Human EGF Receptor (HER) Family and Heregulin Members Are Differentially Expressed in Epidermal Keratinocytes and Modulate Differentiation Isabelle Y. De Potter,* ,1 Yves Poumay,* ,1,2 Karen A. Squillace,† and Mark R. Pittelkow† *De ´partement Histologie–Embryologie, Faculte ´s Universitaires Notre-Dame de la Paix, 61 Rue de Bruxelles, B-5000 Namur, Belgium; and †Department of Dermatology, Mayo Clinic/Foundation, Rochester, Minnesota 55905 Human EGF receptor (HER), also designated HER1, is an activatable tyrosine kinase receptor. HER1 acti- vation regulates cell growth and differentiation in epi- dermal keratinocytes. Expression of other HER family members was investigated in human keratinocytes cultured under autocrine conditions. HER2 and HER3 are expressed and upregulated by confluence, concur- rent with induction of epidermal differentiation. HER4 is not expressed by keratinocytes. Maximum expression of the cognate ligand, heregulin, is ob- served in subconfluent keratinocytes, and the expres- sion of both heregulin  and  isoforms is downregu- lated with confluence. Recombinant heregulin isoforms have no effect on colony formation and ker- atinocyte proliferation, but heregulin  activates ty- rosine phosphorylation of HER2 and HER3, with no effect on HER1, in confluent differentiating keratino- cyte cultures. Also, heregulin  increases HER2/HER3 heterodimerization under those conditions. Treat- ment of confluent cultures by heregulin  correlates with cell signaling and inhibition of epidermal differ- entiation. Together, HER2, HER3, and heregulin con- stitute a potential autocrine–paracrine system in- volved in epidermal homeostasis and repair, as well as in hyperproliferative pathologies. © 2001 Elsevier Science Key Words: keratinocyte; differentiation; heregulin; neuregulin; HER. INTRODUCTION Among the transmembrane receptors containing a cytoplasmic tyrosine kinase domain, the epidermal growth factor (EGF) receptor is the prototypic member of an extended receptor tyrosine kinase family, named the human EGF receptor (HER) family. The EGF re- ceptor is also known as HER1. Three other receptors, HER2, HER3, and HER4, have been identified to date as members of this family. HER2 is the product of the c-erbB-2 oncogene, also designated as the neu oncogene in rat for its involvement in neuroblastomas. HER2 has drawn considerable attention because the receptor has been found to be overexpressed among aggressive subsets of breast and ovarian carcinomas [1]. Although HER2 is closely related to HER1 [2, 3], EGF does not bind or activate HER2 [4]. For some time, no ligand for HER2 had been identified. During this period, the c- erbB-3 gene encoding HER3, the third member of the HER family, was found by reduced stringency hybrid- ization of human genomic DNA [5]. Subsequently, Plowman et al. [6] identified the fourth member of the family (HER4) using degenerated primers and PCR technology to identify the c-erbB-4 gene. Concurrently, ligands that activated HER2 in the absence of effects on HER1 were identified [7–9] and named neu differ- entiation factor (NDF) or heregulin (HRG). With fur- ther investigation, these ligands were also found to activate HER3 and HER4 [10, 11] and an apparent requirement for simultaneous expression of HER3 or HER4, in conjunction with HER2, to induce activation of HER2 by HRG [12] strongly suggested that HRG does not bind directly to HER2. Evidence currently indicates that HRG is bound by HER3 or HER4, where- upon HER3 or HER4 heterodimerizes with HER2 and transactivates these receptors through their tyrosine kinase domains [13, 14]. The involvement of HER1 in the epidermis, specifi- cally in keratinocyte proliferation and differentiation, was suggested by the early observations of EGF-medi- ated responses in developing skin, as well as by a multitude of subsequent in vitro and in vivo studies (for a review, see Ref. [15]). Recently, the critical in- volvement of HER1 was demonstrated in EGF receptor knock-out mice where many epithelia, including epi- dermis as well as hair follicles, are poorly formed and atrophic with markedly attenuated cell proliferation [16 –18]. The epidermis is a tissue composed mainly of prolif- 1 I. Y. De Potter and Y. Poumay were equal contributors to this work. 2 To whom correspondence and reprint requests should be ad- dressed. Fax: +32-81-724272. E-mail: yves.poumay@fundp.ac.be. 0014-4827/01 $35.00 315 © 2001 Elsevier Science All rights reserved. Experimental Cell Research 271, 315–328 (2001) doi:10.1006/excr.2001.5390, available online at http://www.idealibrary.com on