to be essential for growth, differentiation,
and proper functioning of human osteoblastic cells.(58)In
addition, the ERK pathway is a negative regulator of adi-
pogenic differentiation of adipocyte progenitor cells.(49–51)
Consistent with the role of the ERK pathway in both osteo-
genic and adipogenic differentiation, oxysterol-induced
mineralization and inhibition of adipogenic differentiation
were blocked by the inhibitor, PD98059, and the oxysterols
induced a sustained activation of ERK. It must be noted
that, because both individual and combinations of oxys-
terols activated ERK, whereas only the combinations of
oxysterols were able to induce full osteogenic differentia-
tion of M2 cells, ERK activation seems to be an essential
but not sufficient step in mediating the osteogenic effects of
We also show for the first time that the osteogenic oxys-
terols synergistically stimulate BMP2-induced osteogenic
differentiation of MSCs. It is of interest that when BMPs
were originally extracted from bone matrix, they were found
to be associated with lipids.(59)These crudely characterized
lipids potentiated the osteogenic effects of BMP, as evi-
denced by a great reduction in the bone forming activity of
BMP on their removal.(59)This line of evidence and the
previous demonstration of the presence of lipids in healing
fracture callus(60)suggest that lipids such as oxysterols may
play an important role in regulating osteogenesis and the
effects of BMP on this process. These studies suggest that
specific oxysterols may potentiate the osteogenic differen-
tiation of osteoblast precursor cells and that this effect of
oxysterols is in part mediated through a potentiation of the
osteogenic activity of BMPs. Furthermore, the synergistic
effects of osteogenic oxysterols with BMP2 may provide an
exciting new strategy for using BMP2 in local stimulation
of fracture healing. However, because bone turnover is
regulated by a combination of osteoblastic bone formation
and osteoclastic bone resorption, it is important to note that
the putative in vivo osteogenic capacity of oxysterol com-
binations reported here will depend also on their effect on
osteoclastic differentiation/activity and bone resorption.
The possibility of inducing lineage-specific differentia-
tion of MSCs into designated cells and tissues has potential
implications for the prevention and treatment of a number of
disorders that result from age-related tissue deterioration or
from genetic defects.(6)For example, as noted earlier, age-
related osteoporosis seems to be, at least in part, caused by
a decreased osteogenic differentiation of osteoprogenitor
cells.(61–63)Accordingly, the focus of developing new ther-
apeutic approaches that would positively impact osteoporo-
sis has largely shifted from finding additional antiresorption
agents to finding anabolic agents that can enhance bone
formation.(15,16)The possible use of lipid-based treatments
involving oxysterols, rather than protein- or peptide-based
interventions as currently used, introduces a whole new
strategy for creating therapeutics that would potentially
intervene in osteoporosis. Further identification of the
downstream targets of these molecules in MSCs holds great
potential for discovering new and previously unsuspected
ways to regulate the lineage-specific differentiation of key
MSC populations. This could improve the potential for the
use of autologous MSCs in treatment of a variety of con-
nective tissue diseases, as well as in tissue engineering.(6)In
addition, fat cells increase significantly in mesenchymal
tissues with age, in parallel with a decline in osteoblasts,
muscle cells, and other key derivatives of MSCs.(64)The
basis of this apparent shift in MSC differentiation is un-
known, but the ability to reverse this process could signif-
icantly affect the management of age-related disorders.
This work was supported by National Institute on Aging
Pepper Center Grant IP60-AG10415 and National Institutes
of Health Grant HL30568.
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Address reprint requests to:
Farhad Parhami, PhD
University of California Los Angeles Division of
Center for the Health Sciences
10833 Le Conte Avenue
Los Angeles, CA 90095, USA
Received in original form September 15, 2003; in revised form
November 26, 2003; accepted Janaury 9, 2004.
840 KHA ET AL.