Cloning and biological activity of epigen, a novel member of the epidermalgrowth factor superfamily

High throughput sequencing of a mouse keratinocyte library was used to iden tify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen, Epigen encodes a pro tein of 152 amino acids that contains features characteristic of the EGF su perfamily, Two hydrophobic regions, corresponding to a putative signal sequ ence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites. Epigen sh ows 24-37% identity to members of the EGF superfamily including EGF, transf orming growth factor a, and Epiregulin, Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver . Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming grow th factor alpha in several assays. In epithelial cells, Epigen stimulated t he phosphorylation of c-erbB-l and mitogen-activated protein kinases and al so activated a reporter gene containing enhancer sequences present in the c -fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epit helial cells, may constitute a novel molecular target for wound-healing the rapy.