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Published in final edited form as:
Bone. 2010 January ; 46(1): 236. doi:10.1016/j.bone.2009.10.012.
HO-1 expression increases mesenchymal stem cell-derived
osteoblast but decreases adipocyte lineage
Luca Vanella1, Dong Hyun Kim1, David Asprinio3, Stephen J. Peterson2, Ignazio
Barbagallo5, Angelo Vanella5, Dove Goldstein4, Susumu Ikehara6, and Nader G.
Abraham1,2
1Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo,
Ohio
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2Department
of Medicine, New York Medical College, Valhalla, NY
3Department
of Orthopedics, New York Medical College, Valhalla, NY
4Department
of Obstetrics and Gynecology, Columbia University, New York, NY
5Department
of Biological Chemistry, Medical Chemistry and Molecular Biology, University of
Catania, Italy
6First
Department of Pathology, Kansai Medical University, Osaka, Japan
Abstract
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Human bone marrow mesenchymal stem cells (MSC) are pleitrophic cells that differentiate to either
adipocytes or osteoblasts as a result of cross-talk by specific signaling pathways including heme
oxygenase (HO)-1/-2 expression. We examined the effect of inducers of HO-1 expression and
inhibitors of HO activity on MSC differentiation to the osteoblast and adipogenesis lineage. HO-1
expression is increased during osteoblast stem cell development, but remains elevated, at 25 days.
The increase in HO-1 levels proceed an increase in alkaline phosphatase (AP) activity and an increase
in BMP, osteonectin and RUNX-2 mRNA. Induction of HO-1 by osteogenic growth peptide (OGP)
was associated with an increase in BMP-2 and osteonectin. Exposure of MSC to high glucose levels
decreased osteocalcin and osteogenic protein expression, which was reversed by upregulation of the
OGP-mediated increase in HO-1 expression. The glucose mediated decrease in HO-1 resulted in
decreased levels of pAMPK, pAKT and the eNOS signaling pathway and was reversed by OGP. In
contrast, MSC-derived adipocytes were increased by glucose. HO-1 siRNA decreased HO-1
expression but increased adipocyte stem cell differentiation and the adipogenesis marker, PPARγ.
Thus, upregulation of HO-1 expression shifts the balance of MSC differentiation in favor of the
osteoblast lineage. In contrast, a decrease in HO-1 or exposure to glucose drives the MSC towards
adipogenesis. Thus targeting HO-1 expression is a portal to increased osteoblast stem cell
differentiation and to the attenuation of osteoporosis by the promotion of bone formation.
© 2009 Elsevier Inc. All rights reserved.
Address correspondence and reprint requests to: Corresponding authors: Nader G. Abraham, Ph.D., Professor and Chairman, The
University of Toledo College of Medicine, Department of Physiology and Pharmacology, Health Education Bldg., 3000 Arlington
Avenue, Toledo, Ohio 43614-2598 Phone (419) 383-4144, nader.abraham@utoledo.edu.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
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Vanella et al.
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Keywords
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diabetes; osteoporosis; osteoblasts; BMP2
INTRODUCTION
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Human bone marrow-derived mesenchymal stem cells give rise to osteoblastic and adipogenic
lineages. The shift from osteoblastic to adipocyte lineage is a result of cross talk and the various
factors that drive MSC to adipocytes lineages inhibiting osteoblast formation [1]. Diabetes
affects dynamic bone formation in both humans and animals leading to osteopenia and
osteoporosis [2,3] and increased adipogenesis [4-6]. Bone-mineral density and other
biochemical markers of bone turnover are adversely affected in individuals with diabetes [7].
Reduction of bone mass, occurring with increased frequency in individuals with diabetes
mellitus, has been attributed to poor glycemic control, but the pathogenic mechanisms remain
unknown. High concentrations of glucose (hyperglycemia) in diabetics exacerbate this
complication [7-9]. Osteoblasts secrete growth factors including platelet derived growth factor,
insulin like growth factors and bone morphogenetic proteins [10] that are stored in the bone
matrix. Whether these factors are affected by diabetes remains to be seen. Furthermore, the
molecular mechanism underlying osteoblast stem cell differentiation under hyperglycemic
conditions has not been fully elucidated.
In contrast to diabetes, recent studies have shown that several growth factors including
osteogenic growth peptide (OGP) enhance differentiation of mesenchymal stem cells to
osteoblasts [11] and that endothelial growth factor (EGF) and OGP enhance osteoblast cell
proliferation by regulating several signaling pathways. Osteogenic growth peptide is a naturally
occurring tetradecapeptide that is both an anabolic agent and a hematopoietic stimulator [12].
OGP increases osteoblast proliferation, alkaline phosphatase (AP) activity and matrix synthesis
and mineralization. It prevents glucocorticoid-induced apoptosis and the subsequent bone
remodeling alterations that are associated with steroids [13].
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Heme oxygenase-1 (HO-1) plays a major role during bone marrow stem cell differentiation
[14-16]. Heme oxygenase which exists in two forms, HO-1 (inducible) and HO-2
(constitutive), catalyzes the rate-limiting step in heme degradation, resulting in the formation
of carbon monoxide (CO), iron and biliverdin, the latter is subsequently reduced to bilirubin
by biliverdin reductase. Additionally, both CO and nitric oxide (NO) protect against tumor
necrosis factor-induced apoptosis in osteoblasts [17]. In addition, during fracture repair,
activation of HIF-1 and its target genes, VEGF and HO-1, regulate osteoblasto-genesis and
bone reabsorption [18] suggesting a role of HO-1 in bone metabolism. Finally, several lines
of evidence suggest that HO-1 plays an important physiological role in obesity and diabetes.
Increased HO-1 expression decreases adipogenesis in obese animals [4,5,19]. However, the
role of HO-1 expression in mesenchymal stem cell (MSC) development and differentiation
into either osteoblasts or adipocyte stem cells is poorly understood. HO-1 expression and its
role in diabetes and other pathologies is a burgeoning area of research [20]. HO-1 is a target
gene for the prevention of diabetes and obesity. Increased HO-1 expression resulted in higher
adiponectin levels, and improved insulin sensitivity [19].
The goal of the present study was to determine whether HO-1 and HO-2 expression shifts the
balance of MSC-mediated lineage expression towards osteoblast or adipocyte differentiation.
MSC are multipotent cells that have the potential to proliferate and differentiate into a variety
of cell types characteristic of bone, skeletal and cardiac muscle, adipose tissue, and neural cells
[21-24] and are used as a model system to study the effect of HO-1 expression on osteoblast
and adipocyte differentiation. We report here that inducers of HO-1 expression such as OGP,
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affects osteoblast differentiation via increased levels of HO-1, pAKT and eNOS. We
demonstrate that osteoblast differentiation is positively regulated by HO-1 expression, which
was associated with a reduction of reactive oxygen species (ROS), thereby permitting the
restoration of osteoblast markers, specifically, induction of OPG and osteocalcin, while HO-1
expression negatively regulate adipocyte stem cell differentiation.
MATERIALS AND METHODS
Cell culture from BM and differentiation into osteoblasts
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Frozen bone marrow mononuclear cells were purchased from Allcells (Allcells, Emeryville,
CA). After thawing the cells, mononuclear cells were resuspended in an α-minimal essential
medium (α-MEM, Invitrogen, Carlsbad CA) supplemented with 20% heat inactivated fetal
bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1% antibiotic/antimycotic solution
(Invitrogen, Carlsbad, CA). The cells were plated at a density of 1-5X106 cells per 100 cm2
dish. The cultures were maintained at 37 °C in a 5% CO2 incubator, and the medium was
changed after 48h and every 3~4 days thereafter. When the MSCs were confluent, the cells
were recovered by the addition of 0.25% trypsin/EDTA (Life Technologies, Frederick, MD).
Mesenchymal stem cells (MSCs Passage 2-3) were plated in a 60-cm2 dish at a density of
1-2X104 and cultured in α-MEM with 10% FBS for 7 days at 37°C. HO-1 silencing was
achieved by infecting cells two days after seeding with a commercially available lentiviral
siRNA for HO-1, at a viral total of 4-6 transduction units per milliliter; as a control, a siRNA
scrambled mixture was used. siRNA HO-1 was purchased from Sigma-Aldrich, St. Louis, MO.
Beginning at day 7, cultures were switched to differentiation medium in phenol red-free
BGJb (Fitton-Jackson modification) and supplemented with 5% FBS containing 50 μg/ml
ascorbic acid (for appropriate collagen and extracellular matrix production). From day 14
forward, 3 mM β-glycerophosphate (for appropriate mineralization) was added to the
differentiation media. For some studies, cultures were grown entirely in phenol red-free Ham's
F-12 with 5% FBS without the addition of differentiation medium.
HO-1 siRNA transfection
Cells were treated with three different predesigned siRNAs of HO-1 gene (SASI_
Hs01_00035068, SASI_Hs01_00035065 and SASI_Hs01_00035067 from Sigma-Aldrich, St.
Louis, MO). According to the manufacture's protocol, Osteogeneic media containing siRNA
using N-TER (Sigma-Aldrich, St. Louis, MO) was replaced every 48 hours. Briefly,
nanoparticle solution was incubated with 10nM siRNA. After 20 min, cells were treated with
siRNA solution, with or without OGP during osteogenesis for 21 days.
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DNA content and fragmentation
Osteoblasts were plated (4,000 cells/96well) in two plates, for cellular density using
CyQuant™ kit (Molecular Probes, Eugene, OR) and for the fragmentation DNA assay using
Apostain Elisa Kit (Alexis Biochemicals, Inc, Plymouth Meeting, PA) according to the
manufacture's protocol. Cultures were treated with both Osteogeneic media and OGP for 21
days.
Alkaline phosphatase activity
Cells were plated in six-well plates. Cell layers were washed twice with ice-cold phosphatebuffered saline (PBS), then harvested in 1 ml 50 mM Tris-HCl (pH 7.6), sonicated twice on
ice, and then centrifuged at 4°C for 15 min at 1000 xg. The supernatants were stored at −20°
C until analysis for alkaline phosphatase activity was conducted, using p-nitrophenylphosphate
as substrate. Absorbance was read at 405 nm using a microplate reader. Alkaline phosphatase
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activity was expressed as nmol p-nitrophenol released/min per μg DNA. All analyses were
replicated 6 times. Each experiment was repeated two or three times.
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Measurement of DNA content and bone mineralization
Osteoblasts were plated in six-well plates. Cell layers were washed twice with ice-cold PBS,
harvested in 50 mM Tris-HCl (pH 7.6), sonicated, and then centrifuged for 15 min at 1000
xg. The supernatants were analyzed for DNA content by measuring fluorescence at 458 nm
spectrophotometrically, using purified calf thymus as a DNA standard [25]. All analyses were
performed in six replicates. Bone mineralization was determined using Alizarin Red S (SigmaAldrich, St. Louis, MO) staining and phase-contrast microscopy 21 days after treatment. Cell
were incubated with 2% alizarin red at pH 4.2 for 10 minutes and subsequently washed with
distilled water. Subcultured cells were examined by phase-contrast microscopy at 21 days to
determine cell morphology and to verify the presence of mineralized nodules.
Immunoblot analysis
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Osteoblasts or adipocytes were incubated with stimulants in T75 flasks for 24 h. They were
then washed with PBS and trypsinized (0.05% trypsin w/v with 0.02% EDTA). The pellets
were lysed in buffer (Tris-Cl 50 mM, EDTA 10 mM, Triton X-100 1% v/v, PMSF 1%, pepstatin
A 0.05 mM and leupeptin 0.2 mM) and, after mixing with sample loading buffer (Tris-Cl 50
mM, SDS 10% w/v, glycerol 10% v/v, 2-mercaptoethanol 10% v/v and bromophenol blue
0.04%) at a of ratio 4:1, were boiled for 5 min. Samples (10 μg protein) were loaded onto 12%
gels and subjected to electrophoresis (150 V, 80 min). The separated proteins were transferred
to nitrocellulose membranes (Bio-Rad, Hercules, CA; 1 h, 200 mA per gel). After transfer, the
blots were incubated overnight with 5% nonfat milk in TTBS followed by incubation with
1:1000 dilution of the primary antibody for 3 h. Polyclonal rabbit antibodies directed against
the human HO-1, AMPK, pAMPK, peNOS, HO-2 were obtained from Stressgen
Biotechnologies (Victoria, BC). After washing with TTBS, the blots were incubated for 2 h
with secondary antibody (1:5000) and conjugated with alkaline phosphatase. Finally, the blots
were developed using a premixed solution containing 0.56 mM 5-bromo-4-chloro-3-indolyl
phosphate (BCIP) and 0.48 mM nitro blue tetrazolium (NBT) in buffer (Tris-HCl 10 mM, NaCl
100 mM, MgCl2 59.3 μM, pH 9.5). The blots were scanned and the optical density of the bands
was measured using Scion (New York, NY) Image software.
Human bone marrow derived adipocyte mesenchymal stem cells
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Frozen bone marrow mononuclear cells were purchased from Allcells (Allcells, Emeryville,
CA). After thawing the cells were resuspended in an α-minimal essential medium (α-MEM,
Invitrogen, Carlsbad CA) supplemented with 10% heat inactivated fetal bovine serum (FBS,
Invitrogen, Carlsbad CA) and 1% antibiotic/antimycotic solution (Invitrogen, Carlsbad CA).
The cells were plated at a density of 1-5X106 cells per 100 cm2 dish. The cultures were
maintained at 37 °C in a 5% CO2 incubator, and the medium was changed after 48h and every
3-4 days thereafter. When the MSCs were confluent, the cells were recovered by the addition
of 0.25% trypsin/EDTA (Life Technologies, Frederick, MD). MSCs (Passage 2-3) were plated
in a 60-cm2 dish at a density of 1-2X104 and cultured in α-MEM with 10% FBS for 7 days.
The medium was replaced with adipogenic medium, and the cells were cultured for an
additional 21 days. The adipogenic medium consisted of complete culture medium
supplemented with OM EM-high glucose, 10% (v/v) FBS, 10 μg/ml insulin, 0.5 mM
dexamethasone (Sigma-Aldrich, St. Louis, MO), 0.5 mM isobutylmethylxanthine (SigmaAldrich, St. Louis, MO) and 0.1 mM indomethacin (Sigma-Aldrich, St. Louis, MO).
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Oil Red O staining
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For Oil Red O staining, 0.5% Oil Red O solution (Sigma-Aldrich, St. Louis, MO) was used.
Briefly, adipocytes were fixed in 1% formaldehyde, washed in Oil-red O for 20 min, rinsed
with 85% propylene glycol (Sigma-Aldrich, St. Louis, MO) for 3 min, washed in distilled water
and mounted with aqueous mounting medium, [26].
Heme oxygenase activity assay
HO activity was assayed in homogenates of osteoblasts. Cell homogenates were incubated with
50 μM heme, 2 mg/ml rat liver cytosol (as a source of biliverdin reductase), 1 mM MgCl2, 3
units glucose-6-phosphatase dehydrogenase, 1 mM glucose-6-phosphate and 2 mM NADP+
in 0.5 ml 0.1 M potassium phosphate buffer, pH 7.4, for 30 min at 37°C. The reaction was
terminated by placing the tubes on ice and bilirubin was then extracted with chloroform. The
amount of bilirubin generated was determined using a dual-beam scanning spectrophotometer
(Perkin-Elmer, Norwalk, CT, Lambda 17 UV/VIS) and is defined as the difference between
464 and 530 nm (extinction coefficient: 40 mM-1 cm-1 for bilirubin). The results were
expressed as nmol of bilirubin/5×106 cells/h. Tin mesoporphyrin (SnMP), purchased from
Frontier Science, Logan UT, was used to inhibit heme oxygenase activity [27]. Cobalt
protoporphyrin (CoPP) was purchased from Frontier Science, Logan UT to induce HO-1
expression and HO activity [28].
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mRNA isolation and real-time PCR quantification
Total RNA was isolated using Trizol® (Invitrogen, Carlsbad, CA) according to the
manufacturer's instructions. First strand cDNA was synthesized with Roche reverse
transcription reagents. Total RNA (1 μg) was analyzed by real-time PCR.
The quantitative real-time polymerase chain reaction (qRT-PCR) was performed with the
TaqMan gene expression assay on an ABI Prism 7900 sequence analyzer according to the
manufacturer's recommended protocol (Applied Biosystems, Foster City, CA). Each reaction
was run in triplicate. The comparative threshold cycle (CT) method was used to calculate the
amplification fold as specified by the manufacturer. A value of 10 ng of reverse-transcribed
RNA samples was amplified by using the TaqMan Universal PCR Master Mix and TaqMan
gene expression assays (ID Hs01055564_m1 for human BMP-2, ID Hs00231692_m1 for
RUNX2, ID HS99999901_s1 for 18S as an endogenous control; Applied Biosystems).
ELISA assay for osteocalcin and osteoprotegerin
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By using a specific ELISA test, according to the manufacturer's recommendation, we evaluated
the levels of osteocalcin (BioSource International, Inc., Camarillo, CA, USA) and OPG
(osteoprotegerin) in the culture supernatant (BioVendor Laboratory Medicine, Modrice, Czech
Republic).
Statistical analysis
Data are presented as mean ± standard error (SE) for the number of experiments. Statistical
significance (p<0.05) was determined by the Fisher method of multiple comparisons. For
comparison between treatment groups, the null hypothesis was tested by single factor analysis
of variance (ANOVA) for multiple groups or unpaired t-test for two groups.
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RESULTS
Effect of OGP on HO-1 protein levels and HO activity
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The basal levels of HO-1 expression increased during MSC osteoblast-culture growth and
peaked at 20 days following treatment with OGP. HO activity followed a similar pattern but
with a peak at day 15 (p<0.05) and then gradually declining (Figure 1B). HO-2 protein levels
did not change during osteoblast proliferation in absence of OGP (day 5 and 10) or in presence
of OGP (Figure 1A and 1B). To examine whether HO-1 protein expression was associated
with a corresponding increase in osteoblast proliferation and differentiation, the temporal
sequence of differentiation was also determined. Alkaline phosphatase (AP) activity gradually
increased, reaching a maximum at day 20, where it plateaued. OGP treatment significantly
(p<0.05) increased AP levels above control, Figure 1C. A temporal increase in DNA (μg/plate)
followed a pattern similar to that of HO-1 protein. As seen in Figure 1D, DNA accumulation
in control increased to reach a maximum at day 15, and declined to low levels at 25 days. OGP
treatment resulted in significantly (p<0.05) increased levels of DNA at days 10, 15 and 20 over
control.
Effect of OGP on differentiation of mesenchymal stem cells in osteoblastic cells
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PCR quantification of undifferentiated MSCs and of cells at day 21 after culture in
differentiation medium confirmed that the cells were positive for early mature osteoblastic
markers such as BMP-2, RUNX-2, osteonectin and osteocalcin (Figure 2A). The effect of OGP
real time PCR Quantification of osteonectin mRNA indicated a time dependent increase
reaching a maximum at day 15 followed by a decline at day 21. BMP-2 and RUNX-2 exhibited
a similar time-dependent increase that peaked at day 21 of OGP exposure (Figure 2B).
Effect of OGP on eNOS, peNOS and pAKT
To investigate the possibility that differential activation of eNOS, peNOS and the antiapoptotic signaling molecule pAKT could account for the increase in osteoblast cell
proliferation and differentiation, we assessed the levels of these protein after 10 days of culture
in the presence of OGP. As seen in Figure 3, OGP treatment resulted in a significant (p<0.05)
increase in the amount of HO-1 protein. OGP supplementation also resulted in a significant
increase in the anti-apoptotic protein pAKT. Upregulation of HO activity by OGP also resulted
in a significant increase in eNOS and peNOS. It should be noted that the significant increase
in pAKT occurred without any change in AKT (p<0.05, Figure 3).
Effect of OGP on HO-1 expression and pAMPK in the presence of siRNA
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We tested the hypothesis that OGP increases signaling molecules in osteoblasts via an increase
in HO-1. MSC were transduced with HO-1 siRNA to test whether this treatment could inhibit
HO-1 mRNA and the signaling molecule pAMPK expression. The results presented in Figure
4A, strongly support the hypothesis, OGP increased both HO-1 and pAMPK expression in
osteoblasts. HO-1 siRNA, added to osteoblast cells dramatically reduced both HO-1 expression
(Figure 4A) and pAMPK levels (Figure 4B).
Effect of high glucose levels on OPG and osteocalcin levels during osteoblastic
differentiation: Role of OGP
We verified whether suppression of HO-1 as a result of hyperglycemia (glucose 20 mM) can
affect other gene expression. OPG expression (Figure 5A) and osteocalcin secretion (Figure
5B) during differentiation was significantly (p<0.05) decreased when MSC were exposed to
glucose (20 mM) compared to untreated cells. In addition, OGP treatment in the presence of
glucose resulted in an increase in osteocalcin levels after secretion was reduced by a high
glucose concentration. Similarly, glucose exposure showed a significant (p<0.05) reduction in
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osteonectin secretion compared to untreated control cells (data not shown). This is the first
demonstration that OGP can protect osteoblast from hyperglycemia by increasing the levels
of OPG and osteocalcin (Figure 5A and B) during osteoblast cell growth and differentiation.
Inhibition of HO activity by SnMP treatment in the presence of glucose abrogated OGPmediated increases in both OPG and osteocalcin (Figures 5A and B).
Effect of OGP and glucose on HO-1, eNOS, peNOS, pAMPK and pAKT expression
The induction of diabetes by culturing cells in glucose resulted in a significant decrease in
HO-1 protein levels Figure 6A. This decrease was partially restored by culturing the cells in
the presence of OGP. Similarly, eNOS and pAMPK phosphorylation were decreased by
culturing osteoblasts in glucose, Figure 6A. OGP treatment had no effect on AMPK expression.
However, there was a significant (p<0.05) increase in the expression of pAMPK and eNOS
(Figure 6A). As seen in Figure 6B, pAKT phosphorylation was decreased by culturing
osteoblasts in glucose and there was no affect on AKT. The presence of OGP reversed the
effect of glucose and increased activation of pAKT. The changes in protein expression of pAKT
(Figure 6B) mirrored those seen with HO-1 protein expression (Figure 6A). Similarly, MSCs
cultured with SnMP, an inhibitor of HO-1/-2 activity [29], and glucose displayed a significant
p<0.05 decrease in pAKT (Figure 6B) compared to MSC-cultured with OGP and glucose.
Effect of HO-1 induction on osteogenesis, adipogenesis and PPARγ levels
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We used a potent inducer of HO-1, CoPP, to assess the differential effect of HO-1 on
osteogenesis and adipogenesis. CoPP treatment resulted in an increase in bone mineralization
when measured at day 21 (Figure 7A). The increase was significant (p<0.01) when compared
to vehicle. In contrast, the reverse was seen on adipogenesis with a significant (p<0.05) decline
apparent at day 21 (Figure 7B). In agreement with the effect of HO-1 on adipogenesis, Figure
7C, the decline in HO-1 expression at day 10 of adipogenic differentiation (p<0.05) was
accompanied by a significant (p<0.01) increase in the levels of PPARγ (Figure 7C). In addition,
HO-1 siRNA increased the area of lipid droplets, i.e. adipogenesis (Figure8).
DISCUSSION
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In the present study we show, for the first time, that the upregulation of HO-1 increases MSCmediated osteoblasts lineages but decreases adipocytes. The OGP-mediated increase in HO-1
levels increases osteoblast proliferation and differentiation, and is associated with an increase
in osteoblast function, via an increase in AKT. A significant increase in OGP-mediated cell
proliferation was observed while, in contrast, both SnMP, a competitive inhibitor of HO
activity, and HO-1 siRNA, reversed the OGP-mediated effect, suggesting that the effect of
OGP was dependent on an increase in both HO-1 expression and HO activity. To further
explore the functional expression of HO-1 in human MSC-mediated osteoblast lineages, we
measured AP, DNA accumulation and bone mineralization. The increase in HO-1 resulted in
an increase in the rate of AP and DNA accumulation and mineralization as a function of time
when compared to untreated osteoblasts. In addition, osteoblasts cultured in the presence of an
inhibitor of HO-1, as in cells exposed to high glucose, exhibited a decrease in the levels of
BMP-2, osteonectin, pAMPK and eNOS. However, upregulation of HO-1 by OGP in cultured
osteoblasts rescued the hyperglycemia-mediated decrease in BMP-2, HO-1, eNOS and
pAMPK. Previous studies have shown that eNOS was expressed in osteoblasts and that a
deficiency of this enzyme resulted in a significant reduction in bone formation in mice [30].
Thus, the OGP-mediated increase HO-1 and eNOS can be regarded as a pivotal step in bone
metabolism through an ability to modulate osteoblast function. eNOS and NO are stimulators
of the levels of BMP-2 and increase differentiation of osteoblasts [31,32].
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More recently, we reported that HO-1 overexpression in animal models of both type 1 and type
II diabetes attenuates vascular dysfunction via an increase in pAMPK and AKT and a decrease
in oxidative stress [4,28,33]. Diabetes affects the integrity and functionality of bone tissue
[34-36] possibly through increased adiposity [37]. Patients with diabetes frequently show either
low bone mass (osteopenia) or increased bone mineral density with an increased risk of fracture
and an impairment in bone healing [38], presumably, due to stimulation of osteoblast apoptosis
[39], recently reviewed [40].
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Finally, the present data demonstrate a differential effect of HO-1 on MSC-mediated adipocyte
stem cells. We investigated the effect of HO-1 expression on differentiation. A clear induction
of adipogenic transformation was observed upon exposure of MSC to glucose. The capacity
of high glucose to activate adipogenic differentiation has been described in isolated adipocytes
[19] and was shown to be dependent on suppression of HO-1. In agreement with these results
glucose increased adipogenesis and this was assocated with the suppression of HO-1 protein
levels. Glucose has been shown to suppress HO-1 promoter and HO-1 levels [41,42]. High
glucose suppressed HO-1 expression in cell lines [42-44] as well as in animal models [19,45,
46]. In fact, supplementation of high glucose, 20 mM, increased adipogenesis which was
further increased in the presence of HO-1 siRNA (Figure 8). The identification that inhibition
of HO-1 expression increased the MSC-shift towards adipocytes has at least two important
conceptual implications. Firstly, high glucose has an adipogenic potential, secondly, a direct
link exists between the suppression of HO-1 and the increase in adipogenesis and metabolic
syndrome. Upregulation of HO-1 was involved in detecting and decoding a variety of stress
conditions including hyperglycemia and angiotensin II-mediated stress [4]. We recently
showed that HO-1 recruits the EC-SOD to act as an anti-oxidant and to dissipate H2O2 [45]
and in triggering an increase in adiponectin and the signaling pathway pAMPK- pAKT [47].
Additionally, Figure 7 shows that HO-1 expression decreased during differentiation, while
PPARγ levels increased. PPARγ is commonly referred to as the master regulator of
adipogenesis [48,49]. Ectopic expression and activation of PPARγ are sufficient to induce
adipocyte differentiation. Given the role of HO-1 expression in preventing obesity [15], it is
possible that the differential role of HO-1 in adipocytes and osteoblast lineage might represent
a strategy to curb adiposity and increase osteogenesis.
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The results of the present study show that increased HO-1 expression and HO activity are
essential for MSC growth to the osteoblast lineage and is consistent with the role of HO-1 in
hematopoietic stem cell differentiation [14,15,50] in which HO-1 regulates stem cell
differentiation to a number of lineages [14-16]. The HO-1/HO-2 system participates in the
regulation of cell differentiation in osteoblasts and adipocytes in a cell-specific but very
different manner. Although, the basal levels of HO-1 protein and HO activity are needed for
osteoblast cell growth, an increase in the basal level of HO-1 resulted in the enhancement of
osteoblast differentiation. Induction of HO-1 is essential for the resultant increase in pAKT,
pAMPK, peNOS levels and NO bioavailability [5,28,47]. An increase in NO may be necessary
for OGP-mediated osteoblastic acitivity [51]; upregulation of NO was shown to play a positive
role in bone formation [30-32]. Although the mechanism by which HO-1 overexpression
decreased adipocyte differentiation but increased osteoblast differentiation is still unclear, it is
apparent that HO activity and its products, bilirubin, CO, and iron, play a different role in cell
proliferation. More recently, it was shown that the elevation of HO-1-derived CO in endothelial
cells, enhanced endothelial cell proliferation [52,53]. In contrast, increased HO-1 levels caused
a decrease in vascular smooth cells [4]. The effect of HO-1 expression on osteoblasts and
adipocytes is mirrored by the effect of HO-1 on endothelial cells and vascular smooth muscle
cells. In fact, adipocyte stem cells from both obese rats and mice have low levels of HO-1
protein and HO activity, which may reflect an increase in adiposity [4,19]. Upregulation of
HO-1 in obesity decreases adiposity and increases adiponectin [4]. Thus, the site-specific
delivery of HO-1 to adipocytes may play a regulatory role in the prevention of adipocyte
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differentiation in a variety of vascular diseases including metabolic syndrome [4]. Our results
provide direct evidence that HO-1 gene expression has a differential effect on osteoblast and
adipocyte cell proliferation and differentiation. Thus, by manipulating the expression of HO-1,
it will be possible to attenuate the hyperglycemia-mediated inhibition of osteoblast
differentiation, while simultaneously inhibiting adipocyte differentiation and thereby offereing
potential in the management of the metabolic syndrome.
Acknowledgments
This work was supported by NIH grants DK068134, HL55601 and HL34300 (NGA).
List of Abbreviations
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HO-1
heme oxygenase-1
AP
alkaline phosphatase
VEGF
vascular endothelial growth factor
OGP
osteogenic growth peptide
NO
nitric oxide
CO
carbon monoxide
MSC
mesenchymal stem cell
ROS
reactive oxygen species
EGF
endothelial growth factor
REFERENCES
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1. Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrow-derived
mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci 2009;66:236–
53. [PubMed: 18854943]
2. Hamilton EJ, Rakic V, Davis WA, Chubb SA, Kamber N, Prince RL, Davis TM. Prevalence and
predictors of osteopenia and osteoporosis in adults with Type 1 diabetes. Diabet Med 2009;26:45–52.
[PubMed: 19125760]
3. Fowlkes JL, Bunn RC, Liu L, Wahl EC, Coleman HN, Cockrell GE, Perrien DS, Lumpkin CK Jr.
Thrailkill KM. Runt-related transcription factor 2 (RUNX2) and RUNX2-related osteogenic genes are
down-regulated throughout osteogenesis in type 1 diabetes mellitus. Endocrinology 2008;149:1697–
704. [PubMed: 18162513]
4. Abraham NG, Kappas A. Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev
2008;60:79–127. [PubMed: 18323402]
5. Peterson SJ, Drummond G, Hyun KD, Li M, Kruger AL, Ikehara S, Abraham NG. L-4F treatment
reduces adiposity, increases adiponectin levels and improves insulin sensitivity in obese mice. J Lipid
Res 2008;49:1658–69. [PubMed: 18426778]
6. Tuominen JT, Impivaara O, Puukka P, Ronnemaa T. Bone mineral density in patients with type 1 and
type 2 diabetes. Diabetes Care 1999;22:1196–200. [PubMed: 10388989]
7. Yaturu S, Humphrey S, Landry C, Jain SK. Decreased bone mineral density in men with metabolic
syndrome alone and with type 2 diabetes. Med Sci Monit 2009;15:CR5–CR9. [PubMed: 19114969]
8. Inaba M, Terada M, Koyama H, Yoshida O, Ishimura E, Kawagishi T, Okuno Y, Nishizawa Y, Otani
S, Morii H. Influence of high glucose on 1,25-dihydroxyvitamin D3-induced effect on human
osteoblast-like MG-63 cells. J Bone Miner Res 1995;10:1050–6. [PubMed: 7484280]
9. Terada M, Inaba M, Yano Y, Hasuma T, Nishizawa Y, Morii H, Otani S. Growth-inhibitory effect of
a high glucose concentration on osteoblast-like cells. Bone 1998;22:17–23. [PubMed: 9437509]
Bone. Author manuscript; available in PMC 2011 January 1.
Vanella et al.
Page 10
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
10. Lin SS, Landesberg R, Chin HS, Lin J, Eisig SB, Lu HH. Controlled Release of PRP-Derived Growth
Factors Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells. Conf Proc IEEE
Eng Med Biol Soc 2006;1:4358–61. [PubMed: 17947081]
11. Chen ZX, Chang M, Peng YL, Zhao L, Zhan YR, Wang LJ, Wang R. Osteogenic growth peptide Cterminal pentapeptide [OGP(10-14)] acts on rat bone marrow mesenchymal stem cells to promote
differentiation to osteoblasts and to inhibit differentiation to adipocytes. Regul Pept 2007;142:16–
23. [PubMed: 17331598]
12. Spreafico A, Frediani B, Capperucci C, Leonini A, Gambera D, Ferrata P, Rosini S, Di SA, Galeazzi
M, Marcolongo R. Osteogenic growth peptide effects on primary human osteoblast cultures: potential
relevance for the treatment of glucocorticoid-induced osteoporosis. J Cell Biochem 2006;98:1007–
20. [PubMed: 16795077]
13. Weinstein RS, Chen JR, Powers CC, Stewart SA, Landes RD, Bellido T, Jilka RL, Parfitt AM,
Manolagas SC. Promotion of osteoclast survival and antagonism of bisphosphonate-induced
osteoclast apoptosis by glucocorticoids. J Clin Invest 2002;109:1041–8. [PubMed: 11956241]
14. Abraham NG, Nelson JC, Ahmed T, Konwalinka G, Levere RD. Erythropoietin controls heme
metabolic enzymes in normal human bone marrow culture. Exp Hematol 1989;17:908–13. [PubMed:
2767184]
15. Abraham NG. Molecular regulation--biological role of heme in hematopoiesis. Blood Rev 1991;5:19–
28. [PubMed: 2032026]
16. Abraham NG, Lutton JD, Levere RD. Heme metabolism and erythropoiesis in abnormal iron states:
role of delta-aminolevulinic acid synthase and heme oxygenase. Exp Hematol 1985;13:838–43.
[PubMed: 3840094]
17. Chae HJ, Chin HY, Lee GY, Park HR, Yang SK, Chung HT, Pae HO, Kim HM, Chae SW, Kim HR.
Carbon monoxide and nitric oxide protect against tumor necrosis factor-alpha-induced apoptosis in
osteoblasts: HO-1 is necessary to mediate the protection. Clin Chim Acta 2006;365:270–8. [PubMed:
16242122]
18. Zwerina J, Tzima S, Hayer S, Redlich K, Hoffmann O, Hanslik-Schnabel B, Smolen JS, Kollias G,
Schett G. Heme oxygenase 1 (HO-1) regulates osteoclastogenesis and bone resorption. FASEB J
2005;19:2011–3. [PubMed: 16234431]
19. Li M, Kim DH, Tsenovoy PL, Peterson SJ, Rezzani R, Rodella LF, Aronow WS, Ikehara S, Abraham
NG. Treatment of obese diabetic mice with a heme oxygenase inducer reduces visceral and
subcutaneous adiposity, increases adiponectin levels, and improves insulin sensitivity and glucose
tolerance. Diabetes 2008;57:1526–35. [PubMed: 18375438]
20. Wagener FA, Volk HD, Willis D, Abraham NG, Soares MP, Adema GJ, Figdor CG. Different faces
of the heme-heme oxygenase system in inflammation. Pharmacol Rev 2003;55:551–71. [PubMed:
12869663]
21. Ferrari G, Cusella-De AG, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F. Muscle
regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528–30. [PubMed:
9488650]
22. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti
DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science
1999;284:143–7. [PubMed: 10102814]
23. Marie PJ, Fromigue O. Osteogenic differentiation of human marrow-derived mesenchymal stem cells.
Regen Med 2006;1:539–48. [PubMed: 17465848]
24. Barbagallo I, Tibullo D, Di RM, Giallongo C, Palumbo GA, Raciti G, Campisi A, Vanella A, Green
CJ, Motterlini R. A cytoprotective role for the heme oxygenase-1/CO pathway during neural
differentiation of human mesenchymal stem cells. J Neurosci Res 2008;86:1927–35. [PubMed:
18381758]
25. Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem
1980;102:344–52. [PubMed: 6158890]
26. Bavendiek U, Zirlik A, LaClair S, MacFarlane L, Libby P, Schonbeck U. Atherogenesis in mice does
not require CD40 ligand from bone marrow-derived cells. Arterioscler Thromb Vasc Biol
2005;25:1244–9. [PubMed: 15746436]
Bone. Author manuscript; available in PMC 2011 January 1.
Vanella et al.
Page 11
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
27. Drummond GS, Galbraith RA, Sardana MK, Kappas A. Reduction of the C2 and C4 vinyl groups of
Sn-protoporphyrin to form Sn- mesoporphyrin markedly enhances the ability of the metalloporphyrin
to inhibit in vivo heme catabolism. Arch Biochem Biophys 1987;255:64–74. [PubMed: 3592668]
28. Kruger AL, Peterson SJ, Schwartzman ML, Fusco H, McClung JA, Weiss M, Shenouda S, Goodman
AI, Goligorsky MS, Kappas A, Abraham NG. Up-regulation of heme oxygenase provides vascular
protection in an animal model of diabetes through its antioxidant and antiapoptotic effects. J
Pharmacol Exp Ther 2006;319:1144–52. [PubMed: 16959961]
29. Drummond GS, Kappas A. Chemoprevention of neonatal jaundice: potency of tin-protoporphyrin in
an animal model. Science 1982;217:1250–2. [PubMed: 6896768]
30. Armour KE, Armour KJ, Gallagher ME, Godecke A, Helfrich MH, Reid DM, Ralston SH. Defective
bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of
endothelial nitric oxide synthase. Endocrinology 2001;142:760–6. [PubMed: 11159848]
31. Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G. Stimulation
of bone formation in vitro and in rodents by statins. Science 1999;286:1946–9. [PubMed: 10583956]
32. Garrett IR, Gutierrez G, Mundy GR. Statins and bone formation. Curr Pharm Des 2001;7:715–36.
[PubMed: 11405194]
33. Kruger AL, Peterson S, Turkseven S, Kaminski PM, Zhang FF, Quan S, Wolin MS, Abraham NG.
D-4F induces heme oxygenase-1 and extracellular superoxide dismutase, decreases endothelial cell
sloughing, and improves vascular reactivity in rat model of diabetes. Circulation 2005;111:3126–34.
[PubMed: 15939814]
34. Hamada Y, Kitazawa S, Kitazawa R, Fujii H, Kasuga M, Fukagawa M. Histomorphometric analysis
of diabetic osteopenia in streptozotocin-induced diabetic mice: a possible role of oxidative stress.
Bone 2007;40:1408–14. [PubMed: 17251074]
35. Schwartz AV. Diabetes Mellitus: Does it Affect Bone? Calcif Tissue Int 2003;73:515–9. [PubMed:
14517715]
36. Schwartz AV, Sellmeyer DE, Ensrud KE, Cauley JA, Tabor HK, Schreiner PJ, Jamal SA, Black DM,
Cummings SR. Older women with diabetes have an increased risk of fracture: a prospective study.
J Clin Endocrinol Metab 2001;86:32–8. [PubMed: 11231974]
37. Botolin S, Faugere MC, Malluche H, Orth M, Meyer R, McCabe LR. Increased bone adiposity and
peroxisomal proliferator-activated receptor-gamma2 expression in type I diabetic mice.
Endocrinology 2005;146:3622–31. [PubMed: 15905321]
38. Strotmeyer ES, Cauley JA, Schwartz AV, Nevitt MC, Resnick HE, Bauer DC, Tylavsky FA, de RN,
Harris TB, Newman AB. Nontraumatic fracture risk with diabetes mellitus and impaired fasting
glucose in older white and black adults: the health, aging, and body composition study. Arch Intern
Med 2005;165:1612–7. [PubMed: 16043679]
39. Al-Mashat HA, Kandru S, Liu R, Behl Y, Desta T, Graves DT. Diabetes enhances mRNA levels of
proapoptotic genes and caspase activity, which contribute to impaired healing. Diabetes
2006;55:487–95. [PubMed: 16443785]
40. Hofbauer LC, Brueck CC, Singh SK, Dobnig H. Osteoporosis in patients with diabetes mellitus. J
Bone Miner Res 2007;22:1317–28. [PubMed: 17501667]
41. Chang SH, Barbosa-Tessmann I, Chen C, Kilberg MS, Agarwal A. Glucose deprivation induces heme
oxygenase-1 gene expression by a pathway independent of the unfolded protein response. J Biol
Chem 2002;277:1933–40. [PubMed: 11707454]
42. Quan S, Kaminski PM, Yang L, Morita T, Inaba M, Ikehara S, Goodman AI, Wolin MS, Abraham
NG. Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic
rats. Biochem Biophys Res Commun 2004;315:509–16. [PubMed: 14766238]
43. Chang SH, Garcia J, Melendez JA, Kilberg MS, Agarwal A. Haem oxygenase 1 gene induction by
glucose deprivation is mediated by reactive oxygen species via the mitochondrial electron-transport
chain. Biochem J 2003;371:877–85. [PubMed: 12585963]
44. Abraham NG, Kushida T, McClung J, Weiss M, Quan S, Lafaro R, Darzynkiewicz Z, Wolin M. Heme
oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel
endothelial cells. Circ Res 2003;93:507–14. [PubMed: 12933701]
45. Turkseven S, Kruger A, Mingone CJ, Kaminski P, Inaba M, Rodella LF, Ikehara S, Wolin MS,
Abraham NG. Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide
Bone. Author manuscript; available in PMC 2011 January 1.
Vanella et al.
Page 12
NIH-PA Author Manuscript
NIH-PA Author Manuscript
dismutase and catalase in experimental diabetes. Am J Physiol Heart Circ Physiol 2005;289:H701–
H707. [PubMed: 15821039]
46. Di Noia MA, Van DS, Palmieri F, Yang LM, Quan S, Goodman AI, Abraham NG. Heme oxygenase-1
enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental
diabetes. J Biol Chem 2006;281:15687–93. [PubMed: 16595661]
47. Peterson SJ, Kim DH, Li M, Positano V, Vanella L, Rodella LF, Piccolomini F, Puri N, Gastaldelli
A, Kusmic C, L'Abbate A, Abraham NG. The L-4F mimetic peptide prevents insulin resistance
through increased levels of HO-1, pAMPK, and pAKT in obese mice. J Lipid Res 2009;50:1293–
304. [PubMed: 19224872]
48. Schadinger SE, Bucher NL, Schreiber BM, Farmer SR. PPARgamma2 regulates lipogenesis and lipid
accumulation in steatotic hepatocytes. Am J Physiol Endocrinol Metab 2005;288:E1195–E1205.
[PubMed: 15644454]
49. Liu J, Farmer SR. Regulating the balance between peroxisome proliferator-activated receptor gamma
and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylationdefective mutant of beta-catenin inhibits expression of a subset of adipogenic genes. J Biol Chem
2004;279:45020–7. [PubMed: 15308623]
50. Chertkov JL, Jiang S, Lutton JD, Harrison J, Levere RD, Tiefenthaler M, Abraham NG. The
hematopoietic stromal microenvironment promotes retrovirus-mediated gene transfer into
hematopoietic stem cells. Stem Cells 1993;11:218–27. [PubMed: 8318909]
51. Bab I, Gazit D, Chorev M, Muhlrad A, Shteyer A, Greenberg Z, Namdar M, Kahn A. Histone H4related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.
EMBO J 1992;11:1867–73. [PubMed: 1582415]
52. Quan S, Yang L, Abraham NG, Kappas A. Regulation of human heme oxygenase in endothelial cells
by using sense and antisense retroviral constructs. Proc Natl Acad Sci U S A 2001;98:12203–8.
[PubMed: 11593038]
53. Li Volti G, Wang J, Traganos F, Kappas A, Abraham NG. Differential effect of heme oxygenase-1
in endothelial and smooth muscle cell cycle progression. Biochem Biophys Res Commun
2002;296:1077–82. [PubMed: 12207883]
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Figure 1.
A) Effect of OGP (Upper panel) and protein levels in osteoblasts as a function of time during
the periods of proliferation and differentiation. Western blot analyses of HO proteins in
osteoblast homogenates were performed using antibodies against human HO-1 and HO-2
proteins. Blots shown are representative of 3 separate experiments. B) Effect of OGP on HO
activity in isolated cell homogenates of cells harvested after 5, 10, 15, 20 and 25 days of growth.
HO activity in osteoblasts was measured as described in Methods. Results are the mean ± SE;
n=3; *p<0.05 compared to 5 days OGP treated, effect of OGP on AP activity C) and, DNA
accumulation D) AP activity and DNA content were measured as described in the Methods.
Data presented are the result of 3 separate experiments. *p<0.001.
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Figure 2.
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A: Expression of osteoblastic markers in differentiated MSCs by PCR. qRTPCR revealed a
marked increase in BMP-2, RUNX2, osteocalcin and osteonectin at 21 days of osteoblastic
differentiation. Bars represent the mean ± SEM of three independent experiments. *p < 0.05
vs. undifferentiated cells. B) Expression of mRNA of osteonectin, BMP-2 and RUNX-2 with
time of exposure to OPG (10-6M). The results are of 3 independent experiments. *p<0.05 vs
undifferentiated cells.
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Figure 3.
Western blot and densitometry analysis of effect of OGP on HO-1 expression, peNOS, and
eNOS, pAKT, and AKT proteins measured after 21 days of osteoblast differentiation. We
performed quantitative densitometry evaluation of p-eNOS, eNOS, pAKT, AKT in the cells.
*p < 0.05 Control vs. OGP. Each bar represents mean SE± of 3 experiments.
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Figure 4.
A) Western blot analysis of HO in osteoblasts homogenates treated with OGP (10 -6M) for 10
days. In the presence and absence of siRNA HO-1. Immunoblots were performed using
antibodies against human HO-1 protein. Blots shown are representative of 3 separate
experiments, *<0.05 B) Effect of siRNA HO-1 on OGP-mediated pAMPK activation. *p<0.05
and **p<0.01, compared to OGP, n=4.
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Figure 5.
A and B) Effect of Glucose (30 mM), OGP + Glucose and SnMP + Glucose OGP + on
osteoblast marker expression. ELISA shows an increase of OPG A) and osteocalcin B) those
samples treated vehicle solution, control, glucose, glucose + OGP, glucose + OGP + SnMP.
Control bars represent the mean ± SEM of three independent experiments. *p < 0.05 vs.
undifferentiated cells +p<0.05 compared to glucose. Addition of SnMP caused a statistically
significant reduction in the expression of both. p<.05.
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Figure 6.
A) Lysates of osteoblasts cultured in the presence of a high glucose concentration (30 mM)
and OGP were assayed. Representative Western blots are shown for HO-1, eNOS, pAMPK,
AMPK, and actin proteins. Bars represent the mean ± SEM of four independent experiments
B) pAKT and AKT levels were measured in presence of vehicle solution, glucose (30 mM),
glucose + OGP in combination with SnMP. Both CoPP and OGP increased pAKT levels. *p
< 0.05 vs. differentiated cells (Control); †p < 0.05 vs. glucose.
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Figure 7.
Samples were stained as described in Methods to determine A) bone mineralization. CoPP (2.0
μM) was added once every 5 days during culture media change for 21 days. These pictures are
representative of 3 repeated experiments at 16 days, **p<0.01 and *p<0.05 compared to
control. B) Adipogenesis MSC were treated with 2.0 μM CoPP for 14 days and lipid droplets
were stained with Oil red O then measured adipogenesis on 490nm wave length. Levels of
significance: *p<0.05 Control vs CoPP; and C) Effect of HO-1 expression on PPARγ
expression, *p<0.05, **p<0.02, n=4.
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Figure 8.
The effect of HO-1 siRNA on adipogenesis. Lipid droplets area was determined by Oil red O
staining after 21 days. *p<0.05 control vs HO-1 siRNA
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