Report |
Address correspondence to Eisuke Mekada, Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. Tel.: 81-6-6879-8286. Fax: 81-6-6879-8289. email: emekada{at}biken.osaka-u.ac.jp
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Abstract |
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Key Words: ectodomain shedding; ErbB; cardiomyopathy; valvulogenesis; epidermal hyperplasia
The online version of this article includes supplemental material.
Abbreviations used in this paper: EGFR, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; proHB-EGF, membrane-anchored form of HB-EGF; sHB-EGF, soluble form of HB-EGF; TPA, O-tetradecanoylphorbol-13-acetate; tRA, all-trans retinoic acid.
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Introduction |
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Recently, we demonstrated that HB-EGF is critical for proper heart development and function by the analyses of HB-EGF null mice (Iwamoto et al., 2003). However, it remains unclear which forms of HB-EGF are necessary for these process. The relative roles of either proHB-EGF or sHB-EGF and the significance of the control of ectodomain shedding in vivo have yet to be determined. To address these issues, we generated two kinds of mutant mice, by targeted gene replacement, that express either an uncleavable (HBuc) or a transmembrane domaintruncated form (HBtm) of proHB-EGF. Analysis of these mutant lines indicates that proHB-EGF shedding is essential in vivo and that this process must be controlled.
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Results and discussion |
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Uncleavable proHB-EGF mutation causes cardiac dysfunction and heart valve malformation
Recently, we showed that HB-EGF null mice (HBdel/del) have defects in cardiac chamber dilation and cardiac valve malformation (Iwamoto et al., 2003). Therefore, we analyzed the heart phenotype in HBuc/uc mice. Autopsies of HBuc/uc mice revealed massive enlargement of the heart. Histological analysis showed that wall thickness was reduced, accompanied by sporadic fibrosis in 12-wk-old HBuc/uc mice (Fig. 2, AD). These phenotypes are highly similar to those observed in HB-EGF null mice (Iwamoto et al., 2003) and ErbB2 conditional knockout mice (Crone et al., 2002; Ozcelik et al., 2002).
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In addition to the ventricular chamber abnormality, heart valve malformation was also observed in HBuc/uc mice as in HB-EGF null mice (Iwamoto et al., 2003; Jackson et al., 2003) and EGFR knockout mice (Chen et al., 2000). Histological analysis of E17.5 embryonic HBuc/uc hearts showed enlarged semilunar (aortic and pulmonic) and atrioventricular (mitral and tricuspid) valves (Fig. 3, AD). Scoring of cardiac valve size in E17.5 hearts revealed enlarged semilunar and atrioventricular valves in HBuc/uc mice (Fig. 3 E). No overt abnormality was observed in HBlox/lox heart chambers or valves (Iwamoto et al., 2003), indicating that heart abnormalities in HBuc/uc mice were not due to a nonspecific effect of cDNA knock-in.
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Generation of mice expressing soluble proHB-EGF mutant
Although studies of HBuc/uc mice indicated that ectodomain shedding of proHB-EGF and release of sHB-EGF is necessary for proper HB-EGF function in vivo, the physiological importance of the control of proHB-EGF ectodomain shedding remained unclear. To address this issue, we prepared another mouse mutant that only expresses sHB-EGF. The transmembrane domaintruncated mutant (HBtm) was generated by insertion of a stop codon between Leu148 and Pro149 (Fig. 4 A), the major site for proHB-EGF processing (Higashiyama et al., 1992). Mitogenic activity was similar between HB-
TM (product of HB
tm) and WT sHB-EGF derived from proHB-EGF shedding, but HB-
TM is secreted at much higher levels than WT sHB-EGF (Fig. S1, DG, and supplemental Results). The targeting construct for HB
tm is similar to that for HBuc. One allele of the proHB-EGF gene in ES cells was replaced with HB
tm through homologous recombination (Fig. S2, supplemental Results, and supplemental Materials and methods). Chimeric mice carrying HB
tm were generated from these ES clones. Most chimeric founders exhibited abnormally small bodies and thickened skin (Fig. 4, BD). The majority of these mice died before or during the neonatal stage (unpublished data). A few mice, however, survived and were fertile. Most of the resulting F1 heterozygotes carrying the HB
tm allele also died before or during the neonatal stage (unpublished data), displaying a more severe phenotype than that seen in chimeric mice (Fig. 4 E).
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Dysregulated release of HB-EGF induces developmental abnormalities with hyperplasia
The most obvious phenotype of HBtm-carrying mice, distinguishing them from WT mice, was the presence of abnormal elephant-like skin (Figs. 4, BE). HB
tm mice have short lives; thus to further study the epidermal hyperplasia, back skin was transplanted from P3 WT or HB
tm chimeric mice onto the backs of Balb/c nu/nu mice for 2 wk. This transplantation method also allowed for the specific examination of the effects of HB
tm in the skin in an animal with otherwise normal expression and processing of HB-EGF. Histological examination revealed severe epidermal hyperplasia, accompanied by the presence of large, disorganized, hair folliclelike structures (Fig. 5 A). Immunohistochemistry of epidermal marker proteins revealed perturbed differentiation and proliferation of keratinocytes (Fig. 5, BF). Keratin 5, normally expressed by both mitotically active, basal layer keratinocytes and hair follicles, was detected in the suprabasal epidermis of chimeric mice (Fig. 5 D). Keratin 1, normally expressed by differentiating keratinocytes in the suprabasal epidermis, but not by the cells of the hair follicle (Heid et al., 1988), was expressed in the hair folliclelike structures of chimeric mice (Fig. 5 B). Additionally, keratin 10, a differentiating keratinocyte marker, was down-regulated in chimeric mice (Fig. 5 C). Keratin 6, expressed predominantly in hair follicle cells, and at lower levels in the suprabasal epidermis, was detected throughout the epidermis of chimeric mice (Fig. 5 E), consistent with the induction of keratin 6 expression by epidermal hyperproliferation (Werner et al., 1993). The expression of Ki-67, a nuclear mitotic marker, was also increased in the basal layer of chimeric skin compared with levels seen in WT (Fig. 5 F), indicating that HB
tm expression accelerates basal layer keratinocyte proliferation. No overt abnormality was observed in the skin area surrounding the transplanted skin, suggesting that the abnormality in the transplanted skin is due to the action of HB-
TM in a manner of autocrine or paracrine in limited distance, rather than paracrine in long distance.
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The phenotype observed in HBtm mice is likely due to dysregulated secretion of sHB-EGF. In normal conditions, a portion of proHB-EGF molecules are converted to sHB-EGF, but the majority of proHB-EGF molecules on the cell surface seems to be internalized without shedding (Goishi et al., 1995). In the case of HB
tm, most synthesized molecules would be secreted without shedding, resulting in oversecretion of sHB-EGF even though the native HB-EGF promoter regulates gene expression. Therefore, dysregulated secretion of sHB-EGF would result in severe developmental abnormalities. The present study thus confirms the notion that ectodomain shedding of proHB-EGF must be strictly controlled in vivo.
One question regarding the truncated HBtm mutant concerns whether the observed hyperplasia might be due to an intracrine rather than a paracrine mechanism. A transmembrane domaintruncated EGF mutant was reported to activate EGFR in an intracrine manner, as a result of interaction with EGFR within cytoplasmic vesicles, before the molecules reached the cell surface (Wiley et al., 1998). However, this was not the case for HB-EGF. We have performed ex vivo transfection of HB
tm cDNA into mouse embryonic skin (Fig. S3 and supplemental Results, available at http://www.jcb.org/cgi/content/full/jcb.200307035/DC1). Transfection of HB
tm, but not WT proHB-EGF, resulted in embryonic epidermal hyperplasia. CRM197, a protein that specifically inhibits the mitogenic activity of HB-EGF (Mitamura et al., 1995), inhibited HB-
TMinduced hyperplasia. As CRM197 is membrane impermeable, hyperplasia induced by transfection with HB
tm must be mediated by secreted HB-
TM in a paracrine manner.
In conclusion, proHB-EGF shedding and the strict control of this process are essential for the function of this growth factor. Not only HB-EGF, but also other EGF family growth factors and cytokines are synthesized as membrane-anchored forms. The present study suggests that the strict control of ectodomain shedding is essential for the physiological function of these membrane-anchored ligands. This study also indicates that posttranslational regulation, in addition to transcriptional control, is crucial for the function of membrane-anchored growth factors.
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Materials and methods |
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Immunoblotting of tissues
For the detection of HB-EGF and HB-UC in adult skin, back skin from 11-wk-old HBlox/lox and HBuc/uc mice was treated with 800 nmol of tRA for 3 d. 2 h before specimen isolation, the back skin was additionally treated with 8 nmol of TPA. The isolated full back skin was homogenized in lysis buffer (Iwamoto et al., 2003). For detection of HB-TM in neonatal skin, skin from P1 HB
tm heterozygous mice or WT littermates was homogenized in lysis buffer. Details of the immunoblotting procedure using these lysate samples are shown in the supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200307035/DC1).
Histological analysis
Mouse hearts were fixed by perfusion with 4% paraformaldehyde, dehydrated, and embedded in paraffin. 4-µm sections were stained with either hematoxylin-eosin or Azan-Mallory. For immunohistochemical analysis of transplanted skin, specimens of full-thickness skin (23 cm2) were transplanted onto the backs of Balb/c nu/nu mice (8 wk old) and then fixed with adhesive bandages for 1 wk. 2 wk after transplantation, skin specimens were isolated from the grafted skin and subjected to immunohistochemical analysis. Information of the used antibodies for immunohistochemistry, microscopy, and image processing is shown in the supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200307035/DC1).
Echocardiography
Transthoracic echocardiograph was performed with a cardiac ultrasound recorder (SONOS 5500; Hewlett-Packard) with a 15-MHz transducer, as described previously (Iwamoto et al., 2003).
RT-PCR
RNA was isolated from tissues of P14 mice using TRIzol reagent (Invitrogen). Reverse transcription was performed using a reverse transcriptase, ReverTra Dash (TOYOBO). Primer sets used in PCR analyses are shown in the supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200307035/DC1).
Online supplemental material
The supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200307035/DC1. Fig. S1 shows the characterizations of HB-UC and HB-TM. Fig. S2 shows the targeting construct of HBuc and HB
tm and genotypic analyses. Fig. S3 shows ex vivo transfection of HB
tm cDNA into mouse embryonic skin. Supplemental Results, Materials and methods, and References are also presented.
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Acknowledgments |
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This work was supported by the Research for the Future Program of the Japan Society for the Promotion of Science (97L00303 for E. Mekada) and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (12215152 and 14032202 for E. Mekada and 12680705 for R. Iwamoto).
Submitted: 7 July 2003
Accepted: 12 September 2003
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