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β2-AR-induced Her2 transactivation mediated by Erbin confers protection from apoptosis in cardiomyocytes
International Journal of Cardiology 167 (2013) 1570–1577
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β2-AR-induced Her2 transactivation mediated by Erbin confers protection from apoptosis in cardiomyocytes☆ Ming Shi a, 1, Mingzhen Zhao b, 1, Meiru Hu a, Dan Liu c, Hong Cao c, Lu Qian a, Zhengyan Yang b, Yabin Hu a, Ming Yu a, Shuo Yang a, Yuanfang Ma b,⁎, Ning Guo b,⁎⁎ a b c
Department of Pathophysiology, Institute of Basic Medical Sciences, Beijing 100850, PR China Laboratory of Cellular and Molecular Immunology, Medical School of Henan University, Kaifeng 475004, PR China Department of Pathology, Hebei Medical University, Shijiazhuang 050017, PR China
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 11 April 2012 Accepted 14 April 2012 Available online 6 May 2012 Keywords: Erbin β2-AR Her2 Cardiomyocyte Apoptosis
a b s t r a c t Background: Her2 and β2-adrenergic receptor (β2-AR) can form a heterocomplex in cardiomyocytes and agonists can induce Her2 transactivation, which is important for cardiovascular homeostasis. The scaffolding molecules that mediate β2-AR/Her2 interaction are currently unknown. Erbin, a PDZ domain-containing protein is a binding partner of Her2. The C-terminus of β2-AR harbors a PDZ domain-binding motif. Hypothesis of this study is that Erbin may organize the assembly of β2-AR/Her2 complex. Methods: The interaction among β2-AR, Her2 and Erbin was investigated in COS-7, HEK-293 and H9c2 cells and rat brain and heart tissues by coimmunoprecipitation. The β2-AR binding region of Erbin was identified by utilizing the Erbin deletion mutants. The functional significance of Erbin in cardiomyocytes was determined by Erbin silencing, contraction frequency measurement and cellular apoptosis assays. Results: Erbin was able to form a complex with both exogenous and endogenous β2-AR and Her2 in the presence of isoproterenol (ISO). Deletion of the Erbin LRR domain did not affect its binding to β2-AR and Her2, whereas lacking of the PDZ domain lost the ability of Erbin. Silencing of Erbin greatly abrogated ISOinduced activation of ERK. The treatment of H9c2 cells transfected with the Erbin siRNA with ISO caused severe cell apoptosis. Knock-down of Erbin expression in primary neonatal rat cardiomyocytes led to a remarkable reduction of the beating frequency after ISO stimulation. Conclusions: Erbin mediates catecholamine-induced β2-AR/Her2 complexation and promotes catecholamineinduced activation of ERK signaling in cardiomyocytes, conferring protection of cardiomyocytes from apoptosis induced by chronic catecholamine stimulation. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Crosstalk of receptor tyrosine kinases (RTKs) with G-protein coupled receptors (GPCRs) is thought to be important for cardiovascular homeostasis [1]. A fundamental role for the transactivation of Her receptor family in cardioprotection has been recognized [2,3]. It was suggested that GPCR kinases-mediated phosphorylation of β1-AR may transmit an antiapoptotic signal by inducing the transactivation of the epidermal growth factor receptor (EGFR) through a β-arrestin dependent pathway
☆ This work is supported by the National Basic Research Program of China (973 Program, 2010CB911904), the National Natural Science Foundation of China (nos. 30972690, 30901766 and 30800582) and the Beijing National Natural Science Foundation (no. 7122124). ⁎ Corresponding author. Tel.: + 86 3783880398; fax: + 86 3783885036. ⁎⁎ Corresponding author. Tel./fax: + 86 1068213039. E-mail addresses: [email protected] (Y. Ma), [email protected] (N. Guo). 1 Contributed equally as first authors to this work. 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.04.093
[4]. The importance of Her2 in normal cardiac development and physiology was also proposed using cardiac specific Her2 knockout mice [5,6]. Clinical studies have addressed the cardiotoxic effects of the therapeutic antibody Herceptin targeting Her2 [7]. A recent report revealed a potential role of Her2 in ERK1/2 activation induced by multiple GPCR ligands in the heart [8]. However, how GPCR induces Her2 transactivation is not clearly defined. β-adrenergic receptors (β-ARs) are an important class of GPCR family. β-AR mediates the activation of different signaling pathways via assembly of a multi-receptor complex with a variety of receptors and downstream effectors [9]. The PDZ domain-binding motifs at the C-terminus of human β1- and β2-ARs can recruit scaffolding proteins containing PDZ domains [10]. The scaffolding proteins connect the trafficking and signaling molecules including Src, JNK and ERK1/2, modulating physiologic signaling and trafficking of β-ARs in cardiomyocytes [11]. Interaction between the PDZ motif of β1-AR with the postsynaptic density protein 95 (PSD-95), a PDZ domian-containing protein, has been identified in HEK-293 cells and cardiomyocytes [12]. The PDZ motif of β2-AR can bind to a PDZ scaffolding protein
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Na +/H + exchanger regulatory factor under PDGF stimulation in porcine aortic endothelial cells. The PDZ motifs of both β1- and β2-ARs can also couple to Gs and Gi in cardiomyocytes. Erbin is a member of the LAP (leucine-rich repeat and PDZ domain) protein family and the PDZ domain of Erbin binds to the C-terminal region of Her2 [13]. The roles of Erbin in different signaling pathways have been investigated in several previous studies [14–18]. In the present study, we demonstrate that Erbin scaffolds the interaction of β2AR with Her2, promotes β2-AR-mediated Her2 transactivation and confers protection from apoptosis induced by chronic catecholamine stimulation in cardiomyocytes, implicating that Erbin functions as a double modulator in ERK signaling pathway.
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2.7. siRNA targeting Erbin The siRNA specifically targeting Erbin (5′-CACACUGUUGUAUGAUCAACCAU-3′) was designed and chemically synthesized by Invitrogen. Scramble siRNA (Invitrogen) was used as a control. H9c2 cells were plated in 24-well plates at a density of 1.8×105/ml/ well or in 6-well plates (2 ml/well) at a density of 3.6 ×105/ml and cultured for 24 h. The cells were transfected with Erbin specific siRNA or control siRNA (6 pmol/well for 24-well plates or 24 pmol/well for 6-well plates) using Lipofectamine™ RNAiMAX (Invitrogen, 1 μl/well for 24-well plates or 4 μl/well for 6-well plates) when approximately 80% confluent. Freshly isolated primary rat cardiomyocytes were counted and the viability was assessed by Trypan blue dye exclusion method. The cells grown in 3-cm dishes (4× 105/ ml, 2 ml/dish) were transfected with Erbin siRNA or control siRNA (24 pmol/dish) using Lipofectamine™ RNAiMAX (4 μl/dish).
2.8. Transduction 2. Materials and methods 2.1. Cell culture and treatment HEK-293, H9c2 and COS-7 cells are obtained from the American Type Culture Collection. For the treatment with β2-AR agonists, the cells were incubated overnight in a serum-free medium and then treated with 10 ng/ml EGF or 10 μM isoproterenol (ISO) for 10 min.
Freshly isolated primary rat cardiomyocytes were counted and viability was assessed by Trypan blue dye exclusion method. Primary rat cardiomyocytes (4× 105/ml) were plated in 3-cm dishes (2 ml/dish). At the same time, the cells were transduced with the lentiviral vectors expressing Erbin siRNA (pLV-Erbin siRNA, GeneChem) at a MOI of 125. The lentivirus expressing nonspecific siRNA was used as a control.
2.9. Preparation of cytosolic and plasma membrane fractions 2.2. Primary culture of neonatal rat cardiomyocytes All experiments complied with the guidelines of the Chinese Council on Animal Care and were approved by the Animal Care Committee of The Institute of Basic Medical Sciences. Cardiomyocytes were isolated from the ventricles of one-day-old Swiss rats as previously described [19]. The isolated cardiomyocytes were re-suspended in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 20% neonatal calf serum and plated in the dishes for 30 min, which allowed selective attachment of nonmyocytes, mostly fibroblasts. This step was repeated twice. The neonatal rat cardiomyocytes were enriched in the supernatant and used in the following experiments. 2.3. Construction The plasmids containing Myc-Erbin, Myc-ErbinΔLRR, HA-Erbin PDZ, HA-ErbinΔPDZ were kindly provided by Dr. Lin Mei (Institute of Molecular Medicine and Genetics, Medical College of Georgia). The plasmid pcDNA3/Her2 was kindly provided by Dr. Yanjun Jia (Beijing Institute of Transfusion Medicine). The plasmid pcDNA3/β2-AR is a generous gift from Dr. Youyi Zhang (Institute of Vascular Medicine, Beijing Medical University). Full length β2-AR cDNA was amplified by PCR with the primers (forward: 5′-CCCAAGCTTGGGCAACCCGGGAACGGCAGCG-3′; reverse: 5′-CGCGGATCC TTACAGCAGTGAGTCATTTGTACT-3′) from pcDNA3/β2-AR and cloned into the plasmid p3× FLAG-CMV™-10 (Sigma).
HEK-293 cells in 6-well plates (5 × 105/well) were transfected with Her2 and β2AR expression plasmids. After transfection for 48 h, the cells were starved overnight and then treated with 10 μM ISO for 0, 2, 5 and 10 min, respectively. The cytosolic and plasma membrane fractions were isolated by Membrane-Cytosol Extraction Kit (Applygen Technologies) following the manufacturer's instructions.
2.10. Measurement of cardiomyocyte contraction The transfected cardiomyocytes were treated with the designated agonists for 10 min. The spontaneous contractions were observed, the beating frequency from eight randomly selected microscopic fields counted and video images recorded. This experiment was repeated twice.
2.11. Apoptosis assays The H9c2 cells were plated in 6-well plates (6 × 105/well) and transfected with Erbin siRNA or control siRNA as mentioned above. After transfection for 60 h, the cells were treated with 5 μM ISO every 2 h until 10 h. Cellular apoptosis was assayed by DeadEndTM Colorimetric TUNEL System (Promega) after treatment for 10 h.
2.4. Western blot 2.12. Statistical analysis The following antibodies were used for immunoblotting: the antibodies against Her2, β2-AR (Santa Cruz), Na/K-ATPase, HA-Tag, Myc-Tag, p-ERK, ERK and glyceraldehyde-3phosphate dehydrogenase (GAPDH) (Cell Signaling). All experiments were performed in duplicate.
All data were presented as mean ± SD. Student's t test for comparison between 2 groups or one-way ANOVA for comparison of 3 or more groups followed by Turkey's post hoc test. P b 0.05 was considered statistically significant.
2.5. Immunofluorescence and confocal microscopy The HEK-293 cells expressing Flag-β2-AR, HA-Erbin PDZ or HA-ErbinΔPDZ fusion proteins were labeled by the anti-Flag (Sigma) and anti-HA (Cell signaling) antibodies. Binding was detected by Alexa fluor 549-labeled (red) and Alexa fluor 488-labeled (green) secondary antibodies (Invitrogen). Nuclei were stained with 1 μg/ml DAPI (Sigma). Fluorescence images were collected under a laser scanning confocal microscope (LSM 510 META, ZEISS). This experiment was repeated at least twice. 2.6. Transient transfection and immunoprecipitation HEK-293 and COS-7 cells were plated in 24-well plates at a density of 1.2 × 105/ml/ well and cultured for 24 h. The cells were transfected with various plasmids (0.8 μg/ well) using Lipofectamine 2000 (Invitrogen) when approximately 80% confluent. For co-transfection, cells were transfected with 0.8 μg plasmids and 20 pmol siRNA. After transfection for 48 h, the cells were starved overnight and then treated with 10 μM ISO for 10 min. Exogenous Her2 and β2-AR and endogenous Erbin were immunoprecipitated by the anti-Her2, anti-β2-AR (Santa Cruz) and anti-Erbin antibodies [20]. Western blot analysis of the immunoprecipitated protein complexes was performed with the indicated antibodies. For detection of the interaction among endogenous β2-AR, Her2 and Erbin, the lysates from H9c2 cells and the brain and heart tissues isolated from three-month old Swiss rats were prepared and immunoprecipitated by corresponding antibodies. The relative intensities of the bands were densitometrically quantified.
3. Results 3.1. The β2-AR/Her2 complex contains Erbin in rat heart and brain tissues A recent study reveals that Her2 and β2-AR form a complex in heart tissues and suggests that Her2 may act as a coreceptor for GPCR signaling. However, the study also indicates that other unidentified scaffolding molecules are required for Her2 to mediate GPCR ligand-induced ERK1/2 activation [8]. Our study verified the association of Her2 with β2-AR in heart tissues by coimmunoprecipitation (Fig. 1A). It is known that β2-AR can interact with the PDZ domain-containing proteins in cardiomyocytes [10]. A recently identified PDZ domaincontaining protein Erbin can bind to Her2 in epithelia [21]. We speculated that Erbin may also associate with β2-AR, assembling β2-AR/Her2 complex. The data in Fig. 1B confirmed our hypothesis, as Erbin could be coimmunoprecipitated with β2-AR. Further experiments testified the existence of β2-AR, Her2 and Erbin complex in rat heart and brain tissues (Fig. 1C), demonstrating that Erbin is a new binding partner of β2-AR and β2-AR/Her2 complex contains Erbin.
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domain (HA-ErbinΔPDZ) lost the ability of Erbin to interact with β2-AR and Her2 (Fig. 3F, upper panel). To investigate whether the PDZ domain of Erbin impacts intracellular localization of β2-AR, HEK-293 cells were cotransfected with the plasmids expressing Flag-β2-AR and HA-Erbin PDZ or HA-ErbinΔPDZ. Immunofluorescence assays revealed that Flag-β2-AR was distributed in the cell membrane as well as cytoplasm, whereas HA-Erbin PDZ was predominantly diffused in the cytoplasm. After exposure to ISO, Flag-β2-AR and HA-Erbin PDZ were rapidly aggregated to the cell membrane (Fig. 3E, lower panel). HA-ErbinΔPDZ was mainly confined in the cell membrane no matter whether the cells were stimulated with ISO. When the cells expressing exogenous β2-AR and HA-ErbinΔPDZ were stimulated with ISO, no colocalization of these two proteins was observed in the cells (Fig. 3F, lower panel), indicating that the PDZ domain of Erbin is required for ISO-stimulated β2-AR/Her2 complexation. 3.4. Erbin enhances β2-AR-mediated transactivation of ERK Fig. 1. β2-AR/Her2 complex contains Erbin in rat heart and brain tissues. The lysates were prepared from the cardiac and brain tissues of three-month old Swiss rats and coimmunoprecipitation performed with the indicated antibodies. A and B, interactions between Her2 and β2-AR (A) and β2-AR and Erbin (B) in cardiac tissues; C, interactions among β2-AR, Her2 and Erbin in heart and brain tissues.
3.2. Catecholamine stimulates interaction of Erbin with β2-AR and Her2 in HEK-293 and H9c2 cells To evaluate the interaction among β2-AR, Her2 and Erbin under catecholamine stimulation, HEK-293 cells were transfected with the plasmids expressing β2-AR and Her2. The immunoprecipitation assays showed that the association of β2-AR with either Her2 or Erbin was hardly detectable without ISO stimulation (Fig. 2A, B and C). However, Erbin and Her2 were constitutively associated with each other (Fig. 2B). After exposure to 10 μM ISO, Erbin and Her2 could be immunoprecipitated by the antibody against β2-AR (Fig. 2A), so were β2-AR and Erbin by the antibody against Her2 (Fig. 2B). Reciprocal assays also demonstrated that β2-AR and Her2 were immunoprecipitated by the anti-Erbin antibody (Fig. 2C). We next examined the interaction of the endogenous β2-AR, Her2 and Erbin in H9c2 cells. In good agreement with the feature of intermolecular interactions in HEK-293 cells, the association between Her2 and Erbin could be easily detected in the absence of stimulation and β-AR agonist enhanced the interaction among β2-AR, Her2 and Erbin (Fig. 2D, E and F). When the expression of Erbin was silenced by the specific siRNA, the association of β2-AR and Her2 dramatically abolished, indicating that ISO stimulates the formation of β2-AR, Her2 and Erbin complex and that Erbin scaffolds the interaction between β2-AR and Her2. 3.3. The PDZ domain of Erbin is required for β2-AR and Her2 complexation To map the β2-AR-binding region of Erbin, we created a series of Erbin deletion mutants carrying a Myc or hemagglutinin (HA) tag (Fig. 3A). HEK-293 cells were cotransfected with the individual Erbin mutant constructs, together with wild-type β2-AR and Her2 constructs. The expression of the Erbin mutants was confirmed by Western blot with the anti-Myc or anti-HA antibodies (Fig. 3B). Expectedly, Myc-Erbin could bind to the endogenous β2-AR and Her2. ISO stimulation significantly enhanced the interactions of Myc-Erbin with β2-AR and Her2 (Fig. 3C). Deletion of the LRR domain (MycErbinΔLRR) did not affect binding of Myc-Erbin to β2-AR and Her2 (Fig. 3D). Interestingly, the Erbin fragment containing only PDZ domain (HA-Erbin PDZ) could interact with β2-AR and Her2 very effectively and ISO induction markedly promoted the receptor complex formation (Fig. 3E, upper panel). On the contrary, lacking of the PDZ
Fig. 4A showed that silencing of Erbin conspicuously enhanced EGF-induced ERK phosphorylation in H9c2 cells. ISO stimulation significantly induced the phosphorylation of ERK. Surprisingly, Erbin silencing dramatically attenuated the basal phosphorylation level of ERK and abrogated ISO-induced activation of ERK (Fig. 4B). To further evaluate whether catecholamine-induced activation of ERK depends on Erbin-mediated β2-AR/Her2 complexation, COS-7 cells were cotransfected with the plasmids expressing β2-AR, Her2 and HA-Erbin PDZ, together with Erbin siRNA. As shown in Fig. 4C, 5 min exposure of 10 μM ISO to the COS-7 cells overexpressing β2-AR alone resulted in a slight elevation of ERK phosphorylation level, whereas ISO stimulation did not induce ERK phosphorylation in the cells overexpressing Her2 only. When both β2-AR and Her2 were overexpressed, ISO stimulation triggered an extraordinary activation of ERK. The effect was remarkably impaired by silencing of Erbin. Surprisingly, introduction of HA-Erbin PDZ dramatically promoted ISO-induced ERK phosphorylation in the cells overexpressing β2-AR and Her2 simultaneously. In addition, Erbin PDZ also significantly reversed the inhibitory effect of Erbin siRNA on ISO-induced ERK activation. β2-AR and Her2 are transmembrane receptors. Under nonstimulated conditions, both receptors reside on the plasma membrane. Erbin is generally aggregated at the basolateral membrane or associated with lateral junctions in polarized epithelial cells [21]. To form a complex with β2-AR and Her2 after catecholamine stimulation, Erbin should be recruited to the region where β2-AR and Her2 are localized. We treated HEK-293 cells with 10 μM ISO for 0, 2, 5 and 10 min and isolated the cytosolic and plasma membrane fractions. Western blot analysis showed that Erbin in the plasma membrane fraction was prominently increased in a time-dependent manner concomitant with the decline of Erbin protein in the cytosolic fraction (Fig. 4D). These data indicate that Erbin PDZ is essential for the formation of the receptor complex and that Erbin positively promotes Her2-mediated ERK activation by β2-AR agonist in cardiomyocytes. 3.5. Erbin participates in protection from cardiomyocyte apoptosis triggered by chronic ISO stimulation Chronic exposure to catecholamine is associated with cardiomyocyte apoptosis, which contributes to the pathophysiology of myocardial failure [22]. It has been proposed that activation of β1-AR is directly proapoptotic via a Gs-mediated, PKA-dependent mechanism, whereas β2-AR-mediated signaling improves cardiac contractility and protects cardiomyocytes from apoptosis caused by chronic β1-AR stimulation [23]. It has also been reported that ERK signaling pathway plays an important role in the regulation of cardiomyocyte apoptosis [1]. To determine the functional consequences of Erbin in cardiomyocyte apoptosis,
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Fig. 2. ISO stimulates the interactions of β2-AR, Her2 and Erbin in HEK-293 and H9c2 cells. HEK-293 cells were transfected with pcDNA3/Her2 and pcDNA3/β2-AR expression plasmids. After transfection for 48 h, the transfected cells were starved overnight and then treated with 10 μM ISO for 10 min. Co-immunoprecipitations and Western blot analysis were performed. The relative intensities of the bands were densitometrically quantified. A, Erbin and Her2 were coimmunoprecipitated by the anti-β2-AR antibody; B, β2-AR and Erbin by the anti-Her2 antibody; C, β2-AR and Her2 by the anti-Erbin antibody. D, E and F, the interactions among β2-AR, Her2 and Erbin were analyzed by coimmunoprecipitation in H9c2 cells. **P b 0.01 G, H9c2 cells were transfected with Erbin siRNA or control siRNA. Transfected cells were starved and then treated with 10 μM ISO for 10 min. β2-AR and Erbin were coimmunoprecipitated by the anti-Her2 antibody.
H9c2 cells were transfected with the Erbin siRNA and then treated with 10 μM ISO. The treatment with ISO did not cause marked cell apoptosis in H9c2 cells. Without ISO stimulation knockdown of Erbin expression did not alter cell survival either. However, after exposure
to chronic ISO stimulation, severe cellular apoptosis was observed (Fig. 5A). To determine whether disruption of Erbin-mediated β2-AR/Her2 complexation has the potential impact on the cardiomyocyte contractile
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Fig. 3. The PDZ domain of Erbin is required for β2-AR and Her2 complexation. A, schematic constructs expressing Myc-Erbin, Myc-ErbinΔLRR, HA-Erbin PDZ and HA-ErbinΔPDZ. B, HEK-293 cells were transfected with the plasmids. The expression of Myc-Erbin, Myc-ErbinΔLRR, HA-Erbin PDZ and HA-ErbinΔPDZ was analyzed by Western blot. C and D, the transfected cells were starved and treated with 10 μM ISO for 10 min. The interactions among β2-AR, Her2 and Myc-Erbin (C) or Myc-ErbinΔLRR (D) were assessed by immunoprecipitation. E and F, the interactions among β2-AR, Her2 and HA-Erbin PDZ (E) or HA-Erbin ΔPDZ (F) were assessed by immunoprecipitation and immunofluorescence assays at the indicated time points.
function in response to agonist-induced β2-AR activation, the primary neonatal rat cardiomyocytes were transfected with Erbin siRNA and the frequency of spontaneous beating measured. After transfection for 48 h, the beating frequency was remarkably reduced by 31.9% relative to the control cells. The strongest effect on the cardiomyocyte contraction was observed at 72 h post-transfection and the beating rate reduced by 51.8% (Fig. 5B). After 96 h, the cardiomyocyte contraction was slightly restored. To further confirm it, we employed the lentivirus expressing Erbin siRNA and GFP protein. After infection for 96 h, the expression of GFP was observed in ~70–80% primary neonatal rat cardiomyocytes. At the same time, the beating frequency of cardiomyocytes was greatly decreased. However, after an additional 24 h of incubation the beating rate recovered to a previous frequency. At six days post-infection, ISO was added into the cell culture every 2 h and cardiomyocyte contraction
observed after 10 h. In the absence of ISO stimulation, the spontaneous beating of the cells lacking of Erbin appeared to be similar to the control cells (Fig. 5C). After ISO stimulation, the control cells produced a distinct contractile response and the beating rate was increased by 43.3%. While, surprisingly, the spontaneous beating of the cells infected with lentivirus-Erbin siRNA became very week with less rhythmic and the contraction rate was drastically decreased by 88.5% (Fig. 5C). These data implicate a potential mechanism for the requirement of Erbin in cardioprotection under chronic catecholamine stimulation. 4. Discussion It was recently reported that GPCR agonists can induce the transactivation of Her2. However, the interaction of Her2 with GPCR was thought to be indirect and the linkage between them to be unclear
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Fig. 4. Erbin enhances β2-AR-mediated activation of ERK signaling pathway. A and B, H9c2 cells were transfected with Erbin siRNA or control siRNA. The transfected cells were starved and then treated with 10 ng/ml EGF (A) or 5 μM ISO (B) for 10 min. The phosphorylation of ERK was evaluated by Western blot with the antibody recognizing phosphorylated ERK. Detection of GAPDH was used as a control. C, COS-7 cells were cotransfected with the plasmids expressing β2-AR, Her2, HA-Erbin PDZ and Erbin siRNA. The phosphorylation of ERK was detected by Western blot. D, HEK-293 cells were treated with 10 μM ISO for 0, 2, 5 and 10 min and the cytosolic and plasma membrane fractions isolated. The expression of Erbin was analyzed by Western blot. The relative intensities of the bands were densitometrically quantified. *P b 0.05; **P b 0.01.
[8]. Her2 was the first identified partner of Erbin. The C-terminus (YLGLDVPV) of Her2 harbors a class II PDZ-binding consensus (-Ψ-X-Ψ, where Ψ is a hydrophobic amino acid and X any amino acid) [13]. Coincidentally, the C-terminus (DSLL) of β2-AR possesses a class I PDZ-binding motif X–(S/T)–X–(V/I/L) [24]. The primary conclusions of our study are that Erbin mediates catecholamine-induced β2-AR/Her2 complexation in cardiomyocytes and promotes the activation of ERK signaling pathway, conferring the protection of cardiomyocytes from apoptosis induced by chronic catecholamine stimulation. The association of β2-AR with Her2 and related signaling events are critically dependent on Erbin. Knockdown of Erbin expression severely interrupted the formation of β2-AR/
Her2 complex and impaired the phosphorylation of ERK, proving that Erbin as a scaffold organizes the assembly of β2-AR/Her2 complex and mediates the transactivation of ERK stimulated by β2-AR agonists. Our study reveals that the PDZ domain of Erbin is decisively responsible for the assembly of β2-AR/Her2 complexes. Removal of PDZ domain virtually destroyed the scaffolding capacity of Erbin. We noticed that the treatment with ISO caused the accumulation of Erbin to the plasma membrane in a time-dependent manner and induced a prominent colocalization of β2-AR and Erbin at the plasma membrane. Conversely, deficiency of Erbin PDZ domain dramatically disrupted the interaction between β2-AR and Erbin. These data
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Fig. 5. Erbin is involved in protection from cardiomyocyte apoptosis triggered by chronic ISO stimulation. A, H9c2 cells were transfected with Erbin siRNA or control siRNA and then treated with 10 μM ISO every 2 h. Cell apoptosis was assessed by DeadEndTM Colorimetric TUNEL System after treatment for 10 h. B, the primary neonatal rat cardiomyocytes were transfected with Erbin siRNA or control siRNA. After transfection for 48, 72 and 96 h, the beating rate was measured. C, the primary neonatal rat cardiomyocytes were infected with the lentivirus expressing Erbin siRNA or control siRNA. At six days post-infection, 10 μM ISO was added into the cell culture every 2 h and the frequency of spontaneous beating measured after 10 h.
confirm that the PDZ domain of Erbin is essential for the β2-AR/Her2 complexation under catecholamine stimulation. The previous studies demonstrate that Erbin functions as an inhibitor of the MAPK signaling pathway by interfering with the Ras–Raf interaction and the expression of Erbin inhibited EGF- and NGF-induced ERK activation [21]. In contrast to the previous reports, our study show that silencing of Erbin expression completely blocked catecholamineinduced ERK activation, whereas overexpression of Erbin PDZ domain efficiently reversed the effect, suggesting that Erbin positively mediates catecholamine-induced transactivation of ERK in cardiomyocytes and that Erbin exerts dual functions in ERK signaling pathway, either as a negative regulator to suppress EGF-induced ERK activation or as a positive regulator to enhance catecholamine-stimulated ERK activation (Fig. 6). The functions of Erbin were mostly investigated in nerve and tumor cells in the previous studies. Notably, Erbin was highly expressed in cardiac tissues. However, the actions of Erbin in cardiomyocytes have not been explored. The present study provides the first evidence that Erbin plays an important role in cardiomyocytes.
Chronic increase in sympathetic nerve activity is known to have cardiotoxic effects in patients with heart failure. Numerous studies have indicated that persistent β2-AR activation plays an antiapoptotic role by specific linking of β2-AR to Gi involved the activation of MAPK signaling pathways [23]. Clinical studies have pointed out cardiotoxicity associated with Herceptin therapy in the treatment of breast cancer, reflecting the crucial role of Her2-mediated signaling in cardiovascular control and cardiac homeostasis [7]. Our data clearly show that blockage of Erbinmediated β2-AR/Her2 complexation strongly inhibited spontaneous contraction of cardiomyocytes, abolished catecholamine-stimulated contractility response in H9c2 cells and impaired β2-AR-mediated antiapoptotic effect. The scaffolding protein-mediated signaling transduction has been realized as a new mechanism in cardioprotection. For example, β-arrestins bring the members of several MAPK cascades to GPCRs under stimulation and transduce nonclassical signaling events, which contribute to cardioprotective effects against catecholamine toxicity [1]. Our data suggest that Erbin mediates the transition of classic β2-AR signaling to MAPK/ ERK signaling by bridging β2-AR and Her2 following chronic administration of β2-AR agonist and thus antagonizes cardiomyocyte apoptosis. Further exploration of unknown functions of Erbin and related signaling mechanisms in cardiomyocytes may provide the information for the development of selective small molecular drug candidates for treating heart failure. Acknowledgment The authors of this manuscript have certified that they comply with the principles of ethical publishing in the International Journal of Cardiology. References
Fig. 6. Erbin exerts dual functions in ERK signaling pathway. Erbin inhibits EGF-induced ERK signaling pathway by blocking Ras–Raf interaction. In cardiomyocytes Erbin enhances catecholamine-induced transactivation of ERK by promoting β2-AR and Her2 interaction.
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[14] Huang YZ, Zang M, Xiong WC, Luo Z, Mei L. Erbin suppresses the MAP kinase pathway. J Biol Chem 2003;278:1108–14. [15] Dai P, Xiong WC, Mei L. Erbin inhibits RAF activation by disrupting the sur-8–Ras– Raf complex. J Biol Chem 2006;281:927–33. [16] Dai F, Chang C, Lin X, Dai P, Mei L, Feng XH. Erbin inhibits transforming growth factor beta signaling through a novel Smad-interacting domain. Mol Cell Biol 2007;27:6183–94. [17] Zhou Q, Zeng R, Xu C, et al. Erbin inhibits TGF-beta1-induced EMT in renal tubular epithelial cells through an ERK-dependent pathway. J Mol Med (Berl) May 2012;90(5):563–74. [18] McDonald C, Chen FF, Ollendorff V, et al. A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling. J Biol Chem 2005;280:40,301–9. [19] Florian M, Jankowski M, Gutkowska J. Oxytocin increases glucose uptake in neonatal rat cardiomyocytes. Endocrinology 2010;151:482–91. [20] Zhang H, Yu M, Hu M, et al. Overexpression, refolding, purification of Erbin PDZ domain from Escherichia coli and preparation of its polyclonal antibody. Prep Biochem Biotechnol 2008;38:282–93. [21] Dan L, Shi M, Duan H, Han C, Guo N. Erbin, a negative regulator in diverse signal pathways. Curr Protein Pept Sci 2010;11:759–64. [22] Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of beta-adrenergic signaling in heart failure? Circ Res 2003;93:896–906. [23] Zhu W, Woo AY, Zhang Y, Cao CM, Xiao RP. beta-adrenergic receptor subtype signaling in the heart: from bench to the bedside. Curr Top Membr 2011;67:191–204. [24] Valentine CD, Haggie PM. Confinement of beta(1)- and beta(2)-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae. Mol Biol Cell 2011;22:2970–82.
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
β2-AR-induced Her2 transactivation mediated by Erbin confers protection from apoptosis in cardiomyocytes☆ Ming Shi a, 1, Mingzhen Zhao b, 1, Meiru Hu a, Dan Liu c, Hong Cao c, Lu Qian a, Zhengyan Yang b, Yabin Hu a, Ming Yu a, Shuo Yang a, Yuanfang Ma b,⁎, Ning Guo b,⁎⁎ a b c
Department of Pathophysiology, Institute of Basic Medical Sciences, Beijing 100850, PR China Laboratory of Cellular and Molecular Immunology, Medical School of Henan University, Kaifeng 475004, PR China Department of Pathology, Hebei Medical University, Shijiazhuang 050017, PR China
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 11 April 2012 Accepted 14 April 2012 Available online 6 May 2012 Keywords: Erbin β2-AR Her2 Cardiomyocyte Apoptosis
a b s t r a c t Background: Her2 and β2-adrenergic receptor (β2-AR) can form a heterocomplex in cardiomyocytes and agonists can induce Her2 transactivation, which is important for cardiovascular homeostasis. The scaffolding molecules that mediate β2-AR/Her2 interaction are currently unknown. Erbin, a PDZ domain-containing protein is a binding partner of Her2. The C-terminus of β2-AR harbors a PDZ domain-binding motif. Hypothesis of this study is that Erbin may organize the assembly of β2-AR/Her2 complex. Methods: The interaction among β2-AR, Her2 and Erbin was investigated in COS-7, HEK-293 and H9c2 cells and rat brain and heart tissues by coimmunoprecipitation. The β2-AR binding region of Erbin was identified by utilizing the Erbin deletion mutants. The functional significance of Erbin in cardiomyocytes was determined by Erbin silencing, contraction frequency measurement and cellular apoptosis assays. Results: Erbin was able to form a complex with both exogenous and endogenous β2-AR and Her2 in the presence of isoproterenol (ISO). Deletion of the Erbin LRR domain did not affect its binding to β2-AR and Her2, whereas lacking of the PDZ domain lost the ability of Erbin. Silencing of Erbin greatly abrogated ISOinduced activation of ERK. The treatment of H9c2 cells transfected with the Erbin siRNA with ISO caused severe cell apoptosis. Knock-down of Erbin expression in primary neonatal rat cardiomyocytes led to a remarkable reduction of the beating frequency after ISO stimulation. Conclusions: Erbin mediates catecholamine-induced β2-AR/Her2 complexation and promotes catecholamineinduced activation of ERK signaling in cardiomyocytes, conferring protection of cardiomyocytes from apoptosis induced by chronic catecholamine stimulation. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Crosstalk of receptor tyrosine kinases (RTKs) with G-protein coupled receptors (GPCRs) is thought to be important for cardiovascular homeostasis [1]. A fundamental role for the transactivation of Her receptor family in cardioprotection has been recognized [2,3]. It was suggested that GPCR kinases-mediated phosphorylation of β1-AR may transmit an antiapoptotic signal by inducing the transactivation of the epidermal growth factor receptor (EGFR) through a β-arrestin dependent pathway
☆ This work is supported by the National Basic Research Program of China (973 Program, 2010CB911904), the National Natural Science Foundation of China (nos. 30972690, 30901766 and 30800582) and the Beijing National Natural Science Foundation (no. 7122124). ⁎ Corresponding author. Tel.: + 86 3783880398; fax: + 86 3783885036. ⁎⁎ Corresponding author. Tel./fax: + 86 1068213039. E-mail addresses: [email protected] (Y. Ma), [email protected] (N. Guo). 1 Contributed equally as first authors to this work. 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.04.093
[4]. The importance of Her2 in normal cardiac development and physiology was also proposed using cardiac specific Her2 knockout mice [5,6]. Clinical studies have addressed the cardiotoxic effects of the therapeutic antibody Herceptin targeting Her2 [7]. A recent report revealed a potential role of Her2 in ERK1/2 activation induced by multiple GPCR ligands in the heart [8]. However, how GPCR induces Her2 transactivation is not clearly defined. β-adrenergic receptors (β-ARs) are an important class of GPCR family. β-AR mediates the activation of different signaling pathways via assembly of a multi-receptor complex with a variety of receptors and downstream effectors [9]. The PDZ domain-binding motifs at the C-terminus of human β1- and β2-ARs can recruit scaffolding proteins containing PDZ domains [10]. The scaffolding proteins connect the trafficking and signaling molecules including Src, JNK and ERK1/2, modulating physiologic signaling and trafficking of β-ARs in cardiomyocytes [11]. Interaction between the PDZ motif of β1-AR with the postsynaptic density protein 95 (PSD-95), a PDZ domian-containing protein, has been identified in HEK-293 cells and cardiomyocytes [12]. The PDZ motif of β2-AR can bind to a PDZ scaffolding protein
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Na +/H + exchanger regulatory factor under PDGF stimulation in porcine aortic endothelial cells. The PDZ motifs of both β1- and β2-ARs can also couple to Gs and Gi in cardiomyocytes. Erbin is a member of the LAP (leucine-rich repeat and PDZ domain) protein family and the PDZ domain of Erbin binds to the C-terminal region of Her2 [13]. The roles of Erbin in different signaling pathways have been investigated in several previous studies [14–18]. In the present study, we demonstrate that Erbin scaffolds the interaction of β2AR with Her2, promotes β2-AR-mediated Her2 transactivation and confers protection from apoptosis induced by chronic catecholamine stimulation in cardiomyocytes, implicating that Erbin functions as a double modulator in ERK signaling pathway.
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2.7. siRNA targeting Erbin The siRNA specifically targeting Erbin (5′-CACACUGUUGUAUGAUCAACCAU-3′) was designed and chemically synthesized by Invitrogen. Scramble siRNA (Invitrogen) was used as a control. H9c2 cells were plated in 24-well plates at a density of 1.8×105/ml/ well or in 6-well plates (2 ml/well) at a density of 3.6 ×105/ml and cultured for 24 h. The cells were transfected with Erbin specific siRNA or control siRNA (6 pmol/well for 24-well plates or 24 pmol/well for 6-well plates) using Lipofectamine™ RNAiMAX (Invitrogen, 1 μl/well for 24-well plates or 4 μl/well for 6-well plates) when approximately 80% confluent. Freshly isolated primary rat cardiomyocytes were counted and the viability was assessed by Trypan blue dye exclusion method. The cells grown in 3-cm dishes (4× 105/ ml, 2 ml/dish) were transfected with Erbin siRNA or control siRNA (24 pmol/dish) using Lipofectamine™ RNAiMAX (4 μl/dish).
2.8. Transduction 2. Materials and methods 2.1. Cell culture and treatment HEK-293, H9c2 and COS-7 cells are obtained from the American Type Culture Collection. For the treatment with β2-AR agonists, the cells were incubated overnight in a serum-free medium and then treated with 10 ng/ml EGF or 10 μM isoproterenol (ISO) for 10 min.
Freshly isolated primary rat cardiomyocytes were counted and viability was assessed by Trypan blue dye exclusion method. Primary rat cardiomyocytes (4× 105/ml) were plated in 3-cm dishes (2 ml/dish). At the same time, the cells were transduced with the lentiviral vectors expressing Erbin siRNA (pLV-Erbin siRNA, GeneChem) at a MOI of 125. The lentivirus expressing nonspecific siRNA was used as a control.
2.9. Preparation of cytosolic and plasma membrane fractions 2.2. Primary culture of neonatal rat cardiomyocytes All experiments complied with the guidelines of the Chinese Council on Animal Care and were approved by the Animal Care Committee of The Institute of Basic Medical Sciences. Cardiomyocytes were isolated from the ventricles of one-day-old Swiss rats as previously described [19]. The isolated cardiomyocytes were re-suspended in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 20% neonatal calf serum and plated in the dishes for 30 min, which allowed selective attachment of nonmyocytes, mostly fibroblasts. This step was repeated twice. The neonatal rat cardiomyocytes were enriched in the supernatant and used in the following experiments. 2.3. Construction The plasmids containing Myc-Erbin, Myc-ErbinΔLRR, HA-Erbin PDZ, HA-ErbinΔPDZ were kindly provided by Dr. Lin Mei (Institute of Molecular Medicine and Genetics, Medical College of Georgia). The plasmid pcDNA3/Her2 was kindly provided by Dr. Yanjun Jia (Beijing Institute of Transfusion Medicine). The plasmid pcDNA3/β2-AR is a generous gift from Dr. Youyi Zhang (Institute of Vascular Medicine, Beijing Medical University). Full length β2-AR cDNA was amplified by PCR with the primers (forward: 5′-CCCAAGCTTGGGCAACCCGGGAACGGCAGCG-3′; reverse: 5′-CGCGGATCC TTACAGCAGTGAGTCATTTGTACT-3′) from pcDNA3/β2-AR and cloned into the plasmid p3× FLAG-CMV™-10 (Sigma).
HEK-293 cells in 6-well plates (5 × 105/well) were transfected with Her2 and β2AR expression plasmids. After transfection for 48 h, the cells were starved overnight and then treated with 10 μM ISO for 0, 2, 5 and 10 min, respectively. The cytosolic and plasma membrane fractions were isolated by Membrane-Cytosol Extraction Kit (Applygen Technologies) following the manufacturer's instructions.
2.10. Measurement of cardiomyocyte contraction The transfected cardiomyocytes were treated with the designated agonists for 10 min. The spontaneous contractions were observed, the beating frequency from eight randomly selected microscopic fields counted and video images recorded. This experiment was repeated twice.
2.11. Apoptosis assays The H9c2 cells were plated in 6-well plates (6 × 105/well) and transfected with Erbin siRNA or control siRNA as mentioned above. After transfection for 60 h, the cells were treated with 5 μM ISO every 2 h until 10 h. Cellular apoptosis was assayed by DeadEndTM Colorimetric TUNEL System (Promega) after treatment for 10 h.
2.4. Western blot 2.12. Statistical analysis The following antibodies were used for immunoblotting: the antibodies against Her2, β2-AR (Santa Cruz), Na/K-ATPase, HA-Tag, Myc-Tag, p-ERK, ERK and glyceraldehyde-3phosphate dehydrogenase (GAPDH) (Cell Signaling). All experiments were performed in duplicate.
All data were presented as mean ± SD. Student's t test for comparison between 2 groups or one-way ANOVA for comparison of 3 or more groups followed by Turkey's post hoc test. P b 0.05 was considered statistically significant.
2.5. Immunofluorescence and confocal microscopy The HEK-293 cells expressing Flag-β2-AR, HA-Erbin PDZ or HA-ErbinΔPDZ fusion proteins were labeled by the anti-Flag (Sigma) and anti-HA (Cell signaling) antibodies. Binding was detected by Alexa fluor 549-labeled (red) and Alexa fluor 488-labeled (green) secondary antibodies (Invitrogen). Nuclei were stained with 1 μg/ml DAPI (Sigma). Fluorescence images were collected under a laser scanning confocal microscope (LSM 510 META, ZEISS). This experiment was repeated at least twice. 2.6. Transient transfection and immunoprecipitation HEK-293 and COS-7 cells were plated in 24-well plates at a density of 1.2 × 105/ml/ well and cultured for 24 h. The cells were transfected with various plasmids (0.8 μg/ well) using Lipofectamine 2000 (Invitrogen) when approximately 80% confluent. For co-transfection, cells were transfected with 0.8 μg plasmids and 20 pmol siRNA. After transfection for 48 h, the cells were starved overnight and then treated with 10 μM ISO for 10 min. Exogenous Her2 and β2-AR and endogenous Erbin were immunoprecipitated by the anti-Her2, anti-β2-AR (Santa Cruz) and anti-Erbin antibodies [20]. Western blot analysis of the immunoprecipitated protein complexes was performed with the indicated antibodies. For detection of the interaction among endogenous β2-AR, Her2 and Erbin, the lysates from H9c2 cells and the brain and heart tissues isolated from three-month old Swiss rats were prepared and immunoprecipitated by corresponding antibodies. The relative intensities of the bands were densitometrically quantified.
3. Results 3.1. The β2-AR/Her2 complex contains Erbin in rat heart and brain tissues A recent study reveals that Her2 and β2-AR form a complex in heart tissues and suggests that Her2 may act as a coreceptor for GPCR signaling. However, the study also indicates that other unidentified scaffolding molecules are required for Her2 to mediate GPCR ligand-induced ERK1/2 activation [8]. Our study verified the association of Her2 with β2-AR in heart tissues by coimmunoprecipitation (Fig. 1A). It is known that β2-AR can interact with the PDZ domain-containing proteins in cardiomyocytes [10]. A recently identified PDZ domaincontaining protein Erbin can bind to Her2 in epithelia [21]. We speculated that Erbin may also associate with β2-AR, assembling β2-AR/Her2 complex. The data in Fig. 1B confirmed our hypothesis, as Erbin could be coimmunoprecipitated with β2-AR. Further experiments testified the existence of β2-AR, Her2 and Erbin complex in rat heart and brain tissues (Fig. 1C), demonstrating that Erbin is a new binding partner of β2-AR and β2-AR/Her2 complex contains Erbin.
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domain (HA-ErbinΔPDZ) lost the ability of Erbin to interact with β2-AR and Her2 (Fig. 3F, upper panel). To investigate whether the PDZ domain of Erbin impacts intracellular localization of β2-AR, HEK-293 cells were cotransfected with the plasmids expressing Flag-β2-AR and HA-Erbin PDZ or HA-ErbinΔPDZ. Immunofluorescence assays revealed that Flag-β2-AR was distributed in the cell membrane as well as cytoplasm, whereas HA-Erbin PDZ was predominantly diffused in the cytoplasm. After exposure to ISO, Flag-β2-AR and HA-Erbin PDZ were rapidly aggregated to the cell membrane (Fig. 3E, lower panel). HA-ErbinΔPDZ was mainly confined in the cell membrane no matter whether the cells were stimulated with ISO. When the cells expressing exogenous β2-AR and HA-ErbinΔPDZ were stimulated with ISO, no colocalization of these two proteins was observed in the cells (Fig. 3F, lower panel), indicating that the PDZ domain of Erbin is required for ISO-stimulated β2-AR/Her2 complexation. 3.4. Erbin enhances β2-AR-mediated transactivation of ERK Fig. 1. β2-AR/Her2 complex contains Erbin in rat heart and brain tissues. The lysates were prepared from the cardiac and brain tissues of three-month old Swiss rats and coimmunoprecipitation performed with the indicated antibodies. A and B, interactions between Her2 and β2-AR (A) and β2-AR and Erbin (B) in cardiac tissues; C, interactions among β2-AR, Her2 and Erbin in heart and brain tissues.
3.2. Catecholamine stimulates interaction of Erbin with β2-AR and Her2 in HEK-293 and H9c2 cells To evaluate the interaction among β2-AR, Her2 and Erbin under catecholamine stimulation, HEK-293 cells were transfected with the plasmids expressing β2-AR and Her2. The immunoprecipitation assays showed that the association of β2-AR with either Her2 or Erbin was hardly detectable without ISO stimulation (Fig. 2A, B and C). However, Erbin and Her2 were constitutively associated with each other (Fig. 2B). After exposure to 10 μM ISO, Erbin and Her2 could be immunoprecipitated by the antibody against β2-AR (Fig. 2A), so were β2-AR and Erbin by the antibody against Her2 (Fig. 2B). Reciprocal assays also demonstrated that β2-AR and Her2 were immunoprecipitated by the anti-Erbin antibody (Fig. 2C). We next examined the interaction of the endogenous β2-AR, Her2 and Erbin in H9c2 cells. In good agreement with the feature of intermolecular interactions in HEK-293 cells, the association between Her2 and Erbin could be easily detected in the absence of stimulation and β-AR agonist enhanced the interaction among β2-AR, Her2 and Erbin (Fig. 2D, E and F). When the expression of Erbin was silenced by the specific siRNA, the association of β2-AR and Her2 dramatically abolished, indicating that ISO stimulates the formation of β2-AR, Her2 and Erbin complex and that Erbin scaffolds the interaction between β2-AR and Her2. 3.3. The PDZ domain of Erbin is required for β2-AR and Her2 complexation To map the β2-AR-binding region of Erbin, we created a series of Erbin deletion mutants carrying a Myc or hemagglutinin (HA) tag (Fig. 3A). HEK-293 cells were cotransfected with the individual Erbin mutant constructs, together with wild-type β2-AR and Her2 constructs. The expression of the Erbin mutants was confirmed by Western blot with the anti-Myc or anti-HA antibodies (Fig. 3B). Expectedly, Myc-Erbin could bind to the endogenous β2-AR and Her2. ISO stimulation significantly enhanced the interactions of Myc-Erbin with β2-AR and Her2 (Fig. 3C). Deletion of the LRR domain (MycErbinΔLRR) did not affect binding of Myc-Erbin to β2-AR and Her2 (Fig. 3D). Interestingly, the Erbin fragment containing only PDZ domain (HA-Erbin PDZ) could interact with β2-AR and Her2 very effectively and ISO induction markedly promoted the receptor complex formation (Fig. 3E, upper panel). On the contrary, lacking of the PDZ
Fig. 4A showed that silencing of Erbin conspicuously enhanced EGF-induced ERK phosphorylation in H9c2 cells. ISO stimulation significantly induced the phosphorylation of ERK. Surprisingly, Erbin silencing dramatically attenuated the basal phosphorylation level of ERK and abrogated ISO-induced activation of ERK (Fig. 4B). To further evaluate whether catecholamine-induced activation of ERK depends on Erbin-mediated β2-AR/Her2 complexation, COS-7 cells were cotransfected with the plasmids expressing β2-AR, Her2 and HA-Erbin PDZ, together with Erbin siRNA. As shown in Fig. 4C, 5 min exposure of 10 μM ISO to the COS-7 cells overexpressing β2-AR alone resulted in a slight elevation of ERK phosphorylation level, whereas ISO stimulation did not induce ERK phosphorylation in the cells overexpressing Her2 only. When both β2-AR and Her2 were overexpressed, ISO stimulation triggered an extraordinary activation of ERK. The effect was remarkably impaired by silencing of Erbin. Surprisingly, introduction of HA-Erbin PDZ dramatically promoted ISO-induced ERK phosphorylation in the cells overexpressing β2-AR and Her2 simultaneously. In addition, Erbin PDZ also significantly reversed the inhibitory effect of Erbin siRNA on ISO-induced ERK activation. β2-AR and Her2 are transmembrane receptors. Under nonstimulated conditions, both receptors reside on the plasma membrane. Erbin is generally aggregated at the basolateral membrane or associated with lateral junctions in polarized epithelial cells [21]. To form a complex with β2-AR and Her2 after catecholamine stimulation, Erbin should be recruited to the region where β2-AR and Her2 are localized. We treated HEK-293 cells with 10 μM ISO for 0, 2, 5 and 10 min and isolated the cytosolic and plasma membrane fractions. Western blot analysis showed that Erbin in the plasma membrane fraction was prominently increased in a time-dependent manner concomitant with the decline of Erbin protein in the cytosolic fraction (Fig. 4D). These data indicate that Erbin PDZ is essential for the formation of the receptor complex and that Erbin positively promotes Her2-mediated ERK activation by β2-AR agonist in cardiomyocytes. 3.5. Erbin participates in protection from cardiomyocyte apoptosis triggered by chronic ISO stimulation Chronic exposure to catecholamine is associated with cardiomyocyte apoptosis, which contributes to the pathophysiology of myocardial failure [22]. It has been proposed that activation of β1-AR is directly proapoptotic via a Gs-mediated, PKA-dependent mechanism, whereas β2-AR-mediated signaling improves cardiac contractility and protects cardiomyocytes from apoptosis caused by chronic β1-AR stimulation [23]. It has also been reported that ERK signaling pathway plays an important role in the regulation of cardiomyocyte apoptosis [1]. To determine the functional consequences of Erbin in cardiomyocyte apoptosis,
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Fig. 2. ISO stimulates the interactions of β2-AR, Her2 and Erbin in HEK-293 and H9c2 cells. HEK-293 cells were transfected with pcDNA3/Her2 and pcDNA3/β2-AR expression plasmids. After transfection for 48 h, the transfected cells were starved overnight and then treated with 10 μM ISO for 10 min. Co-immunoprecipitations and Western blot analysis were performed. The relative intensities of the bands were densitometrically quantified. A, Erbin and Her2 were coimmunoprecipitated by the anti-β2-AR antibody; B, β2-AR and Erbin by the anti-Her2 antibody; C, β2-AR and Her2 by the anti-Erbin antibody. D, E and F, the interactions among β2-AR, Her2 and Erbin were analyzed by coimmunoprecipitation in H9c2 cells. **P b 0.01 G, H9c2 cells were transfected with Erbin siRNA or control siRNA. Transfected cells were starved and then treated with 10 μM ISO for 10 min. β2-AR and Erbin were coimmunoprecipitated by the anti-Her2 antibody.
H9c2 cells were transfected with the Erbin siRNA and then treated with 10 μM ISO. The treatment with ISO did not cause marked cell apoptosis in H9c2 cells. Without ISO stimulation knockdown of Erbin expression did not alter cell survival either. However, after exposure
to chronic ISO stimulation, severe cellular apoptosis was observed (Fig. 5A). To determine whether disruption of Erbin-mediated β2-AR/Her2 complexation has the potential impact on the cardiomyocyte contractile
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Fig. 3. The PDZ domain of Erbin is required for β2-AR and Her2 complexation. A, schematic constructs expressing Myc-Erbin, Myc-ErbinΔLRR, HA-Erbin PDZ and HA-ErbinΔPDZ. B, HEK-293 cells were transfected with the plasmids. The expression of Myc-Erbin, Myc-ErbinΔLRR, HA-Erbin PDZ and HA-ErbinΔPDZ was analyzed by Western blot. C and D, the transfected cells were starved and treated with 10 μM ISO for 10 min. The interactions among β2-AR, Her2 and Myc-Erbin (C) or Myc-ErbinΔLRR (D) were assessed by immunoprecipitation. E and F, the interactions among β2-AR, Her2 and HA-Erbin PDZ (E) or HA-Erbin ΔPDZ (F) were assessed by immunoprecipitation and immunofluorescence assays at the indicated time points.
function in response to agonist-induced β2-AR activation, the primary neonatal rat cardiomyocytes were transfected with Erbin siRNA and the frequency of spontaneous beating measured. After transfection for 48 h, the beating frequency was remarkably reduced by 31.9% relative to the control cells. The strongest effect on the cardiomyocyte contraction was observed at 72 h post-transfection and the beating rate reduced by 51.8% (Fig. 5B). After 96 h, the cardiomyocyte contraction was slightly restored. To further confirm it, we employed the lentivirus expressing Erbin siRNA and GFP protein. After infection for 96 h, the expression of GFP was observed in ~70–80% primary neonatal rat cardiomyocytes. At the same time, the beating frequency of cardiomyocytes was greatly decreased. However, after an additional 24 h of incubation the beating rate recovered to a previous frequency. At six days post-infection, ISO was added into the cell culture every 2 h and cardiomyocyte contraction
observed after 10 h. In the absence of ISO stimulation, the spontaneous beating of the cells lacking of Erbin appeared to be similar to the control cells (Fig. 5C). After ISO stimulation, the control cells produced a distinct contractile response and the beating rate was increased by 43.3%. While, surprisingly, the spontaneous beating of the cells infected with lentivirus-Erbin siRNA became very week with less rhythmic and the contraction rate was drastically decreased by 88.5% (Fig. 5C). These data implicate a potential mechanism for the requirement of Erbin in cardioprotection under chronic catecholamine stimulation. 4. Discussion It was recently reported that GPCR agonists can induce the transactivation of Her2. However, the interaction of Her2 with GPCR was thought to be indirect and the linkage between them to be unclear
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Fig. 4. Erbin enhances β2-AR-mediated activation of ERK signaling pathway. A and B, H9c2 cells were transfected with Erbin siRNA or control siRNA. The transfected cells were starved and then treated with 10 ng/ml EGF (A) or 5 μM ISO (B) for 10 min. The phosphorylation of ERK was evaluated by Western blot with the antibody recognizing phosphorylated ERK. Detection of GAPDH was used as a control. C, COS-7 cells were cotransfected with the plasmids expressing β2-AR, Her2, HA-Erbin PDZ and Erbin siRNA. The phosphorylation of ERK was detected by Western blot. D, HEK-293 cells were treated with 10 μM ISO for 0, 2, 5 and 10 min and the cytosolic and plasma membrane fractions isolated. The expression of Erbin was analyzed by Western blot. The relative intensities of the bands were densitometrically quantified. *P b 0.05; **P b 0.01.
[8]. Her2 was the first identified partner of Erbin. The C-terminus (YLGLDVPV) of Her2 harbors a class II PDZ-binding consensus (-Ψ-X-Ψ, where Ψ is a hydrophobic amino acid and X any amino acid) [13]. Coincidentally, the C-terminus (DSLL) of β2-AR possesses a class I PDZ-binding motif X–(S/T)–X–(V/I/L) [24]. The primary conclusions of our study are that Erbin mediates catecholamine-induced β2-AR/Her2 complexation in cardiomyocytes and promotes the activation of ERK signaling pathway, conferring the protection of cardiomyocytes from apoptosis induced by chronic catecholamine stimulation. The association of β2-AR with Her2 and related signaling events are critically dependent on Erbin. Knockdown of Erbin expression severely interrupted the formation of β2-AR/
Her2 complex and impaired the phosphorylation of ERK, proving that Erbin as a scaffold organizes the assembly of β2-AR/Her2 complex and mediates the transactivation of ERK stimulated by β2-AR agonists. Our study reveals that the PDZ domain of Erbin is decisively responsible for the assembly of β2-AR/Her2 complexes. Removal of PDZ domain virtually destroyed the scaffolding capacity of Erbin. We noticed that the treatment with ISO caused the accumulation of Erbin to the plasma membrane in a time-dependent manner and induced a prominent colocalization of β2-AR and Erbin at the plasma membrane. Conversely, deficiency of Erbin PDZ domain dramatically disrupted the interaction between β2-AR and Erbin. These data
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Fig. 5. Erbin is involved in protection from cardiomyocyte apoptosis triggered by chronic ISO stimulation. A, H9c2 cells were transfected with Erbin siRNA or control siRNA and then treated with 10 μM ISO every 2 h. Cell apoptosis was assessed by DeadEndTM Colorimetric TUNEL System after treatment for 10 h. B, the primary neonatal rat cardiomyocytes were transfected with Erbin siRNA or control siRNA. After transfection for 48, 72 and 96 h, the beating rate was measured. C, the primary neonatal rat cardiomyocytes were infected with the lentivirus expressing Erbin siRNA or control siRNA. At six days post-infection, 10 μM ISO was added into the cell culture every 2 h and the frequency of spontaneous beating measured after 10 h.
confirm that the PDZ domain of Erbin is essential for the β2-AR/Her2 complexation under catecholamine stimulation. The previous studies demonstrate that Erbin functions as an inhibitor of the MAPK signaling pathway by interfering with the Ras–Raf interaction and the expression of Erbin inhibited EGF- and NGF-induced ERK activation [21]. In contrast to the previous reports, our study show that silencing of Erbin expression completely blocked catecholamineinduced ERK activation, whereas overexpression of Erbin PDZ domain efficiently reversed the effect, suggesting that Erbin positively mediates catecholamine-induced transactivation of ERK in cardiomyocytes and that Erbin exerts dual functions in ERK signaling pathway, either as a negative regulator to suppress EGF-induced ERK activation or as a positive regulator to enhance catecholamine-stimulated ERK activation (Fig. 6). The functions of Erbin were mostly investigated in nerve and tumor cells in the previous studies. Notably, Erbin was highly expressed in cardiac tissues. However, the actions of Erbin in cardiomyocytes have not been explored. The present study provides the first evidence that Erbin plays an important role in cardiomyocytes.
Chronic increase in sympathetic nerve activity is known to have cardiotoxic effects in patients with heart failure. Numerous studies have indicated that persistent β2-AR activation plays an antiapoptotic role by specific linking of β2-AR to Gi involved the activation of MAPK signaling pathways [23]. Clinical studies have pointed out cardiotoxicity associated with Herceptin therapy in the treatment of breast cancer, reflecting the crucial role of Her2-mediated signaling in cardiovascular control and cardiac homeostasis [7]. Our data clearly show that blockage of Erbinmediated β2-AR/Her2 complexation strongly inhibited spontaneous contraction of cardiomyocytes, abolished catecholamine-stimulated contractility response in H9c2 cells and impaired β2-AR-mediated antiapoptotic effect. The scaffolding protein-mediated signaling transduction has been realized as a new mechanism in cardioprotection. For example, β-arrestins bring the members of several MAPK cascades to GPCRs under stimulation and transduce nonclassical signaling events, which contribute to cardioprotective effects against catecholamine toxicity [1]. Our data suggest that Erbin mediates the transition of classic β2-AR signaling to MAPK/ ERK signaling by bridging β2-AR and Her2 following chronic administration of β2-AR agonist and thus antagonizes cardiomyocyte apoptosis. Further exploration of unknown functions of Erbin and related signaling mechanisms in cardiomyocytes may provide the information for the development of selective small molecular drug candidates for treating heart failure. Acknowledgment The authors of this manuscript have certified that they comply with the principles of ethical publishing in the International Journal of Cardiology. References
Fig. 6. Erbin exerts dual functions in ERK signaling pathway. Erbin inhibits EGF-induced ERK signaling pathway by blocking Ras–Raf interaction. In cardiomyocytes Erbin enhances catecholamine-induced transactivation of ERK by promoting β2-AR and Her2 interaction.
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