The Effects of Class-Specific Histone Deacetylase Inhibitors on the Development of Limbs During Organogenesis
The Effects of Class-Specific Histone Deacetylase Inhibitors on the Development of Limbs During Organogenesis
France-He? le` ne Paradis 0
Barbara F. Hales 0
0 Department of Pharmacology and Therapeutics, McGill University , Montreal, QC , Canada H3G 1Y6
Histone deacetylases (HDACs) play a major role in chromatin remodeling, gene regulation, and cellular signaling. While the role of each class of HDAC during normal development is unclear, several HDAC inhibitors are embryotoxic; the mechanisms leading to the teratogenicity of HDAC inhibitors are not known. Here, we investigated the effects of classspecific HDAC inhibitors on the development of organogenesis-stage murine limbs. Timed-pregnant COL2A1-ECFP, COL10A1-mCherry, and COL1A1-YFP CD1 reporter mice were euthanized on gestation day 12; embryonic forelimbs were excised and cultured in vitro for 1, 3, and 6 days in the presence or absence of MS275 (a class I HDAC inhibitor), MC1568 (a class III HDAC inhibitor), Sirtinol (a class II HDAC inhibitor), or valproic acid, our positive control. Fluorescently tagged COL2A1, COL10A1, and COL1A1 served as markers of the differentiation of proliferative chondrocytes, hypertrophic chondrocytes, and osteoblasts, respectively. MS275 and valproic acid caused a reduction in expression of all three markers, suggesting effects on both chondrogenesis and osteogenesis. MC1568 had no effect on chondrocyte markers and mildly inhibited COL1A1 expression at 6 days. Sirtinol had no effect on COL2A1 expression or chondrocyte differentiation 1 day following exposure; however, it caused a drastic regression in limb cartilage and reduced the expression of all three differentiation markers to nearly undetectable levels at 6 days. MS275 and Sirtinol caused a 2.2- and 2.7-fold increase, respectively, in cleaved-caspase 3, a marker of apoptosis, suggesting embryotoxicity. These data demonstrate that inhibition of class I or III HDACs causes severe developmental toxicity and is highly teratogenic. VC The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail:
HDAC inhibitor; MS275; MC1568; Sirtinol; valproic acid; teratogen; limb development; chondrogenesis; osteogenesis; apoptosis
Histone deacetylases (HDACs) are enzymes that remove acetyl
groups from the lysine residues of histone and nonhistone
proteins, leading to effects on both gene expression and cellular
signaling. HDACs are divided into four different classes; classes
I, II, and IV comprise HDAC1?11, whereas SIRT1?7 are members
of class III
(Thiagalingam et al., 2003)
. Inhibitors of class I and
class II HDACs, such as valproic acid (VPA), an anticonvulsant,
and vorinostat (SAHA), an anticancer agent used in the
treatment of cutaneous T-cell lymphoma, are used as therapeutics
(Duvic and Vu, 2007)
. Several other HDAC inhibitors are
currently under development and in clinical trials as anticancer
(reviewed in West and Johnstone, 2014)
. Studies suggest
that HDAC class III inhibitors may have beneficial effects in the
treatment of Parkinson?s disease
(Outeiro et al., 2007; West and
. However, some HDAC inhibitors are
developmental toxicants in animal models, and the embryotoxicity of
others is not known
(Giavini and Menegola, 2014)
. The specific
mechanisms by which HDAC inhibitors cause malformations
(Murko et al., 2013; Paradis and Hales, 2015)
As new therapeutic indications develop, an increasing number
of women of childbearing age may be exposed to HDAC
inhibitors. It is pivotal to decipher the specific roles of individual
HDAC classes during development to predict the effects of their
The four classes of HDACs differ with respect to the
homology of their catalytic domains, structures, and cofactor
(Blander and Guarente, 2004; Taunton et al., 1996)
HDAC1, 2, 3, and 8 are members of class I and homologous to
the yeast RPD3 protein, whereas HDAC4, 5, 6, 7, 9, and 10 are
class II HDACs, homologous to HDA1 (Yang and Seto, 2008).
HDAC11 is the only member of class IV and has mixed
properties, from classes I and II. While classes I, II, and IV are all
Zndependent enzymes, class III HDACs are NAD?-dependent and
are commonly referred to as Sirtuins (SIRT1?7), homologous to
the yeast Sir2
(Thiagalingam et al., 2003)
. The different HDACs
have common targets, but they also have distinct high affinity
substrates; HDAC1 and SIRT1 have a higher affinity for histones,
whereas HDAC6 and SIRT2 target alpha-tubulin
. Mouse knockouts for HDAC1, 3, and 7 are
embryonic lethal early during organogenesis; the knockouts of several
others, such as HDAC2, SIRT1, and 6, are lethal perinatally,
suggesting an important role for these enzymes during
(Chang et al., 2006; Finkel et al., 2009; Montgomery et al.,
. However, little is known about the specific roles of
HDAC classes during development.
The limb has been used as a model system to study
organogenesis for many years
(Neubert and Barrach, 1977)
vertebrate skeleton is formed through a process called endochondral
ossification and requires the formation of a cartilage template
that is subsequently replaced by bone matrix. The cartilage
matrix is made of collagen type 2a1 (COL2A1) and is directly
regulated by the transcription factor SOX9, expressed in proliferative
(Bell et al., 1997)
. These cells differentiate into
hypertrophic chondrocytes, expressing the transcription factor
RUNX2 and the structural protein collagen type 10a1 (COL10A1)
(Ding et al., 2012)
. Ultimately, these cells undergo cell death or
differentiate into osteoblasts that secrete the bone matrix
protein collagen type 1a1 (COL1A1)
(Karsenty and Park, 1995)
. In a
previous study, our group has shown that VPA, a known human
teratogen and inhibitor of class I and II HDACs, caused a
decrease in Sox9, Col2a1, Runx2, and Col10a1 gene expression
(Paradis and Hales, 2013)
. Studies suggest that HDAC signaling
intervenes in different steps of bone formation; HDAC1
modulates NKX3.2 and BMP signaling pathways, both known to play
an important role during chondrogenesis
(Cairns et al., 2012;
Rigueur et al., 2015)
. HDAC1, 3, 4, 5, 6, and 7 interact with RUNX2
(Bradley et al., 2011). HDAC4 knockout mice exhibit premature
chondrocyte hypertrophy in the growth plate, while mice
lacking HDAC2 exhibit a runted phenotype, suggesting congenital
(Montgomery et al., 2007; Vega et al., 2004)
In this study, we investigated the effects of class-specific
HDAC inhibitors on limb development in an in vitro limb bud
culture system using class-specific HDAC inhibitors; MS275
(entinostat) inhibits HDAC1 and 3 (class I), MC1568 inhibits
HDAC4 and 6 (class II), and Sirtinol inhibits SIRT1 and 2 (class
(Beckers et al., 2007; Chiara et al., 2014; Mai et al., 2005)
inhibits both class I and class II HDACs
(Gottlicher et al., 2001)
We used triple transgenic mice expressing fluorescently tagged
chondrogenic and osteogenic markers to readily assess the
effects of our compounds on chondrogenesis and osteogenesis.
MATERIALS AND METHODS
Limb bud cultures and drug treatments.
Col2a1-enhanced cyan fluorescent protein (Col2a1-ECFP),
Col10a1-mCherry, and Col1a1-yellow fluorescent protein
(Col1a1-YFP) CD1 reporter mice were a gift from David L. Butler
(University of Cincinnati, Cincinnati, OH) and David Rowe
(University of Connecticut Health Center, Farmington, CT)
(Maye et al., 2011)
. These mice express fluorescently tagged
markers of cartilage and bone differentiation (Fig. 1). Mice were
housed in the McIntyre Animal Resource Centre (McGill
University, Montreal, QC, Canada), maintained on a 12-h light/
dark cycle, and allowed access to food and water ad libitum. The
mice were mated overnight, and detection of a vaginal plug was
considered gestation day (GD) 0. Timed-pregnant mice were
euthanized by cervical dislocation on GD12, and their embryos
were explanted. The embryonic forelimbs were cultured, as
(Paradis et al., 2012)
. Briefly, limbs were
excised in Hank?s balanced salt solution (HBSS), pooled, and
cultured in vitro in 6 ml culture medium consisting of 75% BGJb
medium (GIBCO BRL Products, Burlington, ON, Canada) and 25%
salt solution supplemented with ascorbic acid (160 lg/ml) and
gentamycin (1 ll/ml, GIBCO BRL Products). Each culture was
gassed with 50% O2, 5% CO2, and 45% N2, and designated
treatments were added; vehicle, sodium valproate (VPA, 3.6 mM,
Sigma Chemical Co., St-Louis, MO, CAS number 1069-66-5)
dissolved in distilled water, or MS275 (entinostat, 2.5 lM,
SelleckChem, Houston, TX, CAS number 209783-80-2), MC1568
(2.5 lM, SelleckChem, CAS number 852475-26-4), or Sirtinol
(50 lM, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, CAS
number 410536-97-9) dissolved in dimethyl sulfoxide.
Concentrations were chosen in accordance with previous
studies and the manufacturers? IC50 recommendations
et al., 2014)
. All animal studies complied with the guidelines
established by the Canadian Council on Animal Care under McGill
University protocol 1825.
Limb morphology and differentiation
Limbs (n ? 5 culture bottles, 5?6 limbs/bottle) were cultured for
6 days with a change of medium on day 3. On days 1, 3, and 6 of
culture, pictures were taken using a Leica DFC450C digital
camera (Leica Microsystems, Wetzlar, Germany) connected to a
Leica M165 Fluorescent Stereo Microscope (Leica Microsystems).
COL2A1-CFP-positive cartilage was quantified using a
morphogenetic scoring system adapted from Neubert and Barrach and
scores were normalized to control
(Neubert and Barrach, 1977)
The proportions of limbs expressing the differentiation markers
COL10A1-mCherry and COL1A1-YFP were quantified at 3 and
Primary cell cultures
Embryonic forelimbs were collected on GD 12 and washed with
low calcium HBSS (HBSS Ca/Mg free [Gibco, Life Technologies,
Burlington, ON], 1 M HEPES pH 7.4, 0.15 M CaCl2). Limbs were cut
and incubated in a collagenase solution (1.5 mg/ml collagenase
type II, 300 mg/ml bovine serum albumin in low Ca HBSS) for 2 h
at 36 C. Cells were sedimented from the suspension by
centrifugation at 1000 g for 5 min and incubated with 2.5 g/ml
Pancreatin for 15 min at room temperature. Cells were then
washed with complete medium 199 (Medium 199, Gibco, Life
Technologies, 26.2 mM NaHCO3, 25 mM HEPES pH7.4, 0.06 mg/ml
gentamicin sulfate, 1% 100X Antibiotic-Antimycotic, 10% fetal
bovine serum) and passed through a 40 lm filter cloth. Cells
were counted with a hemocytometer, and approximately 1 106
cells were plated in a 12-well culture plate for 24 h. Cells were
then treated with dimethyl sulfoxide (vehicle) or Sirtinol (50 lM,
Santa Cruz Biotechnology). Cultures were stopped at designated
times, and protein lysate was extracted. The experiment was
repeated four times.
Whole cell lysates were obtained by sonication in lysis buffer
containing protease inhibitors, consisting of 150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris
pH 7.5, 40 lg/ml bestatin, 0.2 M phenylmethylsulfonyl fluoride,
10 lg/ml leupeptin, and 6 lg/ml aprotinin. Total protein was
extracted and quantified using spectrophotometric Bio-Rad protein
assays (Bio-Rad Laboratories, Mississauga, ON, Canada). Proteins
(15?30 lg per sample) were separated by SDS-PAGE acrylamide
gel electrophoresis and transferred onto polyvinylidene difluoride
membranes (BioSciences Inc., Baie d?Urf e?, QC, Canada). Precision
standards (Bio-Rad Laboratories) were used as molecular weight
markers. Membranes were blocked in 5% nonfat milk in TBS-T
(137 mM NaC1, 20 mM Tris pH 7.4, 0.05% Tween 20) for 1 h at
room temperature, probed overnight at 4 C with primary
antibodies, washed, and incubated with the secondary antibody for
2 h at room temperature. Immunoblotting was done using
polyclonal antibodies against histone 4 acetyl-lys 12 (H4K12Ac, EMD
Millipore, Billerica, MA, 1:5000), alpha-tubulin acetyl-lys40 (Cell
Signaling, Danvers, MA, 1:5000), cleaved-caspase 3 (Cell Signaling,
1:1000), HDAC1 (Cell Signaling, 1:2500), and beta-actin (Santa Cruz
Biotechnology, Inc., 1:10 000). Monoclonal antibodies against
SIRT1 (Abcam, Cambridge, MA, 1:2500) and HDAC6 (Abcam,
1:2500) were used. The secondary antibodies, conjugated to
horseradish peroxidase (HRP), were anti-rabbit (GE Healthcare
Limited, Baie d?Urf e?, QC, Canada, 1:5000), anti-mouse (GE
Healthcare Limited, 1:5000), and anti-goat antibodies (Santa Cruz
Biotechnology, Inc., 1:10 000). Western blots were visualized with
the Enhanced Chemiluminescence Plus Kit (GE Healthcare
Limited) and quantified by densitometry using a ImageJ imaging
software (NIH, Bethesda, MD).
All data sets were analyzed statistically using SigmaPlot 11.0
(Systat Software, San Jose, CA). The Ranked Mann?Whitney
U test and multiple comparisons were used for all data sets,
with Bonferonni?s correction where multiple comparisons
were done within a set. The minimum level of significance was
P < .05.
HDAC inhibitory activity of MS275, MC1568, and Sirtinol in developing limbs
To examine the HDAC inhibition activity of the class-specific
HDAC inhibitors we chose, we used histone 4 acetylation as a
marker of the activity of nuclear HDACs and tubulin acetylation
as a marker of activity in the cytoplasm (Fig. 2). MS275, an
inhibitor of HDAC1 and 3, induced a significant increase in H4k12Ac,
whereas MC1568, an inhibitor of HDAC4 and 6, caused a
hyperacetylation of tubulin. Surprisingly, Sirtinol, an inhibitor of
SIRT1 and 2, did not cause an increase in acetylation of either
target in cultured limb buds. We confirmed the expression of
these targets in the developing limb by western blot
(Supplementary Fig. 1). However, Sirtinol did induce tubulin
hyperacetylation in limb primary cell cultures 3 h after exposure
(Supplementary Fig. 2). The HDAC inhibition activity of VPA in
the limb bud culture model was characterized in a previous
study; VPA induced H4k12 hyperacetylation as early as 1 h
(Paradis and Hales, 2015)
Class-specific HDAC inhibitors have distinct effects on chondrogenesis and osteogenesis
To assess the effects of MS275 on chondrogenesis and
osteogenesis, triple transgenic forelimbs were cultured in vitro in the
presence of MS275 for 6 days. MS275 induced a decrease in CFP
fluorescence (44% decrease in limb score) 1 d following
exposure; changes were also observed at 3 and 6 days (76% and 66%
decrease in score) (Fig. 3). Phalanges were often missing, and
metacarpals were underdeveloped, short, and round.
Hypertrophic chondrocytes and osteoblasts failed to
differentiate, as shown by the complete absence of both mCherry and
In contrast, MC1568 treatment had no effects on
chondrogenesis or on CFP-positive scores and only a mild nonsignificant
effect on mCherry fluorescence at 3 days (Fig. 4). A decrease in
COL1A1-YFP was observed at 6 days, suggesting that inhibition
of HDAC class II affects osteoblast differentiation.
Sirtinol exposure led to a phenotype distinct from that of
either MS275 or MC1568 (Fig. 6). There were no changes in
CFPpositive scores at 1 day and drastic decreases, to 42% and 91%,
at 3 and 6 days, respectively, in Sirtinol-treated limbs.
COL10A1mCherry and COL1A1-YFP fluorescence were also reduced at
both 3 and 6 days. Moreover, qualitative analysis of the
Sirtinoltreated limbs revealed radical effects on limb morphology,
suggesting severe cytotoxicity (Fig 5, bright field).
The effects of VPA on chondrogenesis and osteogenesis are
shown in Figure 6. VPA treatment had a very similar effect to
that of MS275; several digital condensations were missing, and
a rapid decrease in CFP-positive score was observed at 1, 3, and
6 days following exposure (50%, 61%, and 60% decrease in limb
score, respectively); the numbers of limbs exhibiting the
differentiation markers COL10A1-mCherry and COL1A1-YFP were
significantly reduced at both 3 and 6 days.
MS275 and Sirtinol induce activation of caspase 3
Our next goal was to assess the extent of apoptosis in treated
limbs using cleaved-caspase3 as a marker (Fig. 7). Both MS275
and Sirtinol significantly increased the cleavage of caspase 3 at
24 h following exposure, suggesting an increase in cellular death
consistent with the limb phenotype we observed; in contrast,
MC1568 did not increase cleaved caspase 3, suggesting that
class II inhibition is not toxic to the cells. In a previous study
using this model, VPA increased caspase 3 cleavage at 12 and 24 h
(Paradis and Hales, 2015)
Class-specific HDAC inhibitors had distinct effects on limb
development. Inhibition of class I HDACs had highly detrimental
effects on chondrogenesis and osteogenesis and was cytotoxic,
whereas inhibition of class II HDACs had only mild effects on
bone progenitor cell differentiation. Interestingly, inhibition of
class III HDACs with Sirtinol had a delayed effect on
chondrogenesis but was highly cytotoxic. This dramatic toxicity is
interesting since the penetrance of Sirtinol in limb bud cultures is
not clear. We found that treatment with Sirtinol induced the
hyperacetylation of tubulin in cultures of primary limb cells but
did not do this in cultures of the intact limbs. One possibility is
that Sirtinol affects only a subset of limb cells, perhaps those on
the surface of the organ. However, clear effects were observed
on the limb morphology. A similar outcome was observed in a
previous study investigating the effects of quantum dots in the
limb bud culture system (Bigaeva et al., 2014); fluorescent
quantum dots that were localized to the surface of the limb induced
global limb malformations as well as the inhibition of
chondrocyte and osteoblast differentiation. These observations suggest
that the epidermal surface of the limb can affect the
differentiation of the underlying mesenchyme.
MS275 and Sirtinol both triggered programmed cell death in
the developing limb. Teratogen-induced apoptosis in embryonic
tissue has been associated with several birth defects.
VPAinduced apoptosis in the somites of mouse embryos on
gestational day 9 has been associated with an increase in neural
(Di Renzo et al., 2010)
. The severity of the
malformations induced by treatment with hydroxyurea, an anticancer
drug, was correlated with an increase in apoptosis and DNA
fragmentation in specific tissues
(Banh and Hales, 2013;
Schlisser and Hales, 2013)
. Thus, the enhanced apoptosis
induced by HDAC class I and III inhibition in our limb model may
also be observed in other malformation-sensitive tissues and
organs during organogenesis. However, further studies are
needed to investigate the molecular signalling pathways
leading to this increase in cellular death.
The VPA-induced rapid decrease in COL2A1 expression and
decrease in mesenchymal condensation were remarkably
similar to those observed with MS275, suggesting that these may be
mediated through inhibition of HDAC class I, rather than class
II. In a previous study, we showed that VPA exposure induced a
rapid decrease in Sox9 and Runx2 gene expression; these effects
were correlated with HDAC inhibition since valpromide, an
analog of VPA lacking activity as an HDAC inhibitor, was inactive
(Paradis and Hales, 2013)
. Whether Sox9 and Runx2 signaling
pathways are affected by our drug treatments remains to be
The reduction in COL10A1 expression observed in MS-275
exposed limbs suggests that Runx2 signaling is affected by
inhibition of class I HDACs. As mentioned previously, RUNX2
interacts with HDACs and becomes acetylated. However, its
acetylation leads to an increase in its transcriptional activation
that is inconsistent with the decrease in expression of the
downstream targets that we observe in this study
(Jeon et al.,
. The limb skeleton develops through the same process as
other long bones, such as the ribs and vertebrae, via
endochondral ossification. The flat bones of the body, including the
bones of the skull, pelvis, scapula, and mandible, are formed
by intramembranous ossification. Both processes require Runx2
and Col10a1 expression, suggesting that the formation of the
entire skeleton may be affected by the inhibition of this
MS275, MC1568, and Sirtinol target HDAC1/3, HDAC4/6, and
SIRT1/2, respectively. HDAC1 knockout mice are embryonic
lethal prior to limb bud formation
(Montgomery et al., 2007)
In vitro, HDAC1 is involved in a number of pathways that
regulate chondrogenesis, such as Nkx3.2
(Cairns et al., 2012)
HDAC3 knockout mice are runted, have fewer osteoblasts than
their wild-type littermates, and exhibit a decrease in bone
(Razidlo et al., 2010)
. These data are consistent with our
results, suggesting that these members of the class I HDACs play
pivotal roles during limb differentiation.
The effects of MC1568 on limb development are inconsistent
with the phenotype observed in HDAC4 knockout mice, which
exhibit reduced cartilage formation, premature chondrocyte
hypertrophy, and ossification
(Vega et al., 2004)
. MC1568 has been
reported to inhibit matrix metalloproteinase 9 (MMP9) gene
expression in extraembryonic tissue primary cell cultures
et al., 2014)
. MMP9 is a matrix metalloproteinase that is essential
for endochondral ossification; metalloproteinases degrade the
cartilage extracellular matrix to create gaps where the
osteoblasts will deposit the bone matrix
(Ortega et al., 2003)
MC1568 may interfere with this degradation process in our
model, thereby hindering osteoblast differentiation and Col1a1
expression. More studies are needed to assess whether the
MC1568-triggered delay in ossification is transient or
Our results are consistent with the phenotype observed in
SIRT1 knockout mice; both the axial and appendicular skeletons
of these mice exhibit abnormalities, although these are not as
severe as the ones we observed in this study
(Gabay et al., 2013)
This observation suggests that SIRT1 and SIRT2 may have
complementary effects. Alternatively, Sirt1-/- mice may have
compensatory mechanisms with the consequence that a
pharmacologically induced transient inhibition of SIRT1 has a
more detrimental effect than its deletion.
This study provides valuable evidence for the diverse
teratogenic effects of three class-specific HDAC inhibitors on mouse
embryonic skeletal development. We showed that class I and III
inhibitors are highly teratogenic and cause drastic structural
malformations, whereas a class II inhibitor has only minor
effects on ossification. Identifying the molecular pathways and
targets leading to teratogenesis is pivotal to rapidly identify
potential developmental toxicants and prevent their effects.
We thank Dr. David L. Butler (University of Cincinnati,
Cincinnati, OH) and Dr. David Rowe (University of
Connecticut Health Center, Farmington, CT) for providing
the Col2a1-ECFP, Col10a1-mCherry, and Col1a1-YFP reporter
These studies were supported by grants from the Canadian
Institutes of Health Research (CIHR, grant number:
?MOP86511?) awarded to B.F.H. and Fonds de recherche du
Que? bec: Sant e? (FRQS) fellowship awarded to F.P. B.F.H. is a
James McGill Professor.
Supplementary data are available online at http://toxsci.
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