Yohei Hirai

Epimorphin: A mesenchymal protein essential for epithelial morphogenesis

A novel 150 kd protein expressed on the surface of mesenchymal cells of mouse embryonic tissues was identified. A monoclonal antibody to this molecule inhibited various processes of epithelial morphogenesis, such as hair follicle growth and lung epithelial tubular formation, in organ cultures of these tissues. Sequence analysis of cDNA encoding this protein revealed that it had 289 amino acids with a hydrophobic stretch at the C-terminus. NIH 3T3 cells transfected with the cDNA of this protein expressed the exogenous 150 kd protein on their surface. When lung epithelial cells were cocultured with these transfected cells, they showed normal tubular morphogenesis, but not with untransfected NIH 3T3 cells. These results indicate that this protein, termed epimorphin, plays a central role in epithelial-mesenchymal interactions.

Single proteins might have dual but related functions in intracellular and extracellular microenvironments.

The maintenance of organ homeostasis and the control of an appropriate response to environmental alterations require the intimate coordination of cellular functions and tissue organization. An important component of this coordination could be provided by proteins that can have distinct but linked functions on both sides of the plasma membrane. We present a model that proposes that unconventional secretion provides a mechanism through which single proteins can integrate complex tissue functions.

Delivering the message: epimorphin and mammary epithelial morphogenesis

The mammary gland consists of a highly branched tubular epithelium surrounded by a complex mesenchymal stroma. Epimorphin is an extracellular protein that is expressed by mammary mesenchymal cells that directs epithelial morphogenesis. Depending upon the context of presentation--polar versus apolar--epimorphin can selectively direct two key processes of tubulogenesis: branching morphogenesis (processes involved in tubule initiation and extension) and luminal morphogenesis (required for enlargement of tubule caliber). Here, we outline the fundamentals of mammary gland development and describe the function of epimorphin in these processes. We conclude with a review of recent studies that suggest similar morphogenic roles for epimorphin in other glandular organs.

Phosphorylation of claudin-4 is required for tight junction formation in a human keratinocyte cell line.

Extensive studies have identified a large number of the molecular components of cellular tight junctions (TJ), including the claudins, occludin and ZO-1/2, and also many of the physical interactions between these molecules. However, the regulatory mechanisms of TJ formation are as yet poorly understood. In HaCaT, a human epidermal keratinocyte cell line, TJ were newly formed when cells were cultured in the presence of SP600125, a JNK inhibitor. Moreover, claudin-4 was newly phosphorylated during this process. We found that claudin-4 contains a sequence which is phosphorylated by atypical PKC (aPKC). Kinase assay demonstrated that the 195th serine (serine195) of mouse claudin-4 was phosphorylated by aPKC in vitro. The 194th serine (serine194) of human claudin-4 corresponding to serine195 of mouse claudin-4 was phosphorylated in HaCaT cells when TJ were formed, and the phosphorylated claudin-4 co-localized with ZO-1 at TJ. aPKC activity was required for both the claudin-4 phosphorylation and TJ formation in HaCaT. Furthermore, overexpression of mutant claudin-4 protein S195A, which was not phosphorylated by aPKC, perturbed the TJ formation mediated by SP600125. These findings suggest that aPKC regulates TJ formation through the phosphorylation of claudin-4.

Non-classical export of epimorphin and its adhesion to αV-integrin in regulation of epithelial morphogenesis

Epimorphin (also known as syntaxin 2) acts as an epithelial morphogen when secreted by stromal cells of the mammary gland, lung, liver, colon, pancreas and other tissues, but the same molecule functions within the cell to mediate membrane fusion. How this molecule, which lacks a signal sequence and contains a transmembrane domain at the C-terminus, translocates across the plasma membrane and is secreted to become a morphogen, and how it initiates morphogenic events is not clear. Here, we show that epimorphin is secreted through a non-classical mechanism, similar to that previously described for secretion of the leaderless protein FGF1, and we identify the key molecular elements responsible for translocation and secretion from the cell. We also show that secreted epimorphin binds to αv-integrin-containing receptors on target epithelial cells, leading to activation of specific downstream signaling pathways and induction of epithelial morphogenesis. These findings provide key insight into how epimorphin functions as an epithelial morphogen.

Epimorphin Overexpression in the Mouse Mammary Gland Promotes Alveolar Hyperplasia and Mammary Adenocarcinoma

Epimorphin/syntaxin-2 (EPM) is a plasma membrane-anchored protein that has at least two distinct functions depending on its membrane topology: vesicle fusion when localized to the cytoplasmic surface and morphogenic signaling when localized to the extracellular surface. Transgenic mice that express full-length extracellular EPM fused to the NH2-terminal signal sequence of interleukin-2, under the control of the whey acidic protein (WAP) gene promoter, exhibit aberrant mammary gland morphogenesis associated with increased expression of CCAAT enhancer binding protein beta (C/EBPbeta). Here we report that aged nulliparous and uniparous female WAP-EPM transgenic mice develop alveolar hyperplasias and well-differentiated adenocarcinomas that express high levels of C/EBPbeta, keratin-14, matrix metalloproteinase-3, and beta-catenin. This study reveals another pathway in which overexpression and alteration of a normal morphogenic process promote the development of cancer in the mammary gland.

Epimorphin acts to induce hair follicle anagen in C57BL/6 mice

Epimorphin is a mesenchymal morphogen that has been shown to mediate epithelial‐mesenchymal signaling interactions in various organs. We now show that epimorphin functions in hair follicle morphogenesis; using a novel ex vivo organ culture assay, we define a mechanism for epimorphin signaling that may provide insight into general developmental processes. We found that epimorphin was produced by follicular mesenchymal cells and bound selectively to follicular epithelial cells, and that treatment with recombinant epimorphin could stimulate procession of hair follicles from telogen (resting stage) to anagen (growing stage). Based on analyses of epimorphin proteolytic digests that suggested a smaller peptide might be able to substitute for the full‐length epimorphin molecule, we determined that pep7, a 10‐amino acid peptide, was capable of inducing telogen‐to‐anagen transition both in the culture assay and in the mouse. That pep7 showed maximal activity only when modified with specific sulfhydryl‐reactive reagents suggested that a particular structural conformation of the peptide was essential for activity; molecular dynamics studies were pursued to investigate the active peptide structure. These findings define a previously unknown morphogenic process in the hair follicle that may have applications to many other organs.—Takebe, K., Oka, Y., Radisky, D., Tsuda, H., Tochigui, K., Koshida, S., Kogo, K., Hirai, Y. Epimorphin acts to induce hair follicle anagen in C57BL/6 mice. FASEB J. 17, 2037–2047 (2003)

Homology with Vesicle Fusion Mediator Syntaxin-1a Predicts Determinants of Epimorphin/Syntaxin-2 Function in Mammary Epithelial Morphogenesis

We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that, although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.

Epimorphin as a morphogen : does a protein for intracellular vesicular targeting act as an extracellular signaling molecule?

Fig. 1. Scheme of the SNARE model. Along with NSF and SNAP, SNAP receptors on the intracellular vesicle (vSNARE) and on the target membrane (t-SNARE) form a SNARE complex, which triggers membrane docking/fusion for exocytosis. Docking and fusion of secretory vesicles with the plasma membrane are critical processes for the secretion of extracellular molecules. This molecular mechanism has been intensively investigated, especially synaptic vesicle docking and fusion at the active site of the neuronal cell membrane. So far, the SNARE docking/fusion model is the most convincing. The action of the ATPase NSF, and SNAP (soluble NSF attachment protein) cause v-SNARE and t-SNARE, receptors for SNAP on the cytoplasmic surface of the vesicles and the target plasma membrane respectively, to bind to each other. This in turn triggers membrane fusion (Ungermann et al., 1998; Weber et al., 1998; Fig. 1). The primary structure of a neuronal cellspecific membrane protein, HPC-1 (later renamed as syntaxin), determined in 1992 (Inoue et al., 1992; Bennett et al., 1992), contains a C-terminal hydrophobic domain for membrane anchoring, but no N-terminal signal peptide for secretion. Because syntaxin/HPC-1 has cytoplasmic orientation and binding capacity with proteins involved in intravesicular targeting (including v-SNARE proteins) this protein is proposed to be a core molecule in the t-SNARE complex (Fujita et al., 1995). At the same time, membrane proteins similar to syntaxin/

Involvement of epimorphin in the repair of experimental renal fibrosis in mice.

Interaction between epithelial cells and mesenchymal cells is essential in normal organ morphogenesis and in tissue repair after injury. Epimorphin, a mesenchymal protein that regulates epithelial morphogenesis through epithelial–mesenchymal interactions, has recently attracted attention as an important modulator of tissue repair. In this study we analyzed the role of epimorphin in renal fibrosis. We first found a progressive increase in epimorphin expression corresponding to the progression of renal fibrosis in mice with unilateral ureteral obstruction (UUO). To determine whether this expression has a role in the repair or progression of renal fibrosis, we analyzed a model of renal fibrosis repair, the UUO-release (UUO-R) model. Epimorphin expression was increased at 3 and 7 days after the UUO-R rather than on the day of release, but was decreased at 21 days after the release. Inhibition of endogenous epimorphin with anti-epimorphin antibody (MC-1) significantly delayed the repair of fibrosis. When compared with normal-IgG-injected mice, MC-1-injected mice showed significantly decreased renal matrix metalloproteinase (MMP)-2 and MMP-9 expressions by western blotting and increased expression of TGF-β and collagen-I mRNA by real-time RT-PCR. Recombinant epimorphin induced prominent increases in MMP-2 and MMP-9 activities in the culture media of renal interstitial fibroblasts in vitro. These findings indicate that epimorphin has a pivotal role in the repair of renal fibrosis by modulating both extracellular matrix (ECM) degradation and its production.