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Transcript of HDAC4 REGULATES HIF1α LYSINE ACETYLATION AND CANCER ...
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HDAC4 REGULATES HIF1 LYSINE ACETYLATION AND CANCER CELL
RESPONSE TO HYPOXIA
Hao Geng, Chris T. Harvey, Janet Pittsenbarger, Qiong Liu, Tomasz M Beer, Changhui Xue, and
David Z. Qian
From OHSU Knight Cancer Institute
Oregon Health & Science University
Portland, Oregon 97239
Running head: HDAC4 regulates HIF1
Address correspondence to: David Z. Qian, PhD, 3303 SW Bond Avenue, CH14R, OHSU,
Portland, Oregon 97239, Tel: 503-312-5912, Fax: 503-494-6197, Email: [email protected]
Keywords: HDAC4, HIF1Acetylation,
Background: HIF1 is a target of anticancer
therapy.
Results: Lysines within HIF1 N-terminus are
targets of HDAC4 deacetylation. HDAC4
inhibition causes the increase of HIF1 protein
acetylation and decrease of protein stability, which
lead to the reduction of HIF-1 mediated target
gene expressions and activities in cancer cells.
Conclusion: A novel HIF1 regulatory
mechanism by HDAC4.
Significance: HIF-1 can be targeted by HDAC4
inhibition.
Hypoxia-inducible factor 1 alpha (HIF1) is
an essential part of the HIF-1 transcriptional
complex that regulates angiogenesis, cellular
metabolism and cancer development. In VHL-
null kidney cancer cell lines, we previously
reported that HIF1 proteins can be acetylated
and inhibited by histone deacetylase inhibitors
(HDACi) or specific siRNA against HDAC4. To
investigate the mechanism and biological
consequence of the inhibition, we have
generated stable HDAC4 knockdown via
shRNA in VHL-positive normal and cancer cell
lines. We report that HDAC4 regulates HIF1
protein acetylation and stability. Specifically,
the HIF1 protein acetylation can be increased
by HDAC4 shRNA and decreased by HDAC4
overexpression. HDAC4 shRNA inhibits HIF1
protein stability, in contrast, HDAC1 or
HDAC3 shRNA has no such inhibitory effect.
Mutations of the first five lysine residues (lysine
10, 11, 12, 19 &21) to arginine within the
HIF1N-terminus reduce protein acetylation,
but render the mutant HIF1 protein resistant
to HDAC4 and HDACi-mediated inhibition.
Functionally, in VHL-positive cancer cell lines,
stable inhibition of HDAC4 decreases both the
HIF-1 transcriptional activity and a subset of
HIF-1 hypoxia - target gene expression. On the
cellular level, HDAC4 inhibition reduces the
hypoxia-related increase of glycolysis and
resistance to docetaxel chemotherapy. Taken
together, the novel biological relationship
between HDAC4 and HIF1 presented here
suggests a potential role for the deacetylase
enzyme in regulating HIF-1, cancer cell
response to hypoxia, and presents a more
specific molecular target of inhibition.
Hypoxia-inducible factor 1 alpha (HIF1) is an
essential part of the HIF-1 transcriptional complex
that regulates gene expressions critical to cellular
response and adaptation to hypoxia (1,2). HIF-1 is
comprised of an alpha () and a beta () subunit.
The transcriptional activity of HIF-1 is primarily
determined by the availability of HIF1 (3,4). In
addition, transcriptional cofactors including
histone acetyltransferases (HATs) such as p300
and histone deacetylases (HDACs) have been
reported to interact with and influence HIF-1
activity (5-9). However, the relationship between
HIF-1 and these cofactors is not fully understood.
http://www.jbc.org/cgi/doi/10.1074/jbc.M111.257055The latest version is at JBC Papers in Press. Published on September 14, 2011 as Manuscript M111.257055
Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
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Biologically, the HIF1 protein is constantly
synthesized, but rapidly degraded by oxygen under
non-hypoxic conditions. The newly synthesized
HIF1 protein is post-translationally modified by
hydroxylations at proline (P) residues 402 and 564
via oxygen-dependent prolyl hydroxylases (PHDs)
(1). The von Hippel-Lindau (VHL) protein then
binds hydroxylated HIF1, and recruits an E3
ubiquitin ligase complex that targets HIF1 for
26S proteasome mediated degradation (10). There
are also oxygen-independent mechanisms in
HIF1 protein regulation. Hsp90 is a key
molecular chaperon in maintaining HIF1 stability
(11). In contrast, RACK1, Hsp70 and CHIP
promote HIF1 degradation (12,13).
Besides hydroxylation, the HIF1 protein can
also be post-translationally modified by reversible
lysine (K) acetylation (14-16), which can be
pharmacologically modulated by HDAC inhibitors
(HDACi) (16-18). Treating cells with HDACi also
reduces HIF1 on the protein level under
normoxic, hypoxic and hypoxia mimic conditions
(16,19-22). The protein inhibition is dependent on
the 26S proteasomal degradation system, but can
be VHL-independent (16,19). Two of the
acetylation sites within HIF1 protein have been
identified at residues K532 and K674 (14,15).
Functionally, the acetylation at K532 can lead to
VHL-dependent HIF1 protein degradation (14).
In contrast, the acetylation at K674 is required for
HIF-1 transcriptional activity (15).
Currently, the mechanism and the functional
consequence of HIF1 acetylation / deacetylation
at different lysine residues are unclear. While the
identity of HDAC isozymes deacetylating K532 is
unknown (14), the K674 deacetylation is mediated
by a class III HDAC, Sirt1 (15). Previously, we
have shown that the inhibition of class II HDAC
isozymes HDAC4 and HDAC6 via siRNA inhibits
HIF1 protein in VHL-null kidney cancer cell
lines. The HDAC6 siRNA-mediated HIF1
inhibition is thought to be related with the
acetylation of Hsp90, which disrupts the Hsp90
chaperone function toward its client proteins
including HIF1 (16,19). However, the
mechanism for HIF1 inhibition via HDAC4
siRNA is unclear. HDAC4 siRNA does not
increase Hsp90 acetylation, nor disrupts the
interaction between HIF1 and Hsp90/Hsp70
(16). On the other hand, the inhibition of HDAC4,
not HDAC6, increases HIF1 protein acetylation
(16). These results suggest that HDAC4 can
regulate HIF1 protein acetylation and stability.
In this study, we first recapitulated the HIF1
protein acetylation and inhibition by stable
HDAC4 shRNA knockdown in VHL-positive
prostate and liver cancer cell lines. We then
identified that the HIF1 N-terminus lysine
residues are targets of HDAC4, and associated
with HIF1 sensitivity toward HDAC inhibition.
Further, we observed that stable HDAC4
knockdown attenuates cancer cell response and
adaptation to hypoxia in terms of HIF-1 mediated
gene transcriptional upregulation, glycolysis and
chemoresistance.
Cell lines and reagents - Hep3Bc1 is a gift
from Dr. Gregg Semenza at Johns Hopkins
University. It was engineered by stable
transfection of plasmids for hypoxia/HIF-1
response element (HRE)-driving firefly luciferase
reporter (p2.1) and constitutive renilla luciferase
reporter (pSV-renilla) in human liver cancer cell
line Hep3B (23). The prostate cancer C42B cell
line was a gift from Dr. John Isaacs at Johns
Hopkins University. The Hek293 cells were kindly
provided by Dr. Zhengfeng Zhou’s lab at OHSU.
The Hek293T cells were purchased from ATCC.
All cell lines were maintained as previously
described (23,24). Cell culture hypoxic condition
was defined as 1% oxygen, 5% CO2 and 94%
nitrogen. Cycloheximide (CHX), cobalt chloride
(CoCl2), HDACi-SAHA, and MG132 were
purchased from Sigma and Cayman.
Plasmids and transient transfection – Plasmids
encoding for 3×Flag-HIF1, p2.1, and constitutive
pSV-renilla were gifts of Dr. Gregg Semenza at
Johns Hopkins University. Plasmids encoding HA-
HIF1, Flag-HDAC1, and Flag-HDAC4 were
purchased from Addgene (Cambridge, MA).
Transfection was performed using Fugene6
(Roche). All transfections were equalized with
empty vector (Ev) to ensure the same DNA
amount.
Stable shRNA knockdown cell lines – The
pLKO.1-puro vector-based lentiviral transduction
particles containing HDAC1, HDAC3, HDAC4
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and HIF1 shRNA or scramble control were
purchased from Sigma.
Immunoprecipitation (IP) and antibodies – IP
was performed by incubating whole cell lysates
with the primary antibody at 4 degree followed by
incubation with protein A/G plus-agarose beads
(Santa Cruz). For Flag- and HA- based IP, anti-
Flag M2 affinity gel and anti-HA agarose were
purchased from Sigma. The beads were
extensively washed with lysis buffer, and the
associated proteins were eluted and analyzed by
western blots. Key antibodies used for IP and
western blots include anti-HIF1 (R&D Systems),
anti-acetyl-lysine (Ace-K) (Cell Signaling), anti-
Flag (Sigma), anti-HA (Santa Cruz) and anti-
HDAC4 (Cell Signaling).
HIF1 protein half-life measurement using
cycloheximide (CHX) - cells were seeded in 10-cm
culture dishes. HIF1 proteins were induced by
CoCl2 (150 M) for 6 hours. Then, protein
synthesis inhibitor - CHX was added into the
media, and proteins were harvested at the
indicated time points. Western blotting for HIF1
and -tubulin were scanned by LI-COR Odyssey
Fluorescence Scanner, and quantified using LI-
COR odyssey Infrared software.
Site-directed mutagenesis – We used the wild
type 3xFlag HIF1 plasmid (HIF1-wt) as the
template and two-step PCR to generate the mutant
HIF1 (HIF1-mut), which contains the lysine to
arginine mutation at residues K10, K11, K12, K19
and K21. The resulted HIF1-mut plasmid was
sequenced to confirm to mutations.
Real time quantitative RT-PCR – qRT-PCR
was performed as previously described by us (24),
the ΔΔCt method was used to calculate mRNA
transcripts level.
Reporter gene assay - Reporter assays using
Hep3Bc1 were done as described (23). For
transient assays in Hek293, C42B cell lines, cells
were co-transfected by 1.0 g of plasmid p2.1, and
0.1 g of plasmid pSV-renilla. For assays in
Hek293 cells, 20 ng of HIF1-wt or –mut
plasmids were also transfected. 24 hours later,
cells were incubated in normal or hypoxic
incubators overnight and the firefly and renilla
luciferase activities for each sample were
measured as described by the dual-luciferase
activity kit (Promega) (23).
Lactate measurement - Cells were added with
fresh media, cultured under normal and hypoxic
condition for 48 hours. The lactate concentrations
in media were measured with a kit (Biovision),
and all values were adjusted with the total cell
number, and normalized to the value of sh-Scr cell
lines under the normoxic condition.
In vitro deacetylation assay – Hek293T cells
were transfected with plasmid expressing HA-
HIF1 for 48 hours. Then, total HA-HIF1
proteins (including the acetylated and the
unacetylated) were IP-purified using anti-HA
antibodies. Simultaneously, Flag-HDAC1 or
Flag-HDAC4 was overexpressed in HEK293T
cells and IP-purified using anti-Flag M2 agarose.
The agarose beads containing the immunocomplex
of either HDAC4 or HDAC1 were mixed with the
agarose containing HA-HIF1 in deacetylation
buffer (Upstate-Millipore) as described (25,26). At
various time points, the deacetylation was
terminated by adding western blot loading buffer.
The total HA-HIF1 and its acetylation can be
visualized by western blots using anti-HA and
anti-Acetyl-K antibodies.
Statistical analysis - Differences between the
means of unpaired samples were evaluated by the
Student's t-test. P values < 0.05 were considered to
be statistically significant.
RESULTS
HDAC4 regulates HIF1 protein acetylation
and stability – Since most types of cancer cell
have functional VHL, to determine the effect of
HDAC4 inhibition on HIF1 in a VHL-positive
background, we transduced a human prostate
cancer cell line C42B and a variant of human liver
cancer cell line Hep3Bc1 with pseudo-lentivirus
containing shRNA against HDAC4 (sh-HDAC4)
or scramble control (sh-Scr). We incubated the
cells under normal oxygen or hypoxic condition
for 6 hours, and we observed that the HIF1
protein level was significantly reduced in hypoxic
cells of sh-HDAC4 compared to the sh-Scr (Figure
1A). Similar inhibitory results were observed
when we used cobalt chloride (CoCl2) as a
hypoxic mimic (Figure 1B). To measure the
HIF1 acetylation, we treated cells with CoCl2
and the proteasome inhibitor MG132. As
previously seen in the VHL-null cell lines (16), the
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inhibited HIF1 protein due to HDAC4
knockdown can be rescued by MG132 (Figure 1C
IB: HIF1), and there was no significant change in
HIF1 interaction with Hsp90/Hsp70 (data not
shown). Significantly, the HIF1 acetylation level
was higher in the HDAC4 knockdown cells
(Figure 1C IB: Ace-K) than the scramble controls.
To measure the kinetics of HIF1 protein
degradation, we treated sh-HDAC4 and sh-Scr
cells with CoCl2 to stabilize HIF1 protein. Then,
we added cycloheximide (CHX) to arrest protein
synthesis. The degradation rate of existing HIF1
protein was obtained by measuring HIF1 and
tubulin in cell lysates harvested at 0, 15, 30, 60, 90
and 120 minutes after the CHX addition. We
observed that HIF1 protein degraded faster in sh-
HDAC4 cells than in the sh-Scr cells (Figure 1D),
which was confirmed by densitometry of multiple
western blot results (Figure 1E). These data
confirm our ongoing hypothesis that HDAC4
shRNA knockdown causes an increase of HIF1
acetylation and a decrease of HIF1 stability.
Previously we reported that HDAC6 inhibition
can also reduce HIF1 protein level, however,
such inhibition was not associated with HIF1
protein acetylation (16). To further investigate the
differential regulation of HIF1 by HDAC4 and
other HDAC isozymes, similar stable shRNA
knockdown experiments were performed in
Hek293 cells with shRNA against HIF1,
HDAC1, HDAC3, and HDAC4. HDAC4 shRNA
robustly inhibited HIF1 proteins under hypoxic
condition; in contrast, neither HDAC1 nor
HDAC3 shRNA had a significant effect on the
HIF1 protein level (Figure 2A). Previously we
reported that HDAC4 interacts with HIF1 (16).
To further examine the relationship between
HIF1 acetylation level and HDAC4, we
transiently co-overexpressed a hemagglutinin
(HA)-tagged HIF1with either an empty vector or
a Flag-HDAC4 plasmid in Hek293T cells. Co-IP
experiments showed that Flag-HDAC4 interacted
with HA-HIF1 (Figure 2B). Importantly,
HDAC4 overexpression reduced the HA-HIF1
protein basal acetylation (Figure 2B). In addition,
we performed an in vitro deacetylation assay (25)
using overexpressed and IP-purified Flag-HDAC1,
Flag-HDAC4 and HA-HIF1. In the course of 2
hours of deacetylation reaction, we observed that
the level of HA-HIF1 acetylation gradually
decreased by the presence of the HDAC4, but not
by HDAC1 (Figure 2C). These data further
suggest that the HIF1 acetylation level and
protein stability can be regulated by HDAC4.
HDAC4 regulates HIF1 protein stability via
HIF1 N-terminal lysines – To identify the lysine
residues that are deacetylated by HDAC4, we
ectopically overexpressed the full-length Flag-
HIF1 in Hek293 cells with shRNA against
HDAC4 (sh-HDAC4) or scramble control (sh-
Scr). The over expressed Flag-HIF1 proteins
were anti-Flag IP-purified, resolved on SDS-
PAGE gel, digested by trypsin, and subjected to
liquid chromatography – tandem mass
spectrometry analysis (LC-MS/MS) at OHSU
Proteomic Core Facility. MS/MS analysis of the
trypsin-digested Flag-HIF1 peptides did not
reveal a significant change in lysine acetylation
between the sh-Scr and sh-HDAC4 cells. In
repeated experiments, however, trypsin digestion
(cleaves at the un-acetylated lysine) did not
generate a peptide, containing the first 30 amino
acids of HIF1, that was suitable for LC-MS/MS
analysis. There is a cluster of 5 lysine residues
(K10, K11, K12, K19, and K21) within the HIF1
N-terminal amino acids 1-30. We speculated that
this can be a region regulated by HDAC4
deacetylation. Thus we took an alternative
approach. We used the site-directed mutagenesis
to replace all five lysines to arginines in the full-
length Flag-HIF1 wild type (HIF1-wt)
construct. We observed that the acetylation level
of the resulted HIF1 mutant (HIF1-mut) was
significantly lower than HIF1-wt (Figure 3A).
This differential protein acetylation level was also
associated with differential protein stability in the
context of HDAC4. Co-overexpression of HDAC4
with either the HIF1-wt or HIF1-mut showed
that HDAC4 decreased the protein acetylation of
HIF1-wt, but not the HIF1-mut (Figure 3B). On
the other hand, HDAC4 overexpression increased
the protein level of HIF1-wt, but not the HIF1-
mut (Figure 3B). When we overexpressed the
HIF1-wt and –mut constructs in Hek293 cells
with stable sh-Scr or sh-HDAC4, under both
normal and hypoxic mimic conditions, we
observed that HIF1-wt (high in acetylation) was
inhibited by HDAC4 shRNA knockdown
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compared to sh-Scr (Figure 3C). This was
consistent with the results in Figure 2A. In
contrast, HIF1-mut (low in acetylation) was
resistant to the HDAC4 shRNA knockdown
(Figure 3C). Further, we overexpressed the wild-
type or mutant Flag-HIF1 construct in Hek293T
cells, and treated cells with solvent or HDACi-
SAHA. Under both normal and hypoxic mimic
conditions, the HIF1-wt was sensitive to HDACi;
in contrast, the HIF1-mut was resistant to
HDACi-induced protein degradation (Figure 3D).
These data show that the acetylation and stability
of HIF1 can be regulated by HDAC4 via N-
terminal lysines.
HDAC4 regulates HIF-1 protein activity via
HIF1 N-terminal lysines – Using a hypoxia/HIF-
1 response element (HRE)-driving luciferase
reporter (p2.1) assay (23), we also compared the
transcriptional activity of HIF1-wt and HIF1-
mut under non-hypoxic condition, where the
endogenous HIF1 protein is inhibited by oxygen
and does not confound the reporter assay result.
We overexpressed either the HIF1-wt or HIF1-
mut in Hek293 cells along with a firefly luciferase
reporter gene plasmid (p2.1) under the control of
HRE and a constitutive renilla luciferase gene.
The firefly activity can be robustly induced by
HIF1 or hypoxia stimulation. The renilla activity
can be used as a control for equal transfection and
cell density(23). We observed that the HIF1-mut
had a higher transcriptional activity than HIF1-
wt (Figure 4A). Co-overexpression with HDAC4
significantly enhanced the HIF1-wt activity, but
had no effect on HIF1-mut (Figure 4A). This is
consistent with the HIF1 protein level regulation
by HDAC4 in Figure 3B, in which HIF1-wt
protein level was lower than HIF1-mut, but can
be upregulated by HDAC4 overexpression. We
also tested the transcriptional activity of HIF1-wt
and HIF1-mut in HDAC4 shRNA knockdown
conditions by performing reporter gene assay in
Hek293 cells with sh-Scr or sh-HDAC4. Similar to
the protein results in Figure 3C, we observed that
the HIF1-wt transcriptional activity was
significantly reduced in the presence of HDAC4
shRNA knockdown (Figure 4B). In contract, the
HIF1-mut had a higher activity than HIF1-wt,
and was not significantly affected by HDAC4
shRNA (Figure 4B).
The effect of HDAC4 knockdown on HIF-1
activity in cancer cells - To measure the effect of
HDAC4 shRNA knockdown on HIF-1
transcriptional activities in cancer cell lines, we
performed the luciferase reporter assay using
Hep3Bc1 sh-Scr and sh-HDAC4 cells. Previously,
Hep3Bc1 cells had been stably co-transfected with
the HRE-firefly luciferase reporter gene plasmid
(p2.1) and the constitutive renilla luciferase gene
(23). We observed that the sh-Scr cells exhibited
very robust firefly reporter activity due to hypoxia,
in contrast, the sh-HDAC4 cells did not respond to
hypoxia stimulation (Figure 5A). To confirm this,
we transiently co-transfected the HRE-firefly and
constitutive renilla plasmids into C42B cells
containing either stable sh-HDAC4 or sh-Scr, and
performed similar experiments under normal and
hypoxic conditions. Similar to the results in
Hep3Bc1 cell lines, hypoxia upregulated firefly
activity in sh-Scr cells. HDAC4 shRNA
significantly inhibited this upregulation (Figure
5B). Based on these results, we measured the
effect of HDAC4 shRNA on endogenous HIF-1
target gene upregulation in hypoxia by qRT-PCR.
We found that hypoxia significantly upregulated a
subset of HIF-1 target genes including VEGFa,
LDHA, and Glut1 in sh-Scr cell lines, however,
the upregulation was significantly inhibited in the
sh-HDAC4 cells (Figure 5C & 5D).
The effect of HDAC4 knockdown on cellular
response to hypoxia - Based on the reduced
glycolytic gene (LDHA and Glut1) upregulation
by HDAC4 shRNA (Figure 5C), we hypothesized
that HDAC4 shRNA will also affect hypoxia-
induced glycolysis. We measured glycolysis levels
based on the lactate production in the sh-Scr and
sh-HDAC4 cancer cells growing under normal or
hypoxic condition. Indeed, we found that the
lactate levels were significantly increased in the
hypoxic sh-Scr cells, and HDAC4 shRNA
significantly reduced the hypoxia induced lactate
production (Figure 6A). Next, in cell proliferation
assays, we observed that HDAC4 shRNA did not
change the growth rate of the cancer cell lines
under normal oxygen and short-term (48 hours)
hypoxic conditions (Figure 6B). When cells were
cultured in long-term hypoxia (96 hours), C42B
cells didn’t survive regardless the HDAC4 status
(data not shown), but the Hep3Bc1 sh-HDAC4
cells grew significantly slower than the sh-Scr
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cells (Figure 6B). Hypoxia and HIF-1 are
considered as key factors in causing resistance
toward cytotoxic therapies in solid cancer (27,28).
We therefore tested the effect of HDAC4 shRNA
on prostate cancer cell response to docetaxel,
which is a microtubule targeting agent and
standard chemotherapy for late-stage metastatic
prostate cancer patients. We treated C42B sh-Scr
and sh-HDAC4 cells with increasing doses of
docetaxel for 48 hours under normal or hypoxic
condition. Cell viability at each drug concentration
was calculated and adjusted to the solvent control.
We observed that HDAC4 shRNA did not
significantly modify docetaxel efficacy in
normoxic conditions, but significantly inhibited
the hypoxia-induced resistance (Figure 6C).
Since the HIF1 protein plays a pathological
role in human diseases including cancer,
understanding novel mechanisms regulating the
HIF1 and HIF-1 activity is important for
developing more effective and targeted therapies.
In the current study, we determined that a member
of HDACs family – HDAC4 plays an important
role in regulating HIF1 protein N-terminal lysine
acetylation level, stability and HIF-1 activity.
These results provide a mechanistic explanation to
the well-observed phenomena that HDACi can
induce HIF1 protein acetylation, reduce HIF1
protein level, and HIF-1 target gene expression in
cancer cell lines, and angiogenesis in preclinical
mouse xenograft models (16,19,29).
A likely scenario is that the N-terminal lysine
acetylation level of HIF1 protein is in part
regulated by HDAC4. The inhibition of HDAC4
by either HDACi or shRNA causes an
inappropriate increase of HIF1 protein
acetylation (hyperacetylation), which in turn
disrupts the protein stability and leads to the
reduction in HIF1 protein level and HIF-1
activity. In the previous study, we have shown that
the HIF1 protein inhibition due to HDAC4
inhibition requires proteasomal degradation
system, but is independent of VHL, and does not
impact the HIF1 interaction with Hsp70/90 (16).
Currently, the exact molecular detail connecting
the protein acetylation and degradation is
unknown, and warrants further additional studies.
The HIF1 N-terminal sequence is also highly
similar to the HIF2 N-terminus, both are critical
for mediating protein-protein and protein-DNA
interaction. However, we did not observe a
significant HIF2 inhibition in sh-HDAC4 cells.
Based on the novel information regarding the
HIF1 N-terminal lysines and HDAC4 presented
here, we will, in additional studies, investigate in
details whether the N-terminal lysine acetylation
plays a role in HIF1 interaction with DNA and
the additional degradation machinery such as
RACK1, CHIP, and the ubiquitin.
In the current study, the first five lysine
residues in the HIF1 N-terminus collectively
play a pivotal role in HIF1 protein acetylation
and stability in the context of HDAC4. We have
not been able to identify the exact location (s) of
acetylation. Since the single lysine mutants were
not significantly different from the HIF1-wt
(data not shown), it is likely that a combination of
these five lysines (if not all five) is involved.
Based on the results in this and other studies, it
is apparent that multiple lysine residues within the
HIF1 protein can be substrates for multiple
HATs and HDACs. Up to date, HIF1 can be
deacetylated at least at two lysine residues, K532
and K674 by different HDACs (14,15). The K532
deacetylation is likely to be carried out by the
class I and/or II HDACs (14). The K674
deacetylation requires a class III HDAC-Sirt1
(15). In our study, using HIF1 containing point
mutation at K532 or K674 did not indicate a clear
regulation of HDAC4 toward these two lysine
residues (data not shown). It is unclear whether
these regulations occur to HIF1 ubiquitously or
only at specific physiological conditions. Taken
together, these suggest that multiple HDACs are
involved in HIF1 regulation. We speculate that
these specific associations between HDAC
isozymes and deacetylation reflect the biological
complexity and redundancy on HIF-1 regulation.
Further, the acetylation and deacetylation at
different lysine residues within HIF1 can lead to
different biological consequences. Acetylations at
the N-terminus (shown in this study) and at the
oxygen dependent degradation (ODD) domain
(K532) can promote HIF1 protein degradation
and inhibition of downstream HIF-1 activity (14).
In contrast, the acetylation at K674 promotes the
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transcriptional activity of HIF-1 by recruiting the
transcriptional co-factor p300 (15). Recently, it
has been shown that PCAF can act as HIF1
acetylase at K674 (15). Although our primary goal
is to identify the deacetylase of HIF1, we did
observe that endogenous PCAF are well expressed
in all the cell lines, and PACF overexpression
increased HIF1-wt and HIF1-mut acetylation
level. Whether the HIF1 N-terminal lysines are
substrates of PCAF warrants more careful
investigation in additional studies.
In the current study, not all HIF-1 target genes
are downregulated by HDAC4 inhibition. This
suggests that i) the downregulation of HIF1
protein by HDAC4 shRNA is not the only
mechanism responsible for the downregulation of
HIF-1 target gene expression, ii) HDAC4 is only
required for a subset of HIF1 target genes, or iii)
HDAC4 directly regulates a subset of HIF-1 target
genes either independently or in collaboration with
HIF-1. HDAC4 is known to regulate the activity
of transcriptional factors and co-factors via
participating in the formation of transcriptional
complexes including HIF-1 (30-33). Therefore, we
speculate that only a subset of HIF-1 target genes
require HDAC4 as a HIF-1 co-factor to stabilize
HIF1 and to form an effective transcriptional
complex. In HDAC4 knockdown cell lines, the
upregulation of these genes in hypoxia is inhibited.
In addition, the regulation of glycolysis and
cytotoxic stress by HDAC4 in hypoxic cells
suggests a novel role for HDAC4 in cellular
adaptation toward hypoxia. Genetic studies have
shown that HDAC4 regulates cellular proliferation
and differentiation, and is an important
determinant for the development of muscle, bone
and heart (30-32,34). Because the proper
development of these tissues depends on the
availability of oxygen (35), the regulatory role of
HDAC4 in tissue development can potentially
includes the participation in responding and
adapting to the change of oxygen concentrations.
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This work is supported by NIH/NCI grant R01CA149253 01 to D.Z.Q. D.Z.Q. is also a recipient of a
new investigator award from Department of Defense (DOD) Prostate Cancer Research Program.
Abbreviations used: HIF1, hypoxia-inducible factor 1 alpha; HIF1-wt, HIF1 wild type; HIF1-
mut, HIF1-mutant (K to R mutation at K10, 11, 12, 19, 21), HDAC, histone deacetylase; HDAC4,
histone deacetylase isozyme 4; HDACi, histone deacetylase inhibitors; O2, oxygen; HRE, HIF-1 response
element; aa, amino acid; K, lysine; R, arginine; CoCl2, cobalt chloride, IP, immunoprecipitation; IB:
immunoblotting; SAHA, Suberoylanilide Hydroxamic Acid; Glut1, glucose transporter 1; LDHA, Lactate
dehydrogenase A; VEGFa, Vascular endothelial growth factor A; VHL, von Hippel-Lindau; PCAF,
P300/CBP-associated factor.
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Figure 1: HDAC4 regulates HIF1 protein acetylation and stability in cancer cells. (A) Hep3Bc1 cells
with stable sh-Scr (-) or sh-HDAC4 (+) were cultured under normal or hypoxic condition for 6 hours. (B)
C42B sh-Scr and sh-HDAC4 cells were cultured with or without CoCl2 for 6 hours. The HIF1, HDAC4
and tubulin protein levels were measured using whole cell lysates by western blots. (C) Hep3Bc1 sh-Scr
and sh-HDAC4 cells were co-treated with proteasome inhibitor MG132 and CoCl2 for 6 hours. HIF1
was IP-purified (+) from whole cell lysates, and immuno blotted (IB) for HIF1 and its acetylation (ace-
K). (D)Hep3Bc1 sh-Scr and sh-HDAC4 cells were treated with CoCl2 for 6 hours, followed by protein
synthesis inhibitor cycloheximide (CHX) treatment for the indicated times. HIF1 and tubulin protein
levels were measured by western blots. (E) The decay of HIF1 protein level in (D) was quantified by
florescence densitometry. The HIF1 protein band intensity was divided by the tubulin band intensity in
each sample and normalized to the time 0 of CHX treatment. Lines represent mean and standard deviation
of 3 experiments. * P < 0.01, sh-Scr vs. sh-HDAC4 at the indicated time points.
Figure 2: HDAC4 specifically regulates HIF1 proteins. (A) Hek293 cells were transduced with
lentivirus containing shRNA against scramble sequence (sh-Scr), HIF1 (sh-HIF1), HDAC1 (sh-
HDAC1), HDAC3 (sh-HDAC3) or HDAC4 (sh-HDAC4). The indicated cells were cultured in either
normal or hypoxic condition for 6 hours before western blot analysis. (B) An equal amount of HA-HIF1
plasmid was co-overexpressed with either empty vector (-) or Flag-HDAC4 (+). 48 hours after
transfection, whole cell lysates were used for IP with anti-HA antibodies. The level of HA-HIF1 protein
and its acetylation were measured by western blots using anti-HA and anti-acetyl-lysine (Ace-K)
antibodies, respectively. The Flag-HDAC4 was measured by the anti-Flag antibody. (C) The HA-HIF1
plasmid or Flag-HDAC4 or Flag-HDAC1 plasmid was overexpressed in HEK293T cells. 48 hours after
transfection, the HA-HIF1, Flag-HDAC4 and Flag-HDAC1 were IP-purified using anti-HA and anti-
Flag antibodies, respectively. The purified HA-HIF1 was mixed with purified HDAC4 or HDAC1 in
deacetylase buffer for the indicated time in an in vitro deacetylation assay. The resulted proteins were
analyzed by western blotted using anti-HA or anti-ace-K antibodies.
Figure 3: HDAC4 regulates HIF1 protein via HIF1 N-terminal lysines. (A) Flag-HIF1-wt or Flag-
HIF1-mut was overexpressed in Hek293T cells for 48 hours, then HIF1 proteins were IP-purified by
anti-Flag M2 agarose, and analyzed by western blot using anti-HIF1 and anti-ace-K antibodies. (B)
Flag-HIF1-wt and Flag-HIF1-mut were co-overexpressed with Ev (-) or HDAC4 (+) in Hek293T cells.
To control transfection efficiency and specific regulation by HDAC4, all cells were also co-transfected
with a green fluorescent protein (GFP) plasmid. 48 hours after transfection, whole cell lysates (WCL)
were harvested and were analyzed either by western blot using anti-Flag, HDAC4, GFP and tubulin
antibodies or IP-purified by anti-Flag M2 agarose and probed for acetylation. (C) Flag-tagged HIF1-wt
and –mut were overexpressed in Hek293 sh-Scr cells (-) and sh-HDAC4 (+) cells for 48 hours. CoCl2 was
added 4 hours prior to protein harvest to mimic hypoxia. WCL were analyzed by western blots. (D) Flag-
tagged HIF1-wt and –mut were overexpressed in Hek293T cells for 48 hours. 4 hours prior to protein
harvest, CoCl2 was added to mimic hypoxia, and cells were treated with either vehicle (-) or 5 M of
HDACi-SAHA (+). WCL were analyzed by western blot.
Figure 4: HDAC4 regulates HIF-1 transcriptional activity via HIF1 N-terminal lysines. (A) HDAC4,
HIF1-wt, HIF1-mut, or empty vector (Ev) was overexpressed in Hek293 cells as a single agent or in
combination along with HRE-firefly and constitutive renilla luciferase. (B) HIF1-wt, HIF1-mut or Ev
was overexpressed in Hek293 sh-Scr and sh-HDAC4 cells along with HRE-firefly and constitutive renilla
luciferase plasmids. 48 hours later, dual-luciferase activities were measured, and expressed as values
relative to the Ev. *, and ** P < 0.01. All bars represent mean and standard deviation of three
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experiments.
Figure 5: HDAC4 regulates HIF-1 transcriptional activity and target gene expression in cancer cells.
(A) Hep3Bc1 cells containing stable sh-Scr or sh-HDAC4 were cultured under normal and hypoxic
incubators overnight. The ratio of firefly/renilla of all samples was normalized to the value of sh-Scr
under normal condition, and expressed as relative luciferase (luc). * P < 0.001. (B) C42B sh-Scr and sh-
HDAC4 cells were transiently co-transfected with HRE-firefly (p2.1) and constitutive-renilla plasmids.
48 hours after transfection, the transfected cells were cultured under normal or hypoxic condition
overnight. The luciferase activity was measured and calculated as above. ** P < 0.01. (C&D) The
indicated cells were cultured in normal or hypoxia for 24 hours. Selective HIF-1 target gene expressions
were measured by realtime qRT-PCR, and the fold change was expressed as relative values to sh-Scr at
normal condition. # P < 0.01, & P < 0.05. All bars represent mean and standard deviation of three
experiments.
Figure 6: HDAC4 regulates cancer cell response to hypoxia. (A) Lactate levels (indicative of
glycolysis) were measured in sh-Scr and sh-HDAC4 cells in normoxia (n) and hypoxia (h), normalized
with total cell numbers, and expressed as relative fold change to the value of sh-Scr in normoxia. * P <
0.05. (B) Hep3Bc1 sh-Scr and sh-HADC4 cells were cultured under normoxia (n) and hypoxia (h) for 5
days. The viable cells were quantitated at day 1, 3 and 5 of the experiments, and normalized to day 1.
Lines represent mean and standard deviation of three experiments. # P < 0.05, sh-Scr (h) vs. sh-HDAC4
(h) on day 5. (D) C42B sh-Scr and sh-HDAC4 cells were treated with the indicated dose of docetaxel for
48 hours under the normal (n) and hypoxic (h) conditions. For each condition, the viable cells were
counted and compared to the solvent control as % of viability. ** P < 0.05, sh-Scr (h) vs. sh-HDAC4 (h)
at 20 nM and 30 nM of docetaxel. All experiments represent mean and standard deviation of three
experiments.
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Xue and David Z. QianHao Geng, Chris T. Harvey, Janet Pittsenbarger, Qiong Liu, Tomasz M. Beer, Changhui
lysine acetylation and cancer cell response to hypoxiaαHDAC4 regulates HIF1
published online September 14, 2011J. Biol. Chem.
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