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Preosteoblast-specific RUNX2 promotes RANKL membrane association: antagonism by sex steroids through a non-genomic mechanism

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Preosteoblast-specific RUNX2 promotes RANKL membrane association: antagonism by sex steroids through a non-genomic mechanism

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Content PREOSTEOBLAST-SPECIFIC RUNX2 PROMOTES
RANKL MEMBRANE ASSOCIATION:
ANTAGONISM BY SEX STEROIDS THROUGH A
NON-GENOMIC MECHANISM.

by

Jiali Yu

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2015
Copyright 2015 Jiali Yu

I

Acknowledgement

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. Zoltan Tokes and Dr.
Amy Merrill-Brugger.

My sincere thanks also go to Dr. Helty Adisetiyo, Dr. Gillian H Little and Dr.
Anthony Martin for their contributions to this thesis. Their ideas and advices
helped me solve the problems I met in my research.

Finally thank my dear lab mates Dr. Chimge, Eri Champagne, Mirra Liu and
Jie Ji for the first spot of my data and thank my parents for their financial
support to my master study.

II

Table
of
Contents

Acknowledgement
I

List of Tables
IV

List of Figures
V

Abstract
VI

Introduction
1

Objectives
4

Materials and methods
5

Reagents
5

Animals
6

Cell culture
6

Plasmids and transfection
6

RNA extraction and analysis
7

Chromatin immunoprecipitation (ChIP)
7

Immunofluorescence analysis
8

Western blot analysis
8

Alizarin red staining
9

Statistical analysis
9

Results
10

RUNX2 induces GFP-RANKL membrane association, which is
antagonized by both E2 and DHT.
10

Sex steroid-mediated antagonism of RUN2-induced RANKL membrane
association is fast.
12

III

Sex steroid-mediated antagonism of RUNX2-induced RANKL membrane
association occurs via a non-genomic mechanism.
13

Similarities and differences between E2 and DHT in antagonism of
RUNX2- induced gene expression.
15

Expression of endogenous RUNX2 decrease during osteoblast
differentiation.
17

Introduction and validation of exogenous FLAG-RUNX2 expression in
human mesenchymal stem cells.
18

RUNX2 becomes unstable during osteoblast differentiation.
21

Discussion
22

References
26

IV

List of Tables
Table
1.
Reagents
used
in
this
study

Table
2.
Primer
sequences
used
in
qPCR
analysis

V

List of Figures
Figure
1.
RANKL–RANK–OPG
regulatory
pathway
and
osteoclastic
bone

resorption.

Figure
2.
Sex
steroids
antagonize
RUNX2-‐induced
RANKL
membrane

association.

Figure
3.
Time
course
of
sex
steroids-‐mediated
antagonism
of
RUNX2-‐
induced
RANKL
membrane
association.

Figure
4.
Non-‐genomic
effects
of
sex
steroids-‐mediated
antagonism
of

RUNX2-‐induced
RANKL
membrane
association.

Figure
5.
Sex
steroid-‐mediated
attenuation
of
RUNX2
responsive
genes.

Figure
6.
Expression
of
RUNX2
during
osteoblast
differentiation.

Figure
7.
FLAG-‐RUNX2
expression
in
SK11/Rx2
dox

and
SK11
mineralized

ability.

Figure
8.
RUNX2
protein
lost
stability
during
osteoblast
differentiation.

VI

Abstract

Molecular mechanisms underlying the bone-sparing effects of sex steroid
hormones are not fully understood. We show that RUNX2, a master
regulator of osteoblast differentiation and bone formation, promotes
association of the quintessential osteoclastogenic factor RANKL with the
osteoblast membrane, and this is antagonized by both estradiol and
dihydrotestosterone. Sex hormones reversed RUNX2-mediated RANKL
membrane association through a fast, non-genomic mechanism, which was
mimicked by an estrogen dendrimer conjugate (EDC) that cannot enter the
cell. Sex steroids appear to antagonize RUNX2-mediated RANKL
membrane association in early pre-osteoblasts, because, as we show in
several osteoblast cell culture models, RUNX2 expression is lost in mature
osteoblasts due to acquired post-translational instability.

Key words: RUNX2, RANKL, sex steroids, osteoblast differentiation

1

Introduction

Bone is a dynamic tissue regulated by bone resorbing osteoclasts and bone
forming osteoblasts. Osteoblasts make new bone by producing a mineralized
collagenous extracellular matrix. Osteoclasts destroy bone, releasing minerals into
the blood. This balance between bone formation and bone resorption is controlled
strictly in order to maintain bone mass at a constant level. With age, bone
resorption exceeds bone formation. In healthy individuals, however, the resulting
bone loss is minimal and transpires without pathological consequences. In contrast,
an increase in bone turnover rate, such as that observed in postmenopausal women,
often results in accelerated bone loss and pathological fractures. It is estimated
that over 200 million people worldwide suffer from osteoporosis (Cooper et al.,
1992). Approximately 30% of all postmenopausal women have osteoporosis in the
United States. At least 40% of them will sustain one or more fragility fractures in
their remaining lifetime (Melton et al., 1992). The cost of pathological fractures
was almost 17 billion in the USA in 2005 (Burge et al., 2007). Therefore, the
studies on bone turnover play an important role in the treatment of osteoporosis.

Osteoblasts are derived from mesenchymal stem cell in the bone marrow space.
To initiate differentiation towards osteoblasts, these precursor cells express the
master regulator RUNX2, also known as Cbfa1 (Bae et al., 1993). RUNX2 is one
of the three members of mammalian RUNX transcription factors, which share
their evolutionarily conserved DNA-binding domain, located at the N-termius,
with the Drosophila pair rule gene runt (Nusslein-Volhard and Wieschaus, 1980;
van Wijnen et al., 2004). RUNX2 is essential for osteoblasts differentiation.
RUNX2 deficient mice were lack of bone because of the absence of osteoblasts

2

(Otto et al., 1997). Surprisingly, overexpression of Runx2 does not stimulate, but
rather inhibits osteoblasts maturation. In addition, Runx2 over-expression
stimulates bone resorption, which cause osteopenia (Geoffroy et al., 2002; Liu et
al., 2001). Thus, Runx2 expression has to be tightly regulated.

Osteoblasts differentiation is regulated by a range of hormones, cytokines, and
transcription factor (Komori, 2006). Besides Runx2, Bone morphogenetic protein
2 (BMP2) is also one of the most important cytokines that plays important roles in
a variety of cellular functions ranging from embryogenesis, cell growth, and
differentiation to bone development and the repair of bone fractures (Welch et al.,
1998; Yamaguchi et al., 1991). BMP2 exhibits osteogenic action by activating
Smad1/5/8 signaling and regulating the transcription of osteogenic genes,
including distal-less homeobox 5 (Dlx5), which is a key mediator of BMP2-
induced expression of Runx2 (Jang et al., 2012; Lee et al., 2003). Runx2 regulates
the expression of osteoblastic genes, including SPP1, COLIA1, SPARC and
osteocalcin (OCN) by binding to their promoters(Harada and Rodan, 2003;
Komori, 2005).

Expression of RUNX2 in osteoblasts has been reported to induce
osteoclastogenisis. Osteoclasts are differentiated from monocyte cells at or near
the bone surface. Osteoclast precursors from spleen or bone marrow can be
differentiated into mature osteoclasts in vitro when co-cultured with osteoblasts or
their stromal progenitor cells (Takahashi et al., 1988). In these co-cultures,
stroma-derived factors stimulate osteoclast differentiation. The most important
osteoclastogenic factor presented by cells of the osteoblast lineage is receptor
activator of NF-kapa B ligand (RANKL). Binding of RANKL to its receptor
RANK on the osteoclastic cell surface, results in osteoclast differenation and

3

activation, leading to bone resorption (Lacey et al., 1998; Yasuda et al., 1998).
Osteoprotegerin (OPG) is an inhibitor of osteoclastogenesis. It binds to RANKL,
preventing osteoclasts activation by inhibiting the binding of RANK and RANKL
(Theoleyre et al., 2004). The balance between RANKL and OPG determines the
ultimate rate of bone resorption (Figure 1). Postmenopausal osteoporosis is
associated with a high rate of bone remodeling due to an excess of RANKL over
OPG (Lewiecki, 2011).

Figure 1. RANKL–RANK–OPG regulatory pathway and osteoclastic bone resorption. RANKL
expressed by osteoblasts binds to RANK on the pre-osteoclasts cell surface and activates osteoclast
maturation. As a result, bone resorption increases by increasing osteoclasts differentiation and survival.
OPG is also expressed by osteoblasts, which binds to RANKL to inhibit osteoclastogenesis.
Postmenopausal osteoporosis is associated with an excess of RANKL over OPG, which leads to bone
loss(Lewiecki, 2011).

4

Sex hormones regulate bone mass in both men and women. Loss of estrogen at
menopause is a primary contributor to postmenopausal osteoporosis in females
(Eastell, 2005; Riggs et al., 2002). Osteoblast formation assays using
mesenchymal cells from ovariectomized mice suggested that the anti-resorptive
effect of estrogens was mediated by attenuating cell proliferation early in the
osteoblasts lineage (Di Gregorio et al., 2001; Jilka et al., 1998). Although an
increase of bone turnover has been documented at the time of menopause, the high
rate of bone resorption and formation and their potential role in determining bone
mass in the elderly women has still been under investigation (Garnero et al., 1996).
Objectives
This is a follow up study based on the previous finding from our lab, that
estrogens inhibit RUNX2-induced osteoclastogenesis via antagonizing RANKL
membrane association (Martin et al., 2015). The hypothesis in this report states
that RUNX2-induced RANKL membrane association occurs specifically in pre-
osteoblasts, and is antagonized by both estrogens and androgens via a non-
genomic mechanism.

5

Materials and methods
Reagents
Table 1. Reagents used in this study
Name of Materials Sources
Ascorbic acid Sigma-Aldrich, St. Louis, MO
Alizarin Sigma-Aldrich, St. Louis, MO
Amersham Hybond
TM
-P PVDF membranes GE Healthcare, Piscataway, NJ
Aurum
TM
total RNA mini kit Bio-Rad Laboratories, Hercules, CA
Bio-Rad Protein Assay Kit Bio-Rad Laboratories, Hercules, CA
β-glycerophosphate Sigma-Aldrich, St. Louis, MO
bFGF Sigma-Aldrich, St. Louis, MO
Collagenase P. Roche Diagnostics Indianapolis, IN, USA
Charcol stripped fetal bovine serum (CSS) Gemini, West Sacramento, CA
Dexamethasone Sigma-Aldrich, St. Louis, MO
Doxycycline (Dox) Calbiochem, La Jolla, CA
EGF Sigma-Aldrich, St. Louis, MO
Fetuin Calbiochem, Billerica, MA
Fetal bovine serum Gemini BioProducts, West Sacramento, CA
Formadehyde 37% Solution J.T. Baker, Phillipsburg, NJ
iQ™ SYBR® green supermix Bio-Rad Laboratories, Hercules, CA
Insulin Sigma-Aldrich, St. Louis, MO
jetPRIME transfection reagent and buffer Polyplus-Transfection, New York, NY
Mouse monoclonal primary antibody against
FLAG® epitope (M2)
Sigma-Aldrich, St. Louis, MO
PBS (10X) Boston BioProducts, Ashland, MA
Protease inhibitor cocktail Sigma-Aldrich, St. Louis, MO
Rabbit polyclonal antibody against RUNX2
(M70)
Santa Cruz Biotechnology, Santa Cruz, CA
Rabbit polyclonal antibody against RANKL
(FL-317)
Santa Cruz Biotechnology, Santa Cruz, CA
RPMI1640 Mediatech Inc. Manassas, VA
qScript
TM
cDNA supermix Quanta Bioscience, Gaithersburg, MD
Typsin (10X) Gibco, Invitrogen, Carlsbad, CA
αMEM without phenol red Gibco Bioreagent
Vectashield mounting medium for fluorescence
(with DAPI)
Vector Laboratories, Burlingame, CA

6

Animals
Wild-type mice (C57BL/6; JAX® mice) were obtained from the Jackson
Laboratory, Sacramento, CA, USA. All experimental procedures with animals
were approved by the USC Institutional Animal Care and Use Committee.

Cell culture
Newborn Mouse Calvarial Osteoblasts (NeMCOs) were prepared from 1- to 2-day
old newborn wild-type mice by cell digestion with 1 mg/ml collagenase P plus 1X
trypsin (10X) in PBS. Cells were cultured for 3-6 days to confluence prior to use.

NeMCO and MC3T3-E1 cells were maintained in αMEM supplemented with 10%
fetal bovine serum (FBS). ST2 cells were maintained in RPMI1640 supplemented
with 10% FBS. The human embryonic stem cell-derived cell line SK11
(PureStem™, BioTime Inc., Alameda, CA) was maintained in αMEM
supplemented with 5% FBS, 50 µg/ml Fetuin, 10 ng/ml EGF, 1 ng/ml bFGF,
10 µg/ml insulin and 10
−8
M dexamethasone.

For experiments with sex hormones, cells were cultured in αMEM without phenol
red and with 10% CSS charcoal stripped fetal bovine serum (CSS) and 1%
penicillin-streptomycin.

Plasmids and transfection
Lentiviral vector encoding doxycycline (dox)-inducible FLAG-RUNX2 was
constructed by Baniwal et al. (Baniwal et al., 2010) and used to produce lentiviral
particles at the UCLA Vectorcore, Los Angeles, CA, USA. NeMCO and SK11
cells transduced with these lentiviruses are designated NeMCO/Rx2
dox
and

7

SK11/Rx2
dox
. The GFP-RANKL plasmid, a gift from Dr. Hiroshi Suzuki,
University of Tokyo, was introduced into NeMCO along with pcDNA3.0-RUNX2
constructed by Dr. Baniwal or pcDNA3.0 plasmids using the jetPRIME
transfection reagent and buffer according to the manufacturer’s protocol.

RNA extraction and analysis
Total RNA was extracted using Aurum
TM
total RNA mini kit according to the
manufacturer’s protocol and 1 µg RNA was reverse-transcribed using qScript
TM

cDNA supermix to prepare cDNA. Real-time qPCR analysis was performed using
the CFX96
TM
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 GAPDH, which themselves were
not significantly affected by treatment.

Table 2. Primer sequencers used in qPCR analysis
Gene Sequence (5’ to 3’)
OCN ChIP F AAATAGCCCTGGCAGATTCC
R CAGCCTCCAGCACTGTTTAT
Neg ChIP F ATGGTTGCCACTGGGGATCT
R TGCCAAAGCCTAGGGGAAGA
OCN mRNA F ACACTCCTCGCCCTATTGGC
R TGCTTGGACACAAAGGCTGC
RANKL mRNA F GGGGGCCGTGCAGAAGGAAC
R CTCAGGCTTGCCTCGCTGGG
RUNX2 mRNA F ATCACGCCGACCACCCGGC
R GGCTACCACCTTGAAGGCCACG

Chromatin immunoprecipitation (ChIP)
FLAG-RUNX2 ChIP was performed by Dr. Gillian Little as previous described
(Little et al., 2012). Precipitated DNA was analyzed by qPCR using the primers
listed in Table 2.

8

Immunofluorescence analysis
NeMCO co-transfected with the GFP-RANKL and FLAG-RUNX2 expression
vectors were fixed with freshly-made fixation solution (1.23 ml formaldehyde
(37%), 7.5 ml 2X PBS, add sterile water up to 15ml) and incubated with mouse
monoclonal antibodies against FLAG® epitope, followed by the secondary goat
anti-mouse IgG antibody. Cells were finally mounted with Vectashield mounting
medium for fluorescence (with DAPI). FLAG and GFP fluorescence was detected
using Nikon Eclipse Ti microscope.

Western blot analysis
Cell were lysed in a 50 mM Tris-HCL buffer (pH 7.4) containing 150 mM NaCl, 1
mM EDTA, 1% TritonX-100, and fresh protease inhibitor cocktail. Protein
concentrations were measured by Bio-Rad Protein Assay Kit. 20 ug of whole cell
lysate was loaded to SDS-PAGE. Proteins in the gel were transferred to
Amersham Hybond
TM
-P PVDF membranes and detected by mouse monoclonal
FLAG antibody (M2, Sigma), rabbit polyclonal antibody RUNX2 (M70, Santa
Cruz), RANKL (FL-317, Santa Cruz) or Actin (I-19, Santa Cruz).

Microarray analysis
NeMCO/Rx2
dox
cultures were treated with 500 ng/mL dox and/or 10 nM E2 or
DHT for 24 hours. RNA was extracted and expression profiles were analyzed by
microarray analysis and python programming as described (Martin et al., 2015).

9

Alizarin red staining
SK11 cells were plated 25,000/well in the 12-well plate growing in the
differentiation medium (αMEM supplemented with 10% FBS, 50 ng/ml ascorbic
acid, 10 nM β-glycerophosphate and 10
-8
M dexamethasone) for the indicated
days and washed once in PBS. After fixing with 70% ethanol for one hour,
cultures were stained with 1.3mg/mL alizarin for 5 minutes. And then rinsed with
ddH
2
O and air-dried.

Statistical analysis
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.

10

Results
RUNX2 induces GFP-RANKL membrane association, which is antagonized
by both E2 and DHT.

In addition to the crucial role of RUNX2 in osteoblast differentiation, RUNX2 can
induce osteoblast-driven osteoclastogenesis (Geoffroy et al., 2002; Liu et al.,
2001). It is well known that sex steroids, including estradiol (E2), attenuate bone
turnover via regulating osteoblast differentiation and osteoclasts differentiation.
Our previous studies showed that estradiol antagonize RUNX2-induced RANKL
membrane association (Martin et al., 2015). We wanted to know whether another
sex steroid, dihydrotestosterone (DHT) also works the same as estradiol.

In order to test the effect of RUNX2 on RANKL cellular localization in pre-
osteoblasts, we transiently co-transfected primary cultures of Newborn Mouse
Cavarial Osteoblasts (NeMCOs) with a plasmid encoding GFP-RANKL along
with either a FLAG-RUNX2 vector or a pcDNA3.0 control vector. After 48-hour
treatment with vehicle, E2 or DHT, GFP-RANKL was visualized with direct
fluorescence microscopy and FLAG-RUNX2 was visualized by
immunofluorescence using anti-FLAG antibodies. As shown in Figure 2A,
RUNX2 increased RANKL localization at the cell perimeter in the vehicle group.
In the presence of E2 or DHT, however, RUNX2 did not increase RANKL
membrane association. To quantitate RANKL membrane association, we
measured the percentage of the cell perimeter that was labeled with GFP-RANKL.
As shown in Figure 2B, GFP-RANKL was presented on 2.7% of cell surface in
the control and 18.2% with RUNX2 overexpression cells. The percentage went
down to 2% and 1.4% after treating with E2 or DHT, respectively. Thus, both E2
and DHT antagonize RUNX2-induced RANKL membrane association in the pre-

11

osteoblasts, accounting for antagonism of RUNX2-driven osteoblast-induced
osteoclastogenesis (Martin et al., 2015).

Figure 2. Sex steroids antagonize RUNX2-induced RANKL membrane association. NeMCO
cultures were transiently transfected with a plasmid encoding GFP-RANKL along with a plasmid
encoding FLAG-RUNX2 or the pcDNA3.0 vector control. Cultures were treated with vehicle (control),
10 nM of estradiol (E2) or 10 nM of dihydrotestoterone (DHT) for 48 hours. A. Representative cells
(bar=50um) with showing GFP-RANKL in green by direct immunofluorescence and FLAG-RUNX2 in
red by immunofluorescence. Nuclei are stained blue with DAPI. B, The percentage of cell perimeter
occupied by GFP-RANKL was determined in a blinded fashion using the NIS-Element AR3.2 software
(mean±SD for at least 10 cells per condition). Arrows mark membrane-associated GFP-RANKL. C,
NeMCO/Rx2
dox
cultures were treated with 500 ng/ml dox or/and 10 nM DHT for 48 hours. RT-qPCR
(top) and Western blot (bottom) analysis of RANKL expression.

12

Sex steroid-mediated antagonism of RUN2-induced RANKL membrane
association is fast.

Our previous studies showed that E2 decreased RUNX2-induced RANKL
membrane association without changing RANKL expression at the mRNA level
or protein level (Martin et al., 2015), and Figure 2C demonstrates this is also true
for DHT. Because of the similarity between the effects of DHT and E2, we
suspected that their effect on RANKL membrane association might not be
mediated through their nuclear receptors (which presumably regulates different
gene sets), but rather through membrane-associated receptors. If that was the case,
then the effects of E2 and DHT could be expected to occur within minutes to a
few hours. To test this, we performed a time course analysis of the effects of E2
and DHT on RANKL membrane association as in Figure 3A. Up to 50% reduction
of GFP-RANKL membrane association was observed already at the 30-min time
point, and reached 78-85% by 1 hour, approaching the 90-95% inhibition
observed at the 4-hour (Figure 3) and 48-hour (Figure 2) time points. The fast
pace of sex steroid-mediated antagonism of RUN2-induced RANKL membrane
association suggested that E2 and DHT were more likely to perform this function
by binding to their cell membrane receptors instead of cytosolic or nuclear
receptors. Signals transduced by cell surface receptors started in minutes and gene
expression has not been changed, which is consistent with our previous data
showing that RANKL mRNA did not change after treating with E2 (Martin et al.,
2015).

13

Figure 3. Time course of sex steroids-mediated antagonism of RUNX2-induced RANKL membrane
association. NeMCO cultures were co-transfected with FLAG-RUNX2 and GFP-RANKL plasmids as in
Figure 2. Immunofluorescence analysis of control, E2 or DHT treated for the indicated time periods after
two days of transfection. A, Representative images (bar=50um) shown with FLAG-RUNX2 in red and
GFP-RANKL in green. B, RANKL membrane association was assessed as in Figure 2B as percentage of
the cell perimeter labeled with GFP-RANKL.

Sex steroid-mediated antagonism of RUNX2-induced RANKL membrane
association occurs via a non-genomic mechanism.

To definitively test whether E2 antagonized RUNX2-induced RANKL membrane
association through non-genomic mechanism(s), we next employed the estrogen
dendrimer conjugate (EDC), a compound developed at the Katzenellenbogen
laboratory. EDC binds membrane-associated but not nuclear estrogen receptors,

14

and nuclear ER target genes are not activated by EDC treatment (Harrington et al.,
2006).

The effect of EDC on RUNX2-induced RANKL membrane association in
NeMCO cultures was assessed over time as in Figure 4A. Similar to the fast
effects of E2 and DHT, EDC decreased GFP-RANKL membrane association
within 30 minutes of treatment (Figure 4). These results further suggest that
estrogen antagonized RUNX2-induced RANKL membrane association through a
non-genomic pathway without activation of nuclear estrogen receptors.

Figure 4. Non-genomic effects of sex steroids-mediated antagonism of RUNX2-induced RANKL
membrane association. NeMCO cultures were co-transfected with FLAG-RUNX2 and GFP-RANKL
plasmids as in Figure 2. Immunofluorescence analysis of 10 nM control or estrogen dendrimer conjugate
(EDC) treated for the indicated time points after two days of transfection. A, Representative images
(bar=50um) shown with FLAG-RUNX2 in red and GFP-RANKL in green. B, RANKL membrane
association was assessed as in Figure 2B as percentage of the cell perimeter labeled with GFP-RANKL.

15

Similarities and differences between E2 and DHT in antagonism of RUNX2-
induced gene expression.

Besides antagonizing the RUNX2-mediated RANKL trafficking through a fast,
non-genomic mechanism, sex steroids could also regulate bone metabolism by
antagonizing RUNX2-mediated transcription, potentially contributing to their
long-acting anti-resorptive activity. Indeed, both the estrogen receptor and the
androgen receptor physically interact with RUNX2, and these interactions usually
result in mutual inhibition of target genes for RUNX2 and the sex hormone
receptors (Baniwal et al., 2009; Kawate et al., 2007; Khalid et al., 2008). We have
recently established, however, that the outcome of the interaction between
RUNX2 and sex hormone receptors is locus-dependent (Chimge et al., 2012;
Little et al., 2012). Differences between estrogens and androgens in influencing
RUNX2 activity at specific loci may help explain their different effects on bone
metabolism. We therefore decided to compare the influence of E2 on RUNX2 to
the influence of DHT on RUNX2 in osteoblasts genome-wide. NeMCO/Rx2
dox
cultures were treated with vehicle or dox along with E2 or DHT. Global gene
expression was profiled by Beadchip arrays (Illumina). We then compared the
influence of E2 to that of DHT on 82 genes that RUNX2 stimulated by ≥1.2 folds
(p<0.16). As shown in Figure 5A, 61 (74%) of RUNX2-mediated stimulated genes
were attenuated by both E2 and DHT. However, differences between E2 and DHT
were noted. First, 15 of the genes were affected only by DHT and 6 of the genes
were affected only by E2 (Figure 5A). Second, there was a noticeable difference
between the magnitudes by which the two hormones attenuated the RUNX2
response. For example, E2 inhibition of Vwa7 was stronger than DHT, while DHT
inhibition on Cryaa was stronger than E2. These differences may help explain
why the bone-sparing effects of androgens surpass those of estrogens (Frenkel et
al., 2010). More importantly, these results highlight similarities between the

16

RUNX2-realted genomic actions of the two hormones, adding to their similarity in
antagonizing RUNX2-mediated RNAKL trafficking through non-genomic
mechanism(s). In particular, both E2 and DHT antagonized RUNX2-mediated
stimulation of Nppb, Pstpip2 and Vwa7, all of which have been reported to play a
role in membrane trafficking (Cvetkovic et al., 1994; Huang et al., 1998). Their
activation by RUNX2 and its antagonism by E2 and DHT may therefore
contribute to the anti-osteoclastiogenic and the bone-sparing effects of both these
sex hormones.

Figure
5.
Sex
steroid-‐mediated
attenuation
of
RUNX2
responsive
genes.
NeMCO/Rx2
dox

cultures
were
treated
with
500
ng/mL
dox
and/or
10
nM
E2
or
DHT
for
24
hours,
and
mRNA

expression
was
profiled
using
the
Beadchips
platform
(Illumina).
Genes
that
responded
to
RUNX2

by
at
least
1.2
fold
and
were
downregulated
by
E2
or
DHT
were
analyzed.
A,
Veen
diagram

showing
the
overlap
between
the
effects
of
E2
and
DHT.
B,
The
list
of
top
10
genes
that
were
most

downregulated
by
E2
or
DHT.

DvsC,
fold-‐stimulation
by
RUNX2,
DEvsD,
fold
repression
by
E2
in

the
presence
of
RUNX2
(dox);
DAvsD,
fold
repression
in
the
presence
of
the
non-‐aromatizable

androgen
(A)
DHT.

Negative
values
indicate
repression.

17

Expression of endogenous RUNX2 decreases during osteoblast differentiation.

RANKL is expressed throughout the course of osteoblast differentiation, from
mesenchymal precursor cells to mature osteoblasts to matrix-embedded osteocytes
(Kartsogiannis et al., 1999; Mueller and Richards, 2004; Silvestrini et al., 2005).
There is a lot of interest in which cells in the osteoblast lineage contribute the
most RANKL stimulation for osteoclastogenesis. Because RANKL membrane
association depends on RUNX2 (Martin et al., 2015 and Figures 2-4), and because
osteoblasts drive osteoclastogeneis most efficiently through membrane-anchored
(as opposed to secreted) RANKL (Nakashima et al., 2000; Yasuda et al., 1998),
we became interested in the expression pattern of RUNX2 during the course of
osteoblast differentiation. It has previously been suggested that RUNX2 is
expressed in vivo during early stages of osteoblast differentiation and must be
downregulated thereafter (Komori, 2010; Maruyama et al., 2007).

We determined the expression pattern of RUNX2 during osteoblast differentiation
in four tissue culture models including primary cultures of NeMCO, the mouse
mesenchymal stem cell line ST2, a pre-osteoblasts cell line MC3T3-E1 and a
human osteoblast progenitor cell line SK11. Cells were plated and treated with
ascorbic acid and β-glycerophosphate to induce osteoblast differentiation. As
shown by Western blot analysis, RUNX2 was expressed in all these culture
models during the first days in culture, but expression was downregulated towards
the end of the first week, falling to undetectable levels by the second week of
culture (Figure 6). On a technical note, the human SK11 pre-osteoblasts tended to
easily detach from the plates, so 0.1% of gelatin was added to avoid the cell
detachment. Our results indicate that the expression of RUNX2 is lost during

18

osteoblast differentiation, suggesting that RUNX2-driven RANKL membrane
association and osteoclast activation may be specific for premature osteoblasts.

Figure 6. Expression of RUNX2 during osteoblast differentiation. Western blot analysis of RUNX2
on the indicated days. Actin for SK11 and coomassie blue staining for other cell lines were used as
loading control. The arrows indicated endogenous RUNX2 isoform of ~55 kD.

Introduction and validation of exogenous FLAG-RUNX2 expression in
human mesenchymal stem cells.

RUNX2 is tightly regulated in the osteoblastic cells at transcriptional, translational
and post-translational level (Drissi et al., 2000; Prince et al., 2001; Sudhakar et al.,

19

2001; Tou et al., 2003; Xiao et al., 2000). As RUNX2 was lost during the first
week of osteoblast differentiation in culture (Figure 6), we became interested in
the underlying molecular mechanism. To this end, we introduced a doxycycline
(dox) induced FLAG-RUNX2 lentivirus into a human cell line SK11. Western
blot analysis showed the dox induced RUNX2 expression as expected (Figure 7A).
To verify FLAG-RUNX2 was working functionally in the SK11, we performed a
ChIP qPCR assay to prove RUNX2 could bind to the osteocalcin (OCN) promoter
(Figure 7B). Furthermore, we showed that RUNX2 induced expression of its
classical target genes osteocalcin (Figure 7C). We then tested the mineralization
ability of SK11 by alizarin red staining. As shown in Figure 7D, with dox
induction of RUNX2, the mineralization was significantly increased, while the
naïve cultures on the same day did not display any significant calcium deposition.
Mineralization was further promoted by recombinant human BMP2 (rhBMP2) in
the presence, but not in the absence of dox-induced RUNX2 expression.

To determine whether the developmental regulation of RUNX2 expression during
osteoblast differentiation occurred at the transcriptional or post-transcriptional
level, we asked whether the FLAG-RUNX2, expressed in SK11 from a lentiviral
vector, mimicked the downregulation of endogenous RUNX2 (Figure 6).
Sk11/Rx2
dox
cells were treated with dox and expression of exogenous (dox-
induced) FLAG-RUNX2 was determined over time by Western blot analysis. We
found that expression of exogenous FLAG-RUNX2 decreased during osteoblasts
differentiation (Figure 7E) similar to the endogenous protein (Figure 6).

20

Figure 7. FLAG-RUNX2 expression in SK11/Rx2
dox
and SK11 mineralized ability. A, SK11/Rx2
dox
cultures treated with 100 ng/ml dox were subjected to western blot analysis using anti-FLAG antibodies.
Actin shown at the bottom as loading control. B, SK11/Rx2
dox
cultures were treated with 100 ng/ml dox
and subjected to ChIP analysis using FLAG antibodies and precipitated DNA were amplified using
osteocalcin primers in Table 3. C, RT-qPCR analysis of osteocalcin mRNA. D, SK11 naïve or
SK11/Rx2
dox
cultures were treated with 100 ng/ml dox and 10ng/ml rhBMP2 for indicated days. Alizarin
red stained the calcium deposition in the representing wells. E, SK11/Rx2
dox
cultures treated with dox
were subjected to Western blot analysis with anti-FLAG antibodies on the indicated days.

21

RUNX2 becomes unstable during osteoblast differentiation.

Since expression of both endogenous RUNX2 and exogenous FLAG-RUNX2 was
reduced during differentiation, we hypothesized that RUNX2 protein stability
decreased during osteoblast differentiation. So we measured the RUNX2 half-life
by performing a dox withdrawal assay. We treated SK11/Rx2
dox
cells with
100ng/ml dox for 3 days or 5 days and whole cell lysates were collected at the
indicated time points after dox withdrawal. As shown by Western blot analysis,
FLAG-RUNX2 became less stable on day 5 compared to day 3 (Figure 8A). As
control, we measured RUNX2 mRNA during osteoblast differentiation in the
SK11 culture model and found that it did not change significantly in this process
(Figure 8B). Our results suggested that RUNX2 protein becomes relatively
unstable during osteoblast differentiation, contributing to a decrease in its steady
state levels during the firs week of culture.

Figure 8. RUNX2 protein lost stability during osteoblast differentiation. A, SK11/Rx2
dox
cells were
treated with 100 ng/ml dox for 3 days or 5 days. Cell lysates were collected 0 to 12 hours after dox
withdrawal and were subjected to Western Blot analysis with anti-FLAG antibodies. Actin was used as
loading control. B, RT-qPCR analysis of Runx2 mRNA expression on indicated days during SK11
differentiation.

22

Discussion

RUNX2 is detected in pre-osteoblasts, and its expression then decreases during
osteoblast maturation; mature osteoblasts do not express a significant amount of
RUNX2 protein both in vivo and in vitro (Maruyama et al., 2007 and Figure 6).
With the maturation of osteoblasts, RUNX2 decreased at the protein level because
the protein became unstable during the osteoblast differentiation (Figure 8A).
Even though we found RUNX2 protein was regulated at the post-translational
level, the mechanism is still unclear. It is known that RUNX2 is regulated by
phosphorylation, acylation and ubiquitination (Bruderer et al., 2014) and its
stability is regulatd through a ubiquitination-dependent proteasome-medaited
pathway (Tintut et al., 1999). We suspected that the decrease in RUNX2 stability
during osteoblast differentiation results to alterations to components of the
proteasome pathway but this remains a subject for future investigation.

Strict control of RUNX2 likely contributes to balanced bone turnover because
both deficient expression of RUNX2 and overexpression of RUNX2 decrease
bone density (Geoffroy et al., 2002; Otto et al., 1997). Previous results from our
laboratory using both endogenous RANKL and GFP-RANKL indicated that
RUNX2 induced osteoclastogenesis by promoting RANKL membrane association
without affecting RANKL expression (Martin et al., 2015). Based on microarray
data, we had speculated that RUNX2 induced RANKL membrane association
through genes related to protein trafficking. Whether sex steroids antagonize
RUNX2-mediated RANKL membrane association by inhibiting those genes
remains to be investigated, but the present work suggests this may not necessarily
be the case because RANKL membrane association decreased in one hour (Figure
3). The gene expression has not responded to the sex steroids in such a short time.

23

The bone sparing properties of sex steroids, including estrogens and androgens,
have been attribute in part to attenuating bone turnover. The estrogens attenuation
of bone turnover is activated by estrogen receptor α (ERα) through the anti-
resorptive and anabolic effects (Riggs, 2002; Syed and Khosla, 2005). Studies
showed that estrogen increases FAS ligand signaling in the osteoblasts, which
leads to the increase of osteoclasts apoptosis (Krum et al., 2008). Furthermore,
estrogen also attenuated osteoclastogenesis by reducing RANKL trafficking in
osteoblasts (Martin et al., 2015).

Similar to estrogens, androgens also attenuates bone turnover, and this is achieved
via both aromatization-dependent mechanisms (mediated by ERα after
conversions of estrogens to androgens) and aromatization-independent effects
mediated by androgen receptor (AR) (Frenkel et al., 2010). Researchers found the
aromatase deficient male mice had high rate of bone turnover and low bone mass,
which suggested bone protection by ERα through aromatization (Miyaura et al.,
2001; Oz et al., 2000). Interestingly, studies later showed that testosterone also
reduced bone loss when ERα was blocked (Vandenput et al., 2002) suggesting an
aromatization independent pathway mediated by AR. We previously found E2 and
DHT can inhibit osteoblast-driven osteoclastogenesis by antagonizing RANKL
trafficking. However, the mechanisms are still unclear. As shown in this report,
RANKL membrane association reduced in a short time of E2 and DHT treatment
(Figure 3) indicating a non-genomic mechanism. Further supporting this, estrogen
dendrimer conjugates (EDC), which cannot bind to the nuclear ERs, also
decreased RANKL membrane association (Figure 4). In addition, even if RANKL
membrane association was antagonized by estrogen and androgen via non-
genomic effects (Figure 3), it maintained with the longer period of treatment,

24

while the nuclear ER and androgen receptor (AR) have also been activated (Figure
2). With the analysis of E2 and DHT on the expression of RUNX2 stimulated
genes, the two steroids attenuated a large percentage of genes, suggesting the
similar effects of estrogen and androgen reduction in bone turnover (Figure 5).
Apparently, E2 inhibited more genes than DHT, which may indicated that
estrogens in the body plays more important role than androgen in protecting bones.
However, the effects between estrogen and androgen on those genes needed
further verification. It is also possible that estrogen and androgen antagonized
RUNX2-induced RANKL membrane association via both genomic and non-
genomic effects. Since sex steroids inhibited RUNX2 expression in osteoblasts
(Baniwal et al., 2009; Khalid et al., 2008), for the longer exposure to sex steroids,
RUNX2-induced osteoclastogenesis would be inhibited due to the decrease of
RUNX2 expression. Further investigation is needed to assess the relative
contribution of genomic and non-genomic mechanisms in antagonizing RUNX2-
induced osteoclastogenesis.

As RANKL membrane association was induced by RUNX2, we assumed it
occurred only in pre-osteoblasts because RUNX2 is expressed mainly in the early
stage of pre-osteoblasts. In the other word, mature osteoblasts did not express
RUNX2 to induce RANKL membrane associations so the osteoblasts induced
osteoclastogenesis in the immature and mature osteoblasts appear to be under
different control mechanisms.

Since our work was done in the mouse pre-osteoblasts, it will be important to test
whether our observations can be reproduced in human osteoblast culture models.
A human mesenchymal stem cell line SK11 in this study, which is derived from
embryonic stem cells, can differentiate into osteoblasts inducing by osteogenic

25

factors or into chondrocytes inducing by chondrogenic factors (Sternberg et al.,
2013). As a normal diploid human cell culture model for osteoblasts, SK11
presented typical gene expression phenotype under osteogenic condiction (Yu et
al., 2014). With spontaneous mineralization ability, RUNX2 overexpression with
BMP2 promoted SK11 mineralization, which makes SK11 a good model to
investigate human osteogenesis in vitro (Figure 7). However, we were unable to
find the RANKL membrane association in SK11 due to the cell morphology. The
cells are long and thin so it is difficult to distinguish the cytoplasm and membrane.
Modification of the culture conditions or the imaging technique, or employment of
other human culture systems may be necessary to recapitulate the observations we
made with the murine NeMCO culture model.

In summary, postmenopausal women readily develop osteoporosis because bone
turnover is accelerated due to the decrease of estrogen in the body. This kind of
process is partly driven by RUNX2. Our results suggested that RUNX2,
specifically expressed in the early stage of pre-osteoblast differentiation, induced
RANKL membrane association, which caused osteoclastogenesis. As shown in
this report, it is antagonized by sex steroids including E2 and DHT via a non-
genomic mechanism, which may inspire scientists to develop a drug without
targeting the nuclear estrogen receptors or androgen receptors for osteoporosis.
Such drugs would potentially provide skeletal protection without increasing
cancer risk caused by sex hormones.

26

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Abstract (if available)

Abstract Molecular mechanisms underlying the bone‐sparing effects of sex steroid hormones are not fully understood. We show that RUNX2, a master regulator of osteoblast differentiation and bone formation, promotes association of the quintessential osteoclastogenic factor RANKL with the osteoblast membrane, and this is antagonized by both estradiol and dihydrotestosterone. Sex hormones reversed RUNX2‐mediated RANKL membrane association through a fast, non‐genomic mechanism, which was mimicked by an estrogen dendrimer conjugate (EDC) that cannot enter the cell. Sex steroids appear to antagonize RUNX2‐mediated RANKL membrane association in early pre‐osteoblasts, because, as we show in several osteoblast cell culture models, RUNX2 expression is lost in mature osteoblasts due to acquired post‐translational instability.

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