Effects of Thujaplicins on the Promoter Activities of the Human SIRT1 and Telomere Maintenance Factor Encoding Genes
Fumiaki Uchiumi1,2, Haruki Tachibana3, Hideaki Abe4, Atsushi Yoshimori5, Takanori Kamiya4, Makoto Fujikawa3, Steven Larsen2, Shigeo
Ebizuka4 and Sei-ichi Tanuma2,3,6,7*
1Department of Gene Regulation, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda-shi, Chiba-ken 278-8510, Japan
2Research Center for RNA Science, RIST, Tokyo University of Science, Noda-shi, Chiba-ken, Japan
3Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda-shi, Chiba-ken 278-8510, Japan
4Hinoki Shinyaku Co., Ltd, 9-6 Nibancho, Chiyoda-ku, Tokyo 102-0084, Japan
5Institute for Theoretical Medicine, Inc., 4259-3 Nagatsuda-cho, Midori-ku, Yokohama 226-8510, Japan
6Genome and Drug Research Center, Tokyo University of Science, Noda-shi, Chiba-ken 278-8510, Japan
7Drug Creation Frontier Research Center, RIST, Tokyo University of Science, Noda-shi, Chiba-ken 278-8510, Japan
*Corresponding author:
Sei-ichi Tanuma
Department of Biochemistry
Faculty of
Pharmaceutical Sciences
Tokyo University of Science
Tokyo University of Science
2641 Yamazaki, Noda-shi
Chiba-ken 278-8510, Japan Tel:+81-4-7124-1501 Fax:
+81-4-7121-3620 E-mail: tanuma@rs.noda.tus.ac.jp
Received June 30, 2012; Accepted July 06, 2012; Published July 12, 2012
Citation: Uchiumi F, Tachibana H, Abe H, Yoshimori A, Kamiya T, et al. (2012)
Effects of Thujaplicins on the Promoter Activities of the Human SIRT1 and
Telomere Maintenance Factor Encoding Genes. Pharmaceut Anal Acta 3:159.
doi:10.4172/2153-2435.1000159
Resveratrol (Rsv) has been shown to extend the lifespan of diverse range of species to activate Sirtuin (SIRT1)
family proteins, which belong to the class III NAD+ dependent histone de-acetylases (HDACs).The protein deacetylating
enzyme SIRT1 has been implicated in the regulation of cellular senescence and aging processes in
mammalian cells. However, higher concentrations of this natural compound cause cell death. Therefore, novel
compounds that have reduced cellular toxicity will be required for anti-aging therapy, especially for dermatological
treatments. In this study, the Luciferase (Luc) expression vector pGL4-SIRT1 containing 396-bp of the 5’-upstream
region of the human SIRT1 gene was transfected into HeLa S3 cells and Luc assay was performed. The results
showed that treatments with the natural compound, α-, β- and γ-thujaplicins increase the SIRT1 promoter activity
more than that with Rsv. Moreover, we carried out multiple transfection of Luc reporter vectors containing 5’-upstream
regions of various human telomere maintenance factor encoding genes, and observed that β−thujaplicin (hinokitiol)
activates TERT, RTEL, TRF1, DKC1, RAP1 (TERF2IP) and TPP1(ACD) promoters. These results suggest that
that the β−thujaplicin could be used as anti-aging drugs to delay cellular senescence through activating SIRT1 transcription along with strengthening stability of telomeres.
A natural polyphenolic compound Resveratrol (Rsv), which is
known as a stimulator of NAD+-dependent deacetylases sirtuin (SIRT1)
family, sirtuin, elongates lifespan of model animals [1-5]. Previously,
we reported that Rsv moderately activates the human SIRT1 and
TERT promoters inducing telomerase activity in HeLa-S3 cells [6,7].
Moreover, multiple transfection assays showed that promoter activities
of the genes encoding human telomere maintenance factors (shelterin
proteins) [8] are up-regulated by Rsv treatment [9], suggesting that
natural polyphenol compounds, such as Rsv may affect chromosomal
stabilities. Thus, Rsv and its related polyphenols are expected to become
candidate drugs for anti-aging therapeutics. However, it should be
noted that Rsv has cytotoxic effects by inducing apoptotic cell death,
especially when it is used at higher doses [10-12]. Thus, in order to
develop safe drugs with anti-aging effects, searching of alternative
natural compounds that up-regulate SIRT1 and shelterin gene
expression and their induction mechanisms should be investigated.
β-Thujaplicin, which is also known as hinokitiol, is a tropolone
derivative found in the heartwood of cupressaceous plants [13]. It has
been reported to have a variety of biological effects, including induction
of apoptosis [14] and differentiation [15], anti-inflammatory [16], antibacterial
[17] and anti-fungal [18] effects. In this study, we examined
the effects of thujaplicins on the promoter activities of the human
SIRT1 and shelterin-encoding genes by multiple transient transfection
and Luc reporter assay. Here, we show the up-regulating effects of three
types of thujaplicins (a, b and g) on the promoter activities of the human
SIRT1 and shelterin-encoding genes by multiple transient transfection and Luc reporter assay. Here, we show that b-thujaplicin (hinokitiol)
is able to up-regulate these promoter activities. Furthermore, we
propose that b-thujaplicin could be used as one of lead-compounds for
developing anti-aging drugs.
Materials and Methods
Materials
trans-Resveratrol was purchased from Cayman Chem. (Ann
Arbor, MI) [6,7]. α−, β- and γ-Thujaplicins were purchased from
Osaka Chemical Industry Ltd. (Osaka, Japan) [19]. Structures of these
compounds are shown in (Figure 1).
Figure 1:The structure of trans-Resveratrol and thujaplicins.
Cell culture
Human cervical carcinoma (HeLa S3) cells [20] were grown in
Dulbecco’s modified Eagle’s (DME) medium (WAKO-Pure Chemical,
Tokyo, Japan), supplemented with 10% fetal bovine serum (FBS)
(Sanko-Pure Chemical, Tokyo, Japan) and penicillin-streptomycin at
37°C in a humidified atmosphere with 5% CO2.
Construction of Luc reporter plasmids
The Luc reporter plasmid pGL4-SIRT1 carrying 396-bp of the
human SIRT1 promoter region was constructed as described previously
[7]. Other Luc reporter plasmids, which contain 300 to 500-bp of
5’-upstream regions of the human PIF1, RTEL, TRF1, TRF2, TERT,
TERC, TANK1, DKC1, TIN2, POT1, RAP1(TERF2IP) and TPP1(ACD) genes, were constructed as described previously [9,21].
Transient transfection and Luc assay
Plasmid DNAs were transfected into HeLa S3 cells by the DEAEdextran
method [20-22]. The DNA-transfected cells were divided
into at least four dishes. After 24h of transfection, Rsv or thujaplicins
were added to the culture medium. After a further 24 h of incubation,
cells were collected and lysed with 100 μL of 1 X cell culture lysis
reagent, containing 25 mM Tris-phospate (pH 7.8), 2 mM DTT, 2 mM
1,2-diaminocyclohexane-N,N,N’,N’,-tetraacetic acid, 10% glycerol and
1% Triton X-100, then mixed and centrifuged at 12,000 × g for 5 sec.
The supernatant was stored at -80°C. The Luc assay was performed
with a Luciferase assay system (Promega) and relative Luc activities
were calculated as described previously [20-22]. Multiple transfection
of human shelterin promoter-containing Luc reporter plasmids with
96-well culture plate was performed as described previously [8,21].
Results
Effects of thujaplicins on the human SIRT1 promoter
To examine whether the human SIRT1 promoter is affected by
α−, β− ανδ γ−thujaplicins [19], transient transfection and Luc assays
were carried out. Luc activities of pGL4-SIRT1 transfected cells were
normalized to that of non-treated control cells. As shown in Figure
2A, the relative Luc activity of pGL4-SIRT1-transfected cells was
prominently augmented by the addition of Rsv (10 μΜ) ορ α−, β− and
γ-thujaplicins (10 μM) to the culture medium.
Figure 2:Effects of thujaplicins on the human SIRT1 promoter activity. (A) The
Luc reporter plasmid, pGL4-SIRT1 [6,7], was transfected into HeLa S3 cells as
described under Materials and Methods. After 24 h of -thujaplicinσ−, ανδ −,Μ)
ανδ transfection, cells were treated with Rsv (10 M), then harvested after a
further 24 h incubation. (B) A similar (10 -thujaplicin.M of the experiment was
performed as in (A) with 0 to 100 The results show relative Luc activities of the
indicated Luc reporter plasmid-transfected cells relative to those of non-treated
cells. The values are the mean + SD of four independent assays.
To examine the dose-dependent response to β-thujaplicin
(hinokitiol), HeLa S3 cells were treated with 0 to 100 μM of
β-thujaplicin after 24 h of transfection and collected after further 24
h incubation (Figure 2B). The half maximal effective concentration
(EC50) was estimated as 3.1 mM. These results indicate that 10 mM of m-thujaplicin is enough to induce SIRT1 promoter activity equal to Rsv
(10 mM) treatment.
Effect of β−τhujaplicin on the 5’-upstream regions of human
genes encoding telomere maintenance factors
Multiple transcription experiments were carried out with various
Luc reporter plasmids containing 5’-flanking regions of the human
shelterin encoding genes (Figure 3) [9]. By performing the multiple
Luc assay, the effect of b-thujaplicin on these transcription-regulatory
regions were examined. The results showed that b-thujaplicin (10 mM)
could induce up-regulation of relative promoter activities of the RTEL,
TRF1, TRF2, TERT, DKC1, TIN2, RAP1(TERF2IP) and TPP1(ACD) genes (Figure 3). Approximate 1.5 to 2-folds increases as compared
with non-treated cells were observed in a similar manner as the 396-bp
of the SIRT1 promoter (Figure 2A).
Figure 3:The effect of β-thujaplicin on the promoter activities of 5’-upstream
regions of human shelterin encoding genes. Reporter plasmids (10 ng) and
DEAE-dextran were spotted and dried onto each well of the 96-well culture
plate. HeLa-S3 cells (1 x 105/well) were used for transfection and incubated
for further 24 h, then treated with β-thujaplicin (10 μM) for 24 h. Results show
relative Luc activities from various Luc reporter transfected cells compared
with that of pGL4-PIF1 transfected cells.
Discussion
It has been suggested that both cellular senescence and aging
of organisms are accelerated by various factors, such as telomereshortening
[23-25] and DNA damaging reactive oxygen species (ROS)
that are mainly generated from mitochondria [26,27]. Another facet or
aging is the demonstration that caloric restriction elongates lifespans
of organisms [28], suggesting that metabolism regulatory systems
controls lifespan. Genetic analyses of C.elegans showed that several
genes encoding insulin/IGF1 receptor and transcription factor FoxO
play important roles in controlling the lifespan [29]. Moreover, studies
of budding yeast showed that Sir2, a member of the sirtuin proteins
with an NAD+-dependent protein deacetylase activity, has silencing
action on chronological aging of yeast cells [30]. Many proteins,
including PGC-1α, p53, FOXO1, HIF1α, UCP2 and PPARγ, have been reported to be the targets of SIRT1, which is known as mammalian
homologue of Sir2 [31]. Because these protein factors function as
metabolism regulators, SIRT1 could be referred as a key regulator of
healthspan of organisms [31].
In this study, we have examined promoter activities of the 396-bp
5’-flanking region of the human SIRT1 gene to find out its response to
the treatments with three types of thujaplicins (a, b and g) in HeLa-S3
cells. The 396-bp region has no apparent TATA-box but contains
several well known transcription factor binding elements, including
CREB, C/EBPβ, c-ETS, USF, SREBP1, Sp1, GATA and c-MYC binding
motifs [7]. It has been shown that FOXO1, CREB, PPAR proteins and
PARP2 play roles in regulation of the SIRT1 promoter [31]. However,
at present, the Rsv or β-thujaplicin-responsive elements in the SIRT1 promoter region have not been precisely determined. Previously, it
was indicated that the 5’-upstream regions of the WRN, BLM, TERT,
p21 (CDKN1A) and HELB genes possess one or more Sp1/GC-box
elements and that they positively respond to Rsv treatment in HeLa S3
cells [6,32]. The GC-box consensus sequence of the Sp1 transcription
factor binding site is: 5’-(G/T)GGGCGG(G/A)(G/A)(C/T)-3’ or 5’-
(G/T)(G/A)GGCG(G/T)(G/A)(G/A)(C/T)-3’ [33]. It has been shown
that two GC-boxes, 5’-AGGGCGGGGG-3’ and 5’GGGGCGGGTC -3’
(-83 to -74 and -66 to -57, respectively), play important roles in the
SIRT1 promoter activity [34]. As shown in Figure 3, the 5’-upstream
regions of the RTEL, TRF1, TRF2, TERT, DKC1, TIN2, RAP1 and TPP1 genes positively responded to the treatment with β-thujaplicin. All of
the 5’-upstream regions in the Luc-reporter vectors except pGL4-RTEL
have at least one Sp1/GC-box. Although TF-search analysis did not
find Sp1/GC-box, 5’-CGGGCGGGAC-3’, 5’-TTTCCGCCGG-3’ and
5’-TGCGCGCCTC-3’, namely GC-box like sequences are contained
in the pGL4-RTEL. Taken together, the Sp1 binding motif is possibly
one of the candidate elements that respond to β-thujaplicin. Moreover,
Rsv is known to up-regulate cAMP level to activate CREB, which plays
important roles in hormonal metabolism, including that of the insulin
signaling system [35]. The CREB element is located in the 5’-upstream regions of the RTEL [21] and TPP1 [9] genes. This suggests that the
CREB element in the human SIRT1 promoter region (-288 to -281)
may respond to the b-thujaplicin treatment.
It should be noted that β−τhujaplicin (hinokitiol) stabilizes
transcriptional active HIF-1α in HeLa and HepG2 cells to increase
transcription of the VEGF gene [36]. On the other hand, SIRT1 deacetylates HIF-1α to suppress its activity [37]. Therefore, the
induction of SIRT1 gene expression by β−thujaplicin might be
required for reduction of over-stimulated HIF-1α to maintain cellular
homeostasis. Moreover, it has been reported that β-thujaplicin
induces G1 arrest via down-regulation of phosphorylated Rb and Skp2
ubiquitin ligase [38]. This cell cycle arrest is accompanied with an
increase of p27 and p21 protein levels. Although the precise molecular
mechanisms are not known, these biological properties of β-thujaplicin
including transcriptional regulation and antiviral activity [16,36] might
originate from its specific structure (Figure 1) that can act as a chelator
of divalent metal ions [39]. In this study, we observed the up-regulation
of the SIRT1 and shelterin-encoding gene promoter activities by the
treatment of β−thujaplicin. Moreover, comparison of 5’-upstream
regions of those genes suggested that transcription factors, including
Sp1, may control lifespans of organisms responding to β-thujaplicin.
The core structure of the thujaplicins could be applied to design lead
compounds for novel anti-aging drugs, which could simultaneously
activate the SIRT1 and shelterin-encoding gene promoter activities.
Acknowledgements
The authors are grateful to Tsutomu Iijima and Masanao Taniura for their
outstanding technical assistance. This work was supported in part by a Research
Fellowship grant from the Research Center for RNA Science and Drug Creation
Frontier Research Center, RIST, Tokyo University of Science.
Uchiumi F, Tachibana H, Larsen S, Tanuma S (2012) Effect of lignin glycosides extracted from pine cones on the human SIRT1 promoter. Pharm Anal Acta S8: 001.
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