We are studying cell growth and differentiation mechanisms that involve growth factors and adhesion molecules presented at cell-cell contact sites. In particular, we are focusing on the mode of action of HB-EGF, a membrane-anchored EGF family of growth factors, and the molecules to which they bind, namely the tetraspanin family. These proteins function in morphogenesis and tissue maintenance and repair by regulating cell proliferation, migration and adhesion. They are also involved in the growth, invasion and metastasis of cancer cells.
1) Mode of action of HB-EGF
HB-EGF is a member of the EGF family of growth factors and binds to and activates EGFR and ErbB4. It is synthesized as proHB-EGF, a membrane-anchored precursor protein, and is cleaved on the cell surface to yield the soluble growth factor (sHB-EGF). The conversion of proHB-EGF into the soluble form is critical for the activity of this growth factor, and therefore this process is tightly regulated. HB-EGF is secreted by various tissues and cells and functions in several physiological processes. For example, it maintains heart muscle function, suppresses the cell proliferation involved in heart valve and lung alveolar development, promotes the cell migration that participates in wound healing and eyelid closure, supports blastocyst attachment to the uterus during implantation, and promotes the cell proliferation involved in skin hyperplasia. ProHB-EGF is not only a precursor of the soluble form, it is also a biologically active molecule that regulates the growth of neighboring cells in a juxtacrine fashion. How is the conversion of the membrane-anchored form into the soluble form regulated? How does HB-EGF function in the manifold physiological processes that are dependent on this molecule? What roles do sHB-EGF and proHB-EGF play in such physiological processes? Do they participate in pathological processes? These questions are currently being analyzed at the molecular level.
2) Development of anti-cancer drugs targeting HB-EGF
HB-EGF is involved in the growth, invasion and metastasis of various cancers. We are developing new anti-cancer drugs that target HB-EGF, and pre-clinical and clinical studies assessing an anti-HB-EGF monoclonal antibody and a non-toxic mutant protein of diphtheria toxin CRM197 are currently in progress.
3) CD9 and tetraspanin function
CD9, a member of the tetraspanin superfamily, is a membrane protein with four transmembrane domains. It associates with proHB-EGF and upregulates proHB-EGF function. CD9 is also involved in cell signaling, growth, motility, adhesion, and in tumor cell metastasis and sperm-egg fusion. In addition, the Caenorhabditis elegans tetraspanin TSP-15 is essential for the epidermal integrity of the worm. We are analysing the physiological activity of CD9 and other tetraspanins by using genetically engineered mice or C. elegans worms that lack CD9 or other tetraspanins.
Fig.1. Structure of proHB-EGF. |
Fig.2. Tumorigenesis in nude mice explanted with ovarian cancer cells (left) and inhibition of this tumorigenesis by CRM197, a non-toxic mutant of diphtheria toxin (right). |
Fig. 3. HB-EGF KO mice exhibit several tissue abnormalities. Unlike WT mice (a, c, e, g), KO mice (b, d, f, h) show abnormal phenotypes in the heart (a, b), heart valves (c, d), and lung alveoli (e, f), as well as retinoid-induced skin hyperplasia (g, h). HB-EGF KO embryos also show defects in eyelid closure because HB-EGF is normally expressed at the tip of the leading edge of migrating epithelium (i).
Fig.4. Expression of tsp-15 in C. elegans (left) and reduction of tsp-15 function in C. elegans by RNA interference (right), which induces abnormalities of the hypodermis, including dissociation of the cuticle and degeneration of the hypodermis. |
Fig. 5. Entry mechanism of diphtheria toxin. |