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Inhibitory effects of estradiol and SERMs on RUNX2-driven osteoblast differentiation and gene expression

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Inhibitory effects of estradiol and SERMs on RUNX2-driven osteoblast differentiation and gene expression

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Content University
of
Southern
California
Master’s
Thesis

1

Inhibitory effects of estradiol and SERMs
on RUNX2-driven osteoblast differentiation
and gene expression

By
Jie Ji
1

Mentor: Baruch Frenkel
1,2

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2016

1 Department of Biochemistry & Molecular Biology, Keck School of Medicine, University of
Southern California, 90033 CA, USA
2 Departments of Orthopaedic Surgery
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of
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California
Master’s
Thesis

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ACKNOWLEDGEMENTS

I would like to express my deepest appreciation to my supervisor, Dr.
Baruch Frenkel for the tremendous support to my research, for his patience,
motivation, and enthusiasm. I could not complete my degree without his smart
guidance.

I would also like to thank my thesis committee: Dr. Judd Rice and Dr. Janet
Oldak

My sincere thanks also go to Yonatan Amzaleg who generated RT-qPCR
data in this thesis.

Finally thank my dear lab mates Dr. Chimge, Jiali Yu, Eri Champagne,
Mirra Liu, Di ma and Sara Ahmed N Alnassar for the first spot of my data and
thank my parents for their financial support to my master study.

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ABSTRACT
The bone-sparing effects of estradiol (E2), the major female hormone, are
well established, but the underlying molecular mechanisms are complex and not
fully understood. Like E2, Selective estrogen receptor modulators (SERMs) bind
and activate the estrogen receptor (ER). Because they mimic E2 action in bone
cells, but not in breast epithelial cells, SERMs are used as drugs to prevent
postmenopausal osteoporosis. Runx2, a master transcription factor that promotes
both osteoblastogenesis and osteoblast-driven osteoclastogenesis, has been
shown to interact with the ER. In this thesis, we investigated the effects of E2
and two SERMs, raloxifen (Ral) and lasofloxifene (Las), on Runx2-driven gene
expression and osteoblast differentiation. We used ST2/Rx2dox, a mesenchymal
pluripotent cell line, whose treatment with doxycycline (dox) induces expression
of Runx2 and promotes osteoblast differentiation. Despite its positive effects of
bone mass, E2 inhibited differentiation of osteoblasts from their precursor cells
as indicated by inhibition of both alkaline phosphatase activity and expression of
the osteogenic gene markers Osteocalcin (Bone Gla Protein; Bgp) and Bone
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Sialoprotein (BSP). This inhibition was partially attributable to reduction of
RUNX2 protein levels. While inhibitory effects of Ral and Las increased with
their concentration, highest concentration (10
-6
M) of E2 did not give the
strongest inhibitory effect. That indicated two facts that firstly, Ral and Las
interacted with ER through not exactly the same mechanism as E2 did; secondly,
high and low concentration of E2 might affect osteoblastogenesis differently.
Varieties of genes were regulated in this process, and they were expressed
differently under different treatment. For example expressions of CTGF and
Cyr61 were down-regulated by E2 and SERMs, Egr1 and Egr2 were
down-regulated by E2 and Las, while up-regulated by Ral at certain
concentrations. Thus, the effects of E2 and SERMs on Runx2-driven gene
expression is locus- and concentration-dependent, potentially explaining some of
the complexity of changes to bone turnover (formation and resorption) observed
upon estrogen loss in vivo. Furthermore, clear differences between the effects
of E2 and SERMs on Runx2 may explain why SERMs are not as good as E2 in
the preservation of bone mass in postmenopausal women.

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Table of Contents
ACKNOWLEDGEMENTS
................................................................................................
2

ABSTRACT
...................................................................................................................
3

TABLE
OF
CONTENTS
...................................................................................................
5

INTRODUCTION
...........................................................................................................
7

Bone
Metabolsim
............................................................................................................................................
7

RUNX2
.................................................................................................................................................................
8

Estrogens
...........................................................................................................................................................
9

SERMs
..............................................................................................................................................................
12

Effects
of
E2
and
SERMs
on
RUNX2
........................................................................................................
13

Locus-‐specific
effects
of
steroid
hormones
on
RUNX2
....................................................................
14

MATERIALS
AND
METHODS
.......................................................................................
19

Reagents
..........................................................................................................................................................
19

Cell
culture
.....................................................................................................................................................
20

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RNA
extraction
and
analysis
....................................................................................................................
21

Western
blot
analysis
.................................................................................................................................
23

Alkaline
phosphatase
(ALP)
assay
.........................................................................................................
24

RESULTS
....................................................................................................................
25

E2
inhibit
osteoblastogenesis
in
ST2
mesenchymal
stem
cells
...................................................
25

Weak
inhibtion
of
ALP
activity
by
E2
in
committed
osteoblasts
.................................................
27

Inhibitory
effects
of
SERMs
in
ST2
mesenchymal
cells
...................................................................
30

Inhibitory
effect
of
E2
and
SERMs
at
increasing
concentrations
on
ALP
activity
in

ST2/Rx2
dox

cells
..............................................................................................................................................................
33

E2
and
SERMs
down-‐regulate
RUNX2
protein
level
.........................................................................
36

Differential
regulation
of
gene
expression
by
E2,
Ral
and
Las
.....................................................
39

DISCUSSION
...............................................................................................................
47

WORKS
CITED
............................................................................................................
52

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Introduction
Bone Metabolsim
Bones is a connective tissue that functions to support and protect our body
including a variety of important organs. Bone cells ‘built and destroy’ bone
throughout life, a process that helps maintain tissue integrity and balance blood
calcium levels [1]. Bone cells include osteoblasts, osteoclasts, bone lining cells
and osteocytes. Osteoblasts are the fully differentiated cells responsible for the
production of the bone matrix and bone formation. Osteoclasts are large
multinucleated cells which resorb bone [1]. These two are major components
operating in the remodeling process that replaces old with new bone throughout
life.[2]. The balance between these two processes is the key for the maintenance
of bone integrity, and imbalance between the two results in metabolic bone
disease, most commonly osteoporosis [3].
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RUNX2
Osteoblasts for the life-long process of bone remodeling are derived from
meshnchymal stem cells (MSCs) in the bone marrow. The master regulator of
osteoblastogenesis, RUNX2, is expressed by precursor MSCs when they get
osteogenic signals from the environment. Previously also known as Cfba1,
Pebp2αA1, or AML-3, RUNX2 is one of three members of the mammalian
Runt-related family [4, 5], which plays pivotal roles in embryogenesis.
Expression of RUNX2 in MSCs results in their commitment to osteoblast
differentiation. Mice with RUNX2 deficiency lack osteoblasts, fail to form
mineralized bone and die perinatally because they are unable to breath properly
[6]. Although RUNX2 is important for bone formation, it also promotes
osteoclastogenesis in a paracrine manner. Overexpression of RUNX2 in
osteoblasts in vivo results in increased bone resorption, osteopenia and multiple
fractures in transgenic mice [7]. Another study has shown that a dominant
negative (DN) form of RUNX2 did inhibit osteoblatogenesis [8]. Instead, bone
resorption was decreased and bone mass was increased. The roles of RUNX2
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in the osteoblast lineage are mediated by immature cells early in the lineage.
RUNX2 ablation in mature cells expressing Col1a1 has no effect on the skeleton
[9]. In contrast, RUNX2 ablation in less mature cells in the osteoblast lineage,
expressing Prx1 or Osx results in severe defects in bone formation [10]. As
shown by Western analysis of SK11/Rx2
Dox
cells, ST2/Rx2
Dox
cells and
MC3T3-E1/Rx2
Dox
cells, both endogenous and exogenous, Dox-induced RUNX2
became unstable soon after induction of osteoblast differentiation[11]. These
results indicate that RUNX2 only plays its role to initiate MSCs osteoblastic
commitment at a very early stage of osteoblastogenesis. And after the triggering
event, the presence of RUNX2 in osteoblast is unnecessary or even harmful.
In this thesis, I used dox-inducible RUNX2 overexpression system in
mesenchymal cells to induce osteoblast differentiation.

Estrogens
Estrogens are the primary female sex hormone and are responsible for
development and regulation of multiple physiological activities of female and
male. Estrogens are major regulators of bone metabolism and remodeling.
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Previous studies have shown that loss of estrogen signaling decreases bone
mineral density (BMD) and causes fractures. Ovariectomized (OVX) rats had
reduced quality of trabecular bone structures compared with sham-operated rats
and inpaired molars on their maxilla because the lost of estrogen[12]. Treatment
with estradiol (E2) could help OVX rats to recover from surgically created
critical-size defects treated with bovine bone graft faster[13]. And also, estrogen
therapies are effective for preventing osteoporosis in postmenopausal women [14,
15].
Many molecular mechanisms have been suggested to explain the
bone-sparing effect of estrogens. From the aspect of osteoblast protection, E2 is
believed to inhibits osteoblast apopotosis via the inhibition of apoptotic gene
expression and caspase-3/7 [16], and also inhibit the promotion of autophagy
through the ER-ERK-mTOR pathway [17]. From the aspect of osteoclast
elimination, estrogen preserves FasL levels by inhibiting microRNA-181a thus
inducing accelerated osteoclast apoptosis. Loss of all of these and additional
mechanisms at menopause are responsible for osteoporosis and fractures
inflicted on millions of women as a result of the natural depletion of estrogens.
The effect of estrogen on bone is mediated by the estrogen receptor (ER).
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There are two kinds of ER, ERα and ERβ, and both can be activated by several
ligands, in particular estradiol (E2). Knockout of ERα in mice resulted in
decreased BMD in cortical bone, while increased BMD in trabecular bone[18],
which could be attributed to elevated estrogen level after the deletion of ERα.
ERα conditional knock out in mice showed that estrogens differentially protect
female trabecular and cortical bone through activity of ERα in osteoclasts and
osteoblasts, respectively[19, 20]. Interestingly, conditional knockout of ERα in
pre-osteoblasts expressing Prx1 and Osx1, but not Cola1, resulted in cortical
bone loss [20].
Despite the clear bone-sparing effect of estrogens, there is evidence that
estrogens also acts as a negative regulator of osteoblastogenesis. According to
studies from Jilka et al in 1998 and 2001, ovariectomized (OVX) mice had more
colony-forming unit osteoblast (CFU-OB) than sham-operated mice based on the
number of osteoblastic colonies formed in primary MSC cultures (Figure 1A);
and CFU-OB number went down after those primary MSC cultures from the
OVX mice were treated ex vivo with E2 [21, 22]. This result raises the
possibility that estrogens protect bone by inhibiting the process of MSC
differentiation towards osteoblast in an early stage. Although this sounds
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counterintuitive, this idea is supported by the fact that osteoblast differentiation
is associated with paracrine signals that drive osteoclastogenesis of neighboring
cells [23].
SERMs
Selective estrogen receptor modulators (SERMs) are non-steroidal (Figure
1A) artificial molecules that mimic estrogen activity in bone cells while
antagonizing estrogen in breast cells. That means they are endowed the
potential to provide the skeletal benefits of estrogens without the increased risk
of breast cancer. They are commonly prescribed for the prevention of breast
cancer and postmenopausal osteoporosis.
Raloxifene (Ral) (marketed as Evista) is a second generation SERM that
helps to reduce the risk of fracture in postmenopausal women. With a
one-year-treatment of Ral, BMD of lumbar spine and total hip was significantly
increased in study group of postmenopausal women [24].
Lasofoxifene (Las) is also a SERM that decreases markers of bone
resorption and bone formation, slows down bone turnover and results in
increased BMD in postmenopausal women [25]. Pfizer received a
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non-approvable letter from the U.S. Food and Drug Administration as, but Las
was approved in the EU under the brand name Fablyn by the EMEA in March
2009. A clinical study from 2006 have shown that Las may be an effective and
well-tolerated treatment option for the prevention of bone loss in
postmenopausal women and the effects of lasofoxifene were greater than the
responses to raloxifene[26].
Although SERMs are good drugs to treat osteoporosis without raising the
risk of breast cancer, they primarily prevent vertebral fractures while having
limited capability to prevent non-vertebral fractures. In vivo study had shown
that Ral and Las could significantly increase mass and strength of trabecular
bone in OVX rats or aged rats[27, 28], but their abilities to improve the thickness
and strength of cortical bone is limited. That might be related to the inability of
Ral and Las to mimic effects of E2 in MSCs differentiating to pre-osteoblasts.

Effects of E2 and SERMs on RUNX2
Previous results have shown that the activity of RUNX2 is regulated by E2
and SERMs through their binding with ERα. In breast cancer cells, E2
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antagonized the pro-metastatic activities of Runx2, including SNAI2
upregulation, thus inhibited Runx2-induced EMT and invasiveness of BCa
cells[29]. In MC3T3-E1 osteoblastic cells, estradiol-bound ERα strongly
interacted with Runx2 directly through its DNA-binding domain, thus expression
of downstream genes decreased[30]. SERMs also induced ERα-Runx2
interactions but led to various functional outcomes[30].
It has recently been shown that E2 inhibits BMP-induced osteoblast
differentiation in human primary mesenchymal cell cultures, evidenced by
reduced expression of ALP, IGF-1 and BMP2 mRNA expression after E2
treatment[31]. RUNX2 level or activity were not measured in this study, but
since those genes are commonly downstream genes of RUNX2, there is great
chance that RUNX2 was involved in the inhibitory effects of E2 in the human
MSCs.

Locus-specific effects of steroid hormones on RUNX2
Although, as described above, estrogens and SERMs generally antagonize,
RUNX2, there are many exceptions. This is very important because if E2 only
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inhibited RUNX2, this could only explain the reduced bone turnover in the
presence versus absence of estrogens, but it would not explain how E2 improves
the balance between bone resorption and bone formation. Differential effects
of E2 on RUNX2 upon different target genes may ultimately explain how E2
have different effects on RUNX2’s ability to promote osteoblastogenesis versus
osteoblast-driven osteoclastogenesis. Indeed, in a recent paper, Martin et al.[32]
showed that E2 does not antagonize RUNX2 in activating all of its targets, but
can in fact cooperate with RUNX2 in activating some genes. This is not unique
to E2. In another recent paper, Morimoto et al. [33] showed reported a similar
phenomenon for glucocorticoids, hormones that are used to treat patients with
rheumatoid arthritis and induce osteoporosis by as a major side effect[34].
Earlier, it was shown that glucocorticoid receptor binds RUNX2 and
down-regulates expressions of RUNX2 target genes [35], [36]. Genome-wide
analysis, however, has demonstrated that not all RUNX2 target genes were
inhibited by glucocorticoids. In fact, several genes were cooperatively
stimulated by RUNX2 and glucocorticoids, and one of them, Wnt inhibitory
factor 1 (Wif1), was critical for glucocorticoid-mediated suppression of
osteoblast differentiation in the ST2/Rx2
dox
culture system [33]. Although the
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glucocorticoids and estrogens do not inhibit and stimulate RUNX2 at the same
target genes, they share the principle of affecting RUNX2 in a locus-dependent
manner. Therefore, in the present study, we analyzed effects of RUNX2 on
multiple target genes, and in fact observed locus-dependent effects.
Furthermore, the effects also depended on the ER ligand, with different effects
observed for E2, raloxifene and lasofloxifene.

Hypothesis and experimental models
Based on the background provided above, we hypothesized that E2, and
possibly and SERMs, would inhibit MSC differentiation into osteoblasts. We
tested this hypothesis in the ST2/Rx2
dox
culture system, in which RUNX2
expression is induced by dox (Figure 1B). We further hypothesized that this
inhibition will be absent or weaker in pre-osteoblasts, which are already
committed to the bone phenotype, and we used the pre-osteoblast cell line
MC3T3-E1 to test this hypothesis (Figure 1B). Last, we also used the
ST2Rx2
dox
system to test the inhibition of RUNX2 at the gene expression level.
Using alkaline phosphatase assay to measure development of the bone phenotype,
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and RT-qPCR to measure gene expression, we report here that both E2 and
SERMs inhibit RUNX2-driven osteoblast differentiation and expression of select
RUNX2 target genes. Other genes, however, are not inhibited and some exhibit
differential response to E2 versus SERMs. The inhibition of RUNX2 by E2
may be critical for E2-mediated attenuation of cortical bone turnover.
Furthermore, failure of SERMs to mimic E2 in locus-specific modulation of
RUNX2 activity may explain why they do not prevent non-vertebral osteoporotic
fractures.
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Fig 1 A. Chemical structures of E2, Ral and Las Ral, illustrating that SERMs are small molecule
chemicals that do not share with E2 the backbone of steroid hormones. Still, SERMs can interact
with estrogen receptor. B. Schematic representation of osteoblast differentiation in the ST2 and
MC3T3-E1 models, illustrating the hypothesized early stage in which E2 inhibits RUN
osteoblasts(
MC3T3(
fibroblasts(
ST2/Rx2
dox
(
Dox((RUNX2)(
E2(
pre=osteoblasts(
mesenchymal(cells(
Estrodial( Raloxifene( Lasofoxifene(
adipocytes(
commiFed=mesenchymal(cells(
A"
B"
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Materials and Methods
Reagents
Table 1. Reagents used in this study
Name of Materials Sources
Alkaline Phosphatase Detection Kit
Aurum
TM
total RNA mini kit
Merck Millipore Products, Temecula, CA
Bio-Rad Laboratories, Hercules, CA
Bio-Rad Protein Assay Kit Bio-Rad Laboratories, Hercules, CA
Charcol stripped fetal bovine serum (CSS) Gemini, West Sacramento, CA
Doxycycline (Dox)
Estradiol (E2)
Calbiochem, La Jolla, CA
Sigma-Aldrich, St. Louis, MO
Fetal bovine serum Gemini BioProducts, West Sacramento, CA
Formadehyde 37% Solution J.T. Baker, Phillipsburg, NJ
iQ™ SYBR® green supermix
Lasofoxifene tartrae
Bio-Rad Laboratories, Hercules, CA
Sigma-Aldrich, St. Louis, MO
Mouse monoclonal primary antibody
against FLAG® epitope (M2)
Sigma-Aldrich, St. Louis, MO
Protease inhibitor cocktail
Raloxifene hydrochloride
RPMI1640
RPMI1640 without phenol red
SensoLyte pNPP Alkaline Phosphatase
Assay Kit
Sigma-Aldrich, St. Louis, MO
Sigma-Aldrich, St. Louis, MO
Gibco, Invitrogen, Carlsbad, CA
Gibco, Invitrogen, Carlsbad, CA
AnaSpec, Fremont, CA
Typsin (10X) Gibco, Invitrogen, Carlsbad, CA
aMEM
aMEM without phenol red
Facility core, USC
Facility core, USC
Quanta Bioscience, Gaithersburg, MD

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Cell culture
ST2 cells are bone-marrow derived mesenchymal stem cells that have the
ability to become osteoblast-like cells. ST2 cells used in this study were
transduced in our lab with lentiviruses encoding Dox inducible FLAG-RUNX2
[23]. When ST2/Rx2
Dox
cells are treated with Dox, RUNX2 protein is expressed
to initiate osteoblast differentiation.
MC3T3-E1 cells are pre-osteoblasts that has the potential to differentiation
into osteoblasts by the induction of ascorbic acid and BGP or other
bone-promoting molecules such as BMP2
ST2/ Rx2
Dox
cells were maintained in RPMI1640 (Mediatech Inc. Manassas,
VA) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin (P/S). MC3T3-E1 cells were maintained in αMEM
supplemented with 10% FBS and 1% P/S.
For experiments with sex hormones, cells were cultured in RPMI or αMEM
without phenol red and with 10% CSS charcoal stripped fetal bovine serum (CSS)
and 1% P/S.
E2 or SERMs were diluted with ethanol or DMSO to prepare stock
solutions increasing concentration of from 10
-7
M to 10
-3
M. They were added to
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culture media at a volume of 1:1000 to yield final concentrations of 10
-10
M to
10
-6
M. Control cultures were treated with ethanol and DMSO, so that all cultures
were incubated with ethanol and DMSO at final concentrations of 2uL/mL each.

RNA extraction and analysis
Total RNA was extracted using AurumTM total RNA mini kit according to
the manufacturer’s protocol and 1 µg RNA was reverse-transcribed using
qScriptTM cDNA supermix to prepare cDNA. Real-time qPCR analysis was
performed using the CFX96TM RT-PCR system and the iQ™ SYBR® Green
Supermix according to the manufacturer’s protocol. The primers used for qPCR
are listed in Table 2. Data were normalized for the mRNA levels of 18S and
PPIA, which themselves were not significantly affected by treatment.
All qPCR assays were performed in triplicate and results are presented as
the
mean±SD. P<0.05 was considered significant using Student’s t test.
Reverse-transcription, Real-time qPCR experiment and data analysis was
performed by Yonatan Amezaleg.
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Table 2. Primer sequencers used in qPCR analysis
Gene Sequence (5’ to 3’)
Cyr61a F AAG AGG CTT CCT GTC TTT GGC
R AGG ACG CAC TTC ACA GAT CC
BSP F AGG AAG AGG AGA CTT CAA ACG A
R AAA AGT CTG TGC TTG GGG TG
CTGF F CTG CTG TGC ATC CTC CTAC C
R CCA TAG CAG GCC GGG TG
18s F GTA ACC CGT TGA ACC CCA TT
R CCA TCC AAT CGG TAG TAG CG
PPIA F GT GCC AGG GTG GTG ACT TT
R CGT TTG TGT TTG GTC CAG CAT
ALP F CAG CGA GGG ACG AAT CTC AG
R GGA GAC GCC CAT ACC ATC TC
Greb1 F CAG CCT TGG GTA TCT TGC CA
R TAC CTT CGT GCT TGG CTC TG
Egr1 CAC CTG ACC ACA GAG TCC TTT
AGG CTG AAA AGG GGT TCA GG
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Egr2 CCT AGG CTC AGT TCA ACC CC

TTG ATC ATG CCA TCT CCC GC
OC
CCA CAC AGC AGC TTG GCC CAG A

GGG CTT GGC ATC TGT GAG GTC AGA G
SP7
CCC CAG CTG CCT ACT TAC CC

CTA TTG CCA ACC GCC TTG GG
DUSP5
AGT GTG GAA AGC CCG TTC TC

CTT CTT CCC TGA CAC AGT CAA T

Western blot analysis
Cells were lysed in RIPA cell lysis buffer and a protease inhibitor mix was
added (1:100). Protein concentrations were measured by Bio-Rad Protein Assay
Kit. 20 ug of whole cell lysate was subjected to SDS-PAGE. Proteins in the gel
were transferred to Amersham HybondTM-P PVDF membranes and detected by
mouse monoclonal FLAG antibody (M2, Sigma) and goat anti mouse secondary
antibody (SC-2005, Santa Cruz). Beta actin was detected using rabbit polyclonal
antibody (I-19, Santa Cruz) and donkey anti goat secondary antibody, (SC-2033,
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Santa Cruz).
Alkaline phosphatase (ALP) assay
ST2/Rx2
Dox
cells were plated (12,500 cells/well in 24-well plates) in phenol
red-free RPMI1640 media supplemented with 10% CSS and 1% P/S for the
indicated days. After washing with PBS 3 times, cells were lysed with 0.01 M
Tris-Saline buffer with 0.2% Triton, scraped and vortex for 5s each. Cell lysates
were centrifuged and supernatants were collected. ALP activities were measured
using SensoLyte pNPP Alkaline Phosphatase Assay Kit.

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Results
E2 inhibit osteoblastogenesis in ST2 mesenchymal stem cells
Because E2 is beneficial for the skeleton, its negative effect on the number
of CBU-OBs [21, 22] was surprising. E2 administration to other osteoblast
systems did not reproduce the negative effect observed by Jilka et al. [37, 38]
Here, we treated ST2/Rx2
Dox
cells with E2 and then added Doxycyline (Dox) to
induce RUNX2 and initiate osteoblast differentiation (Figure 2A).
Preliminary experiments indicated that ALP was hardly detectable
under any treatment condition before Day 6, and so we decided to test the effect
of Dox and E2 on ALP activity on Days 6 and 8. As shown in Figure 2B, when
cells were treated with Dox, to induce RUNX2, they responded with high levels
of ALP on Day 6 and Day 8. The treatment of E2 alone did not change cells’
ALP activity. But for group of cells that were treated with Dox as well as E2,
their ALP activities dropped greatly compared to Dox-only treatment group after
6 days treatment, and even more significantly after 8 days.
We also investigated the effect of E2 on osteoblastogenesis in ST2
mesenchymal cells at the gene expression level. We collected RNA from cells
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treated with Dox and/or and measured the expression of two osteogenic genes,
Osteocalcin (OC) and Bone Sialoprotein (BSP) by RT-qPCR after 24 hours of
Dox treatment.

Fig 2 E2 inhibit ALP activity and mRNA level of osteogenic genes in ST2 mesenchymal stem cells
A. The scheme of protocol A. ST2/Rx
DOX
cells were treated with phenol red free RPMI1640 media and
charcoal strip serum for two days in advance, then E2 10
-9
M was added to the media. We wait another
3 hours to make sure estrogen receptors are activated by E2, and then we added Dox 200ng/mL to
induce exogenous RUNX2. B. ALP activity was measured after 6 or 8 days of Dox treatment. Every
treatment was done in triplicate. C. RNA was collected after 6 hours of Dox. RT-qPCR was conducted
using primers targeting osteoclacin and BSP in triplicate. For statistical analysis we used one-tailed
student t test. *, p<0.01, **p<0.05,***p<0.001
C
D
E
DE
0.0
0.2
0.4
0.6
Day 6
ng#p%Nitrophenol#/#mg#Protein
*
C
D
E
DE
0.0
0.2
0.4
0.6
0.8
1.0
Day 8
ng#p%Nitrophenol#/#mg#Protein
C
D
E
DE
**
Protocol'A'
ALP'
Pla+ng'(in'CSS)'
3h'
'
hormones'
Day:';2'
Dox'
0'
RUNX2'Western'
Gene'expression'
'24'hr'
A"
B"
C"
C
D
E
DE
0
100
200
300
400
500
Osteocalcin
Relative mRNA
**
C
D
E
DE
0
10
20
30
BSP
Relative mRNA
DE
E
D
C *
8' 6'
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As illustrated in Figure 2A. The results were very consistent with the ALP
activity assay. OC and BSP expression was strongly induced by Dox alone
treatment, and this induction was significantly weaker in the presence of E2
(Figure 2C). Thus, the inhibitory effect on ALP activity observed on days 6
and 8 likely reflects antagonism of RUNX2-driven gene expression soon after
the induction of RUNX2 (day 1).
Inhibition of osteoblast differentiation by E2 was counter-intuitive
because estrogens are well known for their bone-sparing effects. In fact, others
have reported positive or no effect of E2 on osteoblast differentiation[39, 40].
We reasoned that E2 might inhibit osteoblast differentiation only when
administered to cells before their commitment to the osteoblast phenotype.

Weak inhibtion of ALP activity by E2 in committed osteoblasts
To address the possibility that E2 only inhibits osteoblast differentiation
when administered to cells before their commitment to the osteoblast phenotype,
we first treated MC3T3-E1 cells, which are already committed to the osteoblast
phenotype, with E2. Indeed, E2 did inhibited ALP activity in the MC3T3-E1
University
of
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California
Master’s
Thesis

28

cultures only minimally and insignificantly (Figure 3A). We further addressed
this notion by administration of E2 to ST2/Rx2
dox
after commitment to the
osteoblast phenotype. First we showed that one day of Runx2 induction was
sufficient to induce a significant level of commitment as demonstrated by ALP
activity measured on day 8 (Figure 3B, C). Then, we treated cells with E2 one
day after dox-mediated Runx2 induction and measured ALP activity on day 6
and day 8 (Figure 3D, Schematics of Protocol B). As shown in Figure 3E, E2
inhibited Dox-induced ALP acitvy, but this inhibition here (with uncommitted
ST2 mesenchymal cells; Protocol B) was weaker than in protocol A, when Dox
and E2 treatment commenced on the same day (Figure 2). We also we checked
the expression of osteogenic genes OC and BSP in committed ST2 mesenchymal
cells as well. Interestingly, their expression was more inhibited in Protocol B
than in Protocl A.

University
of
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California
Master’s
Thesis

29

Fig 3 Weak inhibition of committed cells A. MC3TC-E1 cells were cultured in phenol red-free
α-MEM medium supplemented with 10% CSS. 50 ug/mL Ascorbic acid and BGP were added to
induce osteoblast differentuation and 10
-9
M E2 (or ethanol vehicle) was added along with dox.. ALP
activity was measured on day 6. Experiment was done in triplicate. B,C. ST2/RX2
Dox
cells were treated
Veh
E2
0.0
0.2
0.4
0.6
0.8
MC3T3
ng#p%Nitrophenol#/#mg#Protein
Veh
E2
NS
0"
0.2"
0.4"
0.6"
0.8"
1"
1.2"
Control" Day"1" Day"2" Day"3" Day"4" Day"8"
0"
ALP"
Staining"
8"
1"day"Dox"
"2"days"Dox"
"3"days"Dox"
"4"days"Dox"
8"days"Dox"
Rela>ve"ALP"ac>vity"
measured"by"ng"pE
Nitronphenol"/"mg"Protein""
A" B"
C"
C
D
E
DE
0.0
0.1
0.2
0.3
0.4
ng#p%Nitrophenol#/#mg#Protein
*
Day 6
C
D
E
DE
0.0
0.2
0.4
0.6
0.8
Day 8
ng#p%Nitrophenol#/#mg#Protein
C
D
E
DE
*
C
D
E
DE
0
500
1000
1500
Osteocalcin
Relative mRNA
*
C
D
E
DE
0
2
4
6
BSP
Relative mRNA
C
D
E
DE
*
Dox$
RUNX2$Western$
Gene$expression$
6h$
hormones$
ALP$
Pla;ng$(in$CSS)$
Day:$C2$ 1$
$24$hr$
Protocol$B$
D"
E" F"
8$ 6$
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of
Southern
California
Master’s
Thesis

30

with Dox (200ng/mL) for the durations indicated by the red bars. Then Dox were withdrawn from the
media at described in the scheme. ALP activity was measured by both histochemical staining (B) and
p-Nitronphenol substrate assay (C) after 8 days of Dox treatment. Every treatment was done in
triplicate. D. ST2/RxDOX cells were subjected to treatment Protocol B. In this protocol, cells are
treated with phenol red free RPMI1640 media and CSS for two days in advance, then Dox (200ng/mL)
was added to the media for two days, and only then we added E2 (10
-9
M). E. ALP activity was
measured by substrate after 6 or 8 days of Dox treatment. Every treatment was done in triplicate. F.
RNA were collected after 6 hours of Dox treatment. RT-qPCR was conducted using primers targeting
OC and BSP in triplicate. For statistical analysis we used one-tailed student t test. *, p<0.01,
**p<0.05,***p<0.001

Inhibitory effects of SERMs in ST2 mesenchymal cells
SERMs are clinically used to prevent osteoporosis by substituting for
endogenous estrogen in postmenopausal women. None of them, however, is as
good as estrogen in preventing fractures. When we were looking at the inhibitory
effects of E2/SERMs on ALP activity, we found that it seems like Ral and Las
have a much stronger effect than E2
In this experiment, we used two concentrations of the SERMs 10
-6
M and
10
-7
M, which were different from the concentration of E2 (10
-9
M), but were
concentrations that are commonly used to treat bone cells by investigators [41,
42]. Expecting to explore the effect of SERMs on ST2 mesenchymal cells, we
University
of
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California
Master’s
Thesis

31

used them in the same experiment as we had used E2 with. Gene expressions and
ALP activities were assayed in different day with protocol A or protocol B
treatment separately.
As expected, ALP activity in ST2 mesenchymal stem cells was inhibited by
both Ral (Figure 4A) and Las (Figure 4B), in both protocol A (top) and protocol
B (bottom), on both day 6 and day 8. When compared with the inhibitory effect
of E2 in ST2 stem mesenchymal cells (Figure 2), SERMs’ ability to inhibit
osteoblastogenesis was stronger. Specifically, while E2 inhibited 20% ALP
activity, Ral inhibited 20% to 60% percent and Las inhibited about 50% ALP
activity on day 6 and day 8. And this difference between E2 and SERMs was
more significant in protocol B than protocol A.
In general, the inhibitory effects of SERMs on ALP activity (Figure 4) were
paralleled by inhibitory effects on OC and BSP. When treated with Ral or Las
with Dox, osteogenic genes were less expressed compared with Dox alone group
except the expression of BSP in protocol A is not. Expressions of OC in protocol
B were more inhibited than in protocol A, while expressions of BSP were
inhibited more significant in protocol A than in protocol B. But unlike ALP
activities, there was not evidence that the mRNA expression inhibitory effect of
University
of
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California
Master’s
Thesis

32

SERMs was stronger than E2.

Fig 4 SERMs inhibit ALP activity and mRNA level of osteogenic genes in ST2 mesenchymal stem
cells ST2/RxDOX cells were treated according to protocol A or protocol B. Instead of E2, we used Ral
and Las at the concentration of 10
-6
M or 10
-7
M to conduct this experiment.(ABCD). ALP activity was
measured by substrate after 6 or 8 days of Dox treatment according to protocol A or B. Every treatment
was done in triplicate. (EFGH). RNA were collected after 6 hours of Dox. RT-qPCR was conducted
using primers targeting OC and BSP in triplicate. For statistical analysis we used one-tailed student t
test. *p<0.01*, **p<0.05, ***p<0.001

C
D
Ral
DRal
0
100
200
300
400
500
prtocol A Osteocalcin
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Ral
LRal
0
500
1000
1500
protocol B Osteocalcin
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Las
DLas
0
100
200
300
400
500
protocol A Osteocalcin
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Las
DLas
0
500
1000
1500
protocol B Osteocalcin
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Ral
DRal
0
10
20
30
protocol A BSP
ng#p%Nitrophenol#/#mg#Protein
***
C
D
Ral
DRal
0
2
4
6
protocol B BSP
ng#p%Nitrophenol#/#mg#Protein
*
protocol A BSP
ng#p%Nitrophenol#/#mg#Protein
C
D
Las
DLas
0
20
40
60
C
D
Las
DLas
0
2
4
6
protocol B BSP
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Ral
DRal
0.0
0.2
0.4
0.6
protocol A Day 6
ng#p%Nitrophenol#/#mg#Protein
**
C
D
Ral
DRal
0.0
0.1
0.2
0.3
0.4
protocol B Day 6
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Ral
DRal
0.0
0.2
0.4
0.6
0.8
1.0
protocol A Day 8
ng#p%Nitrophenol#/#mg#Protein
**
C
D
R
DRal
0.0
0.2
0.4
0.6
0.8
protocol B Day 8
ng#p%Nitrophenol#/#mg#Protein
***
C
D
Las
DLas
0.0
0.2
0.4
0.6
protocol A Day 6
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Las
DLas
0.0
0.1
0.2
0.3
0.4
protocol B Day 6
ng#p%Nitrophenol#/#mg#Protein
*
C
D
Las
DLas
0.0
0.2
0.4
0.6
0.8
1.0
protocol A Day 8
ng#p%Nitrophenol#/#mg#Protein
**
C
D
L
DLas
0.0
0.2
0.4
0.6
0.8
protocol B Day 8
ng#p%Nitrophenol#/#mg#Protein
**
A"
C" D"
B"
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of
Southern
California
Master’s
Thesis

33

Inhibitory effect of E2 and SERMs at increasing concentrations on
ALP activity in ST2/Rx2
dox
cells
In Figures 2,3 and 4, ST2 cells were treated with E2 (10
-9
M), Ral (10
-6
M)
and Las (10
-6
M) at concentrations typically used in the literature[41]. We
suspected that some of the differences observed between E2, Ral and Las might
be attributable to the different concentrations used. Because there is no
standard assay to compare the activities of E2, Ral and Las, and because they are
have substantially different structures (Figure 1A), we decided to compare their
effects on RUNX2-driven ALP activity and gene expression across a range of
increasing concentrations.
We initially compared the effects of E2 and SERMs on ALP in ST2/Rx2
dox
cultures when administered either just before (protocol A; see Figure 2) or a day
after (Protocol B; see Figure 3) dox-mediated induction of Runx2. This
experiment was performed with E2 and SERMs at increasing concentrations
from 10
-10
M to 10
-6
M, which included most concentrations used in the literature
for E2, ral and las [41]. MC3T3-E1 cells were included as control. As shown in
Figure 5, when E2/SERMs treatment commenced just before Dox (3 hours)
(protocol A), a great drop of ALP activity could be observed, even with the
University
of
Southern
California
Master’s
Thesis

34

lowest concentration of 10
-10
M (fig 4, AB), in both day6 and day 8.

Fig 5 The effect of E2 and SERMs on ST2 mesenchymal cells and MC3TC-E1 cells osteoblast
differentiation shown by ALP activities (A\B). ST2 ／RX2
Dox
cells were treated with E2/SERMs 3
hours before treated with Dox (200ng/mL) according to protocol A. (C\D). ST2RX2
Dox
cells were
treated with E2/SERMs 24 hours after treated with Dox (200ng/mL) according to protocol B. All cells
were treated with Dox for 6 or 8 days. (E) MC3T3 cells were treated with differentiation media and
E2/SERMs for 6 days. The concentration of E2/SERMs ranges from 10
-6
M to 10
-10
M. Every treatment
was done in triplicate. All results are normalized to 1.

And with protocol B, we also found that E2/SERMs showing the ability to
inhibit the differentiation of committed-osteoblast with all concentrations (Figure
Protocol'B'
0'
0.5'
1'
1.5'
w/o' 10^010' 10^09' 10^08' 10^07' 10^06'
0'
0.5'
1'
1.5'
w/o' 10^010' 10^09' 10^08' 10^07' 10^06'
Rela8ve'ALP'ac8vity'measured'by'ng'p0Nitronphenol'/'mg'Protein''
0'
0.5'
1'
1.5'
w/o' 10^010' 10^09' 10^08' 10^07' 10^06'
E'
R'
L'
DE'
DR'
DL'
0'
0.5'
1'
1.5'
w/o' 10^010' 10^09' 10^08' 10^07' 10^06'
Day'6'
Day'8'
Day'8'
Day'6'
A"
B"
C"
D"
0'
0.5'
1'
1.5'
2'
w/o' 10^010' 10^09' 10^08' 10^07' 10^06'
E"
Protocol'A' MC3T3'
University
of
Southern
California
Master’s
Thesis

35

5, CD), only this inhibition is not as strong as the effect E2/SERMs on
uncommitted ST2 mesenchymal cells, especially of E2 treatment on day 6.
Results of MC3T3-E1 cells were quite consistent with previous data.
Not surprisingly, E2/SERMs barely shows any significant inhibition of ALP
activity with nearly all concentrations (Figure 5 E). Except for the two SERMs at
the concentration of 10
-6
M, which is the highest concentration in all experiments.
With 10
-6
M of Ral and Las but not E2, we can observe the activity dropped by
about 50%.
Then we compared E2, Ral and Las at all concentrations. For uncommitted
ST2 mesenchymal cells (treated in with protocol A) (Figure 4 AB), there were
very small differences between E2 and SERMs on day 6, with relatively higher
concentration (10
-8
to 10
-6
M). And these differences even became bigger in day
8: ALP activities of cells under E2 of all concentrations are higher than the one
under Ral and Las of all concentrations.
For those committed ST2 mesenchymal cells (treated with protocol B)
(Figure 4 CD), on day 6, low dose (10
-10
M to 10
-9
M) E2 treatment had almost
no inhibition on their ALP activities. But with the same dose of Ral and Las on
the same day, we already can observe the significant drop of ALP activity. And
University
of
Southern
California
Master’s
Thesis

36

the same result repeated itself on day 8. The ALP activity of E2 treatment once
again is much higher than the on of Ral/Las treatment.
Another phenomenon showed up here and interested us was that in protocol
A, E2 treatment, the inhibitory effect was strong at low concentrations, then
became weaker at medium concentrations and almost null at high concentrations.
This suggests that E2 has two different effects thorough different mechanisms
probably. With low concentration of E2, osteoblast differentiation was inhibited,
while with high concentration of E2, an anabolic mechanism was activated and
antagonized the inhibitory effect driven by the lower concentrations. But this
kind of anabolic effect was not significant show in SERMs treatment with either
protocol. Except of protocol B, day 8, Ral treatment, which gave an increase of
ALP activity at concentrations of 10
-8
M and 10
-7
M.

E2 and SERMs down-regulate RUNX2 protein level
Since the ALP assays indicated significant inhibition of RUNX2-mediated
MSCs differentiation with E2/SERMs treatment, we wondered how these
treatments affected the level of RUNX2. Thus we treated cells with E2/SERMs
University
of
Southern
California
Master’s
Thesis

37

with a range of concentrations from 10
-10
to 10
-6
M according to protocol A and
protocol B, and collected proteins after 24 hours of E2/SERMs and Dox
treatment. After cell lysis and quantification of total protein concentrations, we
ran SDS-PAGEs and used mouse-anti-FLAG antibody to detect the level of
exsogenous RUNX2 that was induced by Dox. Remakably, the level of
exsogenous RUNX2 was strongly decreased by the treatment of E2/SERMs in
both protocol A and B, even though the protein was coded by an exogenous
cassette[23].
Under protocol A, in which E2/SERMs were added 3 hours before Dox, the
level of RUNX2 showed an remakable >90% reduction (Figure 6A). Comparing
the 3 drugs, the effects of ral and las seemed stronger than E2, although this
difference remains to be validated. Importantly, the maximum inhibitory
effects on E2, ral and las was already observed at the lowest concentration of
10
-10
M. Increasing concentrations did not inhibit RUNX2 levels further
(Figure 6).
University
of
Southern
California
Master’s
Thesis

38

Figure 6 E2/SERMs inhibits exogenous RUNX2 expression in ST2/Rx2
Dox
cells detected by
western blot. A. E2/SERMs were treated 3 hours in advance of Dox (200 ng/mL) treatment with the
indicated concentrations of E2, ral or las, from 10
-10
M to 10
-6
M. Cells were harvested and lysed for
Western analysis after 24 hours of Dox treatment. B. Cells were treated with E2/SERMs for 24 hours
after Dox (200 ng/mL) treatment. Cells were harvested and lysed for Western analysis after 6 hours of
E2/SERMs treatment.

When E2/SERMs were treated 24 hours after Dox, this reduction appeared
to be less significant (Figure 6B). Under E2 treatment, there are almost no
reduction with the concentration of 10
-10
M and 10
-9
M. But very weak reductions
Ral$$
Flag'Runx2$
Ac.n$
Veh$$$$$$$$$$10$$$$$$$$$$9$$$$$$$$$$8$$$$$$$$$$$$$7$$$$$$$$$$$$$$6$$$$$$$('log$M)$
E2$$
Flag'Runx2$
Ac.n$
Las$ Flag'Runx2$
Ac.n$
Ral$
Flag'Runx2$
Ac.n$
E2$
Flag'Runx2$
Ac.n$
Las$ Flag'Runx2$
Ac.n$
Veh$$$$$$$$$$10$$$$$$$$$$9$$$$$$$$$$8$$$$$$$$$$$$$7$$$$$$$$$$$$$$6$$$$$$$('log$M)$
B"
A"
University
of
Southern
California
Master’s
Thesis

39

from 10
-8
to 10
-6
M. This result is very important because it suggests that the
existence of E2 will cause the reduction of RUNX2 level.
Under Ral treatment, there was apparently a does-dependent RUNX2
reduction effect: the level of RUNX2 with 10
-10
M seems have no difference
with vehicle, but form 10
-9
M to 10
-6
M, less and less RUNX2 remained. Same
thing happened with Las. Except 10
-10
M of Las already reduced the level of
exogenous RUNX2 protein, and the protein level continually decreased with the
concentration of Las increased.
This western-blot data is very consistent with our previous ALP activity
data. It suggests that the way E2/SERMs regulate RUNX2 level is not decrease
its expression but accelerates its degradation. Because since we were detecting
the Dox-inducible exogenous RUNX2, its expression will be ceaseless since
there is Dox in the media.

Differential regulation of gene expression by E2, Ral and Las
Ral is the only SERM approved in the USA for the prevention of
postmenpausal osteoporosis. Although Las is not approved for this indication
University
of
Southern
California
Master’s
Thesis

40

in the USA, it is reported to outperform Ral[26] and it is widely used in Europe
for this indication. To gain insight into differences between mechanisms of
action of Ral vs. Las, we first conducted a preliminary RNA-seq experiment
trying to identify genes that respond differently to Ral vs. Las in Dox-treated
ST2/Rx2
dox
cultures. While the results of this experiment are too preliminary to
show, they allowed us to focus on several gene candidates for thorough
RT-qPCR analysis under three different protocols with each of E2, Ral and Las
employed at increasing concentrations, from 10
-10
M to 10
-6
M (Figures 7-9)
CTGF and Cyr61 are both immediate early genes belonging to the CNN
family that may be involved in regulating osteoblastogenesis[43]. When
commenced just three hours before a 24-hr of Dox treatment, ral and las
treatment had different effects on Cry61 and CTGF mRNA expression (Figure
7A, B). The mRNA level of Cry61 appeared to raised after Ral and Las
treatment, and remain the same after E2 treatment, except the concentration of
10
-9
M, which give a big increasing peak. But for CTGF, the 3 drugs gave very
similar result at control and concentration for 10
-10
to 10
-8
M. When
concentrations increase to 10
-7
M and 10
-6
M, E2 and Ral inhibited mRNA
expression while Las increased it.
University
of
Southern
California
Master’s
Thesis

41

Figure 7 Modulation of RUNX2-driven gene expression by E2, ral and las (Protocol A)
ST2/RX2Dox cells were first treated with E2/SERMs for 3 hours then Dox was added for additional 24
hours before RT-qPCR analysis of the indicated genes. Data is Mean±SD (n=3).

Dusp5 or dual-specificity phosphatase 5 primarily expressed in angioblasts
CTGF
Veh 10 9 8 7 6
0.01
0.10
1
10
Relative mRNA
- Log M
Greb1
Veh 10 9 8 7 6
0.01
0.10
1
10
100
Relative mRNA
- Log M
veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
Osteocalcin
Cyr61
Veh 10 9 8 7 6
0.01
0.10
1
10
100
Relative mRNA
- log M
Egr1
Veh 10 9 8 7 6
0.1
1
10
Relative mRNA
- Log M
veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
ALP
veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
SP7
Dusp5
Veh 10 9 8 7 6
0.001
0.010
0.100
1
10
- Log M
Relative mRNA
E2
Ral
Las
Egr2
Veh 10 9 8 7 6
0.01
0.10
1
10
Relative mRNA
- Log M
veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
BSP
A" B" C"
J"
E" F"
G" H" I"
D"
University
of
Southern
California
Master’s
Thesis

42

and the lost of it may result in endothelial cell apoptosis[44]. Dusp5 can also
inhibit osteoclastogenesis and thus attenuate autoimmune arthritis via the
regulation of Th17/Treg cell balance[45]. When treated with E2/SERMs then
Dox for 24 hours (Figure 7C), Las reduced Dusp5 expression at low
concentrations and raised Dusp5 mRNA level with high concentrations while
Ral and E2 reduced Dusp5 mRNA level in general.
Greb1 (short for Growth regulation by estrogen in breast cancer 1) is
known to be activated by ERα in breast epithelial cells[46]. In Dox-treated
ST2/Rx2
dox
cells, Greb1 mRNA was robustly regulated by E2 and Ral, but the
effects of las were weak (Figure 7D) . E2 strongly and dose-dependently
stimulated Greb1 expression, up to 20-fold (with 10
-6
E2) compared to control
cultures treated with dox and no E2. In contrast, Ral stimulated Greb1
expression by up to only 5-fold (at 10
-10
M and 10
-6
M), and Las did not stimulate
Greb1 expression at any concentration. Interestingly, the stimulation of Greb1 by
E2 and Ral was no longer observed at high concentrations of 10
-7
M and 10
-6
M.
Egr1 and Egr2 (early growth response 1 and 2) share high homology and
are often regulated in parallel [47]. When treated with E2, expressions of
Egr1/2 was inhibited at both low (10
-10
M) and high concentrations (10
-7
M, 10
-6

University
of
Southern
California
Master’s
Thesis

43

M), but significantly stimulated at intermediate concentrations (10
-8
M) (Figure
7E,F). With the treatment of Ral, Egr1 rose at concentrations of 10
-10
M to 10
-8
M,
depressed at concentrations of 10
-7
M and 10
-6
M; Egr2 was depressed by high
concentration. With Las, the expression of Egr1/2 was induced in general.
We also measured expressions of a four osteogenic genes (OC, ALP, BSP
and SP7) as control (Figure 7 G to J). And all of their expressions are similar.
When we conducted the 24 hours Dox treatment, significant drop of OC
mRNA expression level was happened with all concentrations of all drugs in
general, except a few outliners (Figure 7G). After excluded those outliners, we
might describe the trend of OC mRNA expression level as decreased with
increasing drug concentration, especially in E2 and Ral treatment. In Las
treatment, this pattern was not appearing.
In general, osteogenic genes were less expressed with SERMs treatments,
especially with Las treatment; and less expressed in high concentrations of E2
than in low concentrations of E2. Except the concentration of 10
-8
M, it seemed
that 10
-8
M of E2 and Ral sometimes gave the highest osteogenic genes’
expression.

University
of
Southern
California
Master’s
Thesis

44

Figure 8. Modulation of RUNX2-driven gene expression by E2, ral and las (Protocol B).
Genes that were regulated by E2/SERMs after 36 hours Dox treatment ST2/RX2Dox cells were
first treated with Dox for 24 hours then treated with Dox and E2/SERMs for 12 hours before mRNA
were collected. RT-qPCR analysis were done in triplicate.

Except the previous described treatment, we also had two others treatment
that treat the treatment of Dox last 30 (Figure 9) and 36 hours (Figure 8), while
CTGF
Veh 10 9 8 7 6
0.001
0.010
0.100
1
10
100
- Log M
Relative mRNA
Greb1
Veh 10 9 8 7 6
0.001
0.010
0.100
1
10
100
Relative mRNA
- Log M
veh 10 9 8 7 6
0.001
0.010
0.100
1
10
100
- log M
Relative mRNA
Osteocalcin
Egr1
Veh 10 9 8 7 6
0.0001
0.001
0.01
0.1
1
10
100
1000
Relative mRNA
- Log M
veh 10 9 8 7 6
0.1
1
10
100
- Log M
Relative mRNA
ALP
veh 10 9 8 7 6
0.01
0.10
1
10
100
- Log M
Relative mRNA
SP7
Dusp5
Veh 10 9 8 7 6
0.1
1
10
100
1000
- Log M
Relative mRNA
E2
Ral
Las
Egr2
Veh 10 9 8 7 6
0.0001
0.001
0.01
0.1
1
10
100
1000
Relative mRNA
- Log M
veh 10 9 8 7 6
0.0001
0.0010
0.0100
0.1000
1
10
100
- Log M
Relative mRNA
BSP
Veh 10 9 8 7 6
0.001
0.010
0.100
1
10
100
- Log M
Relative mRNA
Cry6
A"
B" C"
J"
E" F"
G" H" I"
D"
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of
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California
Master’s
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45

E2/SERMs were involved at the last 6 and 12 hours. The result of Figure 8 is
very interesting because after 36 hours of Dox treatment, all genes expressions
were changed into the same pattern. Which were no response to E2, but dramatic
rose up by Ral and Las treatment at certain concentrations mostly 10
-10
M and
10
-8
M. We were interested in this because in figure 5, ALP activities of ST2
mesenchymal cells were affected by E2 in a very weak way but inhibited
significantly by Ral and Las. Those ‘dull to E2 by sensitive to SERMs’ patterns
might be related.
The result of 30 hours Dox treatment was very different from 36 hours Dox
treatment.
When cells were treated with Dox for 30 hours, the existence of E2 strongly
inhibited the expression of CTGF and Cyr61 mRNAs (Figure 9 A,B). And it
seems that those two genes are more sensitive to E2 compared to Ral and Las.
Almost in every concentration, E2 caused the strongest inhibition. Between Ral
and Las the effect of Las is more inhibiting than Ral. Ral did not inhibit
expressions of Cyr61 and CTGF mRNA except at the level of 10
-7
M. E2 and Ral
inhibited Expressions of Dusp at all concentrations, but induced by Las at the
concentration of 10
-10
M and 10
-8
M. E2 and Ral induced expressions of Greb1
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of
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California
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46

(Figure 9 D) at low concentrations and repressed it at high concentrations, while
Las repressed it all the time. Again, expressions of Egr1 and Egr2 (Figure 9 E
and F) were very similar. E2 and Las caused very small and in general decreased
fluctuat while Ral gave a big increasing peak at 10
-8
M

Figure 9 Short-term (6-hour) modulation of RUNX2-driven gene expression by E2, ral and las
(Protocol B). ST2/RX2Dox cells were first treated with Dox for 24 hours then treated with Dox and
E2/SERMs for 6 hours before mRNA were collected. RT-qPCR analysis were done in tripli
CTGF
Veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
GREB
Veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
Veh 10 9 8 7 6
0.001
0.010
0.100
1
10
- Log M
Relative mRNA
Osteocalcin
CYR61
Veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
Egr1
Veh 10 9 8 7 6
0.0001
0.0010
0.0100
0.1000
1
10
Relative mRNA
- Log M
Veh 10 9 8 7 6
0.1
1
10
- Log M
Relative mRNA
ALP
Veh 10 9 8 7 6
0.1
1
10
- Log M
Relative mRNA
SP7
DUSP
Veh 10 9 8 7 6
0.01
0.10
1
10
- Log M
Relative mRNA
E2
Ral
Las
Egr2
Veh 10 9 8 7 6
0.01
0.10
1
10
Relative mRNA
- Log M
Veh 20 9 8 7 6
0.0001
0.0010
0.0100
0.1000
1
10
- Log M
Relative mRNA
BSP
A" B" C"
J"
E" F"
G" H" I"
D"
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of
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California
Master’s
Thesis

47

Discussion
Counter to intuitive thinking, increased rates of osteoblast differentiation
from mesenchymal precursor cells is not always beneficial for the skeleton. In
fact, osteoblastogenesis is strongly coupled to signals that osteoblasts produce to
promote osteoclastogenesis, and accelerated bone turnover is usually associated
with osteoporosis. The key to pharmacological protection of our bones is
favorable regulation of the balance between osteoblastogenesis and
osteoclatogenesis. Estrogens actually protect bone while decreasing bone
turnover (formation and resorption). In this thesis, we have successfully
demonstrated one mechanism by which estrogens likely slow down bone
turnover, i.e., they inhibited RUNX2, ALP activity and expression of osteogenic
genes. Also, one reason why SERMs are not as effective as estrogens in
preventing osteoporosis may be that they do not mimic estrogens in
pre-osteoblasts: they inhibit RUNX2 and ALP activities more than E2 and they
do not inhibit some genes that E2 does. Thus, SERMs might fail to mimc E2 in
“correctly” regulating the balance between RUNX2-mediated osteoblastogenesis
and RUNX2-mediated osteoblast-driven osteoclastogenesis.
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of
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California
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Osteoblasts build up and strengthens the bone but osteoblastogenesis might
not. In fact,, RUNX2 expressed during osteoblastogenesis might induce
osteoclastogenesis[32] that is bad to bone. So there are two jobs of estrogen
regarding osteoblast: protecting already existing osteoblasts from apoptosis[16],
and inhibiting mesenchymal progenitor cells from starting the process of
osteoblastogenesis. Early commitment of progenitor cells to the osteoblast
phenotype is the stage of osteoblastogenesis when RUNX2 is predominantly. So
when mesenchymal progenitors are treated with E2 or SERMs, they immediately
reduced RUNX2 thus to attenuating osteoblastogenesis. After the onset of
RUNX2 expression, MSCs are committed and produce more RUNX2. When
E2/SERM treatment commences before this RUNX2 self-promoting cycle, they
are inhibit the process of RUNX2 expression and osteoblast differnetiation. But
if E2/SERMs are added to a system where RUNX2 is already expressed, they are
less inhibitory. This explains the difference between the results we obtained with
protocol A versus protocol B.
Whereas E2 is an endogenous estrogen that is physiologically produced and
maintains bone integrity, SERMs are human-made small molecules that interact
with estrogen receptors[48]. However, our work demonstrates that binding of
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SERMs to estrogen receptors does not have the same effects as E2 in
pre-osteoblasts. E2 moderately inhibited osteoblastogenesis. Presumably, this
level of inhibition does not interfere with osteoblastogenesis but does interfere
with the coupled osteoclastogenesis. But SERMs do not achieve this fine balance.
Like E2, they inhibited RUNX2 (Figure 4), but the resulting decrease in
osteoblasts differentiation, as determined based on ALP activity, was more
aggressive. This may be the reason why SERMs are not as good as E2 in
clinically preventing osteoporosis. This is particularly true for prevention of
non-vertebral fractures .
It brings us back to the key word about bone: balance. Too large or too
small scale of osteoblastogenesis (or in other words, too high or too low bone
turnover rate) will break the balance, resulting in bone loss. Only appropriate
lelve of osteoblastogenesis is the desired situation. Of course SERMs did not
inhibit too much osteoblastgenesis to result in bone loss, they just eliminate
osteoblast number a little bit more than the best situation, and that make them
less effective than estrogen.
We also tested some gene expressions of ST2 mesenchymal cells treated
with E2/SERMs. It turned out that gene expressions regulated by E2 and
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of
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SERMS are highly concentration- and locus- dependent. Some genes were
activated at certain concentrations while other genes were inhibited. Those
concentration-dependent effects make doses of drug very critical when treating
or preventing osteoporosis.
Different genes were regulated differently and show locus-specific
expressions. These locus-specific effects has also been shown in
glucocorticoid-treated osteoblasts [33], and also in the effect of estrogen on
primary pre-osteoblasts[32]. Thorough investigation of the locus- and
concentration-dependent effects of various steroid hormone receptors on
RUNX2 activity during commitment of mesenchymal progenitors to the
osteoblast phenotype may help evaluate currently available SERMs and develop
new SERMs.

Further Studies. Since SERMs are drugs used to treat patients, we are more
interested in the effect of E2/SERMs on human cells. So the next step, we are
going to use SM30/Rx
Dox
cells, which aer human mesenchymal stem cells, and
repeated all those experiments we did on murine MSCs. If we could have the
same result, those data will be more helpful to understand the mechanism of E2,
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and to improve the structure of SERMs.
We have conducted a RNA-seq experiment to get unbiased picture
comparing effects of E2 and SERMs with or without Dox-induced RUNX2. We
used very few percent of this RNA-seq data, and we are going to explore those
data more thoroughly, to find other genes that could explain the effects of
E2/SERMs. There are many other regulators there on osteoblastogenic RUNX2
pathway. We are also interested in the relation of estrogen to other factors in the
RUNX2 pathway, and may conduct new experiments to investigate them.
And also we are not only interested in the two drugs we used in this
experiment, Ral and Las. We are intending to analyze more SERMs such as
Tamoxifene, ICI180 and Bazoxifene.
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Works Cited
1. Bilezikian, J.P., L.G. Raisz, and G.A. Rodan, Principles of bone biology.
1996, San Diego: Academic Press. xx, 1398 p.
2. Parfitt, A.M., Osteonal and hemi-osteonal remodeling: the spatial and
temporal framework for signal traffic in adult human bone. J Cell Biochem, 1994.
55(3): p. 273-86.
3. Geoffroy, V., et al., High bone resorption in adult aging transgenic mice
overexpressing cbfa1/runx2 in cells of the osteoblastic lineage. Mol Cell Biol, 2002.
22(17): p. 6222-33.
4. Bae, S.C., et al., Isolation of PEBP2 alpha B cDNA representing the mouse
homolog of human acute myeloid leukemia gene, AML1. Oncogene, 1993. 8(3): p.
809-14.
5. Westendorf, J.J., Transcriptional co-repressors of Runx2. J Cell Biochem,
2006. 98(1): p. 54-64.
6. Otto, F., et al., Cbfa1, a candidate gene for cleidocranial dysplasia syndrome,
is essential for osteoblast differentiation and bone development. Cell, 1997. 89(5): p.
765-71.
7. Liu, W., et al., Overexpression of Cbfa1 in osteoblasts inhibits osteoblast
maturation and causes osteopenia with multiple fractures. J Cell Biol, 2001. 155(1): p.
157-66.
8. Maruyama, Z., et al., Runx2 determines bone maturity and turnover rate in
postnatal bone development and is involved in bone loss in estrogen deficiency. Dev
Dyn, 2007. 236(7): p. 1876-90.
9. Takarada, T., et al., An analysis of skeletal development in osteoblast-specific
and chondrocyte-specific runt-related transcription factor-2 (Runx2) knockout mice. J
Bone Miner Res, 2013. 28(10): p. 2064-9.
10. Takarada, T., et al., Genetic analysis of Runx2 function during
intramembranous ossification. Development, 2016. 143(2): p. 211-8.
11. Yu, J.L., et al., Initial Characterization of Osteoblast Differentiation and
Loss of RUNX2 Stability in the Newly Established SK11 Human Embryonic Stem
Cell-Derived Cell Line. J Cell Physiol, 2015. 230(2): p. 237-41.
12. Du, Z., et al., Estrogen Deficiency-Associated Bone Loss in the Maxilla: A
Methodology to Quantify the Changes in the Maxillary Intra-radicular Alveolar Bone
in an Ovariectomized Rat Osteoporosis Model. Tissue Eng Part C Methods, 2015.
University
of
Southern
California
Master’s
Thesis

53

21(5): p. 458-66.
13. de Almeida, J.M., et al., Effects of oestrogen deficiency and 17beta-estradiol
therapy on bone healing in calvarial critical size defects treated with bovine bone graft.
Arch Oral Biol, 2015. 60(4): p. 631-41.
14. Palacios, S., et al., Perspective on prescribing conjugated
estrogens/bazedoxifene for estrogen-deficiency symptoms of menopause: a practical
guide. Maturitas, 2015. 80(4): p. 435-40.
15. Fan, J.Z., et al., Estrogen improves the proliferation and differentiation of
hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway.
Mol Cell Biochem, 2014. 392(1-2): p. 85-93.
16. Bradford, P.G., et al., Estrogen regulation of apoptosis in osteoblasts.
Physiol Behav, 2010. 99(2): p. 181-5.
17. Yang, Y.H., et al., Estradiol inhibits osteoblast apoptosis via promotion of
autophagy through the ER-ERK-mTOR pathway. Apoptosis, 2013. 18(11): p. 1363-75.
18. Sims, N.A., et al., Deletion of estrogen receptors reveals a regulatory role for
estrogen receptors-beta in bone remodeling in females but not in males. Bone, 2002.
30(1): p. 18-25.
19. Nicks, K.M., et al., Deletion of Estrogen Receptor Beta in Osteoprogenitor
Cells Increases Trabecular but Not Cortical Bone Mass in Female Mice. J Bone Miner
Res, 2016. 31(3): p. 606-14.
20. Almeida, M., et al., Estrogen receptor-alpha signaling in osteoblast
progenitors stimulates cortical bone accrual. J Clin Invest, 2013. 123(1): p. 394-404.
21. Jilka, R.L., et al., Loss of estrogen upregulates osteoblastogenesis in the
murine bone marrow. Evidence for autonomy from factors released during bone
resorption. J Clin Invest, 1998. 101(9): p. 1942-50.
22. Di Gregorio, G.B., et al., Attenuation of the self-renewal of transit-amplifying
osteoblast progenitors in the murine bone marrow by 17 beta-estradiol. J Clin Invest,
2001. 107(7): p. 803-12.
23. Baniwal, S.K., et al., Runx2 promotes both osteoblastogenesis and novel
osteoclastogenic signals in ST2 mesenchymal progenitor cells. Osteoporos Int, 2012.
23(4): p. 1399-413.
24. Abdelazim, I.A., et al., Effect of raloxifene hydrochloride on bone mineral
density and bone turnover in Kuwaiti postmenopausal women with osteoporosis. Arch
Osteoporos, 2014. 9: p. 189.
25. Eastell, R., et al., Effects of 3 years of lasofoxifene treatment on bone
turnover markers in women with postmenopausal osteoporosis. Bone, 2012. 50(5): p.
1135-40.
University
of
Southern
California
Master’s
Thesis

54

26. McClung, M.R., et al., Prevention of bone loss in postmenopausal women
treated with lasofoxifene compared with raloxifene. Menopause, 2006. 13(3): p.
377-86.
27. Takeda, S., et al., Combination treatment with eldecalcitol (ED-71) and
raloxifene improves bone mechanical strength by suppressing bone turnover and
increasing bone mineral density in ovariectomized rats. Bone, 2013. 53(1): p. 167-73.
28. Ke, H.Z., et al., Lasofoxifene (CP-336,156), a selective estrogen receptor
modulator, prevents bone loss induced by aging and orchidectomy in the adult rat.
Endocrinology, 2000. 141(4): p. 1338-44.
29. Chimge, N.O., et al., Regulation of breast cancer metastasis by Runx2 and
estrogen signaling: the role of SNAI2. Breast Cancer Res, 2011. 13(6): p. R127.
30. Khalid, O., et al., Modulation of Runx2 activity by estrogen receptor-alpha:
implications for osteoporosis and breast cancer. Endocrinology, 2008. 149(12): p.
5984-95.
31. Kim, R.Y., et al., Estrogen Modulates Bone Morphogenetic Protein-Induced
Sclerostin Expression Through the Wnt Signaling Pathway. Tissue Eng Part A, 2015.
21(13-14): p. 2076-88.
32. Martin, A., et al., Estrogens antagonize RUNX2-mediated osteoblast-driven
osteoclastogenesis through regulating RANKL membrane association. Bone, 2015. 75:
p. 96-104.
33. Morimoto, E., et al., Glucocorticoids Hijack Runx2 to Stimulate Wif1 for
Suppression of Osteoblast Growth and Differentiation. J Cell Physiol, 2016.
34. Tanaka, I., [Glucocorticoid and Bone. Efficacy and safety of bisphosphonate
in treatment of glucocorticoid induced osteoporosis]. Clin Calcium, 2014. 24(9): p.
1371-8.
35. Chang, D.J., et al., Reduction in transforming growth factor beta receptor I
expression and transcription factor CBFa1 on bone cells by glucocorticoid. J Biol
Chem, 1998. 273(9): p. 4892-6.
36. Koromila, T., et al., Glucocorticoids antagonize RUNX2 during osteoblast
differentiation in cultures of ST2 pluripotent mesenchymal cells. J Cell Biochem, 2014.
115(1): p. 27-33.
37. Wang, J., et al., Equol promotes rat osteoblast proliferation and
differentiation through activating estrogen receptor. Genet Mol Res, 2014. 13(3): p.
5055-63.
38. Yin, X., et al., ERbeta induces the differentiation of cultured osteoblasts by
both Wnt/beta-catenin signaling pathway and estrogen signaling pathways. Exp Cell
Res, 2015. 335(1): p. 107-14.
University
of
Southern
California
Master’s
Thesis

55

39. Kim, H., et al., Estrogen stimuli promote osteoblastic differentiation via the
subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells. J Bone Miner Metab,
2015. 33(1): p. 30-9.
40. Shang, Z.Z., et al., Differentially expressed genes and signalling pathways
are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-beta estradiol. Int
J Oral Sci, 2014. 6(3): p. 142-9.
41. Noda-Seino, H., et al., Estradiol and raloxifene induce the proliferation of
osteoblasts through G-protein-coupled receptor GPR30. J Endocrinol Invest, 2013.
36(1): p. 21-7.
42. Giner, M., et al., Alendronate and raloxifene affect the
osteoprotegerin/RANKL system in human osteoblast primary cultures from patients
with osteoporosis and osteoarthritis. Eur J Pharmacol, 2011. 650(2-3): p. 682-7.
43. Lau, L.F., Cell surface receptors for CCN proteins. J Cell Commun Signal,
2016. 10(2): p. 121-7.
44. Holmes, D.A., et al., Dusp5 negatively regulates IL-33-mediated eosinophil
survival and function. EMBO J, 2015. 34(2): p. 218-35.
45. Moon, S.J., et al., Dual-specificity phosphatase 5 attenuates autoimmune
arthritis in mice via reciprocal regulation of the Th17/Treg cell balance and inhibition
of osteoclastogenesis. Arthritis Rheumatol, 2014. 66(11): p. 3083-95.
46. Nwachukwu, J.C., et al., Predictive features of ligand-specific signaling
through the estrogen receptor. Mol Syst Biol, 2016. 12(4): p. 864.
47. Chen, Z., et al., ERK1 and ERK2 regulate chondrocyte terminal
differentiation during endochondral bone formation. J Bone Miner Res, 2015. 30(5): p.
765-74.
48. Ohta, K., et al., Synthesis and biological evaluation of novel
m-carborane-containing estrogen receptor partial agonists as SERM candidates.
Bioorg Med Chem Lett, 2015. 25(16): p. 3213-6.

Abstract (if available)

Abstract The bone-sparing effects of estradiol (E2), the major female hormone, are well established, but the underlying molecular mechanisms are complex and not fully understood. Like E2, Selective estrogen receptor modulators (SERMs) bind and activate the estrogen receptor (ER). Because they mimic E2 action in bone cells, but not in breast epithelial cells, SERMs are used as drugs to prevent postmenopausal osteoporosis. Runx2, a master transcription factor that promotes both osteoblastogenesis and osteoblast-driven osteoclastogenesis, has been shown to interact with the ER. In this thesis, we investigated the effects of E2 and two SERMs, raloxifen (Ral) and lasofloxifene (Las), on Runx2-driven gene expression and osteoblast differentiation. We used ST2/Rx2dox, a mesenchymal pluripotent cell line, whose treatment with doxycycline (dox) induces expression of Runx2 and promotes osteoblast differentiation. Despite its positive effects of bone mass, E2 inhibited differentiation of osteoblasts from their precursor cells as indicated by inhibition of both alkaline phosphatase activity and expression of the osteogenic gene markers Osteocalcin (Bone Gla Protein

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Asset Metadata

Creator Ji, Jie (author)

Core Title Inhibitory effects of estradiol and SERMs on RUNX2-driven osteoblast differentiation and gene expression

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/22/2018

Defense Date 06/17/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag estradiol,lasofoxifene,OAI-PMH Harvest,osteoblast,raloxifene,RUNX2,SERMs

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Frenkel, Baruch (committee chair), Oldak, Janet (committee member), Rice, Judd (committee member)

Creator Email jieji@usc.edu,seasonapple510@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-277428

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