No abstract

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.

We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)β and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPβ, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPβ expression.

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.

Epimorphin is representative of a unique class of stromal membrane-anchored proteins that plays distinct functions depending on its membrane topology. When exposed extracellularly, this molecule acts as a morphoregulator for various tissues including hair follicle epithelia. Previous study identified its functional domain (the pep7 domain: SIEQSCDQDE) for hair follicular morphogenesis followed by the successful generation of a chemically modified active peptide. Here, we report optimization of this peptide by the introduction of sequential mutations and subsequent structural determination. We found that three residues from the C-terminus are dispensable, and alternation of the seventh amino acid to an Alanine residue enhanced activity. To favour the biologically active conformation, epsilon-Acp (NH(CH(2))(5)CO) linked to a Cysteine residue was connected at the N-terminus followed by the introduction of an intramolecular disulphide bridge, the modification process of which could be included in the peptide synthesis. The obtained modified peptide, termed 'EPM (epimorphin-derived) peptide', has a Mw of 950 Da and exerts an inductive effect on hair follicle regeneration at a concentration of approximately 0.00001% or even lower. The action of this EPM peptide was more apparent in mice treated with 1% minoxidil, suggesting its potential clinical benefit as a new type of hair-regenerating agent.