BMB
BMB Rep. 2015; 48(6): 319-323
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Reports
Contributed Mini Review
The role of microRNAs in cell fate determination of mesenchymal
stem cells : balancing adipogenesis and osteogenesis
1,
2
Hara Kang * & Akiko Hata
1
Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, Korea, 2Cardiovascular
Research Institute, University of California, San Francisco, CA 94158, USA
Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into adipocytes, osteoblasts, or chondrocytes. A mutually inhibitory relationship exists between osteogenic and adipogenic lineage commitment and differentiation. Such cell fate decision is regulated by several signaling
pathways, including Wnt and bone morphogenetic protein
(BMP). Accumulating evidence indicates that microRNAs
(miRNAs) act as switches for MSCs to differentiate into either
osteogenic or adipogenic lineage. Different miRNAs have
been reported to regulate a master transcription factor for osteogenesis, such as Runx2, as well as molecules in the Wnt or
BMP signaling pathway, and control the balance between osteoblast and adipocyte differentiation. Here, we discuss recent
advancement of the cell fate decision of MSCs by miRNAs and
their targets. [BMB Reports 2015; 48(6): 319-323]
INTRODUCTION
MSCs are capable of differentiate into several distinct cell
types, including osteoblasts and adipocytes. The osteogenesis
and adipogenesis of MSCs maintain a homeostasis under physiological conditions. It is often found that the signal, which
promotes one cell fate, actively represses the alternative fate
(1). The balance between osteogenic and adipogenic differentiation is tightly regulated by multiple signaling pathways.
Dysregulation of this balance is known to lead to various human diseases, such as osteopotosis which is often associated
with a significant increase in adipocytes accumulation at an
expense of bone loss (2). On the contrary, patients with a high
bone mass phenotype often exhibit reduced fat tissue volume
(3). Therefore, clear understanding of the control mechanism
of maintenance of this balance between osteogenic and adipo*Corresponding author. Tel: +82-32-835-8238; Fax: +82-32-835-0763;
E-mail: harakang@incheon.ac.kr
http://dx.doi.org/10.5483/BMBRep.2015.48.6.206
Received 24 September 2014
Keywords: Adipogenesis, Mesenchymal stem cells, MicroRNAs,
Osteogenesis
genic differentiation of MSCs is of great importance to elucidate the pathogenesis and a development of novel and effective therapies for bone diseases.
The adipogenic and osteogenic differentiation from MSCs is
regulated by multiple regulatory factors and signaling pathways, such as the Wnt/-catenin, TGF/BMPs/Smads, Notch,
JAK/STAT, MAPK, phosphatidylinositol-3 kinase (PI3K)/Akt and
Hedgehog pathways (4-6). Osteoblast development is governed by the activation of Wnt/-catenin signaling. Wnt signaling through Frizzled and its co-receptors, low-density lipoprotein receptor-related protein (LRP) 5/6, inhibits the Axin
/GSK3/APC complex, and -catenin accumulates in the nucleus, which then directly regulates osteoblast activity (7).
Transcription factors, such as Runt-related transcription factor
2 (Runx2) and Osterix (Osx), lead to the terminal osteoblast
differentiation, which is characterized by the calcification of
the extracellular matrix (5). Alkaline phosphatase (ALP), osteopontin (Opn) and osteocalcin (Ocn) are involved in the mineralization process. BMP signaling is also a central signaling
pathway involved in the induction of osteogenic differentiation
and regulation of bone formation. Specifically, BMP-2 is the
most frequently studied ligand of BMPs that promotes osteogenic commitment and terminal osteogenic differentiation in
MSCs. Gene regulation mediated by several transcription factors play a critical role to form mature adipocytes from MSCs
(8, 9). CCAAT/enhancer binding protein (C/EBP) and activate C/EBP and peroxisome proliferator-activated receptor
(PPAR) to coordinate the expression of adipogenic genes
characteristic of terminally differentiated adipocytes. PPAR is
regarded as a master transcriptional regulator of both adipocyte differentiation and lipid storage in mature adipocytes.
miRNAs are evolutionarily conserved short (19-25 nt) noncoding RNAs that mainly regulate gene expression in a posttranscriptional manner. miRNAs function via partially complementary base pairing with the 3’-untranslated region (UTR) of
target mRNAs. miRNA and target mRNA pairing typically results in gene silencing via translational repression and/or destabilization of mRNA (10). Many studies suggest that miRNAs
critically regulate fate decisions of stem cells, including self-renewal and differentiation. Conversely, miRNAs also critical
during the reprogramming of differentiated somatic cells to
generate induced pluripotent stem (iPS) cells (11). During a
ISSN: 1976-670X (electronic edition)
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microRNAs for balancing adipogenesis and osteogenesis
Hara Kang and Akiko Hata
pluripotent state, transcription factors which are required to
promote cellular differentiation are downregulated by miRNAs.
Once decision to exit from a pluripotent state is made, lineage-specific miRNAs are induced, which inhibit transcription
factors specific for the pluripotent state, such as Sox2, Oct4
and Nanog.
Emerging evidence suggests that miRNAs are involved in
regulating the differentiation and cell fate decisions of MSCs
(12). In human bone marrow-derived MSCs, silencing of Dicer
or Drosha, two key enzymes in the miRNA biogenesis pathway, inhibits both osteogenic and adipogenic differentiation
(13). Recently, miR-196a, -29b, -2861, -3960 and -335-5p are
reported to enhance osteogenic differentiation (14-17), while
miR-26a, -133, -135, -141 and -200a could impede osteogenic
differentiation (18-20), and miR-143, -24, -31, -30c and -642a-3p
are involved in regulating adipogenesis (21-24). Although
many miRNAs have been identified to regulate either adipogenesis or osteogenesis, only a few were implicated in both
processes and play a role in balancing these two cell fates.
This review focuses on miRNAs that function as mediators of
the balance between the adipogenesis and osteogenesis of
MSCs. These miRNAs determine the adipogenic versus osteogenic fates of MSCs by modulating Wnt or BMP signaling via
the repression of components of the signaling pathway or regulating key transcription factors in the differentiation of MSCs,
such as Runx2 (Table 1).
miRNAs THAT DETERMINE ADIPOGENIC
DIFFERENTIATION
Each member of the miR-30 family (miR-30a-e) is differentially
regulated during adipocyte and osteoblast differentiation (25).
miR-30e is the most prominently regulated during adipogenesis and osteogenesis (26). miR-30e is induced in the mesenchymal cell line C3H10T1/2 and the pre-adipocyte 3T3-L1
in response to treatment of adipocyte-inducing medium.
Conversely, the expression of miR-30e is reduced in the
mouse stromal line ST2 and pre-osteoblast MC3T3-E1 after
treatment of osteocyte-inducing medium. The overexpression
of miR-30e promotes pre-adipocytes to differentiate into ma-
ture adipocytes, along with increased expression of adipocyte-specific transcription factors, such as PPARc, C/EBP and
C/EBP (26). The overexpression of miR-30e inhibits osteoblast
differentiation, characterized by reduced expression of pro-osteogenic transcription factors, such as Runx2, Osx, Ocn, ALP
and bone sialoprotein (BSP). The inhibition of the endogenous
miR-30e represses the differentiation of pre-adipocytes and potentiates the osteoblast differentiation (26). LRP6 is shown to
be a direct target of miR-30e (26). The knockdown of LRP6 in
3T3-L1 cells downregulates -catenin/T-cell factor (TCF)-mediated
gene expression and potentiates the differentiation into mature
adipocytes. These results demonstrate that miR-30e controls
the balance of adipocyte differentiation and osteoblast differentiation by modulating the canonical Wnt signaling (Fig. 1).
The levels of miR-30c and miR-30d are also increased during
adipocyte differentiation, but decreased during osteoblast differentiation similar to miR-30e (25). miR-30c and miR-30d are
found to target Smad1, a signal transducer of BMP signaling
pathway, and inhibit BMP-mediated osteoblast differentiation.
Therefore, miR-30c and miR-30d are also mediators to balance
the osteogenesis and adipogenesis via regulating BMP signaling (Fig. 1).
miRNA expression profiling in human adipose-derived mesenchymal stem cells (hADSCs) find that the miR-17 cluster of
family of miRNAs, miR-17-5p, miR-106a and miR-20a, are
downregulated when the cell undergoes osteogenic differentiation while upregulated during adipocyte differentiation
(27). The overexpression of miR-17-5p and miR-106a inhibits
the ALP activity, mineralization and expression of the osteogenic transcription factors, such as Runx2, Osx, Opn and Ocn.
The downregulation of the endogenous miR-17-5p and miR-106a
Table 1. miRNAs that reciprocally regulate the differentiation of adipocytes and osteoblasts
miRNA
Osteogenic switch
(Increased expression
during osteogenesis)
Adipogenic switch
(Increased expression
during adipogenesis)
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Target mRNA
miR-21
miR-22
Sox2, Spry2
HDAC6
miR-204
miR-211
miR-17-5p
miR-106a
miR-30e
miR-637
Runx2
Rnux2
BMP2
BMP2
LPR6
Osx
Fig. 1. miRNAs that control signaling governing osteogenesis and
adipogenesis. BMP and Wnt signaling pathways have been demonstrated to preferentially induce the osteogenesis of MSCs at the
expense of adipogenesis. miR-17-5p/miR-106a and miR-30c/miR-30d
inhibit BMP signaling by targeting key components of the pathway, such as BMP2 and Smad1, respectively. miR-30e inhibits
Wnt signaling via the repression of LPR6, a key coreceptor of
Wnts.
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microRNAs for balancing adipogenesis and osteogenesis
Hara Kang and Akiko Hata
promotes osteogenic differentiation and suppresses the adipogenic differentiation in hADSCs (27). BMP2 is identified as a
direct target of miR-17-5p and miR-106a (27). Therefore,
miR-17-5p and miR-106a balance the osteogenic and adipogenic lineage commitment in hADSCs by modulating BMP signaling (Fig. 1).
Runx2 is identified as a key transcription factor that regulates
osteogenesis and chondrogenesis (28, 29). Regulation of Runx2
also affects the adipogenic potential of MSCs. miRNAs that
regulate MSC differentiation via the modulation of Runx2 were
investigated. miR-204 and miR-211 are induced during adipocyte differentiation, which downregulate Runx2 expression
(30) (Fig. 2). miR-204 and miR-211 act as endogenous repressors of Runx2 in MSCs (30). The perturbation of miR-204 resultes in upregulation of osteogenesis and downregulation of
adipogenesis, characterized by suppression of adipocyte marker genes, such as adipocyte protein 2 (aP2), adipsin and PPAR
(30). Conversely, when miR-204 was overexpressed, the expression levels of aP2, adipsin and PPAR are increased,
which adipocyte differentiation is promoted and osteoblast differentiation is inhibited (30). However, miR-204 inhibitor did
not reverse the decrease of Runx2 levels during adipocyte differentiation, although miR-204 perturbation did significantly
affect the Runx2 levels. This finding suggests that Runx2 expression is not exclusively regulated by miRNAs in MSC
differentiation.
Osx, as a downstream of Runx2, is induced by BMP2 in
MSCs and required for the differentiation of pre-osteoblasts into mature osteoblasts (31, 32). The cartilage is formed normally in Osx-null embryos, but they completely lack bone for-
Fig. 2. miRNA switch of mesenchymal stem cell fate. The differentiation of an MSC into either an adipocyte or osteoblast can be
controlled by miRNA switches. miR-21 and miR-22 switch on osteogenesis, while miR-204, miR-17-5p, miR-106a, miR-30e and
miR637 switch on adipogenesis.
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mation (33). miR-637 is shown to target Osx (34). The expression of miR-637 is increased during adipocyte differentiation, and decreased during osteoblast differentiation. The
expression of adipogenic markers, such as PPAR, C/EBP and
sterol regulatory element-binding protein 1c (SREBP-1c), are
significantly increased in miR-637-overexpressing MSCs, but
are decreased in response to a miR-637 inhibitor. Moreover,
the levels of both BMP2 and Runx2 are downregulated by
miR-637 and upregulated by inhibition of miR-637. These results indicate that miR-637 promotes the adipogenesis and
suppresses the osteogenesis of MSCs, and maintains the balance of these two cell fates.
miRNAs THAT PROMOTE OSTEOGENIC
DIFFERENTIATION
miR-22 is also found to regulate the adipogenic and osteogenic differentiation in hADSCs (35) (Fig. 2). The expression of
miR-22 is decreased during adipogenic differentiation but increased during osteogenic differentiation. Consistently, the overexpression of miR-22 in hADSCs inhibits the accumulation of
lipid droplets and represses the expression of adipogenic transcription factors and adipogenic-specific genes. Conversely,
the enhanced ALP activity and matrix mineralization, as well
as the increased expression of osteo-specific genes, indicate a
positive role of miR-22 in regulating osteogenic differentiation.
Histone deacetylase 6 (HDAC6), a co-repressor of Runx2 (36),
is identified as a target of miR-22. Silencing endogenous
HDAC6 expression in hADSCs enhances osteogenesis but represses adipogenesis, suggesting a role of the miR-22-HDAC6
axis which in turn activates Runx2 activity and osteogenic
differentiation.
The ERK-MAPK signaling pathway plays a pivotal role in initiating and maintaining cell differentiation (37). The elimination of ERK activity is sufficient to maintain the self-renewal
ability of embryonic stem cells, and the inhibition of MAPK
signaling can convert terminally differentiated cells to a pluripotent state (37, 38). The ERK-MAPK signaling pathway has also been shown to be a major regulator of adipogenesis and osteogenesis in MSCs (39). Sprouty 1 and 2 (Spry1 and Spry2)
are negative regulators of the ERK signaling pathway, and
Spry2 is identified as a target of miR-21. miR-21 expression is
elevated during adipogenesis and osteogenesis (40). These results suggest that miR-21 plays a critical role in maintaining
the duration of the ERK-MAPK signaling pathway by repressing
Spry2 expression to increase the differentiation potential of
MSCs.
Furthermore, miR-21 targets Sox2 (41). Sox2 is one of four
genes used to promote iPS cells and repress cell differentiation
in concert with Oct4 and Nanog (42). The expression of osteogenic markers, such as Ocn and Runx2, is increased in MSCs
when miR-21 is overexpressed. These results demonstrate that
miR-21 not only suppresses the pluripotency but also accelerates osteogenic differentiation (Fig. 2).
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microRNAs for balancing adipogenesis and osteogenesis
Hara Kang and Akiko Hata
CONCLUSIONS AND PERSPECTIVES
The differentiation of mesenchymal stem cells into a particular
lineage is tightly regulated, and a malfunction in this regulation could lead to pathological consequences. Specifically,
an inverse relationship exists between the osteogenic and adipogenic lineage commitment and differentiation, suggesting a
switch between these two processes. Recent miRNA expression profiling studies during both the adipogenic and osteogenic differentiation of MSCs have found several miRNAs
with an inverse expression pattern between adipogenesis and
osteogenesis. These miRNAs act as switches during the fate determination of MSCs by regulating molecular signaling pathways, such as Wnt//-catenin and BMP signaling, and multiple
transcription factors. Therefore, modulation of levels of these
miRNAs could serve as novel therapies for osteogenesis- or
adipogenesis-related disorders. Further understanding of the
miRNAs that modulate signaling pathways other than Wnt or
BMP, including the TGF, Notch, JAK/STAT, PI3K/Akt and
Hedgehog signaling pathways during MSC differentiation will
provide more complete picture of the mechanisms of the cell
fate decision in MSCs.
ACKNOWLEDGEMENTS
This work was supported by the Incheon National University
Research Grant in 2013.
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