An efficient two-step synthesis of 3-amino-1-benzhydrylazetidine

Article abstract of DOI:10.1002/jcb.20940

A streamlined process for the synthesis of 3-amino-1-benzhydrylazetidine is described. Commercially available 1-benzhydrylazetidin-3-ol was reacted with methanesulfonyl chloride in the presence of triethylamine in acetonitrile, upon quench with water, the

Full text of DOI:10.1002/jcb.20940

Journal of Cellular Biochemistry 99:478–484 (2006)
Regulation of dHAND Protein Expression by All-Trans
Retinoic Acid Through ET-1/ETAR Signaling in H9c2 Cells
Weixin Li and Yong Li*
Laboratory of Development Molecular Biology, Department of Nutrition and Food Hygiene,
School of Public Health, Peking University Health Science Center, Beijing 100083, China
Abstract
dHAND is thought to be a cardiac-restricted transcription factor during embryonic development.
Vertebrate heart development involves many transcription factors such as Nkx2.5, GATA, and tbx5. All-trans retinoic acid
(AtRA), the oxidative metabolite of vitamin A, can regulate the expression of these factors to affect embryonic heart
development. However, the action of atRA on the expression of dHAND is rarely reported. To clarify whether atRA
regulate the dHAND expression, we exposed cultured H9c2 cells (rat embryonic cardiomyocytes) to atRA and detected
the protein expression of dHAND by Western blot analysis. We observed atRA can regulate the dHAND expression in a
dose- and time-dependent manner. AtRA also inhibited endothelin-1 (ET-1) expression in a time-dependent manner.
Further studies revealed that pretreatment with 10 mM BQ-123, a selective endothelin-1 receptor (ETAR) antagonist, for 2 h
can significantly counteract the inhibition of 5 mM atRA treatment for 2 h of dHAND mRNA and protein expression. Taken
together, these results suggest that atRA regulates dHAND expression by ET-1/ETAR signal transduction pathway in H9c2
cells. The mechanism of ET-1/ETAR signaling in controlling the level of dHAND protein is to reduce the levels of dHAND
mRNA. It is possible for atRA to exert its cardiac teratogenesis during vertebrate embryonic development in this way.
J. Cell. Biochem. 99: 478–484, 2006. ß 2006 Wiley-Liss, Inc.
Key words: all-trans retinoic acid; H9c2 cells; dHAND; ET-1; signal
Vertebrate heart development involves many
transcription factors such as dHAND, Nkx2.5,
and GATA [Lyons, 1996]. The proper temporal
and spatial expression of these factors governs
the normal development of the vertebrate heart.
Any situation affecting correct expression of
these heart specific transcription factors may
influence cardiac development. It has been
reported that a deficiency of vitamin A and its
active derivatives such as retinoic acid (RA)
results in congenital heart defects [Smith et al.,
1998]. RA is necessary for early fetal cardiogen-
esis. Supplementing with excess vitamin A or
RA during the gestational period leads to
neonatal cardiac malformations [Tembe et al.,
1996; Collins and Mao, 1999].
Retinoic acid may regulate vertebrate cardi-
ogenesis by affecting some transcription factors
such as Nkx2.5 [Pellizzer et al., 2004], GATA-4,
and tbx5 [Liberatore et al., 2000]. dHAND is a
basic helix-loop-helix (bHLH) transcription fac-
tor. It is expressed in mesodermal and neural
crest-derived structures during heart develop-
ment. dHAND plays an essential role in the
formation of the right ventricle, trabeculae, and
neural crest-derived aortic arches. It is also
involved in the regulation of chamber specifica-
tion, cardiac looping, and the cardiac neural
crest [Srivastava et al., 1997]. During the zone
of polarizing activity (ZPA) formation in verte-
brate limb development, RA acts early to
activate expression of dHAND as an upstream
signal [Mic et al., 2004]. All-trans retinoic acid
(AtRA) suppresses endothelin-1 (ET-1) mRNA
expression in cultured endothelial cells [Yokota
et al., 2001]. ET-1 is a 21-amino acid peptide.
It has been found to be produced and secreted
by diverse types of cells and to elicit a wide
Abbreviations used: ET-1/ETAR, endothelin-1/endothelin-1
receptor; atRA, all-trans retinoic acid; bHLH, basic helix-
loop-helix; ZAP, zone of polarizing activity.
Grant sponsor: National Natural Science Foundation,
People’s Republic of China; Grant number: 30371208.
*Correspondence to: Prof. Yong Li, Department of Nutri-
tion and Food Hygiene, School of Public Health, Peking
University, Beijing 100083, China.
E-mail: liyong@bjmu.edu.cn
Received 31 December 2005; Accepted 2 March 2006
DOI 10.1002/jcb.20940
ß 2006 Wiley-Liss, Inc.

Regulation of dHAND by atRA in H9c2 Cells
479
spectrum of biological effects. ETA receptors are
preferentially activated by ET-1 and mediate
biological actions of ET-1 [Chandan et al., 2003].
ET-1 mainly acts as a locally-active autocrine/
paracrine factor rather than as a circulating
hormone [Giannessi et al., 2001]. Many studies
[Charite et al., 2001; Mina, 2001; Ivey et al.,
2003; Yanagisawa et al., 2003; Fukuhara et al.,
2004] have indicated that ET-1/endothelin-1
receptor (ETAR) signaling is crucial for the exp-
ression of dHAND in embryogenesis. dHAND
serves as a downstream target for ET-1/ETAR
signaling.
All the facts and studies mentioned above
prompted us to think that it is possible for RA to
regulate the expression of dHAND by ET-1/
ETAR signaling during vertebrate heart devel-
opment. AtRA may exert its cardiac teratogen-
esis in this way. Most previous studies used
were practiced by vertebrate whole-embryo
culture and explant cultures but rarely done
on cultured cells in vitro. In this article, we
aimed to explore whether atRA regulates the
expression of dHAND in H9c2 cells (rat embryo-
nic cardiomyocytes) and if so, its underlying
mechanism(s). We present data showing that
atRA inhibits the expression of dHAND in H9c2
cells in a time- and dose-dependent manner.
AtRA may suppress the dHAND expression by
ET-1/ETAR signaling. The mechanism of ET-1/
ETAR signaling in controlling the level of
dHAND protein is to reduce the levels of
dHAND mRNA. These results may be helpful
for interpreting the mechanisms of cardiac
teratogenesis by atRA and vitamin A.
supplemented with 10% FCS, 100 U/ml of
penicillin and 100 mg/ml streptomycin, in an
atmosphere of 5% CO2/95% humidified air.
H9c2 cells were grown to approximately 80%
confluence, at which point the medium was
changed to serum-free DMEM. Twenty-four
hours later, different doses of atRA [dissolved
in dimethyl sulfoxide (DMSO)] were added and
cells were incubated in the dark for various
times and different doses for different purposes.
Western Blot
After treatment with or without atRA, H9c2
cells were rapidly chilled on ice, washed twice
with ice-cold washing buffer (10 mM Tris-HCl,
150 mM NaCl, 1 mM Na3VO4 [pH7.5]), and then
lysed in 300 ml (per 106 cells) lysis buffer [10 nM
Tris (pH7.4), 300 nM NaCl, 1 nM EDTA, 10 mM
MgCl2, 2 mM DTT, 5 mM phenylmethysulfonyl
fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin,
and 0.5% Nonidet P-40]. The lysis products were
centrifuged at 12,000g for 15 min, the super-
natent was collected, and protein concentra-
tions were measured by Bradford method.
Samples of H9c2 cell extract were subjected to
SDS/PAGE and then electrophoretically trans-
ferred to a PVDF membrane (Millipore Co.).
Non-specific binding was blocked by incubating
the membranes with phosphate buffered saline
containing 1% BSA and Tween-20 (PBST).
Western blotting was performed using 1:200
dilution of dHAND or propre-endothelin-1 anti-
body as the primary, a 1:10,000 dilution of
horseradish peroxidase-conjugated anti-anti-
body as the secondary and an ECL detection
system. The membranes were exposed to Kodak
XAR film. Quantification of the relative abun-
dance of dHAND or endothelin-1 protein was
done by volume densitometric analysis of the
films using SmartView software (Shanghai
FURI Science & Technology Co., Ltd). All the
values were normalized to the values obtained
with b-actin to correct for differences in loading
and transfer efficiency.
MATERIALS AND METHODS
Materials
All-trans retinoic acid, Dulbecco’s modified
Eagle’ medium (DMEM), bovine serum albumin
(BSA), BQ-123, Western blotting luminol
reagent were purchased from Sigma (St.Louis,
MO). Fetal calf serum (FCS) was from HyClone.
The polyclonal antibodies to dHAND and b-
actin were from Santa Cruz Biotechnology
(Santa Cruz, CA). The anti-Propre-endothelin-
1 IgG was obtained from Phoenix Pharmaceu-
ticals, Inc.
RNA Isolation and Northern Blot Analysis
After H9c2 cells were treated with 5 mM atRA
for 2 h in the presence or absence of 10 mM
BQ-123, total cellular RNA was isolated with
Trizol reagent (Invitrogen) following the recom-
mended protocol. Twenty micrograms of total
RNA was size-fractioned on formaldehyde-
agarose gel (1%), transferred onto a nylon
H9c2 Cell Line and Culture
H9c2 cells from the American Type Culture
Collection (ATCC, CRL-1446) were maintained
by our laboratory. Cells were cultured in DMEM

480
Li and Li
membrane (Hybond Nþ; Amersham Biosciences)
by capillary blotting in the presence of 20 ꢀ SSC
(300 mM sodium chloride and 300 mM sodium
citrate), and then immobilized by incubating
the membrane for 2 h at 808C. Blots were hy-
bridized with random prime-labeled rat cDNA
probes for dHAND or glyceraldehyde-3-phos-
phate dehydrogenase (GAPDH). The probes
were prepared as previously described [Thomas
et al., 1998] and were labeled with [32P] dCTP by
a Prime-a-Gene labeling system (Promega). The
membrane was washed with 0.1 ꢀ SSC contain-
ing 1% sodium dodecyl sulfate (SDS) at 428C for
30 min and then exposed to X-ray film at ꢁ708C.
Blots of specific mRNA bands were detected by
autoradiography and analyzed with a densit-
ometer (Shanghai FURI Science & Technology
Co., Ltd). The level of expression of dHAND
mRNA was quantified and was normalized to
the GAPDH signal.
Fig. 1. The time course of effects of atRA on dHAND protein
expression. (A) H9c2 cells were exposed to atRA (5 mM) for the
times indicated. The protein expression of dHAND was deter-
mined by Western blotting. (B) The protein levels of dHAND
were quantified by densitometric scanningand normalized to the
levels of b-actin. Data represent the average fold of controls from
six independent experiments (mean ꢂ SD). *P < 0.05 versus
control.
Statistical Analysis
Data are presented as mean ꢂ SD (standard
deviation). The data were evaluated by un-
paired Student’s t-test or one-way ANOVA
using SPSS 13.0 (SPSS, Inc., Chicago, IL)
program. Following the one-way ANOVA, the
least significant difference (LSD) test or the
Student-Newman-Keuls (S-N-K) q test was per-
formed as a post hoc multiple comparision.
P < 0.05 was considered statistically significant.
RESULTS
The Regulation by AtRA of dHAND Protein
Expression in H9c2 Cells
To investigate the effects of atRA on the
protein expression of dHAND, we employed the
Western blot assay. AtRA (5 mM) significantly
inhibited the protein expression of dHAND
after atRA treatment for 2, 6, 12, and 24 h
(P < 0.05; Fig. 1). When H9c2 cells were exposed
to atRA for 2 h, the dHAND protein expression
is the lowest among all the treatment groups
compared with control cells that were treated
only with vehicle (indicated as 0 mM). AtRA also
inhibited the dHAND protein expression in a
dose-dependent manner (Fig. 2). AtRA (5 mM
and 10 mM) inhibited the dHAND protein
expression by 88.51% and 89.53%, respectively
compared with 0 mM atRA as the control
(P < 0.05). The level of dHAND following 24 h
exposure to RA increased compared with the 2 h
treatment (P < 0.05).
Fig. 2. AtRA inhibits the protein expression of dHAND in a
dose-dependent manner. (A) H9c2 cells were treated with
different doses of atRA for 2 h. Cell extracts were subjected to
10% SDS–PAGE and immunoblotted with an antibody specific
for dHAND. (B) The protein levels of dHAND were quantified by
densitometric scanning and normalized to the levels of b-actin.
Data represent the average fold of controls from six independent
experiments (mean ꢂ SD). *P < 0.05 versus control.

Regulation of dHAND by atRA in H9c2 Cells
481
Fig. 3. The time course of effects of atRA on ET-1 protein
expression. (A) H9c2 cells were exposed to atRA (5 mM) for the
times indicated. The protein expression of ET-1 was determined
by Western blotting. (B) The protein levels of ET-1 were quan-
tified by densitometric scanning and normalized to the levels of
b-actin. Data represent the average fold of controls from six
independent experiments (mean ꢂ SD). *P < 0.05 versus control.
Fig. 4. Role of ET-1/ETA signal pathway in the atRA-induced
decrease in dHAND expression. (A) H9c2 cells were exposed to
atRA (5 mM) for 2 h, in the presence or absence of BQ-123
(10 mM). Cell extracts were subjected to 10% SDS–PAGE and
immunoblotted with an antibody specific for dHAND. (B) The
protein levels of dHAND were quantified by densitometric
scanning and normalized to the levels of b-actin. Data represent
the average fold of controls from six independent experiments
(mean ꢂ SD). a, b, different letters represent P ꢂ 0.05 versus each
other.
The Regulation by AtRA of ET-1 Protein
Expression in H9c2 Cells
Based on the effects of atRA on dHAND
protein expression, we treated H9c2 cells by
5 mM atRA for different times and found the
ET-1 protein level was lowered versus control
after atRA treatment for 2, 6, 12, and 24 h
(P < 0.05; Fig. 3). When H9c2 cells were exposed
to atRA for 2 h, the ET-1 protein expression is
the lowest among all the treatment groups
compared with control cells that were treated
only with vehicle (indicated as 0 mM). ET-1
protein level also increased after 24 h of RA
treatment compared with the 2 h treatment
(P < 0.05).
treatment can significantly attenuate the inhi-
bition by atRA of dHAND protein level (P <
0.05; Fig. 4). This indicates atRA exert its
inhibition of dHAND protein expression by
ET-1/ETA signal pathway.
The Role of ET-1/ETA Signaling in the Regulation
of AtRA to dHAND mRNA Expression
The Role of ET-1/ETA Signaling in the Regulation
of AtRA to dHAND Protein Expression
Based on the results mentioned above, we
pretreated H9c2 cells with 10 mM BQ-123 for 2 h
and then exposed the cells to 5 mM atRA for 2 h.
We found 5 mM atRA treatment for 2 h decreased
the dHAND mRNA level by 71.57% while 10 mM
BQ-123 pretreatment for 2 h before 5 mM atRA
treatment can significantly attenuate the inhi-
bition of atRA to dHAND mRNA level (P < 0.05;
Fig. 5). This indicates atRA exerts its inhibition
of dHAND mRNA expression by ET-1/ETA
signal pathway.
To test whether ET-1/ETA signal pathway
plays a role in the regulation of atRA to dHAND
protein expression, H9c2 cells were pretreated
with 10 mM BQ-123 for 2 h and then exposed to
5 mM atRA for 2 h. BQ-123 is a kind of selective
blocking agent for ETA receptor and can block
ET-1/ETA signal transduction pathway effec-
tively. We found 5 mM atRA treatment for 2 h
decreased the dHAND protein level while 10 mM
BQ-123 pretreatment for 2 h before 5 mM atRA

482
Li and Li
HAND2, Th2, and exd [Srivastava et al., 1997].
In the developing heart, dHAND is initially
expressed in the precardiac mesoderm. It is also
expressed throughout the linear heart tube,
but becomes restricted predominantly to the
future right ventricular compartment during
cardiac looping [Srivastava et al., 1995, 1997].
dHAND mutants exhibit hypoplasia of the
right ventricle, branchial arches, and aortic
arch arteries [Srivastava, 1999]. RA, derived
from the nutrient vitamin A, has potent cardiac
teratogenicity. RA receptors are expressed
throughout heart. A major function of RA
is transcriptional regulation through its nuclear
receptors. RA-responsive genes are found in all
cardiac compartments (myocardium, endocar-
dium, cushion mesenchyme). Various cardiac
malformations can be observed in RA receptor
null mutant mice [Smith and Dickman, 1997]. It
has been reported that RA affects heart devel-
opment by regulating transcription factors such
as Nkx2.5, GATA, and tbx5. But whether RA
regulates dHAND to affect heart development
still remains to be elucidated. Mic et al. [2004]
proved that RA acts early to activate the ex-
pression of dHAND during ZPA in vertebrate
limb development. This caused us to assume
that atRA may regulate dHAND expression in
embryonic cardiomyocytes. Our results also
indicate that atRA regulates the expression of
dHAND in a time- and dose-dependent manner
in cultured H9c2 cells. AtRA (5 mM) treatment
for 2 h inhibited the expression of dHAND to the
maximal degree. High levels of atRA may exert
their cardiac teratogenesis by suppressing
dHAND expression. There is an apparent
increase in the level of dHAND following 24 h
exposure to RA compared with the 2 h treat-
ment. This is caused by the time course effects of
atRA on the protein levels of ET-1.
Fig. 5. Northern blot analysis of dHAND mRNA in H9c2 cells.
(A) H9c2 cells were exposed to atRA (5 mM) for 2 h, in the
presence or absence of BQ-123 (10 mM). Total RNA was
extracted from cells and Northern blot analyses were performed.
(B) The mRNA levels of dHAND were quantified by densito-
metric scanning and normalized to the mRNA levels of GAPDH.
Data represent the average fold of controls from six independent
experiments (mean ꢂ SD). a, b, different letters represent
P ꢂ 0.05 versus each other.
DISCUSSION
The present study demonstrated that atRA
suppressed dHAND protein expression in a
time- and dose-dependent manner in cultured
H9c2 cells. AtRA (5 mM) inhibited the expres-
sion of ET-1 protein time-dependently. AtRA
(5 mM) treatment for 2 h inhibited the expres-
sion of dHAND protein and ET-1 protein to the
greatest extent. If blocked ETA receptors using
their specific repressor BQ-123 beforehand, we
observed the dHAND protein expression increa-
sed compared with that of only 5 mM atRA
treatment for 2 h. BQ-123 (10 mM) also can
significantly attenuate the inhibition of atRA to
dHAND mRNA level. AtRA appears to regulate
the protein expression of dHAND through the
ET-1/ETAR signaling pathway. The mechan-
ism of ET-1/ETAR signaling in controlling the
level of dHAND protein is to reduce the level of
dHAND mRNA.
We next explored the possible mechanism(s)
of the inhibitory effect of atRA on dHAND
protein expression. Endothelins (ETs, including
ET-1, ET-2, and ET-3) is a peptide family of 21
amino acids that were originally isolated from
the culture medium of porcine aortic endothelial
cells [Yanagisawa et al., 1988]. ETs exerts a
variety of biological effects including vasocon-
striction and cell proliferation via G protein-
coupled endothelin receptors type A and B
[Kurihara et al., 1999; Kedzierski and Yanagi-
sawa, 2001]. ETA receptors exist mainly on
vascular smooth muscle cells and respond to
ET-1. Many types of cells can produce and
dHAND is a bHLH transcription factor cloned
in 1995. It was given different names such as

Regulation of dHAND by atRA in H9c2 Cells
483
secrete ET-1 which mainly acts as a locally-
active autocrine/paracrine factor. Signalings
through ET receptors are essential for normal
embryonic development of subsets of neural
creast cell derivatives. ET-1, as well as ETA
receptor, also has an important role in cardio-
vascular development, as observed by the
variety of abnormalities related to neural
crest-derived tissues in mouse embryos defi-
cient in a member of the ET-1/ETAR pathway
[Giannessi et al., 2001]. RA, leptin, prostaglan-
dins, hypoxia, etc. are modulators of the ET
system. Yokota et al. [2001] have proved that
atRA suppressed ET-1 mRNA expression in
cultured endothelial cells. Consistent with this,
we further observed that atRA inhibited the
expression of ET-1 in a time-dependent manner.
AtRA (5 mM) treatment for 2 h can significantly
inhibited the expression of ET-1 in cultured
H9c2 cells. While the level of ET-1 following 24 h
exposure to RA apparently increased in com-
pared with that of the 2 h treatment. This may
be the adaptive response of cells to extraneous
stimulating factors. It is possible for cells to
initiate some potential protective mechanism(s)
unknown to us. Taking cues from the consis-
tency of the time course effects of atRA on
dHAND and ET-1 protein expressions, we
assumed that there should be some linkage(s)
between the expression of dHAND and ET-1 in
H9c2 cells.
It has been proved that ET-1 can regulate
dHAND expression during embryonic develop-
ment. dHAND is a ET-1-dependent transcrip-
tion factor. In ET-1 mutant embryos, dHAND
expression in the branchial arches is down-
regulated, implicating it as a transcriptional
effector of ET-1 action [Charite et al., 2001]. ET-
1/ETAR and subsequent epithelial signals are
sequentially involved in branchial arch devel-
opment by maintaining dHAND and Dlx6 ex-
pression [Fukuhara et al., 2004]. Yet the role of
ET-1/ETAR signaling in the regulation of atRA
to dHAND expression in heart development still
remains to be determined. We tentatively used
BQ-123, a selective inhibiter of ETAR, to block
the ET-1/ETAR signaling pathway before atRA
treatment in cultured H9c2 cells and observed
the dHAND protein expression was signifi-
cantly increased compared with that of only
atRA treatment. This caused us to hypothesize
that atRA first inhibited ET-1/ETAR signaling,
while cells produced and secreted ET-1 to act on
themselves and cells in the peripheral region
adjacent to them. With the inhibition of ET-1/
ETAR signal, dHAND protein expression
reduced correspondingly. AtRA may exert its
teratogenic action and lead to cardiac malfor-
mations in this way eventually. Therefore, we
came to the conclusion that atRA regulates the
expression of dHAND through ET-1/ETAR
signaling pathway in cultured H9c2 cells.
Further exploration indicated that the mechan-
ism of ET-1/ETAR signaling in controlling the
level of dHAND protein is to reduce the levels of
dHAND mRNA.
These results may have significance in illu-
minating the signal transduction pathways of
atRA in embryonic developing heart. They
may also be helpful to understand the cardiac
teratogenesis of atRA and its underlying
mechanism(s). Vertebrate heart development
is a very complicated course involving many
cellular and molecular events. Various related
protein-signaling molecules interact with each
other and form a complex network fine-tuning
cardiac development temporally and spatially.
Factors in vivo and in vitro affecting these
molecules will cause a delicate imbalance of this
network and influence heart development. We
prove that atRA suppresses dHAND expression
through ET-1/ETAR signal transduction path-
way in cultured H9c2 cells. AtRA may induce
cardiac malformations in vertebrate developing
embryos. Further studies are needed to explore
whether there are other signaling molecules
such as Dlx6, etc. functioning as segments of the
signaling pathway from ET-1 to dHAND. AtRA
may also regulate dHAND expression through
other pathways. The regulation by atRA of
dHAND may be different in different types of
cells and tissues. All these prospective ques-
tions may generate future research projects.
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