Received: 13 January 2017
|
Accepted: 3 April 2017
DOI: 10.1002/jcp.25945
ORIGINAL RESEARCH ARTICLE
HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy Chih-Yang Huang1
|
Fa-Lun Lee2 | Shu-Fen Peng3 | Kuan-Ho Lin4 |
Ray-Jade Chen5 | Tsung-Jung Ho6,7 | Fu-Jen Tsai6 | Vijaya V. Padma8 | Wei-Wen Kuo3,* | Chih-Yang Huang2,6,9,* 1 Translation
Research Core, China Medical University Hospital, China Medical University, Taichung, Taiwan
2 Graduate
Institute of Basic Medical Science, China Medical University, Taichung
3 Department
of Biological Science and Technology, China Medical University, Taichung, Taiwan
4 Emergency
Department, China Medical University Hospital, Taichung, Taiwan
5 Department
of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei
6 School
of Chinese Medicine, China Medical University, Taichung, Taiwan
7 Chinese
Medicine Department, China Medical University Beigang Hospital, Taiwan
Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of earlystage heart failure (HF). Inhibition of extracellular signal-regulated kinases (ERK) efficaciously suppressed angiotensin II (ANG II)-induced cardiomyocyte hypertrophy and apoptosis by blocking insulin-like growth factor II receptor (IGF-IIR) signaling. However, the detailed mechanism by which ANG II induces ERK-mediated IGF-IIR signaling remains elusive. Here, we found that ANG II activated ERK to upregulate IGF-IIR expression via the angiotensin II type I receptor (AT1R). ERK activation subsequently phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation by glycogen synthase kinase III (GSK3) at serine 303. Moreover, we found that ANG II mediated ERK/GSK3-induced IGF-IIR protein stability by downregulating the E3 ubiquitin ligase of IGF-IIR RING finger protein CXXVI (RNF126). The expression of RNF126 decreased following ANG II-induced HSF1S303 phosphorylation, resulting in IGF-IIR protein stability and increased cardiomyocyte injury. Inhibition of GSK3 significantly alleviated ANG IIinduced cardiac hypertrophy in vivo and in vitro. Taken together, these results suggest that HSF1 phosphorylation stabilizes IGF-IIR protein stability by downregulating RNF126 during
of Biotechnology, Bharathiar University, Coimbatore, India
cardiac hypertrophy. ANG II activates ERK/GSK3 to phosphorylate HSF1, resulting in RNF126
9 Department
hypertrophy. HSF1 could be a valuable therapeutic target for cardiac diseases among
8 Department
of Health and Nutrition Biotechnology, Asia University, Taichung Correspondence Chih-Yang Huang, PhD, Translation Research Core, China Medical University Hospital, China Medical University, Taichung, Taiwan, 40402, R.O.C. Email:
[email protected]
degradation, which stabilizes IGF-IIR protein expression and eventually results in cardiac hypertensive patients. KEYWORDS
cardiac hypertrophy, HSF1, hypertension, IGF-IIR, RNF126
Funding information Ministry of Health and Welfare, Taiwan, Grant number: MOHW106-TDU-B-212113004
1 | INTRODUCTION
dysfunction. Our previous studies have reported that angiotensin II (ANG II) is involved in the development of cardiac hypertrophy and
Apoptosis has been implicated in a wide variety of cardiovascular
apoptosis by regulating insulin-like growth factor-II receptor (IGF-IIR)
disorders, including myocardial infarction and hypertension-related
expression (Chen et al., 2009; Chu et al., 2009; Huang et al., 2014; Lee
heart failure (HF), suggesting that activation of apoptotic pathways
et al., 2006). IGF-IIR is a multifunctional protein receptor that binds
contributes to cardiomyocyte loss and, subsequently, cardiac
insulin-like growth factor II (IGF-II) to activate downstream signaling pathways, leading to pathological hypertrophy and activation of
*Chih-Yang Huang and Wei-Wen Kuo contributed equally in this paper.
J Cell Physiol. 2017;9999:1–11.
the mitochondria-mediated apoptotic pathway (Chen et al., 2015;
wileyonlinelibrary.com/journal/jcp
© 2017 Wiley Periodicals, Inc.
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1
2
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ET AL.
Huang et al., 2016). Furthermore, IGF-II and IGF-IIR positively
anti-apoptotic protein BCL-2 was clearly decreased in a dose-dependent
correlated with the progression of pathological hypertrophy after
manner (Figure 1a). Moreover, the level of p-HSF1Ser303 was also clearly
complete abdominal aorta ligation and may play a critical role in ANG
upregulated, suggesting that the activity of HSF1 was inhibited
II-induced cardiomyocyte apoptosis (Chu et al., 2009).
when ANG II was administered. These effects also occurred in a
The MAPK pathway (mitogen-activated protein kinases, originally
time-dependent manner during ANG II treatment (Figure 1b).
called extracellular signal-regulated kinases) cascade Ras/Raf/MEK/
To further confirm that ANG II enhanced IGF-IIR expression and
ERK are involved in a variety of cellular and physiological processes
membrane translocation, we examined the levels of IGF-IIR in the
(Busca, Pouyssegur, & Lenormand, 2016; Plotnikov, Zehorai, Procac-
cytoplasm and plasma membrane by subcellular fractionation follow-
cia, & Seger, 2011). However, the small GTP-binding proteins Ras and
ing ANG II treatment. We found that IGF-IIR was simultaneously
ERK are associated with the progression of cardiomyocyte hypertro-
increased in the cytoplasm and plasma membrane following ANG II
phy (Liu et al., 2016). HSF1 is considered a cardioprotective factor that
treatment, suggesting that ANG II indeed promoted plasma membrane
controls the expression of heat shock protein (HSP), which is
translocation of IGF-IIR (Figure 1c). We further verified the plasma
responsible for the stress response in cardiomyocytes (Griffin, Valdez,
membrane level of IGF-IIR following ANG II treatment by enzyme-
& Mestril, 2004; Peng et al., 2010; Yin, Xi, Wang, Eapen, & Kukreja,
linked immunosorbent assay (ELISA) (Figure 1d). These results showed
2005; Yu et al., 2012); however, HSF1 activation has also been
the upregulation of plasma membrane IGF-IIR expression following
reported to enhance HF (Krishnamurthy, Kanagasabai, Druhan, &
ANG II exposure in H9c2 cardiomyoblasts.
Ilangovan, 2012; Vedam et al., 2010). Doxorubicin-induced reactive
ANG II has been reported to activate downstream signaling via the
oxygen species (ROS) activated HSF1 to increase HSP25 expression,
angiotensin type 1 and type 2 receptors (AT1R and AT2R, respectively)
and HSP25 then transactivated p53 to increase Bax expression, thus
to elicit various biological responses via the MAPK pathway (George,
leading to HF (Vedam et al., 2010). Therefore, the role of HSF1 in
Thomas, & Hannan, 2010; Herichova & Szantoova, 2013). Therefore,
cardiomyocytes is still debated.
we silenced AT1R and AT2R to determine which ANG II receptor
In this pioneering study, we first found that phosphorylated HSF1S303
induces ERK activation. As shown in Figure 2a, the knockdown of
and HSF1S307 maintain IGF-IIR protein stability by downregulating its E3
AT1R decreased ANG II-induced ERK activation, whereas the
ligase ring finger protein 126 (RNF126) during hypertension-induced HF.
knockdown of AT2R did not influence the effects of ANG II on
ANG II activated ERK to phosphorylate HSF1Ser307 and subsequently
ERK activation. These results suggest that ANG II promoted ERK
phosphorylate HSF1Ser303 via GSK3β. HSF1 phosphorylation at these
phosphorylation via AT1R. Similarly, treatment with the AT1R blocker
sites trigger a decrease in the E3 ligase RNF126 to prevent IGF-IIR protein
losartan alleviated ANG II-induced ERK activation but the
degradation. Therefore, IGF-IIR translocates to the membrane for
AT2R blocker PD123319 did not (Figure 2b).
downstream hypertrophy and apoptosis signaling in ANG II-stimulated cardiomyocytes and hypertensive hearts.
To assess whether the IGF-IIR expression was mediated by MAPK kinases via AT1R following ANG II treatment, we treated the cells with MEK (the upstream kinase of ERK) and the ERK inhibitors U0126 and PD98059. IGF-IIR expression was significantly reduced when the MEK inhibitor U0126 and the ERK inhibitor PD98059 were administered even with the
2 | RE SULTS
exposure to ANG II (Figure 2c). Next, we estimated the surface IGF-IIR expression under these kinase inhibitor challenges. As anticipated, surface
2.1 | ANG II stimulates the IGF-IIR apoptotic pathway and the phosphorylation of HSF1S303 in cardiomyoblasts
IGF-IIR expression decreased after the addition of ERK kinase inhibitors
Our previous studies have demonstrated that ANG II promotes IGF-IIR
2.2 | MEK-ERK regulated HSF1 phosphorylation at serine 303/307 following ANG II treatment
expression and cardiomyocyte apoptosis by inhibiting HSF1 DNA-binding activity via JNK-mediated SIRT1 degradation (Huang et al., 2014, 2016).
(Figure 2d). These results demonstrate that the MEK/ERK pathway may participate in the process of IGF-IIR membrane translocation.
Moreover, we found that ERK and p38 MAPK also participated in the
We then tested whether the MEK/ERK signaling pathway was involved in
IGF-IIR signaling pathway (Huang et al., 2014). Cytosolic phosphorylated
HSF1 phosphorylation following ANG II treatment. As shown in Figure 2e,
HSF1Ser303, which has been demonstrated to inhibit HSF1 activity (Chu,
treatment with ERK and MEK inhibitors alleviated ANG II-induced
Soncin, Price, Stevenson, & Calderwood, 1996; Kline & Morimoto, 1997;
HSF1 phosphorylation. Interestingly, we found that the reduction of ANG
Knauf, Newton, Kyriakis, & Kingston, 1996), accumulated with the
II-induced HSF1 phosphorylation was located at serine 303/307
increase dosage of ANG II (Huang et al., 2014). Therefore, we aimed to
(Figure 2e). Moreover, p-HSF1S303 and p-HSF1S307 accumulated in the
Ser303
in the IGF-IIR signaling pathway
cytoplasm following ANG II treatment (Figure 2f). Consistent with the
following ANG II treatment. As shown in Figure 1a, the expression levels
results of immunoblotting, we found that pHSF1S303 was predominantly
of IGF-IIR apoptotic pathway-related proteins, such as IGF-IIR, AT1R,
accumulated in the cytoplasm and that pHSF1S307 gradually translocated
ERK1/2, p-ERK1/2, Gαq, and cleaved caspase-3 (cCasp-3) as well
into cytoplasm following ANG II treatment (Figure 2g). These
as the pro-apoptotic protein BAX were significantly increased in a
results suggested that ANG II promoted pHSF1S303 and pHSF1S307 via
dose-dependent manner following ANG II treatment. However, the
MEK/ERK signaling, which leads to their accumulation in the cytoplasm.
elucidate the role of p-HSF1
HUANG
ET AL.
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3
ANG II stimulated ERK, p-HSF1S303 and IGF-IIR-induced apoptotic pathways in cardiomyoblasts. (a) H9c2 cardiomyoblast cells were treated with different doses of ANG II (100, 200, or 400 nM) for 24 hr. Protein expression was detected by immunoblotting. Quantification of the results is shown (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001. (b) H9c2 cardiomyoblast cells were treated with 200 nM ANG II for different durations (3, 6, 12, 24, 36, or 48 hr). Protein expression was detected by immunoblotting. Quantification of the results is shown (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001. (c) H9c2 cardiomyoblast cells were treated with increasing concentrations of ANG II (100, 200, or 400 nM) for 24 hr, and the cell lysates were fractionated into cytoplasmic and membrane proteins. The expression of IGF-IIR was measured by immunoblotting. Protein expression was detected by immunoblotting. Quantification of the results is shown on the right (n = 3). * p < 0.05 and ** p < 0.01. (d) The membrane levels of IGF-IIR in H9c2 cardiomyoblast cells were detected by ELISA following treatment with increasing concentrations of ANG II for 24 hr. Quantification of the results is shown (n = 3). * p < 0.05. These data were obtained from at least three independent experiments, and the values represent the mean ± S.D
F I G UR E 1
2.3 | ANG II-induced ERK activation phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation at serine 303 by GSK3
confirm whether HSF1 phosphorylation was decreased (Figure 3b). Consistent with the results of kinase inhibition, we found that ERK deficiency significantly suppressed the phosphorylation of HSF1 both at serine 303 and serine 307, but GSK3β deficiency only suppressed
Previous studies have indicated that ERK1 phosphorylates HSF1 at
phosphorylation at serine 303. Additionally, ERK and GSK3β inhibition
serine 307 and leads to further phosphorylation by glycogen synthase
clearly alleviated ANG II-induced cardiomyocyte hypertrophy
kinase 3 (GSK3) at serine 303 (Chu et al., 1996; Wang, Grammatikakis,
(Figure 3c). Taken together, these results indicated that the ERK-
Siganou, & Calderwood, 2003). Therefore, we treated the cells with
GSK3β cascade indeed regulated ANG II-induced cardiac hypertrophy
ERK and GSK3 inhibitors in conjunction with the ANG II challenge
via HSF1 phosphorylation in H9c2 cardiomyoblasts.
to determine whether ERK/GSK3 activation is involved in regulating IGF-IIR expression. As expected, the ERK inhibitor suppressed the phosphorylation of HSF1 both at serine 303 and serine 307, but the GSK3 inhibitor (active wild GSK3 and inactive phosphorylated GSK3Ser9)
only
suppressed
phosphorylation
at
serine
2.4 | ThERK/GSK3β cascade participates in ANG II-induced IGF-IIR protein stability
303
To determine the mechanism by which ANG II-induced ERK
(Figure 3a). These results suggested that GSK phosphorylates
activation promotes IGF-IIR upregulation, we detected IGF-IIR
HSF1S307 downstream of ERK-mediated HSF1S303 phosphorylation.
mRNA expression by RT-PCR following ANG II treatment
We next silenced these two kinases by small interfering RNA to
(Figure 4a). We found that the ANG II significantly upregulated
4
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ET AL.
ANG II-induced ERK activation phosphorylates HSF1 at serine 307 and serine 303. (a) H9c2 cardiomyoblast cells were transfected with 10 nM siCtrl, siAT1R, or siAT2R for 24 hr and then treated with 200 nM ANG II for 24 hr. Protein expression was detected by immunoblotting. Quantification of the results is shown (n = 3). * p < 0.05. (b) H9c2 cardiomyoblast cells were treated with 1 µM Losartan (AT1R inhibitor) and 1 µM PD123319 (AT2R inhibitor) with or without ANG II for 24 hr. Protein expression was detected by immunoblotting. Quantification of the results is shown (n = 3). * p < 0.05. (c) H9c2 cardiomyoblast cells were treated with the ERK inhibitor PD98059 (10 µM) and the MEK inhibitor U0126 (20 µM) with or without ANG II for 24 hr. Protein expression levels of IGF-IIR, ERK, and apoptotic markers were measured by immunoblotting. Quantification of the results is shown on the right (n = 3). * p < 0.05, and ** p < 0.01. (d) H9c2 cardiomyoblast cells were treated with the ERK inhibitor PD98059 (10 µM) and the MEK inhibitor U0126 (20 µM) with or without ANG II for 24 hr. The membrane IGF-IIR protein levels in H9c2 cells were detected by ELISA. Quantification of the results is shown (n = 3). * p < 0.05. (e) H9c2 cardiomyoblast cells were treated with the ERK inhibitor PD98059 (10 µM) and the MEK inhibitor U0126 (20 µM) with or without ANG II for 24 hr. The protein expression of p-HSF1Ser230, p-HSF1Ser303, p-HSF1Ser307, and total HSF1 was measured by immunoblotting. Quantification of the results is shown on the right (n = 3). ** p < 0.01, and *** p < 0.001. (f) H9c2 cardiomyoblast cells were treated with ANG II (100, 200, or 400 nM) for 24 hr, and then, the cells were fractionated into cytosolic and nuclear proteins. Protein expression was detected by immunoblotting. Quantification of the results is shown on the right (n = 3). ** p < 0.01. (g) H9c2 cardiomyoblast cells were treated with different doses of ANG II (200 and 400 nM) for 24 hr and fixed with 4% paraformaldehyde. Immunofluorescence staining using antibodies against p-HSF1S303 and p-HSF1S307 was observed using a confocal microscope. Quantification of the results is shown on the right (n = 3). * p < 0.05. These data were obtained from at least three independent experiments, and the values represent the mean ± S.D F I G UR E 2
IGF-IIR mRNA levels. However, ERK/MEK inhibition did not
ANG II treatment reduces ubiquitin-conjugated IGF-IIR protein levels
influence IGF-IIR mRNA levels. Similarly, knockdown of ERK or
following MG132 treatment, suggesting that ANG II promotes IGF-IIR
GSK3β did not influence ANG II-induced IGF-IIR mRNA upregulation
protein stability by inhibiting ubiquitin (Ub)-conjugation. Moreover,
(Figure 4b). The results suggested that the ERK and GSKβ pathways
transfection with HA-Ub promoted Ub-conjugated IGF-IIR protein
might be involved in IGF-IIR protein stability.
under MG132 treatment (Figure 4d). These results suggested that
We then treated the cells with the proteasome inhibitor MG132 and evaluated IGF-IIR protein stability (Figure 4c). We found that
ANG II promotes IGF-IIR protein stability through ERK and GSK3β signaling.
HUANG
ET AL.
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5
ANG II-induced ERK activation phosphorylates HSF-1 at serine 307 and leads to a secondary phosphorylation by GSK3β at serine 303. (a) H9c2 cardiomyoblast cells were treated with ANG II alone or combined with the ERK inhibitor PD98059 (10 µM) or the GSK3 inhibitor LiCl (20 µM) for 24 hr. Protein expression was detected by immunoblotting. Quantification of the results is shown on the right (n = 3). * p < 0.05 and ** p < 0.01. (b) H9c2 cardiomyoblast cells were transfected with 10 nM control siRNA, siERK, or siGSK3 for 24 hr and then treated with 200 nM ANG II for another 24 hr. Protein expression was detected by immunoblotting. Quantification of the results is shown on the right (n = 3). ** p < 0.01 and *** p < 0.001. (c) H9c2 cardiomyoblast cells were treated with the ERK inhibitor PD98059 (10 µM) or the GSK3 inhibitor LiCl (20 µM) with or without 200 nM ANG II for 24 hr. Then, the cells were fixed with 4% paraformaldehyde for immunofluorescence staining. The hypertrophic cardiomyocytes were identified by F-actin staining. A total of 100 cells were scored. Quantification of the results is shown (n = 3). * p < 0.05 and ** p < 0.01. These data were obtained from at least three independent experiments, and the values represent the mean ± S.D F I G UR E 3
2.5 | ANG II induced IGF-IIR protein accumulation through the inhibition of the E3 ligase RNF126 Early studies have indicated that the ubiquitin E3 ligase RNF126
2.6 | GSK3β inhibitor reduced IGF-IIR-mediated heart damage in spontaneously hypertensive rat (SHR) with similar efficacy as ARB therapy
regulates the retrograde sorting of the IGF-IIR (Wang et al., 2003).
To further confirm our hypothesis in vivo, we evaluated protein
Therefore, we tested the expression of RNF126 following ANG II
expression in the heart tissue of spontaneously hypertensive rats
treatment. We found that RNF126 is significantly downregulated
(SHR) before and after administration of angiotensin II receptor
following ANG II administration (Figure 5a).
blockers (ARBs; irbesartan) and LiCl by intraperitoneal injection
We then examined whether ANG II increased IGF-IIR protein
(Figure 6a).
accumulation through RNF126. We found that the interaction
The results of echocardiography showed that ejection fraction (EF)
between RNF126 and IGF-IIR was significantly decreased following
and fractional shortening (FS) were significantly lower in the SHR control
ANG II treatment (Figure 5b). Moreover, we found that knockdown of
group (Table 1 and Figure 6d). However, after 12 weeks of ARBs and
RNF126 conversely promoted the upregulation of IGF-IIR expression
GSK3 inhibitor administration, the heart functions were clearly improved
(Figure 5c). Finally, we found that knockdown of RNF126 reduced Ub-
(Table 1). We also found that cardiac arrangement was disordered in the
conjugated IGF-IIR protein accumulation following MG132 treatment
SHR group. However, ARBs and GSK3 inhibitor treatment alleviated that
(Figure 5d). We then transfected with the dominant negative HSF1
phenomenon (Figure 6b). Furthermore, high IGF-IIR expression was also
constructs, HSF1S303A, HSF1S303Q, HSF1
, to
observed by IHC staining in the SHR group (Figure 6b). The numbers of
observe the expression of RNF126 and IGF-IIR (Figure 5e). We found
TUNEL+ cardiomyocytes were significantly increased in the SHR group
that these dominant negative HSF1 mutants significantly alleviated
(Figure 6b,c). However, treatment with ARBs and GSK3 inhibitor
ANGII-induced RNF126 decrease and IGF-IIR protein accumulation.
attenuated hypertension-induced cardiomyocyte apoptosis. These
These results showed that decrease of RNF126 by ANG II promoted
results indicate that administration of ARBs or GSK3 inhibitor significantly
IGF-IIR protein accumulation for the apoptotic pathway.
alleviated hypertension-induced cardiomyocyte apoptosis, which
S307A
, and HSF1
S307Q
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ET AL.
ANG II induced ERK/GSK3 activation to repress IGF-IIR protein degradation. (a) H9c2 cardiomyoblast cells were treated with the ERK inhibitor PD98059 (10 µM) or the MEK inhibitor U0126 (20 µM) with or without 200 nM ANG II for 24 hr. mRNA expression was detected by RT-PCR. Quantification of the results is shown (n = 3). * p < 0.05. (b) H9c2 cardiomyoblast cells were transfected with 10 nM control siRNA, siERK, or siGSK3 for 24 hr and then treated with 200 nM ANG II for another 24 hr. mRNA expression was detected by RT-PCR. Quantification of the results is shown (n = 3). *** p < 0.001. (c) H9c2 cardiomyoblast cells were treated with ANG II for 24 hr and then co-treated with the proteasome inhibitor MG132 (5 µM) for 6 hr. Cell lysates were immunoprecipitated using antibodies against IGF-IIR. Protein expression was detected by immunoblotting. (d) H9c2 cardiomyoblast cells were transfected with HA-Ubiquitin for 24 hr and then treated with ANG II for 24 hr. Then, cells were co-treated with 5 µM MG132 for 6 and 9 hr. The cell lysates were immunoprecipitated using antibodies against IGF-IIR. Protein expression was detected by immunoblotting. These data were obtained from at least three independent experiments, and the values represent the mean ± S.D F I G UR E 4
indicates that ERK-mediated HSF1 phosphorylation plays a vital role in
IGF-IIR protein expression and downstream apoptotic pathways by
IGF-IIR-induced cardiac dysfunction.
phosphorylating HSF1 during hypertension-induced HF in vitro and in vivo. Phosphorylation of HSF1 at the serine 303 and serine 307 residues by ERK and GSK3 has been reported to repress HSF1 transcriptional activity (Chu et al., 1996; Kline & Morimoto, 1997).
3 | DISCUSSION
Kline and Morimoto (1997) found that ERK1 phosphorylates HSF1 at serine 307 and subsequently leads to a secondary phosphorylation by
In this study, we demonstrated that ANG II induced ERK/GSK3
GSK3 at serine 303. These two phosphorylation sites are located
activation through AT1R, leading to HSF1 phosphorylation at serine
within the regulatory domain and repress HSF1 transcriptional
303 and serine 307. HSF1 phosphorylation at these sites suppressed
activity. Moreover, Xavier et al. (2000) found that GSK3β-mediated
IGF-IIR protein degradation via RNF126, resulting in cardiac
HSF1 phosphorylation negatively regulates both DNA-binding and
hypertrophy and apoptosis. These findings provide a well-
transcription activity, suggesting that HSF1 function is modulated by
characterized mechanism that may aid in the development of a
posttranslational modifications. Consistent with these observations,
therapeutic strategy for regulating hypertension-induced HF through
we found that MEK/ERK/GSK3β specifically phosphorylated HSF1 at
the ERK/GSK3-HSF1-RNF126-IGF-IIR pathway (Figure 7).
serine 303 and serine 307, which is indicative of suppressed HSF1
A growing body of evidence has shown that the MEK1-ERK1/2
transcriptional activity. Our results showed that the phosphorylation
cascade promotes cardiac hypertrophy and cardiomyocyte apoptosis
of HSF1 at different residues is coordinated to regulate HSF1 function
(Bueno et al., 2000; De Windt et al., 2000; Huang et al., 2017).
and distribution. Furthermore, our earlier studies have indicated that
Consistent with these findings, our recent studies have indicated that
JNK degrades SIRT1 to enhance HSF1 acetylation, which is companied
the knockdown of ERK clearly reduced IGF-IIR expression to alleviate
with HSF1 phosphorylation at serine 303 (Huang et al., 2014).
ANG II-induced cardiac hypertrophy and apoptosis (Huang et al.,
Eventually, the inactive HSF1 proteins result in abnormal transcrip-
2014) and doxorubicin-induced cardiotoxicity (Huang et al., 2017). In
tional regulation in response to cellular stress, thereby resulting in
these findings, we found that ERK amplification positively regulated
severe HF.
HUANG
ET AL.
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7
ANG II induced IGF-IIR protein accumulation through E3 ligase RNF126 suppression. (a) H9c2 cardiomyoblast cells were treated with ANG II (25, 50, 100, 200, or 400 nM) for 24 hr. Protein expression was detected by immunoblotting. Quantification of the results is shown (n = 3). * p < 0.05 and ** p < 0.01. (b) H9c2 cardiomyoblast cells were treated with ANG II for 24 hr. The cell lysates were immunoprecipitated with antibodies against IGF-IIR. Protein expression was detected by immunoblotting. (c) H9c2 cardiomyoblast cells were transfected with RNF126 (10 nM) and scrambled siRNA (10 nM) for 48 hr. Protein expression was detected by immunoblotting. Quantification of the results is shown on the right (n = 3). ** p < 0.01. (d) H9c2 cardiomyoblast cells were transfected with siRNA against RNF126 (10 nM) for 48 hr and then treated with 5 µM MG132 for 9 hr. The cell lysates were immunoprecipitated with antibodies against IGF-IIR. Protein expression was detected by immunoblotting. (e) H9c2 cardiomyoblast cells were transfected with the dominant negative mutants of HSF1, HSF1S303A, HSF1S303Q, HSF1 S307A, and HSF1 S307Q for 24 hr and then treated with 100 nM ANG II for 24 hr. These data were obtained from at least three independent experiments, and the values represent the mean ± S.D F I G UR E 5
The ubiquitin proteasome system is central to the regulation of a
suppressed IGF-IIR protein degradation via RNF126, leading to
number of intracellular sorting pathways in mammalian cells,
cardiac hypertrophy and apoptosis. These findings may provide a
including quality control at the endoplasmic reticulum as well as
therapeutic strategy for regulating hypertension-induced HF
the internalization and endosomal sorting of cell surface receptors.
through
Recent studies have identified that the ubiquitin ligase RNF126
AT1R and GSK3 antagonists.
the
ERK/GSK3-HSF1-RNF126-IGF-IIR
pathway
by
regulates the retrograde sorting of IGF-IIR (Smith & McGlade, 2014). Moreover, the RING finger domain of RNF126 is required to regulate IGF-IIR levels, suggesting that the ubiquitin ligase activity of
4 | M ATERIA LS AN D METH ODS
RNF126 is required for IGF-IIR sorting and stability. Here, we identified that ANG II decreases RNF126, resulting in IGF-IIR protein accumulation and HSF1 phosphorylation to induce cardiomyocyte
4.1 | Cell culture and transient transfection
hypertrophy. However, the detailed mechanism by which HSF1
H9c2 cardiomyoblast cells derived from embryonic BD1X rat heart
phosphorylation influences RNF126-mediated IGF-IIR levels needs
tissue were obtained from American Type Culture Collection (ATCC,
to be elucidated in the future.
Manassas, VA) and cultured in Dulbecco’s modified essential medium
Taken together, these studies indicated that ANG II-induced
supplemented with high glucose, 10% fetal bovine serum, 2 mM
ERK/GSK3 activation through AT1R resulted in HSF1 phosphory-
glutamine, 100 U/ml penicillin and 1 mM sodium pyruvate in
lation at serine 303 and serine 307. HSF1 phosphorylation
humidified air (5% CO2) at 37°C.
8
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ET AL.
GSK3 enhanced IGF-IIR-induced hypertrophy and apoptosis in SHR hearts. (a) The schematic procedure of ARB and LiCl administration. (b,c) After treating SHR rats with ARB or LiCl for 12 weeks, the cardiac characteristics of the different groups were examined via echocardiography and hematoxylin–eosin staining (HE staining). Echocardiographic assessments of the cardiovascular structure and function were performed on the following groups: control WKY rat group (WKY), SHR rats (SHR), ARB-treated SHR rats (ARB), and LiCltreated SHR rats (GSK3β inhibitor). Hematoxylin and eosin staining was performed on heart ventricles. The expression of IGF-IIR and TUNEL+ cardiomyocytes was evaluated by immunohistochemistry (IHC) and the TUNEL assay, respectively. Quantification of TUNEL+ cardiomyocytes from each group is shown (n = 3 per group). (d) Quantification of fractional shortening (FS%, n = 5 per group) in the left ventricle of the heart is shown below. * p < 0.05. These data were obtained from at least three independent experiments, and the values represent the mean ± S.D. F I G UR E 6
The cells were grown to 80% confluence on the day of
To generate a dominant negative mutant of HSF1, we performed
transfection. The plasmids and siRNAs were transfected using the
PCR using Flag-HSF1 WT plasmids as the template according to the
PureFection transfection reagent according to the manufacturer’s
manufacturer’s instructions (QuikChange II Site-Directed Mutagenesis
instructions (System Biosciences, Mountain View, CA). The siRNA
Kit, Agilent Technologies, Santa Clara, CA). The primer sequences
against
were as follows:
AT1R
(SASI_Rn01_00081485,
sequence
start
1236),
AT2R (SASI_Rn02_00259530, sequence start 837), GSK3β (SASI_Rn01_00035805, sequence start 562), and RNF126 (SASI_Rn01_00085667, sequence start 333) were purchased from Sigma (St. Louis, MO). Flag-HSF1 was purchased from (Addgene, Cambridge, MA).
5′-HSF1
S303A(5’-ctctgaggcggggcggggggctcctc-3’);
3′-HSF1
S303A (5’-gaggagccccccgccccgcctcagag-3′),
5′-HSF1
ctctacccggggggcctgaggcgggctg-3’), and 3′-HSF1 S307A (5’-cagcccgcctcaggccccccgggtagag-3’).
S307A
(5’-
HUANG
ET AL.
TABLE 1
|
9
Cardiac characteristics of the GSK3 inhibitor in SHR rats following GSK3 inhibitor treatment SHR WKY (n = 6)
Vehicle (n = 5)
ARB (n = 5)
GSK3β inhibitor (n = 3)
Tibia length (mm)
40.9 ± 2.08
40.4 ± 1.18
39.9 ± 1.26
40.0 ± 1.02
WHW (g)
1.07 ± 0.19
1.25 ± 0.11
1.11 ± 0.14
1.09 ± 0.11
LVW (g)
0.70 ± 0.08
0.86 ± 0.09
0.72 ± 0.05
0.71 ± 0.04
LVW/TL (×100)
1.71
2.13
1.80
1.78
EF (%)
75.11 ± 1.24
64.96 ± 2.67*
72.35 ± 2.27**
69.15 ± 2.77**
FS (%)
39.3 ± 1.32
31.62 ± 1.89*
37.06 ± 1.77**
34.32 ± 2.18**
Values are presented as the mean ± SD. WHW, whole heart weight; LVW, left ventricular weight; LVW/Tibia, left ventricular weight normalized by tibial length; EF, ejection fraction; and FS, fractional shortening. *Indicates a significant difference compared to the WKY group at p < 0.05. **Indicates a significant difference compared to the SHR group at p < 0.05.
4.2 | Western blot analysis and immunoprecipitation A total of 30 µg of total lysates or 10 mg of subcellular fractions was separated using 6–12% SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes (GE Healthcare, Amersham, UK). The membranes were blocked using 5% non-fat milk and blotted
with specific antibodies overnight at 4°C. Then, these protein signals were measured using horseradish peroxidase-conjugated secondary antibodies (1:10,000; GE Healthcare) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA). Densitometric analysis of the immunoblots was performed using the AlphaImager2200 digital imaging system (Digital Imaging System, Commerce, CA). The digital images were processed in Adobe Photoshop 7.0. Each blot was stripped using Restore Western Blot Stripping Buffer (Pierce, Page County, IA) and incubated with other antibodies. The results were analyzed and quantified by the ImageJ software (NIH, Bethesda, MD). Immunoprecipitations were performed using H9c2 cell lysates and the PureProteome Protein G Magnetic Bead System (Millipore) according to the manufacturer’s instructions (Lin, Fan, Maa, & Leu, 2015). A total of 300 µg of cell lysate was prepared. The lysate was combined and allowed to interact with 2 µg of a specific primary antibody, and the mixture was incubated on a rotator at 4°C overnight. Immunoprecipitated proteins were eluted from the magnetic beads at 95°C for 5 min and separated by SDS-PAGE.
4.3 | Antibodies and reagents The following antibodies were used in this study: anti-IGF-IIR, antiERK1/2, anti-phospho-ERK1/2, anti-phospho-GSK-3β (serine 9), anti GSK-3β, anti-β-actin, anti- Gαq/11, anti-ANP, anti-BNP, and antiHDAC1 from Santa Cruz Biotechnology (Santa Cruz, CA). All of the secondary antibodies (HRP-conjugated anti-rabbit, mouse, and goat) were purchased from Santa Cruz Biotechnology. All reagents were ANG II induced cardiac hypertrophy via ERK/GSK3βmediated HSF1 phosphorylation and destabilized the E3 ligase RNF126 to promote IGF-IIR signaling. ANG II challenge activates ERK to phosphorylate HSF1Ser307, which subsequently triggers GSK3ß to phosphorylate HSF1Ser303. Phosphorylation at these sites leads to the cytosolic translocation of HSF1, which in turn reduces the level of the E3 ligase protein RNF126. Once RNF126 is degraded by ANG II, IGF-IIR is highly expressed and translocates into the membrane to induce caspase-3 activation and, eventually, cardiac hypertrophy and cardiomyocyte apoptosis. Moreover, supplementation with the GSK3ß inhibitor LiCl alleviates ANG IIinduced cardiomyocyte apoptosis via the suppression of the IGF-IIR apoptotic signaling pathway by inhibiting HSF1 phosphorylation F I G UR E 7
purchased from Sigma (St. Louis, MO).
4.4 | RNA extraction Total RNA was extracted using the Directzol RNA MiniPrep Kit (Zymo Research Corporation, Irvine, CA) according the manufacturer’s instructions. Briefly, 1 mg of total RNA was incubated with 0.5 µg of oligo dT (MD.Bio., Taipei, Taiwan) at 70°C for 15 min. Then, the RNA was mixed with buffer containing 0.25 mM dNTPs (MD. Bio.), 20 U of RNasin I Plus RNase Inhibitor (Promega, Madison, WI), and 20 U of M-MLV Reverse Transcriptase (Promega) and incubated at 42°C for
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90 min for cDNA synthesis. This mixture was then used for specific
ACKNOWLEDGMENTS
cDNA amplification in a GeneAmp PCR system 2400 (Perkin-Elmer,
This study is supported in part by Taiwan Ministry of Health and
Waltham, MA).
Welfare Clinical Trial and Research Center of Excellence (MOHW106TDU-B-212-113004).
4.5 | Measurement of surface IGF-IIR expression Cells were seeded onto 12-well plates 1 day before treatment with siRNAs or drugs. After treatment, the cells were washed using PBS and
CONFLICT OF INTEREST The authors declare no conflict of interest.
fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were blocked with 5% goat serum and incubated with a
REFERENCES
mouse anti-IGF-IIR antibody (ab2733, Abcam) overnight at 4°C. After
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staining with the primary antibody, the cells were incubated with a rabbit anti-mouse HRP-conjugated antibody for 1.5 hrs at room temperature. Finally, the cells were washed and incubated with the HRP substrate (1-step ultra TMB solution; Pierce, Rockford, IL) for 30 min. The reaction was stopped by 1 M sulfuric acid and measured at 550 mm.
4.6 | Indirect immunofluorescence and confocal microscopy Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X–100 for 15 min at room temperature before staining with a specific antibody (Liua et al., 2015). Then, the cells were washed and stained with Alexa 546 rabbit anti-mouse IgG secondary antibodies (Invitrogen, Carlsbad, CA). Images were captured using a Leica SP2 Confocal Spectral Microscope. The images were processed using Adobe Photoshop.
4.7 | Experimental animals and intraperitoneal injection of anti-hypertension drugs All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85–23, revised 1996) under a protocol approved by the Animal Research Committee of China Medical University in Taichung, Taiwan. Female SHRs and normotensive control Wistar-Kyoto (WKY) rats were used in our experiments. The rats were housed at a constant temperature (22°C) on a 12 hr light/dark cycle with food and tap water. The animals were arranged into five groups: WKY rats, SHRs, SHRs treated with irbesartan (SHR/ARB) and SHRs treated with LiCl (127 mg/kgw/day). Each group contained five animals. The ARB drug irbesartan (40 mg/kg/day; Merck, Sao Paulo, Brazil) was dissolved in Cremophor EL, and the other drugs were dissolved in drinking water.
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How to cite this article: Huang C-Y, Lee F-L, Peng S-F, et al. HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy. J Cell Physiol. 2017;9999:1–11. https://doi.org/10.1002/jcp.25945
