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A novel Fbxo25 acts as an E3 ligase for destructing cardiac specific transcription factors
Biochemical and Biophysical Research Communications 410 (2011) 183–188
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A novel Fbxo25 acts as an E3 ligase for destructing cardiac specific transcription factors Jae-Woo Jang 1, Won-Young Lee 1, Jae-Ho Lee, Sung-Hwan Moon, Chang-Hoon Kim ⇑, Hyung-Min Chung ⇑ Stem Cell Research Laboratory, Department of Developmental Biology, CHA University, Seoul 135-907, Republic of Korea
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Article history: Received 25 April 2011 Available online 7 May 2011 Keywords: Fbxo25 Nkx2-5 Isl1 Ubiquitin E3 ligase Cardiomyocytes
a b s t r a c t Alterations in ubiquitin–proteasome system (UPS) have been implicated in the etiology of human cardiovascular diseases. Skp1/Cul1/F-box (SCF) ubiquitin E3 ligase complex plays a pivotal role in ubiquitination of cardiac proteins. However, a specific ubiquitin E3 ligase responsible for the destruction of cardiac transcription factors such as Nkx2-5, Isl1, Mef2C, and Tbx5 remains elusive to date. Here, we show that a novel F-box containing Fbxo25 is cardiac-specific and acts as an ubiquitin E3 ligase for cardiac transcription factors. Fbxo25 expression was nuclei-specific in vitro and cardiomyocytes. Expression level of Fbxo25 was higher in a fetal heart than an adult. Moreover, Fbxo25 expression was increased along with those of cardiac-specific genes during cardiomyocyte development from ESCs. Fbxo25 expression facilitated protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C. Especially, Fbxo25 ubiquitinated Nkx2-5, Isl1, and Hand1. Altogether, Fbxo25 acts as an ubiquitin E3 ligase to target cardiac transcription factors including Nkx2-5, Isl1, and Hand1, indicating that cardiac protein homeostasis through Fbxo25 has a pivotal impact on cardiac development. Ó 2011 Published by Elsevier Inc.
1. Introduction Ubiquitin–proteasome system (UPS) regulates the majority of about 80% of intracellular proteins in eukaryote cells [1]. Additionally, it controls cell signaling and protein quality in cardiac development and diseases [2]. The ubiquitin E3 ligases are responsible for substrate destruction from E2 conjugating enzymes to a lysine residue [3]. Thus, the E3 ligases are the most numerous and diversified components in UPS for discriminating own substrates. Skp, Cullin, F-box (SCF)-containing complex is a multi-protein E3 ubiquitin ligase complex. The F-box proteins are the components that contain protein interaction domains for binding the ubiquitination targets [3,4]. More than 68 different F-box-containing proteins have been identified in humans [5]. SCF E3 ligases are known to participate in various important cellular processes, but the biological functions of the majority of the F-box proteins remain uncharacterized except for Atrogin-1/Fbxo32 functions in skeletal muscle and heart [6–8]. Though Fbxo25 is expressed in heart, brain, liver, and kidney, its biological function is undefined [9]. The expressions of cardiac genes are controlled by cardiac-specific transcription factors such as Nkx2-5, Tbx5, Gata4, Mef2C,
⇑ Corresponding authors. Address: Department of Developmental Biology, CHA University, CHA Bio and Diostech Co., Ltd., 605-21 Yeoksam-1 dong, Gangnam gu, Seoul 135-907, Republic of Korea. Fax: +82 02 3468 3373. E-mail addresses: [email protected] (C.-H. Kim), [email protected] (H.-M. Chung). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Published by Elsevier Inc. doi:10.1016/j.bbrc.2011.05.011
Hand1, Hand2, and other factors. They bind specific DNA sequences in cardiac gene promoters regulating gene transcription. They are involved in cardiogenesis as well as hypertrophic cardiomyocyte growth [10]. Nkx2-5 is a cardiac homeobox transcription factor, which plays a key role in regulating many tissue specific gene expressions. Nkx2-5 null mice show abnormal cardiac looping [11], indicating that Nkx2-5 function is necessary for cardiac formation. Isl1 is a member of the LIM-homeodomain transcription factor family expressed early in this cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development [12]. Isl1 is a direct transcriptional target of forkhead transcription factors such as Foxa2 and Foxf1 in the second heart field [13]. Hand1 is expressed in the developing ventricular chambers and plays an essential role in cardiac morphogenesis. Additionally, Hand1 acts as a mediator of cardiac development [14] and interacts with Mef2 for the activation of cardiac specific gene expressions [15]. Tbx5 is a T-box-containing transcription factor that is important for limb and heart development. TBX5 mutations cause Holt–Oram syndrome which is characterized by congenital heart defects, conduction system abnormalities, and upper limb deformities [16,17]. Tbx5 has been reported to interact with Gata4, Gata6, and Mef2C which are cardiac transcription factors required for heart development [18,19]. Moreover, Tbx5 associates with Nkx2-5 to promote cardiomyocyte differentiation [20]. In consistency with the above notion, various cardiac transcription factors make up complex networks of cardiac transcriptional regulation and development. However, the regulation of protein expression levels in cardiac specific transcription factors is still unknown.
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Today, death due to myocardial infarction is increasing throughout the world. In order to overcome difficulties in heart transplantation owing to the lack of availability and survivability, the use of embryonic stem cells (ESCs) is currently under study to develop regenerative medicine. ESC-derived cardiomyocytes can be used for drug screening purposes regarding cardiac diseases and for the model of heart developmental biology. Therefore, more attention must be given to understand molecular mechanisms via cardiac specific transcription factors. In the present study, we demonstrated that Fbxo25 expression is cardiomyocyte-specific and acts as an ubiquitin E3 ligase to target cardiac specific transcription factors.
0.1% diethyl pyrocarbonate-treated water. RNA concentration was determined by a NANO-drop. A reverse-transcription reaction was performed with 1 lg RNA using SuperScript III Reverse Transcriptase (Invitrogen) after incubating with DNaseI for eliminating contaminated genomic DNA. The synthesized cDNA was amplified by Platinum PCR Master Mix (Invitrogen) using the proper primer sets (Supplementary Table 1). Amplification conditions were as follows: 5 min at 95 °C, followed by optimal cycles of denaturing (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 30 s) with a final extension at 72 °C for 5 min. PCR products were visualized by electrophoresis on a 2% agarose gel with ethidium bromide.
2. Materials and methods
2.5. Immunocytochemistry
2.1. Human and mouse embryonic stem cell (ESC) culture
Contracting cardiomyocytes were mechanically dissected using a glass micropipette. Mouse adult hearts and fetal hearts were frozen-sectioned for immunostaning. Contracting clusters or tissue sections were fixed with ice-cold 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, then blocked by 3% bovine serum albumin in PBS for 30 min for the inhibition of nonspecific antibody binding. Primary antibodies were used as follows; anti-rabbit Tbx5 (42-6500, Invitrogen), antirabbit Fbxo25 (AV43118, Sigma), or anti-mouse sarcomere and myosin (MF20, Developmental Studies Hybridoma Bank, University of Iowa). The sections were incubated with the appropriate antibodies overnight at 4 °C. Samples were washed three times with PBS and incubated with the following secondary antibodies: Alexa Fluor 488 or 594 conjugated goat anti-mouse IgG (488: A11001, 594: A-11005, Invitrogen) or Alexa Fluor 488 or 594 conjugated goat anti-rabbit IgG (488: A-11008, 594: A-11012) at RT for 1 h. Cell nuclei were stained with DAPI at RT for 10 min. Cells were mounted and immediately examined under a LSM 510 META confocal laser scanning microscope (Carl Zeiss).
Mouse OG2 ES cell lines [21] were cultured and maintained in an undifferentiated state on a feeder layer of mitomycin C (10 lg/ml for 2 h)-treated CF1 mouse embryonic fibroblasts (MEFs) in low glucose Dulbecco’s modified Eagle’s minimal essential medium (DMEM), supplemented with 15% fetal bovine serum (FBS), 1% mercaptoethanol, 1% GlutaMAX-1, 1% non-essential amino acids and 1000 U/ml recombinant mouse leukemia inhibitory factor (LIF). Culture medium was replaced daily. hESC H9 lines [22] were cultured and differentiated to cardiomyocytes as described in Supplementary Materials and methods [23,24]. 2.2. Induction of Cardiomyocyte differentiation from embryonic stem cells Cardiomyocyte differentiation was induced by embryoid body (EB) formation in a suspension culture. After washing with phosphate-buffered saline (PBS), undifferentiated ESCs were dissociated using 0.125% trypsin–ethylenediaminetetraacetic acid. Detached colonies were suspension-cultured for 5 days in differentiation medium including 15% heat-inactivated FBS in DMEM without LIF. On day 5, EBs were plated onto gelatin-coated 35 mm culture dishes for cardiomyocyte induction. Aggregates were observed daily by light microscopy to monitor the morphology and number of spontaneously beating clusters which were identified as cardiomyocytes. 2.3. Plasmids, Western blotting, and immunoprecipitation Full length Fbxo25, Hand1, Nkx2-5, Isl1, Mef2C, Tbx5, and Ub were amplified using human cDNA by RT-PCR cloning technique. Amplified fragments were cloned to the proper epitope tagging expression vectors. These plasmids were transfected to 293T and COS-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturers’ manual. Two days after transfection, cells were washed by PBS and lyzed with RIPA buffer. Cell lysates were used for Western blotting or further experiments. Fifty lg proteins from each lysate was subjected to SDS–polyacrylamide gel electrophoresis, transferred onto PVDF membrane and probed with proper antibodies. Proteins were detected using HRP conjugated secondary antibodies and ECL reagents. For immuneprecipitation, cell lysates were mixed with proper 2 lg antibodies plus 40 ll protein A-Sepharose 4B in RIPA buffer and incubated at 4 °C overnight. The bound beads were washed 3 times with RIPA buffer and analyzed by Western blotting.
3. Results 3.1. Nuclear localization of Fbxo25 with cardiac specific transcription factors F-box containing Fbxo25 is expressed in several tissues [25]. Previous results show that Fbxo25 is localized mainly at the nuclei [9,25]. To examine the localization of Fbxo25, we performed the immunocytochemistry using GFP-Fbxo25 expression in vitro cells. When GFP-Fbxo25 was expressed to 293T cells, GFP signals were largely detectable at the nuclei (Supplementary Fig. S1A) as shown in previous data [25]. Tbx5, Nkx2-5, and Hand1 are cardiac-specific transcription factors for heart development. To examine the cellular distribution of Fbxo25 with Tbx5, Nkx2-5, and Hand1, we conducted the immunocytochemistry experiments using Myc-, GFPor mRFP-tagged transgenes. Tbx5, Nkx2-5, Hand1 as well as Fbxo25 were expressed at the nuclei (Supplementary Fig. S1B, C, D). The localization patterns of Tbx5 and Nkx2-5 were matched with that of Fbxo25 (Supplementary Fig. S1B, C). However, GFP-Hand1 localized differently with mRFP-Fbxo25 (Supplementary Fig. S1D). Unlike the previous report, we didn’t observe Cajal body-like subnuclear localization of Fbox25 probably due to the use of different experiment conditions [25]. Together, novel cardiac Fbxo25 expresses at the nuclei along with Tbx5, Nkx2-5, and Hand1. 3.2. Cardiac expression of Fbxo25 and cardiac transcription factors in heart
2.4. Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturers’ protocol. RNA pellets were dissolved in
A previous report showed that Fbxo25 is highly expressed in adult heart, brain, liver and kidney, but undetectable in adult skeletal muscle, lung, and spleen by Northern blotting [9]. Whether
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Fbxo25 is expressed in early cardiac tissues was under question. To test this idea, we investigated cardiac expression of Fbxo25 proteins in a fetal heart using immunostaining. Endogenous Fbxo25 was detectable in cardiac cells labeled with fast and slow sarcomeric MHCs by MF20, indicating that fetal cardiomyocytes express Fbxo25 (Fig. 1A). In addition, Tbx5 was also expressed in cardiomyocytes stained by MF20 (Fig. 1B). Unfortunately, colocalization between Fbxo25 and Tbx5 in fetal cardiomyocytes could not be examined due to the antibody problem possessing same host-specificity of both antibodies, anti-rabbit. However, it is tempting to speculate that Fbxo25 distributes along with Tbx5 in cardiomyocytes based on the overlay of sarcomeric MHCs. To overcome the antibody problem used in immunostaining, we investigated mRNA expression patterns of Fbxo25, Tbx5, and Nkx2-5 in several tissues such as MEFs, fetal heart, adult heart, ESCs, and adult liver using RT-RCR. Fbxo25 was expressed in the tested tissues as reported previously (Fig. 1C) [9]. As expected, Tbx5 and Nkx2-5 were highly cardiac-specific. In contrast, the expression level of Fbxo25 was higher in a fetal than adult heart. This pattern was similar to those of Tbx5 and Nkx2-5, indicating that Fbxo25 has a physiological rel-
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evance in cardiac development. Collectively, our results demonstrate that Fbxo25 expression is cardiac-specific. 3.3. Fbxo25 is expressed in cardiomyocytes derived from ESCs Our data indicates that Fbxo25 is expressed in cardiomyocytes as well as other tissues. Thus, we investigated the expression pattern of Fbxo25 during cardiomyocyte differentiation from ESCs using OG2-ESCs, which express Oct4 promoter-driven GFP to directly monitor stemness by fluorescence [21]. Cardiomyocytes were differentiated from OG2-ESCs using EB formation in 15% FBS condition without LIF. Maturation was proportional to the decrease of Oct4 expression based on GFP fluorescence (Fig. 2A). GFP fluorescence was detectable in non-beating clusters, but not in beating clusters, indicating that cardiomyctes are differentiated from fully differentiated stem cells (Supplemental video). Formation of visceral endoderm-like cells are necessary for cardiomyocyte development, but GFP expressing clusters may not meet this criterion [21]. Expression patterns of cardiac-specific genes were monitored during cardiomyocyte differentiation from ESCs by
Fig. 1. Cardiac expression of Fbxo25 along with Tbx5 and Nkx-5. (A) Fbxo25 expression in cardiomyocytes at embryonic day 12.5. Frozen sectioned heart tissues from E12.5 embryos were obtained using cryostat. Anti-mouse MF20 antibody labeled both fast and slow sarcomeric MHCs. Endogenous Fbxo25 was stained by anti-rabbit Fbxo25 antibody. DAPI was used for staining cardiac nuclei. (B) Tbx5 expression in cardiomyocytes at embryonic day 12.5. Anti-rabbit Tbx5 antibody was used for staining of cardiac Tbx5. Cardiac MHCs were labeled by anti-mouse MF20. (C) RT-PCR data showed the expressions of Fbxo25, Tbx5, and Nkx2-5 in MEFs, fetal heart, adult heart, ESCs, and adult liver. RNAs were isolated from MEFs, ESCs, embryonic heart at day 12.5, adult heart and liver by TRIzol following manufacturer’s protocol. Primer information used in RT-PCR was shown in Supplementary Table 1.
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Fig. 2. Fbxo25 expression is proportional to ESC-derived cardiomyocyte differentiation. (A) Maturation of cardiomyocytes from ESCs correlated to the decrease of Oct4 expression. Beating clusters didn’t express GFP, whereas GFP expressing clusters showed no contraction. Cardiomyocytes were obtained by spontaneous differentiation of EB formation using OG2 ESCs as described in Materials and methods. Images were acquired by inverted fluorescent microscopy at the indicated differentiation time. (B) Expression patterns of Oct4, Fbxo25, and other cardiac specific genes during cardiomyocyte development from ESCs. Samples were collected at the indicated time and RNAs were isolated using TRIzol. Information of used primers is revealed in Supplementary Table 1. (C) Tbx5 and Fbxo25 expressions in beating cardiomyocytes. Beating clusters were collected and replated on gelatin-coated dishes. Fbxo25 or Tbx5 were labeled with anti-rabbit Fbxo25 antibody or anti-rabbit Tbx5 together with anti-mouse MF20 antibody, respectively. (D) Human FBXO25 or TBX5 expressions in cardiomyocytes derived from hESCs. Cardiomyocytes were collected from beating clusters using 0.25% Trypsin–EDTA and replated on gelatin-coated dishes. After DAPI stains nuclei, images were acquired by confocal microscope.
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RT-PCR experiments. Similar to the reduction of GFP fluorescence, Oct4 was downregulated during cardiomyocyte differentiation. In contrast, cardiac-specific genes such as Myh6, Myh7, Nkx2-5, Tbx5 as well as Fbxo25 were upregulated (Fig. 2B). Moreover, Fbxo25 expression was higher in beating than non-beating clusters similar to those of other cardiac genes. Immunostainings of Fbxo25 and Tbx5 was also conducted in cardiomyocytes from ESCs. As shown in Fig. 2C, Fbxo25 and Tbx5 were stained in sarcomeric MHCs expressing cells, suggesting that Fbxo25 is expressed in ESC-derived cardiomyocytes. hESCs-derived cardiomyocytes also expressed FBXO25 and TBX5 (Fig. 2D). Interestingly, human FBXO25 was expressed at the nuclei and cytosol, indicative of the broad cellular distribution. Collectively, this result demonstrates that Fbxo25 is a novel cardiac-specific gene. 3.4. Fbxo25 activates protein degradation of cardiac transcription factors Fbxo25 contains an F-box domain that allows a multi-protein complex E3 ubiquitin ligase system with Skp1, Cullin1 and
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Rbx1, thus Fbxo25 may execute E3 ligase activity against certain proteins expressed in cardiomyocytes. Based on the colocalization between Fbxo25 and cardiac transcription factors in cardiomyocytes, we hypothesized whether Fbxo25 targets the proteins of cardiac transcription factors via SCF complex. To address this idea, we examined the protein expression patterns of cardiac transcription factors influenced by Fbxo25. As shown in Fig. 3A, Fbxo25 facilitated protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C in comparison to the absence of Fbxo25. This data suggested that Nkx2-5, Isl1, Hand1 are eliminated by an Fbxo25-mediated ubiquitination pathway. To test the notion, we conducted in vitro ubiquitination assays showing that Fbxo25 activated the degradation of Nkx2-5 via ubiquitination pathway (Fig. 3B). Furthermore, proteasome inhibitor lactacystin induced the accumulation of more ubiquitined-Nkx2-5, implicative of Fbxo25-mediated Nkx2-5 degradation. Fbxo25 also facilitated the ubiquitination of Isl1 and Hand1 (Fig. 3C, D) but lactacystin treatment didn’t accumulate ubiquitined Isl1 and Hand1. As a result, the degradation of Nkx2-5, Isl1, and Hand1 is required for Fbxo25-mediated UPS.
Fig. 3. Fbxo25 facilitates protein degradation of cardiac transcription factors by UPS. (A) Fbxo25 reduced protein expression levels of cardiac transcription factors. MycHand1, Myc-Isl1, Flag-Mef2C, Myc-Nkx2-5 and/or GFP-Fbxo25 were transfected to 293T cells. (B) Fbxo25 ubiquitinated Nkx2-5. Myc-Nkx2-5, HA-Ub and/or GFP-Fbxo25 were transfected to 293T cells using Lipofectamine 2000. For the inhibition of proteasome, 5 lM lactacystin was treated for 5 h. Two days after transfection, ubiquitination assays were done using immunoprecipitation with anti-Myc followed by immunoblotting with anti-HA for the detection of ubiquitination as in Materials and methods. (C) Isl1 was degraded by Fbxo25 in ubiquination assay. Myc-Isl1, HA-Ub and/or GFP-Fbxo25 were transfected to 293T cells. (D) Hand1 was ubiquitinated by Fbxo25 in ubiquination assay. Myc-Hand1, HA-Ub and/or GFP-Fbxo25 were transfected to 293T cells.
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4. Discussion The purpose of this study was to identify that Fbxo25 expression is cardiac-specific and acts as an ubiquitin E3 ligase for cardiac transcription factors. Fbxo25 belongs to the F-box only family of proteins composed of 77 and 84 different proteins in mouse and human to date by our data analysis, respectively. F-box proteins like Fbxo32 make up E3 ubiquitin–ligase complex with Skp1, Cullin1 and Rbx1 [9]. Thus, SCF complex proteolyzes certain proteins via UPS. A previous report showed that Fbxo25 is expressed in an adult heart, brain, liver, and kidney by Northern experiment [9]. However, Manfiolli et al. was unable to demonstrate protein expression of Fbxo25 in an adult heart [25]. Our antibody against Fbxo25 recognizes endogenous Fbxo25 in a fetal and adult heart as well as cardiomyoctes from ESCs. However, we cannot prove the colocalization between Fbxo25 and Tbx5 in cardiac tissues due to the antibody problem concerning same host species, antirabbit. Fbxo25 labeling nuclei are overlaid to sarcomeric MHCs staining cardiac cells, which represent cardiomyocytes, similar to Tbx5 (Figs. 1 and 2). It’s likely that Fbxo25 codistributes along with cardiac-specific transcription factor such as Nkx2-5, Isl1, Hand1 as well as Tbx5 in cardiomyocytes. Unlike Fbxo32, Fbxo25 is expressed in the heart rather than skeletal muscle [9]. The expression level of Fbxo25 is higher in a fetal than adult heart (Fig. 1). Fbxo25 is upregulated with cardiomyocyte differentiation (Fig. 2). These patterns of Fbxo25 are similar to those of other cardiac specific genes in heart (Fig. 2) [26]. Fbxo25 activates protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C (Fig. 3). Thus, Fbxo25 might be down-regulated to maintain protein homeostasis during heart development. In summary, Fbxo25 is likely to be expressed in cardiomyocytes in the presence of Tbx5 and other cardiac transcription factors. Cardiac Fbxo25 belongs to the F-box only family as an ubiquitin E3 ligase for the distruction of cardiac transcription factors. Thus, Fbxo25 is a novel E3 ligase responsible for cardiomyocyte development. Cardiac UPS including Fbxo25 has a pivotal impact on cardiac development. Acknowledgments We are very grateful to Dr. Jeong-Tae Do (Laboratory of Stem Cell and Developmental Biology, CHA Stem Cell Institute, CHA University, 605-21 Yoeksam 1-dong, Gangnam-gu, Seoul 135-081, Republic of Korea) for providing OG2-ESCs. This research was supported by a grant (SC-3110) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.05.011. References [1] M.H. Glickman, A. Ciechanover, The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction, Physiol. Rev. 82 (2002) 373–428. [2] M.S. Willis, W.H. Townley-Tilson, E.Y. Kang, J.W. Homeister, C. Patterson, Sent to destroy: The ubiquitin proteasome system regulates cell signaling and protein quality control in cardiovascular development and disease, Circ. Res. 106 (2010) 463–478. [3] A. Hershko, A. Ciechanover, The ubiquitin system, Annu. Rev. Biochem. 67 (1998) 425–479.
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A novel Fbxo25 acts as an E3 ligase for destructing cardiac specific transcription factors Jae-Woo Jang 1, Won-Young Lee 1, Jae-Ho Lee, Sung-Hwan Moon, Chang-Hoon Kim ⇑, Hyung-Min Chung ⇑ Stem Cell Research Laboratory, Department of Developmental Biology, CHA University, Seoul 135-907, Republic of Korea
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Article history: Received 25 April 2011 Available online 7 May 2011 Keywords: Fbxo25 Nkx2-5 Isl1 Ubiquitin E3 ligase Cardiomyocytes
a b s t r a c t Alterations in ubiquitin–proteasome system (UPS) have been implicated in the etiology of human cardiovascular diseases. Skp1/Cul1/F-box (SCF) ubiquitin E3 ligase complex plays a pivotal role in ubiquitination of cardiac proteins. However, a specific ubiquitin E3 ligase responsible for the destruction of cardiac transcription factors such as Nkx2-5, Isl1, Mef2C, and Tbx5 remains elusive to date. Here, we show that a novel F-box containing Fbxo25 is cardiac-specific and acts as an ubiquitin E3 ligase for cardiac transcription factors. Fbxo25 expression was nuclei-specific in vitro and cardiomyocytes. Expression level of Fbxo25 was higher in a fetal heart than an adult. Moreover, Fbxo25 expression was increased along with those of cardiac-specific genes during cardiomyocyte development from ESCs. Fbxo25 expression facilitated protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C. Especially, Fbxo25 ubiquitinated Nkx2-5, Isl1, and Hand1. Altogether, Fbxo25 acts as an ubiquitin E3 ligase to target cardiac transcription factors including Nkx2-5, Isl1, and Hand1, indicating that cardiac protein homeostasis through Fbxo25 has a pivotal impact on cardiac development. Ó 2011 Published by Elsevier Inc.
1. Introduction Ubiquitin–proteasome system (UPS) regulates the majority of about 80% of intracellular proteins in eukaryote cells [1]. Additionally, it controls cell signaling and protein quality in cardiac development and diseases [2]. The ubiquitin E3 ligases are responsible for substrate destruction from E2 conjugating enzymes to a lysine residue [3]. Thus, the E3 ligases are the most numerous and diversified components in UPS for discriminating own substrates. Skp, Cullin, F-box (SCF)-containing complex is a multi-protein E3 ubiquitin ligase complex. The F-box proteins are the components that contain protein interaction domains for binding the ubiquitination targets [3,4]. More than 68 different F-box-containing proteins have been identified in humans [5]. SCF E3 ligases are known to participate in various important cellular processes, but the biological functions of the majority of the F-box proteins remain uncharacterized except for Atrogin-1/Fbxo32 functions in skeletal muscle and heart [6–8]. Though Fbxo25 is expressed in heart, brain, liver, and kidney, its biological function is undefined [9]. The expressions of cardiac genes are controlled by cardiac-specific transcription factors such as Nkx2-5, Tbx5, Gata4, Mef2C,
⇑ Corresponding authors. Address: Department of Developmental Biology, CHA University, CHA Bio and Diostech Co., Ltd., 605-21 Yeoksam-1 dong, Gangnam gu, Seoul 135-907, Republic of Korea. Fax: +82 02 3468 3373. E-mail addresses: [email protected] (C.-H. Kim), [email protected] (H.-M. Chung). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Published by Elsevier Inc. doi:10.1016/j.bbrc.2011.05.011
Hand1, Hand2, and other factors. They bind specific DNA sequences in cardiac gene promoters regulating gene transcription. They are involved in cardiogenesis as well as hypertrophic cardiomyocyte growth [10]. Nkx2-5 is a cardiac homeobox transcription factor, which plays a key role in regulating many tissue specific gene expressions. Nkx2-5 null mice show abnormal cardiac looping [11], indicating that Nkx2-5 function is necessary for cardiac formation. Isl1 is a member of the LIM-homeodomain transcription factor family expressed early in this cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development [12]. Isl1 is a direct transcriptional target of forkhead transcription factors such as Foxa2 and Foxf1 in the second heart field [13]. Hand1 is expressed in the developing ventricular chambers and plays an essential role in cardiac morphogenesis. Additionally, Hand1 acts as a mediator of cardiac development [14] and interacts with Mef2 for the activation of cardiac specific gene expressions [15]. Tbx5 is a T-box-containing transcription factor that is important for limb and heart development. TBX5 mutations cause Holt–Oram syndrome which is characterized by congenital heart defects, conduction system abnormalities, and upper limb deformities [16,17]. Tbx5 has been reported to interact with Gata4, Gata6, and Mef2C which are cardiac transcription factors required for heart development [18,19]. Moreover, Tbx5 associates with Nkx2-5 to promote cardiomyocyte differentiation [20]. In consistency with the above notion, various cardiac transcription factors make up complex networks of cardiac transcriptional regulation and development. However, the regulation of protein expression levels in cardiac specific transcription factors is still unknown.
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Today, death due to myocardial infarction is increasing throughout the world. In order to overcome difficulties in heart transplantation owing to the lack of availability and survivability, the use of embryonic stem cells (ESCs) is currently under study to develop regenerative medicine. ESC-derived cardiomyocytes can be used for drug screening purposes regarding cardiac diseases and for the model of heart developmental biology. Therefore, more attention must be given to understand molecular mechanisms via cardiac specific transcription factors. In the present study, we demonstrated that Fbxo25 expression is cardiomyocyte-specific and acts as an ubiquitin E3 ligase to target cardiac specific transcription factors.
0.1% diethyl pyrocarbonate-treated water. RNA concentration was determined by a NANO-drop. A reverse-transcription reaction was performed with 1 lg RNA using SuperScript III Reverse Transcriptase (Invitrogen) after incubating with DNaseI for eliminating contaminated genomic DNA. The synthesized cDNA was amplified by Platinum PCR Master Mix (Invitrogen) using the proper primer sets (Supplementary Table 1). Amplification conditions were as follows: 5 min at 95 °C, followed by optimal cycles of denaturing (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 30 s) with a final extension at 72 °C for 5 min. PCR products were visualized by electrophoresis on a 2% agarose gel with ethidium bromide.
2. Materials and methods
2.5. Immunocytochemistry
2.1. Human and mouse embryonic stem cell (ESC) culture
Contracting cardiomyocytes were mechanically dissected using a glass micropipette. Mouse adult hearts and fetal hearts were frozen-sectioned for immunostaning. Contracting clusters or tissue sections were fixed with ice-cold 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, then blocked by 3% bovine serum albumin in PBS for 30 min for the inhibition of nonspecific antibody binding. Primary antibodies were used as follows; anti-rabbit Tbx5 (42-6500, Invitrogen), antirabbit Fbxo25 (AV43118, Sigma), or anti-mouse sarcomere and myosin (MF20, Developmental Studies Hybridoma Bank, University of Iowa). The sections were incubated with the appropriate antibodies overnight at 4 °C. Samples were washed three times with PBS and incubated with the following secondary antibodies: Alexa Fluor 488 or 594 conjugated goat anti-mouse IgG (488: A11001, 594: A-11005, Invitrogen) or Alexa Fluor 488 or 594 conjugated goat anti-rabbit IgG (488: A-11008, 594: A-11012) at RT for 1 h. Cell nuclei were stained with DAPI at RT for 10 min. Cells were mounted and immediately examined under a LSM 510 META confocal laser scanning microscope (Carl Zeiss).
Mouse OG2 ES cell lines [21] were cultured and maintained in an undifferentiated state on a feeder layer of mitomycin C (10 lg/ml for 2 h)-treated CF1 mouse embryonic fibroblasts (MEFs) in low glucose Dulbecco’s modified Eagle’s minimal essential medium (DMEM), supplemented with 15% fetal bovine serum (FBS), 1% mercaptoethanol, 1% GlutaMAX-1, 1% non-essential amino acids and 1000 U/ml recombinant mouse leukemia inhibitory factor (LIF). Culture medium was replaced daily. hESC H9 lines [22] were cultured and differentiated to cardiomyocytes as described in Supplementary Materials and methods [23,24]. 2.2. Induction of Cardiomyocyte differentiation from embryonic stem cells Cardiomyocyte differentiation was induced by embryoid body (EB) formation in a suspension culture. After washing with phosphate-buffered saline (PBS), undifferentiated ESCs were dissociated using 0.125% trypsin–ethylenediaminetetraacetic acid. Detached colonies were suspension-cultured for 5 days in differentiation medium including 15% heat-inactivated FBS in DMEM without LIF. On day 5, EBs were plated onto gelatin-coated 35 mm culture dishes for cardiomyocyte induction. Aggregates were observed daily by light microscopy to monitor the morphology and number of spontaneously beating clusters which were identified as cardiomyocytes. 2.3. Plasmids, Western blotting, and immunoprecipitation Full length Fbxo25, Hand1, Nkx2-5, Isl1, Mef2C, Tbx5, and Ub were amplified using human cDNA by RT-PCR cloning technique. Amplified fragments were cloned to the proper epitope tagging expression vectors. These plasmids were transfected to 293T and COS-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturers’ manual. Two days after transfection, cells were washed by PBS and lyzed with RIPA buffer. Cell lysates were used for Western blotting or further experiments. Fifty lg proteins from each lysate was subjected to SDS–polyacrylamide gel electrophoresis, transferred onto PVDF membrane and probed with proper antibodies. Proteins were detected using HRP conjugated secondary antibodies and ECL reagents. For immuneprecipitation, cell lysates were mixed with proper 2 lg antibodies plus 40 ll protein A-Sepharose 4B in RIPA buffer and incubated at 4 °C overnight. The bound beads were washed 3 times with RIPA buffer and analyzed by Western blotting.
3. Results 3.1. Nuclear localization of Fbxo25 with cardiac specific transcription factors F-box containing Fbxo25 is expressed in several tissues [25]. Previous results show that Fbxo25 is localized mainly at the nuclei [9,25]. To examine the localization of Fbxo25, we performed the immunocytochemistry using GFP-Fbxo25 expression in vitro cells. When GFP-Fbxo25 was expressed to 293T cells, GFP signals were largely detectable at the nuclei (Supplementary Fig. S1A) as shown in previous data [25]. Tbx5, Nkx2-5, and Hand1 are cardiac-specific transcription factors for heart development. To examine the cellular distribution of Fbxo25 with Tbx5, Nkx2-5, and Hand1, we conducted the immunocytochemistry experiments using Myc-, GFPor mRFP-tagged transgenes. Tbx5, Nkx2-5, Hand1 as well as Fbxo25 were expressed at the nuclei (Supplementary Fig. S1B, C, D). The localization patterns of Tbx5 and Nkx2-5 were matched with that of Fbxo25 (Supplementary Fig. S1B, C). However, GFP-Hand1 localized differently with mRFP-Fbxo25 (Supplementary Fig. S1D). Unlike the previous report, we didn’t observe Cajal body-like subnuclear localization of Fbox25 probably due to the use of different experiment conditions [25]. Together, novel cardiac Fbxo25 expresses at the nuclei along with Tbx5, Nkx2-5, and Hand1. 3.2. Cardiac expression of Fbxo25 and cardiac transcription factors in heart
2.4. Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturers’ protocol. RNA pellets were dissolved in
A previous report showed that Fbxo25 is highly expressed in adult heart, brain, liver and kidney, but undetectable in adult skeletal muscle, lung, and spleen by Northern blotting [9]. Whether
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Fbxo25 is expressed in early cardiac tissues was under question. To test this idea, we investigated cardiac expression of Fbxo25 proteins in a fetal heart using immunostaining. Endogenous Fbxo25 was detectable in cardiac cells labeled with fast and slow sarcomeric MHCs by MF20, indicating that fetal cardiomyocytes express Fbxo25 (Fig. 1A). In addition, Tbx5 was also expressed in cardiomyocytes stained by MF20 (Fig. 1B). Unfortunately, colocalization between Fbxo25 and Tbx5 in fetal cardiomyocytes could not be examined due to the antibody problem possessing same host-specificity of both antibodies, anti-rabbit. However, it is tempting to speculate that Fbxo25 distributes along with Tbx5 in cardiomyocytes based on the overlay of sarcomeric MHCs. To overcome the antibody problem used in immunostaining, we investigated mRNA expression patterns of Fbxo25, Tbx5, and Nkx2-5 in several tissues such as MEFs, fetal heart, adult heart, ESCs, and adult liver using RT-RCR. Fbxo25 was expressed in the tested tissues as reported previously (Fig. 1C) [9]. As expected, Tbx5 and Nkx2-5 were highly cardiac-specific. In contrast, the expression level of Fbxo25 was higher in a fetal than adult heart. This pattern was similar to those of Tbx5 and Nkx2-5, indicating that Fbxo25 has a physiological rel-
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evance in cardiac development. Collectively, our results demonstrate that Fbxo25 expression is cardiac-specific. 3.3. Fbxo25 is expressed in cardiomyocytes derived from ESCs Our data indicates that Fbxo25 is expressed in cardiomyocytes as well as other tissues. Thus, we investigated the expression pattern of Fbxo25 during cardiomyocyte differentiation from ESCs using OG2-ESCs, which express Oct4 promoter-driven GFP to directly monitor stemness by fluorescence [21]. Cardiomyocytes were differentiated from OG2-ESCs using EB formation in 15% FBS condition without LIF. Maturation was proportional to the decrease of Oct4 expression based on GFP fluorescence (Fig. 2A). GFP fluorescence was detectable in non-beating clusters, but not in beating clusters, indicating that cardiomyctes are differentiated from fully differentiated stem cells (Supplemental video). Formation of visceral endoderm-like cells are necessary for cardiomyocyte development, but GFP expressing clusters may not meet this criterion [21]. Expression patterns of cardiac-specific genes were monitored during cardiomyocyte differentiation from ESCs by
Fig. 1. Cardiac expression of Fbxo25 along with Tbx5 and Nkx-5. (A) Fbxo25 expression in cardiomyocytes at embryonic day 12.5. Frozen sectioned heart tissues from E12.5 embryos were obtained using cryostat. Anti-mouse MF20 antibody labeled both fast and slow sarcomeric MHCs. Endogenous Fbxo25 was stained by anti-rabbit Fbxo25 antibody. DAPI was used for staining cardiac nuclei. (B) Tbx5 expression in cardiomyocytes at embryonic day 12.5. Anti-rabbit Tbx5 antibody was used for staining of cardiac Tbx5. Cardiac MHCs were labeled by anti-mouse MF20. (C) RT-PCR data showed the expressions of Fbxo25, Tbx5, and Nkx2-5 in MEFs, fetal heart, adult heart, ESCs, and adult liver. RNAs were isolated from MEFs, ESCs, embryonic heart at day 12.5, adult heart and liver by TRIzol following manufacturer’s protocol. Primer information used in RT-PCR was shown in Supplementary Table 1.
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Fig. 2. Fbxo25 expression is proportional to ESC-derived cardiomyocyte differentiation. (A) Maturation of cardiomyocytes from ESCs correlated to the decrease of Oct4 expression. Beating clusters didn’t express GFP, whereas GFP expressing clusters showed no contraction. Cardiomyocytes were obtained by spontaneous differentiation of EB formation using OG2 ESCs as described in Materials and methods. Images were acquired by inverted fluorescent microscopy at the indicated differentiation time. (B) Expression patterns of Oct4, Fbxo25, and other cardiac specific genes during cardiomyocyte development from ESCs. Samples were collected at the indicated time and RNAs were isolated using TRIzol. Information of used primers is revealed in Supplementary Table 1. (C) Tbx5 and Fbxo25 expressions in beating cardiomyocytes. Beating clusters were collected and replated on gelatin-coated dishes. Fbxo25 or Tbx5 were labeled with anti-rabbit Fbxo25 antibody or anti-rabbit Tbx5 together with anti-mouse MF20 antibody, respectively. (D) Human FBXO25 or TBX5 expressions in cardiomyocytes derived from hESCs. Cardiomyocytes were collected from beating clusters using 0.25% Trypsin–EDTA and replated on gelatin-coated dishes. After DAPI stains nuclei, images were acquired by confocal microscope.
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RT-PCR experiments. Similar to the reduction of GFP fluorescence, Oct4 was downregulated during cardiomyocyte differentiation. In contrast, cardiac-specific genes such as Myh6, Myh7, Nkx2-5, Tbx5 as well as Fbxo25 were upregulated (Fig. 2B). Moreover, Fbxo25 expression was higher in beating than non-beating clusters similar to those of other cardiac genes. Immunostainings of Fbxo25 and Tbx5 was also conducted in cardiomyocytes from ESCs. As shown in Fig. 2C, Fbxo25 and Tbx5 were stained in sarcomeric MHCs expressing cells, suggesting that Fbxo25 is expressed in ESC-derived cardiomyocytes. hESCs-derived cardiomyocytes also expressed FBXO25 and TBX5 (Fig. 2D). Interestingly, human FBXO25 was expressed at the nuclei and cytosol, indicative of the broad cellular distribution. Collectively, this result demonstrates that Fbxo25 is a novel cardiac-specific gene. 3.4. Fbxo25 activates protein degradation of cardiac transcription factors Fbxo25 contains an F-box domain that allows a multi-protein complex E3 ubiquitin ligase system with Skp1, Cullin1 and
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Rbx1, thus Fbxo25 may execute E3 ligase activity against certain proteins expressed in cardiomyocytes. Based on the colocalization between Fbxo25 and cardiac transcription factors in cardiomyocytes, we hypothesized whether Fbxo25 targets the proteins of cardiac transcription factors via SCF complex. To address this idea, we examined the protein expression patterns of cardiac transcription factors influenced by Fbxo25. As shown in Fig. 3A, Fbxo25 facilitated protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C in comparison to the absence of Fbxo25. This data suggested that Nkx2-5, Isl1, Hand1 are eliminated by an Fbxo25-mediated ubiquitination pathway. To test the notion, we conducted in vitro ubiquitination assays showing that Fbxo25 activated the degradation of Nkx2-5 via ubiquitination pathway (Fig. 3B). Furthermore, proteasome inhibitor lactacystin induced the accumulation of more ubiquitined-Nkx2-5, implicative of Fbxo25-mediated Nkx2-5 degradation. Fbxo25 also facilitated the ubiquitination of Isl1 and Hand1 (Fig. 3C, D) but lactacystin treatment didn’t accumulate ubiquitined Isl1 and Hand1. As a result, the degradation of Nkx2-5, Isl1, and Hand1 is required for Fbxo25-mediated UPS.
Fig. 3. Fbxo25 facilitates protein degradation of cardiac transcription factors by UPS. (A) Fbxo25 reduced protein expression levels of cardiac transcription factors. MycHand1, Myc-Isl1, Flag-Mef2C, Myc-Nkx2-5 and/or GFP-Fbxo25 were transfected to 293T cells. (B) Fbxo25 ubiquitinated Nkx2-5. Myc-Nkx2-5, HA-Ub and/or GFP-Fbxo25 were transfected to 293T cells using Lipofectamine 2000. For the inhibition of proteasome, 5 lM lactacystin was treated for 5 h. Two days after transfection, ubiquitination assays were done using immunoprecipitation with anti-Myc followed by immunoblotting with anti-HA for the detection of ubiquitination as in Materials and methods. (C) Isl1 was degraded by Fbxo25 in ubiquination assay. Myc-Isl1, HA-Ub and/or GFP-Fbxo25 were transfected to 293T cells. (D) Hand1 was ubiquitinated by Fbxo25 in ubiquination assay. Myc-Hand1, HA-Ub and/or GFP-Fbxo25 were transfected to 293T cells.
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4. Discussion The purpose of this study was to identify that Fbxo25 expression is cardiac-specific and acts as an ubiquitin E3 ligase for cardiac transcription factors. Fbxo25 belongs to the F-box only family of proteins composed of 77 and 84 different proteins in mouse and human to date by our data analysis, respectively. F-box proteins like Fbxo32 make up E3 ubiquitin–ligase complex with Skp1, Cullin1 and Rbx1 [9]. Thus, SCF complex proteolyzes certain proteins via UPS. A previous report showed that Fbxo25 is expressed in an adult heart, brain, liver, and kidney by Northern experiment [9]. However, Manfiolli et al. was unable to demonstrate protein expression of Fbxo25 in an adult heart [25]. Our antibody against Fbxo25 recognizes endogenous Fbxo25 in a fetal and adult heart as well as cardiomyoctes from ESCs. However, we cannot prove the colocalization between Fbxo25 and Tbx5 in cardiac tissues due to the antibody problem concerning same host species, antirabbit. Fbxo25 labeling nuclei are overlaid to sarcomeric MHCs staining cardiac cells, which represent cardiomyocytes, similar to Tbx5 (Figs. 1 and 2). It’s likely that Fbxo25 codistributes along with cardiac-specific transcription factor such as Nkx2-5, Isl1, Hand1 as well as Tbx5 in cardiomyocytes. Unlike Fbxo32, Fbxo25 is expressed in the heart rather than skeletal muscle [9]. The expression level of Fbxo25 is higher in a fetal than adult heart (Fig. 1). Fbxo25 is upregulated with cardiomyocyte differentiation (Fig. 2). These patterns of Fbxo25 are similar to those of other cardiac specific genes in heart (Fig. 2) [26]. Fbxo25 activates protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C (Fig. 3). Thus, Fbxo25 might be down-regulated to maintain protein homeostasis during heart development. In summary, Fbxo25 is likely to be expressed in cardiomyocytes in the presence of Tbx5 and other cardiac transcription factors. Cardiac Fbxo25 belongs to the F-box only family as an ubiquitin E3 ligase for the distruction of cardiac transcription factors. Thus, Fbxo25 is a novel E3 ligase responsible for cardiomyocyte development. Cardiac UPS including Fbxo25 has a pivotal impact on cardiac development. Acknowledgments We are very grateful to Dr. Jeong-Tae Do (Laboratory of Stem Cell and Developmental Biology, CHA Stem Cell Institute, CHA University, 605-21 Yoeksam 1-dong, Gangnam-gu, Seoul 135-081, Republic of Korea) for providing OG2-ESCs. This research was supported by a grant (SC-3110) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.05.011. References [1] M.H. Glickman, A. Ciechanover, The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction, Physiol. Rev. 82 (2002) 373–428. [2] M.S. Willis, W.H. Townley-Tilson, E.Y. Kang, J.W. Homeister, C. Patterson, Sent to destroy: The ubiquitin proteasome system regulates cell signaling and protein quality control in cardiovascular development and disease, Circ. Res. 106 (2010) 463–478. [3] A. Hershko, A. Ciechanover, The ubiquitin system, Annu. Rev. Biochem. 67 (1998) 425–479.
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