Interrelationship of Runx2 and estrogen pathway in skeletal tissues.
ABSTRACT Two key molecules in skeletal tissues are bone formation master transcription factor Runx2 and the steroid hormone estrogen. It is well known that these two molecules play pivotal roles in bone homeostasis; however, the functional interaction between Runx2 and estrogen synthesis in skeletal tissues is largely unknown. Recent studies have indicated that there is a positive relationship between Runx2 and the estrogen biosynthesis pathway. In this review, a possible functional link between Runx2 and estrogen synthesis pathway in skeletal tissues will be discusses as well as the biological significance of this interaction.
- SourceAvailable from: Eui-Sic Cho[Show abstract] [Hide abstract]
ABSTRACT: Fibroblast growth factor-4 (FGF4) is expressed in embryonic stages and in adult tissues, where it plays critical roles in modulating multiple cellular functions. However, the exact roles of FGF4 on proliferation and differentiation of embryonic stem cells (ESCs) are not completely understood. Exogenous addition of FGF4 stimulated proliferation of mouse ESCs (mESCs), as proven by the increases in DNA synthesis and cell cycle regulatory protein induction. These increases were almost completely inhibited by pre-treating cells with anti-FGF4 antibody. FGF4 also activated c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK) signaling, but not p38 kinase. Blockage of JNK signaling by SP600125 or by transfection with its specific siRNA significantly inhibited FGF4-stimulated cell proliferation through the suppression of c-Jun induction and activator protein-1 (AP-1) activity. However, ERK or p38 kinase inhibitor did not affect FGF4-stimulated proliferation in mESCs. FGF4 suppressed osteogenic differentiation of mESCs by inhibiting expression of transcription factors involved in bone formation. Further, exogenous FGF4 addition stimulated proliferation of human periodontal ligament stem cells (hPDLSCs) and bone marrow mesenchymal stem cells (BMMSCs) via activation of ERK signaling. FGF4 also augmented mineralization of hPDLSCs, but not of BMMSCs. Collectively, it is suggested that FGF4 triggers proliferation of stem cells by activating MAPK-mediated signaling, while it affects differently osteogenic differentiation according to the origins of stem cells.PLoS ONE 01/2013; 8(8):e71641. · 3.73 Impact Factor
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ABSTRACT: Background: Periodontal ligament fibroblasts (PLF) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. We hypothesized that PLF induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GF) and skin fibroblasts (SF). Methods: PLF and GF were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SF were purchased from ATCC. mES cells were incubated with culture supernatants of these fibroblasts or co-cultured directly with the cells. Proliferation and mineralization in mES cells was determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured. Results: In co-cultures, PLF stimulated proliferation of mES cells more effectively than GF or SF. Similarly, the addition of culture supernatant of PLF induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor-4 (FGF4) or the JNK inhibitor SP600125. Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of ES cells in the order PLF > GF > SF. These activities of PLF were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pre-treatment of mES cells with the ERK inhibitor PD98059 or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin. Conclusion: PLF regulate the proliferation and osteogenic differentiation of mES cells more strongly than GF and SF via the secretion of FGF through a mechanism that involves MAPK-mediated signaling.Journal of Periodontology 06/2013; · 2.40 Impact Factor
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ABSTRACT: Fibroblast growth factor-7 (FGF7) is known to regulate proliferation and differentiation of cells; however, little information is available on how FGF7 affects the differentiation of embryonic stem cells (ESCs). We examined the effects of FGF7 on proliferation and osteogenic differentiation of mouse ESCs. Exogenous FGF7 addition did not change the proliferation rate of mouse ESCs. In contrast, the addition of FGF7 facilitated the dexamethasone, ascorbic acid, and β-glycerophosphate (DAG)-induced increases in bone-like nodule formation and calcium accumulation. FGF7 also augmented mRNA expression of runt-related transcription factor-2 (Runx2), osterix, bone sialoprotein (BSP), and osteocalcin (OC) in the presence of DAG. FGF7-mediated increases in the mineralization and bone-specific gene expression were almost completely attenuated by pretreating with anti-FGF7 antibody. FGF7 treatment accelerated the DAG-induced activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in the cells. A pharmacological inhibitor specific to ERK, but not to JNK or p38 kinase, dramatically suppressed FGF7-mediated mineralization and accumulation of collagen and OC in the presence of DAG. This suppression was accompanied by the reduction in Runx2, osterix, BSP, and OC mRNA levels, which were increased by FGF7 in the presence of DAG. Collectively, our results suggest that FGF7 stimulates osteogenic differentiation, but not proliferation, in ESCs, by activating ERK/Runx2 signaling.Molecular and Cellular Biochemistry 10/2013; 382(1-2):37-45. · 2.33 Impact Factor
*Corresponding author. Tel: 82-53-420-4823; Fax: 82-53-422-1466;
Received 26 September 2011
Keywords: Aromatase, Estrogen pathway, Runx2
Interrelationship of Runx2 and estrogen pathway in skeletal
Jae-Hwan Jeong & Je-Yong Choi*
Department of Biochemistry and Cell Biology, School of Medicine, WCU Program, Kyungpook National University, Daegu 700-422, Korea
Two key molecules in skeletal tissues are bone formation master
transcription factor Runx2 and the steroid hormone estrogen. It
is well known that these two molecules play pivotal roles in
bone homeostasis; however, the functional interaction between
Runx2 and estrogen synthesis in skeletal tissues is largely
unknown. Recent studies have indicated that there is a positive
relationship between Runx2 and the estrogen biosynthesis path-
way. In this review, a possible functional link between Runx2
and estrogen synthesis pathway in skeletal tissues will be dis-
cusses as well as the biological significance of this interaction.
[BMB reports 2011; 44(10): 613-618]
Bone is a dynamic tissue which is continuously resorbed by
osteoclasts and formed by osteoblasts in a process called bone
remodeling throughout the lifetime of the organism. Bone re-
modeling is the replacement of old bone tissue with new bone
tissue in local bone areas, which is called the bone remodel-
ing compartment (BRC), and this process is responsible for a
gain in bone mass and change in skeletal form (1). BRC com-
prises the basic multicellular unit (consisting of osteoclasts, os-
teoblasts and osteocytes), the canopy of bone-lining cells and
the associated capillaries. The remodeling process within BRC
involves the coupling of bone formation and bone resorption.
Runx2 and estrogen have been shown to play important roles
in this process. However, the relationship between Runx2 and
estrogen biosynthesis in bone is still not clearly understood.
Function of Runx2 and estrogen in bone tissues
The transcription factor Runx2, also known as Pebp2A, Cbfa1,
Osf2 and AML3, plays essential roles in osteoblast differentiation
and function (2). Ablation of Runx2 function in mice results in a
complete lack of both intramembranous and endochondral ossifi-
cation and shows an absence of osteoblast differentiation, as well
as retardation of chondrocyte differentiation (3-5). Furthermore,
many in vitro studies have shown that Runx2 is a positive regu-
lator for the expression of bone matrix genes, including type I col-
lagen, osteopontin, bone sialoprotein, osteocalcin, and matrix
metalloproteinase 9 (6-10). The heterozygous mutation of Runx2
results in cleidocranial dysplasia, a dominantly inherited devel-
opmental disorder of bone, in both mice and humans (3, 4,
11-13). Moreover, Runx2 accelerates chondrocyte differentiation
in response to the upregulation of Runx2 target genes, which in-
clude the indian hedgehog (Ihh) and type X collagen (Col10A1)
(14, 15). Runx2 also plays an important role in estrogen defi-
ciency-mediated bone resorption in adult mice (16). Together,
these studies demonstrate that Runx2 acts as a master regulator of
bone formation by modulating the activity of a cohort of target
genes not only in early growth but also in postnatal bone develop-
ment and maintenance stages.
Estrogen is essential in skeletal growth and development and
the maintenance of bone health in both men and women (17).
Estrogen synthesis starts from cholesterol and converts an-
drogens like testosterone and androstenedione into estrogens by
aromatase at the final step (Fig. 1). Deficiency of estrogen by mu-
tation of aromatase (18-20), or mutation of estrogen receptor al-
pha (ERα) could lead to unfused epiphyses, osteopenia and even
osteoporosis (21). Estrogen deficiency after menopause has been
associated with increased bone turnover, which results in a re-
duced bone mass and an increased fracture risk. One of the key
roles of estrogen is to modulate osteoclast generation and life
span, which is revealed by a loss of expected increase of the
number of osteoclasts in bone than expected and further de-
crease of cancellous bone after loss of estrogens in mono-
cyte/macrophage cell lineage- or osteoclast lineage-specific ERα
conditional knockout mice (22, 23). Although the stage specific
roles of Fas ligand (FasL) during osteoclastogenesis (24) and os-
teoclast apoptosis (23) remain controversial, estrogen mediated
upregulation of FasL through ERα in osteoclasts or osteoblasts
has been shown to induce osteoclast apopotosis (22, 25). In ad-
dition, estrogen inhibits osteoclast formation through the upre-
gulation of osteoprotegerin production (26). Therefore, mutation
or ablation of genes in the pathway related to estrogen synthesis
disrupts bone homeostasis (27).
Invited Mini Review
Interrelationship of Runx2 and estrogen pathway in skeletal tissues
Jae-Hwan Jeong and Je-Yong Choi
Fig. 1. Estrogen synthesis pathway. The biosynthesis of estrogens from
cholesterol involves many enzymes and the final step is catalyzed by
the enzyme aromatase (CYP19). CYP11A, cholesterol side-chain cleav-
age enzyme; CYP17, 17α-hydroxylase and 17,20-lyase; CYP21, 21-
hydroxylase; CYP11B1, 11β-hydroxylase; CYP11B2, aldosterone syn-
thase; CYP19, aromatase; StAR, steroidogenic acute regulatory protein;
SULT2A1, dehydroepiandrosterone-sulfotransferase; 17-OHPreg, 17α-
hydroxypregnenolone; 17-OHProg, 17α-hydroxyprogesterone; 17βHSD3,
17β-hydroxysteroid dehydrogenase type 3; 3βHSD2, 3β-hydroxysteroid
dehydrogenase type 2. Cartoon was modified from Pezzi, et al. (35).
Fig. 2. Genome structure of aromatase gene and Runx binding sites
of aromatase promoter found in various human tissues. (A) Structure
of the human aromatase gene. Promoter (P) I.4 and I.6 are indicated
as arrows. (B) Using the Transfac database and the TF search, the
Runx binding site was identified in tissue-specific promoters that are
alternatively used in various tissue types. Ellipses indicate the posi-
tion of the Runx binding site.
Alternative usage of multiple aromatase promoters in various
Aromatase catalyzes the conversion of androgen to estrogen and
is encoded by a single gene (CYP19) in human. The human ar-
omatase gene is comprised of a 30-kb coding region containing
nine exons and a 93-kb regulatory region containing 10 un-
translated exons I and is located in the chromosome 15q21.2 re-
gion (28-30). Aromatase is expressed in many tissues including
the ovaries (31), muscle, skin, adipose tissue (32-34), placenta
(35), and bone (36-40). The unusually large regulatory region
contains 10 tissue-specific promoters that are alternatively used
in various cell types (28, 29). Each promoter is regulated by a dis-
tinct set of regulatory sequences in the DNA and transcription
factors, which bind to these specific sequences. The promoters
specific for the ovary tissues (PII), bone tissues (I.4 and I.6), brain
(I.f), endothelial cells (I.7), fetal tissues (I.5), and placenta (2a, I.1)
are localized in tandem ~0, 73, 1, 33, 36, 43, 78, and 93 kb up-
stream of the ATG translational start site in exon II. The majority
of aromatase transcripts in bone cells and tissues contain exon
1.4 and exon 1.6 in the 5'-untranslated region (5’-UTR) (Fig. 2).
Expression of aromatase in bone tissues
Multiple usage in the aromatase promoter has been well estab-
lished and among them aromatase exon I.4 and I.6 show high
activity in bone and bone cells. Indeed, osteoblast-specific ar-
omatase gene expression in transgenic mice increases bone
mass (41). Several groups have shown that bone tissue, as well
as osteoblast-like cells in primary culture, expresses aromatase
and that this expression is up-regulated in some physiological
or pathological conditions such as bone fracture or osteopo-
rosis, probably under conditions in which osteogenesis is
stimulated (39, 42, 43).
With regard to the expression of aromatase in osteoblasts,
many factors have been identified as a positive regulator for ar-
omatase gene expression. For example, TGF-β1 enhances the
expression of aromatase through promoter I.4 (43). Dexametha-
sone stimulates aromatase activity as well as the expression of ar-
omatase mRNA in isolated human osteoblast cells (36). Oncos-
tatin M and Forskolin strongly stimulates aromatase expression
together with dexamethasone in human osteoblasts (44, 45).
IL-1β is one of the most potent stimulators of aromatase ex-
pression in osteoblast-like cells obtained from the human fetus
and the presence of dexamethasone is necessary for the in-
duction of aromatase by this cytokine (39). IL-1β also directly en-
hances the mRNA expression and activity of aromatase in the
human osteoblast cell line, HOS (46). Several potential cyto-
Interrelationship of Runx2 and estrogen pathway in skeletal tissues
Jae-Hwan Jeong and Je-Yong Choi
Fig. 3. Interrelationship between Runx2 and the estrogen pathway.
Runx2 increases aromatase gene expression and results in estrogen
production. Upregulated estrogen can inhibit Runx2 transactivation
function through ERα.
kines such as IL-1β, TNFα and TGFβ regulate the aromatase ac-
tivity of osteoblast-like cells in the presence of dexamethasone
(43, 47). Moreover, 1,25-Dihydroxyvitamin D3 enhances the en-
zyme activity and gene expression of aromatase in the presence
of dexamethasone (48). Recently, Runx2 was shown to stimulate
aromatase gene expression in skeletal tissues (49). In bone and
osteoblast cells, positive regulation of aromatase activity by glu-
cocorticoid, Vitamin D3, and Runx2 may contribute to the local
production of estrogens, thus leading to protective effects
against osteoporosis especially after menopause.
The importance of the aromatase in bone has been well
characterized by two groups using aromatase deficient mice
and both have reported osteoporotic phenotypes in these mice
although the sexual dimorphic response reported by these two
groups was somewhat inconsistent (50-52).
Interrelationship between Runx2 and estrogen synthesis
Runx2 is a master regulator of osteoblast differentiation. Consis-
tent with its role in the physiological control of osteoblasto-
genesis, Runx2 anabolically modulates bone formation and con-
trols transcriptional signaling pathways that are linked to PTH
(16), glucocorticoids (53) and vitamin D3 (54). Runx2 is also a
strong positive regulator of aromatase gene expression in osteo-
blastic cells (49). Runx2 was directly interact with the aromatase
gene promoter and stimulated aromatase gene expression in a
transient transfection assay. Estrogen production was elevated by
forced expression of Runx2 in several osteoblastic cells. In con-
trast, estrogen production was decreased in bone marrow stro-
mal cells derived from Runx2 heterozygous mice. Aromatase ex-
pression in the perichondrial and periosteal area was drastically
decreased in Runx2 null mice (49). Therefore, aromatase gene
expression was well correlated with the level of Runx2 ex-
pression in osteoblastic cells. Runx2 mediated increase in cel-
lular estrogen production through upregulation of bone-specific
aromatase gene expression may contribute to the maintenance of
bone mass in conjunction with endocrine effects. Indeed, both
cortical bone thickness and trabecular bone mineral density
were lower in Runx2 heterozygous mice compared to wild type
mice. Together, these results indicate that aromatase is a down-
stream target the Runx2 gene, and that Runx2 functions as a pos-
itive regulator for aromatase gene expression and estrogen
Positive and negative feedback loop between Runx2 and
estrogen synthesis pathway
Recently, ERα was shown to directly interact with Runx2 and
modulate its transcriptional function in the presence of estrogen
(55). Interestingly, ERα inhibited Runx2 transactivation function
and this inhibition was further enhanced in the presence of
17β-estradiol (49). These results suggest that local production of
estrogen by Runx2 through upregulation of aromatase in bone
can be regulated by both a positive and negative feedback loop,
which can further contribute to the maintenance of bone ho-
meostasis (Fig. 3). In addition to the interrelationship between
Runx2 and ERα, Runx2 also controls the expression of genes in-
volved in sterol/steroid metabolism and GPR30/GPER, a cell sur-
face G-protein-coupled receptor for estrogen, which are im-
portant for osteoblast proliferation (56, 57). Runx2 is expressed
in not only skeletal tissues but also in nonskeletal tissues such as
ovary, testis and brain (58). Because the expression of Runx2 is
not confined to skeletal tissues, the functional significance of
Runx2 may also include modulation of aromatase gene ex-
pression in these new organs.
Aromatase, a key enzyme for estrogen synthesis from androgen,
is a Runx2 downstream target gene and local production of es-
trogen in bone may in part be due to the regulation of Runx2
mediated aromatase gene expression. Collectively, these results
indicate a possible functional link between aromatase and
Runx2 and a component of a physiological regulatory network
between Runx2 and the estrogen pathway that can contribute to
skeletal development and bone homeostasis.
This study was supported by grants from the Korea Health 21 R&D
Project (Ministry of Health, Welfare, and Family Affairs, Republic
of Korea, A010252), The Korean Ministry of Education, Science
and Technology (The Regional Core Research Program/ Anti-aging
and Well-being Research Center), and WCU program through the
National Research Foundation of Korea funded by the Ministry of
Education, Science and Technology (R32-10064).
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