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Cardiovascular Research 2007 75(1):139-147; doi:10.1016/j.cardiores.2007.03.014
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Copyright © 2007, European Society of Cardiology

Transgenic overexpression of the secreted, extracellular EGF-CUB domain-containing protein SCUBE3 induces cardiac hypertrophy in mice

Hsin-Yu Yanga,1, Ching-Feng Chenga,b,1, Bambang Djokoa, Wei-Shiung Liana,c, Cheng-Fen Tua, Ming-Tzu Tsaia, Yen-Hui Chena, Chien-Chang Chena, Chien-Jui Chengd and Ruey-Bing Yanga,e,*

aInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
bDepartment of Pediatrics, Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan
cDepartment of Animal Science and Technology, National Taiwan University, Taiwan
dGraduate Institute of Medical Sciences and Department of Pathology, School of Medicine, Taipei Medical University, Taipei, Taiwan
eInstitute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan

* Corresponding author. Institute of Biomedical Sciences, Academia Sinica, 128, Academia Road, Sec. 2, Taipei 11529, Taiwan. Tel.: +886 2 2652 3943; fax: +886 2 2785 8847. rbyang{at}ibms.sinica.edu.tw

Received 5 September 2006; revised 26 February 2007; accepted 13 March 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Objective The aim of this study was to investigate in a transgenic animal model the cardiac expression and function of a novel extracellular protein SCUBE3 [signal peptide-CUB (complement proteins C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor)-like domain-containing protein 3].

Methods and results Real-time quantitative reverse transcriptase (RT)-PCR analysis showed that SCUBE3 is expressed in the ventricular myocardium. Male transgenic (TG) mice overexpressing SCUBE3 appeared normal during development, from birth to adulthood. However, echocardiography and histopathological examination revealed significant cardiac hypertrophy in TG animals as they aged, at 8 months. Interestingly, left-ventricle hypertrophy occurred more rapidly and more severely under pressure overload in TG mice, as compared to age-matched wild-type littermates. Induced SCUBE3 expression, together with elevated transforming growth factor (TGF)-β1 level under pressure overload, may account for the accelerated onset and progression of cardiac hypertrophy in TG mice. Furthermore, biochemical and molecular studies revealed that the carboxyl-terminal portion of SCUBE3 could physically interact with TGF-β1 and promote the TGF-β1-mediated transcriptional activation.

Conclusion This report is the first demonstration that SCUBE3 may play a role in the regulation of cardiac growth and remodeling responses, possibly through the stabilization of the TGF-β1 signaling pathway.

KEYWORDS Transgenic animal models; Echocardiography; Hypertrophy; Myocytes; Extracellular matrix


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
SCUBE3 (signal peptide-CUB-EGF-containing protein 3) is a novel, secreted, extracellular protein belonging to an evolutionarily-conserved SCUBE gene family [1–4]. To date, three distinct members have been identified and designated SCUBE1–3 by the order of their discovery in mammals [1–4]. These genes encode polypeptides of approximately 1000 amino acids and share a conserved protein domain organization, including an amino-terminal signal peptide, followed by 9 copies of EGF-like repeats and one CUB domain residing at the carboxyl terminus. In addition, one spacer region with less conserved sequence homology is centered between the EGF-like repeats and the CUB domain. A recent search of the protein families database (Pfam) (http://pfam.wustl.edu/) further identified a repeated motif of six cysteine residues with unique and regular spacing located in the spacer region close to the CUB domain [5,6] (Fig. 2A). Yet, the functional significance of this motif is currently unclear.


Figure 2
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  		Fig. 2 Generation of the SCUBE3 TG mice. A, Domain organization of SCUBE3. SCUBE3 protein is composed of signal peptide (SP), nine copies of EGF-like repeats (E), a spacer region, three cysteine-rich (CR) repeats [5,6], and one CUB domain. The small "Y" symbol marks five potential N-glycosylation sites [2]. B, Transgene construct. A 1.8-kb fragment of rat Col1a1 promoter was used as a specific promoter to control the expression of human SCUBE3 cDNA [2]. Two copies of the chicken globin DNase I hypersensitive site 4 (HS4) region were added and served as an insulator to protect the SCUBE3 minigene from position effects [11]. Note the inclusion of a Flag epitope tag for detection of the transgene expression. The transgene construct is flanked by two unique BssHII sites. C, Southern blot analysis of two transgenic founders and a wild-type control mouse. Using as a probe a 400-bp internal fragment covering the spacer region of SCUBE3 cDNA, the SCUBE3 transgene was revealed after EcoRI–XbaI digestion as a 3-kb band and the endogenous gene as a 7.0-kb band. D, Quantification of the expression of the transgene. RNAs were extracted from kidney, femur or heart isolated from 2-month-old WT and TG mice. The level of expression was determined by real-time quantitative RT-PCR, normalized to the expression level of GAPDH (glyceraldehyde-3 phosphate dehydrogenase) used as an internal control. E, Protein expression of the transgene. Western blot analysis of protein extracts from the femur of the wild-type (WT) littermate or transgenic (TG) mice with anti-Flag monoclonal antibody used to detect the SCUBE3 transgenic protein (arrow).

 
These SCUBE genes have been shown to be expressed prominently in various developing tissues, including gonads, the central nervous system, and limb buds during mouse embryogenesis, which implies their potential roles during development [3,4]. However, SCUBE genes are also predominantly expressed in adult tissues [1,2,4]. For example, SCUBE1 is highly enriched in the endothelium [1] and platelets [7], and SCUBE2 mRNA was found in a broader spectrum of tissues and cell types, including endothelial, smooth-muscle, and renal mesangial cells, and fibroblasts [1,4]. In contrast, SCUBE3 seems to be expressed in a more restricted fashion: high expression in cultured primary osteoblasts, but low expression in cardiac tissue and vascular cells [2]. When overexpressed in human embryonic kidney (HEK)-293T cells, SCUBE3 is a secreted glycoprotein that can form either homo- or hetero-oligomers tethered to the cell surface [2]. On the basis of its secretory nature, SCUBE3 was proposed to function locally or distantly in a paracrine or endocrine fashion [2]. However, the exact functions of SCUBE3 remain elusive.

In this report, we describe our further characterization of the cardiac expression of SCUBE3 and investigation of its function in vivo by use of a SCUBE3-overexpressing transgenic (TG) mouse model.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1 Animal studies
Our investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All surgical procedures were performed according to the protocols approved by the Institutional Animal Care and Utilization Committee, Academia Sinica.

2.2 Real-time quantitative RT-PCR (TaqMan) analysis
Human SCUBE3 mRNA expression was measured by real-time quantitative RT-PCR (Applied Biosystems PRISM 7700) by use of a panel of cDNAs from various regions of normal human hearts (Clontech). Primers used for human SCUBE3 and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) were as described [2]. Normalization was to GAPDH mRNA levels as controls in parallel TaqMan reactions. The relative expression ratio of SCUBE1 transcript to GAPDH transcript was calculated on the basis of a mathematical model as described [8]. Real-time RT-PCR was performed in duplicate for each sample. Each experimental group contained five animals.

2.3 Generation of SCUBE3 transgenic mice
The transgene construct contains the Flag-tagged coding sequences of human SCUBE3 cDNA fused to the polyadenylation signal of the bovine growth hormone gene and under control of the rat 1.8-kb Col1a1 gene promoter [9,10]. In addition, two copies of the chicken globin DNase I hypersensitive 4 region were introduced downstream of the minigene cassette. This sequence has previously been demonstrated to ensure expression of a transgene independent of its insertion site [11]. The 7.8-kb transgene was released by digestion with BssHII and microinjected into the pronuclei of one-celled embryos (FVB strain) by standard techniques.

Southern blot hybridization with a SCUBE3 cDNA probe was used to identify transgenic progeny. Eight mice were found to be positive for the presence of the transgene, and two founder males were mated to FVB females to establish the transgenic lines TG1 and TG2. Transgenic offspring were identified by PCR analysis of genomic tail DNA with transgene-specific primers. Heterozygous TG mice were bred to generate homozygous TG and wild-type (WT) mating pairs, then subsequently bred for phenotype analysis. Since the two lines exhibited similar transgene expression and cardiac phenotypes, only data derived from the TG2 line is presented.

2.4 Transverse aortic banding (TAB)
TAB was performed as described [12]. Briefly, male mice were anesthetized with isoflurane (3%) without intubation. A longitudinal skin incision (~2–3 cm) was made across the suprasternal notch. A 4-mm longitudinal cut was made in the proximal portion of the sternum. A bent 27-gauge needle was placed next to the aortic arch, and a 6-0 silk suture was tied around the needle and the aorta twice. After ligation, the needle was removed quickly. After the surgery, the mice were allowed to recover on a warming pad until fully awake, which normally took less than 5 min. The sham-operated group went through the same procedure, except the aorta was not ligated. For experiments, each animal group involved 5 animals.

2.5 Electrocardiography, echocardiography, and blood pressure measurement
Mice were anesthetized with avertin (2.5%, 20 µl/g body weight, i.p.), and heart electrical conduction was recorded by use of Dual BIO Amp hardware and Power Lab software (AD Instruments, Castle Hill, Australia) via needle electrodes subcutaneously implanted in each limb. Digital recordings (16 bit, 2 kHz/channel) were analyzed by use of the Chart v5.0 program (AD Instruments).

For echocardiography, mice were anesthetized with phenobarbital (50 mg/kg body weight, i.p.) and measurements involved the Phillips ATL HDI5000 instrument equipped with a 15-MHz probe. Posterior wall thickness, end-systolic and end-diastolic left-ventricular diameters were measured digitally on the M-mode tracings and averaged from 3 cardiac cycles. Fractional shortening was calculated.

Blood pressure was measured in conscious young adult male TG and WT mice by a non-invasive computerized tail-cuff method (Model MK-2000 instrument, Muromachi Kikai Co., Tokyo, Japan).

2.6 Histology and histomorphometry
Mice were euthanized under methoxyflurane anesthesia before histopathological examination. After perfusion with normal saline, the heart was excised and rinsed in ice-cold PBS. Tissues were either snap-frozen in liquid nitrogen for RNA and protein analysis or fixed in 4% paraformaldehyde at 4 °C for 24 h and embedded in paraffin. Sections were taken at the midventricular level, which was confirmed by the presence of papillary muscle in the cavity and the semicircular shape of the right-ventricular free wall. Sections were stained with hematoxylin/eosin or Sirius red to detect fibrillar collagen in heart sections (see Supplemental Materials and methods). For morphometrical analysis, microphotographs of ventricular sections from TG and WT mice were taken at 200x magnification, and the transversal cardiomyocyte diameters were calculated by measurement of 100 cells per specimen (5 hearts in both the TG and WT groups) in the region of the cell nucleus.

2.7 Cell culture, construction of expression plasmids, and transfection
HEK-293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were seeded in 6-well plates overnight before transfection. The epitope-tagged versions of SCUBE3 were constructed as described [2]. The transfection was performed with use of Lipofectamine 2000 reagent (Invitrogen).

2.8 Immunoprecipitation and western blot analyses
Two days post-transfection, cell lysates were clarified by centrifugation at 10,000 xg for 20 min at 4 °C. Samples underwent immunoprecipitation, followed by western blot analysis as described [2].

2.9 Luciferase activity assays
Human HepG2 cells (3x105 cells per well) were seeded into 24-well plates and transfected on the following day with the SCUBE3 expression plasmid or empty vector, together with 0.4 µg of the luciferase reporter 3TP-lux [13] and 0.01 µg of the Renilla luciferase reporter vector as an internal control. Transfected cells were maintained in medium containing 0.1% fetal calf serum for 24 h. Luciferase activity was measured following 24-h treatment with TGF-β1 (10 ng/ml) by use of the dual reporter system (Promega). Data are expressed as relative luciferase activity (firefly luciferase activity divided by Renilla luciferase activity).

2.10 Statistical analysis
Data are expressed as mean±SEM. Differences between groups were analyzed by unpaired Student's t-test. A p<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
3.1 Cardiac expression of SCUBE3
We previously showed by northern blot analysis that SCUBE3 mRNA is expressed at a lower level in the heart [2]. To further determine the cardiac expression of SCUBE3, we examined the regional expression profile of SCUBE3 in the human heart by real-time quantitative RT-PCR analysis. In assessing a panel of cDNAs derived from different regions of normal hearts for SCUBE3 expression, we found that SCUBE3 mRNA is relatively enriched in the ventricle, interventricular septum (IVS), and apex, but low in level in the atrium, auricle, or atrioventricular node (Fig. 1). Together, these data demonstrate that SCUBE3 is selectively expressed in the ventricular myocardium.


Figure 1
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  		Fig. 1 Cardiac expression of SCUBE3. Expression of human SCUBE3 was determined by TaqMan analysis in cDNA samples derived from a panel of different parts of normal human heart, primary osteoblasts, and umbilical cord endothelial cells. Expression levels were normalized to GAPDH. Experiments were performed twice in duplicate with similar results. L, left; R, right; IVS, interventricular septum; AV, atrioventricular.

 
3.2 Generation of transgenic mice overexpressing SCUBE3
To further understand the function of SCUBE3 in vivo, we generated transgenic mice overexpressing SCUBE3 under the control of a type I collagen (Col1a1) promoter. The Col1a1 promoter has been shown to direct strong expression of green fluorescent protein to bone and isolated tail tendons and lower expression in other type I collagen-producing tissues [9,10]. As shown in Fig. 2B, we prepared a construct containing the Flag-tagged coding sequences of human SCUBE3 cDNA fused to the polyadenylation signal of the bovine growth hormone gene and under control of the rat 1.8-kb Col1a1 gene promoter. The Flag epitope tag was added for easy detection of the transgene product (Fig. 2B). Transgenic mice were generated by injection of the DNA construct into pronuclei from FVB mouse embryos. As shown in Fig. 2C, southern blot hybridization with a SCUBE3 cDNA probe identified mice positive for the presence of the transgene. Of eight male founders positive for the presence of the transgene, two founders were mated to FVB females to establish the transgenic lines, TG1 and TG2. Because the 2 lines displayed similar transgene expression and cardiac phenotypes (see below), we present only the data derived from the TG2 line.

To further confirm the SCUBE3 transgenic (TG) expression in the targeted tissues, we performed quantitative real-time RT-PCR and found the SCUBE3 TG mRNA highly expressed in long bone tissues, with a lower expression in the heart or kidney (Fig. 2D). Consistent with this finding, western blot analysis with the anti-Flag monoclonal antibody readily detected the overexpressed Flag-tagged SCUBE3 protein in bone lysate (Fig. 2E).

3.3 SCUBE3 TG mice show baseline cardiac hypertrophy at 8 months
SCUBE3 TG mice appeared normal during embryonic development from birth to adulthood (2 months old) as compared to nontransgenic wild-type (WT) littermates. Since SCUBE3 is a ventricle-enriched gene and is targeted to the heart in TG mice (Fig. 2D), we thus took serial measurements by electrocardiography (ECG) and echocardiography at 1-month intervals to follow the changes in cardiac structure and function in vivo. At 8 months, TG mice showed an abnormal repolarization ECG pattern with an obvious depression of the ST-T segment (Supplemental Fig. 1A), which is usually seen in patients with acute coronary syndrome associated with ventricular hypertrophy or ischemia [14,15].

Echocardiography was further utilized to validate the in vivo cardiac structure/function in male TG and WT mice (illustrated in Supplemental Fig. 1B). In agreement with the ECG finding, echocardiography showed that left-ventricular septum (IVS) and posterior wall thickness (LVPW) in both diastolic and systolic phases were significantly greater in TG than WT mice (p<0.01, Table 1). Likewise, tissue histology confirmed severely thickened left-ventricular heart walls and septum in TG mice as compared to WT mice and showed concentric hypertrophy (Fig. 3A and B). Consistently, we found TG mice with an elevated heart-to-body weight ratio, a mean of 144% of that of WT mice (p<0.01) by 8 months (Fig. 3E). Histopathological examination showed typical large nuclei in the TG group, confirming myocyte hypertrophy (Fig. 3D). In addition, multiple foci of classic myocyte disarray were observed in TG mouse tissue, a change not present in the WT heart (Fig. 3C and D). As well, further morphometric analysis demonstrated significantly larger left-ventricular cardiac myocyte diameters in TG than WT mice (Fig. 3F). RT-PCR analysis revealed significantly elevated mRNA expression of three hypertrophic marker genes (atrial natriuretic factor (ANF), {alpha}-skeletal actin, and β-myosin heavy chain) in TG mice as compared to WT mice (Supplemental Fig. 2). Furthermore, TG and WT mice showed no differences in heart rate or systolic or diastolic blood pressure as demonstrated by tail-cuff measurement in awake animals (Supplemental Fig. 3).


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  		Table 1 Echocardiographic parameters for cardiac structure and function for 8-month-old WT and TG mice

 

Figure 3
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  		Fig. 3 Cardiac hypertrophy observed at organ and myocyte levels in TG mice. (A–D) Hematoxylin and eosin staining of the heart. Panels A and C represent an 8-month-old WT control mouse; panels B and D, an 8-month-old TG animal. Each section was taken at the midventricular level, which was confirmed by the presence of papillary muscle in the cavity and the semicircular shape of the right-ventricular free wall. Panels C and D show high-power magnification of a section from the left ventricle in the WT or TG mice, respectively. A, B: bar=1.0 mm; C, D: bar=50 µm. (E) Heart weight-to-body weight ratios determined in TG mice and WT littermates at 8 months. (F) Mean left-ventricular cardiomyocyte diameters were determined from 100 myocytes in each animal. Values are mean±SEM (n=5 animals in each group). *, p<0.01.

 
In addition, left-ventricular diameter of diastolic (LVIDd) or systolic (LVIDs) dimensions, fractional shortening (%FS, an index of systolic function), and ratio of E to A transmitral flow velocity (E/A, an indicator of diastolic function) were all maintained in the TG mice (Table 1). Together, these results suggest that the TG mice exhibited cardiac hypertrophy with well-preserved left-ventricular function.

3.4 Effect of pressure overload on cardiac phenotype of TG mice
To further evaluate the role of transgenic SCUBE3 under pathological conditions, we performed TAB in male TG and WT mice. TAB produces pressure overload as a stimulus for the development of left-ventricular hypertrophy, resembling the clinical scenario observed in patients with hypertension or aortic stenosis [16]. After one week of TAB, TG and WT mice showed similar pressure gradient across the banding site (TG vs WT; 24.8±1.4 vs 25.4±1.2 mm Hg). Although IVS and LVPW thickness increased significantly in response to pressure overload in both TG and WT mice, the degree of the increase in these indicators of cardiac hypertrophy was markedly greater in the banded TG animals as compared with banded WT mice (Fig. 4). Consistent with this finding, severe cardiac hypertrophy in the banded TG mice was further confirmed by their 3-fold higher expression of ANF mRNA (a molecular marker for cardiac hypertrophy) as compared to banded WT mice (Fig. 4). Quantitative analysis of Sirius red-stained sections indicated increased fibrosis in both banded TG and WT mice, compared with their respective sham-treated counterparts (Supplemental Fig. 4). An increase of ~30% in cardiac collagen deposition was observed in banded TG mice but was not significant as compared with that in banded WT mice (1.11±0.2 vs 1.43±0.18%; p=0.07, Supplemental Fig. 4). Together, these results demonstrate that pressure overload accelerates ventricular hypertrophic response in TG mice as compared to WT mice.


Figure 4
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  		Fig. 4 Effects of pressure overload on LV hypertrophy in WT and TG mice. TG and WT mice were subjected to either TAB or sham operation for one week. IVS (top panel), LVPW thickness (middle panel), and ventricular ANF mRNA level (bottom panel) were measured by echocardiography or TaqMan analysis, respectively. Values are mean±SEM (n=5 per group). *, p<0.05; **, p<0.01.

 
3.5 Up-regulation and interaction between SCUBE3 and TGF-β1
Since TGF-β1 is an important mediator of hypertrophic growth response of the heart induced by pressure overload or angiotensin II [17,18], we then investigated whether TGF-β1 is directly involved in TAB-triggered left-ventricular hypertrophy in our TG mouse model. Consistent with the literature [17,19,20], TG and WT mice showed the same degree of increase (40%) in TGF-β1 expression in response to TAB as compared to their respective sham-treated controls (Fig. 5). Interestingly, similar to TGF-β1, ventricular SCUBE3 was up-regulated in both TG and WT mice under pressure overload, with a greater than 50% increase as compared to sham-treated controls (Fig. 5).


Figure 5
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  		Fig. 5 Cardiac TGF-β1 and SCUBE3 mRNA are co-regulated under pressure overload. After 1 week of TAB or sham operation, the ventricular tissues derived from TG or WT animals were measured for mRNA expression of TGF-β1 (top panel) or SCUBE3 (bottom panel) by TaqMan analysis. Values are mean±SEM (n=5 animals in each group). *, p<0.05.

 
Because SCUBE3 and TGF-β1 are simultaneously up-regulated during pressure overload and because both proteins are expressed in cardiomyocytes [20] and could be secreted into the extracellular environment [2,21,22], we next tested whether SCUBE3 could interact with TGF-β1 and form a complex. HEK-293T cells were transfected with an HIS epitope-tagged TGF-β1 expression construct alone or in combination with a series of Flag-tagged SCUBE3 deletion constructs (Fig. 6A). Two days after transfection, cell lysates underwent immunoprecipitation with the anti-HIS monoclonal antibody, and the precipitates were analyzed by immunoblotting with anti-Flag monoclonal antibody to determine the protein association. As shown in Fig. 6B, immunoprecipitation with anti-HIS antibody resulted in the co-immunoprecipitation of the SCUBE3 full-length (FL), D1, D3 or D4 deletion protein, which suggests that SCUBE3 could form a complex with TGF-β1 through its carboxy and/or amino-terminal domain.


Figure 6
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  		Fig. 6 SCUBE3 could interact and modulate the TGF-β1-mediated signaling. A, Domain organization of SCUBE3-FL, -D1, -D3, and -D4 constructs used in this study. FL, full-length (amino acids 1–993); D1, D3 and D4, deletion 1 mutant (amino acids 1–803), deletion 3 mutant (amino acids 1–401), and deletion 4 mutant (amino acids 633–993), respectively. B, Interaction between SCUBE3 and TGF-β1. The HIS-tagged TGF-β1 construct was transfected alone or in combination with the expression plasmids encoding indicated Flag-tagged SCUBE3 proteins in HEK-293T cells. Two days later, cell lysates underwent immunoprecipitation (IP) and western blotting (WB) with antibodies as indicated. C, Effect of ectopic SCUBE3 expression on 3TP-lux reporter transcription activity. HepG2 cells were transfected with 3TP-lux reporter, together with SCUBE3 (S3)-FL, -D4 or empty vector. After 20 h, cells were treated with TGF-β1 (10 ng/ml), or left untreated. Luciferase activity was assayed 24 h later, and normalized to Renilla luciferase with the pRL-TK used as an internal control plasmid. Each data point represents the mean±SEM. The experiments were performed twice in duplicate.

 
Because of the direct physical interaction between SCUBE3 and TGF-β1, we then evaluated whether SCUBE3 affects the TGF-β1-mediated transcriptional activity. The effects of ectopic SCUBE3 expression were evaluated on the TGF-1β-responsive 3TP-lux reporter in human HepG2 cells [13]. We cotransfected HepG2 cells with the 3TP-lux reporter and expression plasmids encoding either SCUBE3-FL or D4, or the empty vector, and transfected cells were treated with TGF-β1 (10 ng/ml) or left untreated for 24 h. As shown in Fig. 6C, treatment with TGF-β1 increased the basal level of 3TP-lux reporter transcription by 3-fold in cells cotransfected with vector plasmid, whereas ectopic expression of SCUBE3-FL had virtually no effect on TGF-β1 signaling. Coexpression of SCUBE3-D4 significantly promotes the TGF-β1-induced transcriptional activation in HepG2 cells (Fig. 6C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
SCUBE3 is the third and probably the last member of the newly-discovered, secreted SCUBE protein family [2]. Although the full-length cDNA was originally isolated from cultured primary osteoblasts, SCUBE3 mRNA was also found in the heart and vascular cells [2]. However, neither the precise regional expression nor the functions of SCUBE3 in the heart have been addressed. In the present study, we demonstrated that SCUBE3 is a ventricle-enriched gene and unraveled its potential roles in maintaining cardiac homeostasis and modulating cardiac hypertrophic response during pressure overload in the transgenic animal model. The cardiac phenotype was not likely caused by the disruption of endogenous genes, because pressure overload-induced ventricular hypertrophy has been independently confirmed in two different lines at the heterozygous state.

Although the SCUBE3 TG mice reached adulthood and showed no apparent phenotype up to 2 months of age, significant cardiac hypertrophy was observed in male TG animals as they aged, at 8 months. Initially, left-ventricular hypertrophy in aging TG mice was suggested by an abnormal ECG tracing with a deep ST-T wave depression (Supplemental Fig. 1A), which was further confirmed by echocardiographic (Table 1) and histomorphologic analyses at the organ and myocyte levels (Figs. 4 and 5Go). However, no difference in heart rate and arterial blood pressure was observed between the TG and WT littermates (Supplemental Fig. 3), which indicated that cardiac hypertrophy in the TG mice was not the compensatory effect caused by the hemodynamic changes.

We have previously reported that recombinant SCUBE3 is a secreted glycoprotein that could be proteolytically processed by a serum-associated proteinase in vitro [2]. Consistent with this finding, western blot analysis identified a similar proteolytic fragment of ~65 kDa from tissue lysates derived from the TG animals (Fig. 1E). In addition, ELISA specific to the Flag epitope estimated the concentration of the transgenic Flag.SCUBE3 protein at 124±41 ng/ml in plasma from the TG mice, but the protein was undetectable in the WT counterparts. Because 2 novel CUB domain-containing growth factors (PDGF-C and -D) require the proteolytic removal of the CUB domain to release the biologically active growth factor core domain [23–26], such cleavage might represent a potential regulatory mechanism for SCUBE3 in vivo. However, this suggestion requires further investigation.

One interesting observation is that left-ventricular hypertrophy occurred more rapidly and more severely in male TG mice than WT littermates in response to a pathological stimulus (i.e., increased hemodynamic load by aortic banding; Fig. 4). Although TGF-β1 was up-regulated to a similar degree both in TG and WT mice under pressure overload, the banded TG mice showed an additional 2-fold increase in SCUBE3 mRNA level than banded WT mice (Fig. 5), which may account for the rapid remodeling and severity of cardiac hypertrophy observed in the TG mice under pressure overload (Fig. 5). Together, these data suggest that elevated levels of SCUBE3 above a certain threshold (e.g., >3-fold increase in SCUBE3 compared to the baseline level) in combination with a high TGF-β1 level may contribute to the accelerated onset and progression of cardiac hypertrophy. Consistent with this notion, the phosphorylated and total protein levels of Smad2, a well-known TGF-β downstream signaling molecule [27], were elevated in TG hearts as compared to WT hearts under pressure overload (Supplemental Fig. 5). Similarly, cardiac remodeling up-regulating Smad2 expression has been recently reported in a rat model of myocardial infarction [28,29].

Recent genetic analysis identified that the zebrafish orthologue of the mammalian SCUBE2 gene functions upstream of Hedgehog ligands or through a parallel pathway during zebrafish development [5,6,30]. Although the exact mechanism of SCUBE2 action remains unknown, a nonsense mutation results in a functional null protein lacking the six cysteine repeat motif and CUB domain, which indicates that this carboxyl-terminal region is essential for SCUBE2 function [5,6,30]. Consistent with this notion, our study showed that the same domain composition in the SCUBE3-D4 deletion construct, but not the SCUBE3-FL, could enhance the transcriptional activation mediated by TGF-β1 (Fig. 6). Together, these results suggest that SCUBE3 may be synthesized as a latent form that requires a proteolytic activation to release the carboxyl terminus from the amino-terminal EGF-like repeats to exert their distinct biological functions. In support of this notion, our recent study demonstrated a limited proteolysis of SCUBE1 in human platelets by releasing the amino-terminal EGF-like repeats to function as an adhesive module for platelet–platelet and platelet–subendothelial matrix interactions [7].

Our data suggest a physical interaction between SCUBE3 and TGF-β1 when overexpressed in HEK-293T cells (Fig. 6B); however, it remains to be confirmed whether or not such an interaction indeed occurs in vivo. In addition, further studies will be needed to understand the molecular mechanism by which SCUBE3 or its derived fragment regulates the TGF-β1-mediated signal transduction. However, by analogy with the functions of heparin sulfate proteoglycans in binding and regulating the turnover of ligands that act at the cell surface [31], growth factors or cytokines bound to SCUBE3 may be protected from inactivation by protease and have a longer lifetime locally in the cardiac tissues or in the circulation, thus being more potent in transducing its downstream signaling. Therefore, the extracellular SCUBE3 protein may serve as an important component of the regulatory mechanisms for active TGF-β1 bioavailability, systemically or locally in cardiac tissues, under baseline conditions and during pathological stresses. This paradigm has been reported in a recent study showing a crucial role for an extracellular matrix protein Emilin1 in the maintenance of cardiovascular homeostasis by binding specifically to the proTGF-β1 precursor and modulating TGF-β1 availability and signaling in the extracellular space [32].

Although our previous study showed that SCUBE3 appeared to be expressed in an osteoblast-enriched fashion [2], we found here no abnormality in the skeletal structure in TG mice under normal conditions. However, some bone phenotypes may arise if TG mice are stimulated or subjected to stress, or under a pathological state. In summary, our results show, for the first time, that SCUBE3 is selectively expressed in the ventricular myocardium and may be involved in the maintenance of myocyte integrity/growth or the compensatory response to myocardial stress. In future studies, ventricle-specific transgenic overexpression or deletion of SCUBE3 will be helpful for elucidating the role of SCUBE3 in ventricular myocardial function during development or pathological processes.

Time for primary review 29 days


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.03.014.


   Acknowledgements
 
We thank Dr. Alexander C. Lichtler (University of Connecticut Health Center) for providing the rat type I collagen promoter, Drs. Chi-Kuang Wang and Hsian-Jean Chin (Transgenic Core Facility, Academia Sinica) for their generous help and advice on the transgene construct, and Yueh-Hsing Su and Bo-Tsung Wu for their excellent technical assistance. This study was supported by National Council Grants NSC 94-2314-B-303-009 (to C.-F.C), NSC 95-2627-B-001-002, NSC 95-2752-B-006-003-PAE, and NSC 95-2752-B-001-002-PAE (to R.-B.Y).


   Notes
 
1 These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

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