Heparin-Binding Epidermal-Growth-Factor-Like Growth Factor Activation of Keratinocyte ErbB Receptors Mediates Epidermal Hyperplasia, a Prominent Side-Effect of Retinoid Therapy  James Varani, Mary Zeigler, Michael K. Dame  Journal of Investigative Dermatology  Volume 117, Issue 6, Pages 1335-1341 (December 2001) DOI: 10.1046/j.0022-202x.2001.01564.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Topical ROL and RA induce epidermal thickening in human skin. Volunteers were treated for 7 d with vehicle or with 1.0% ROL under occlusion or 0.1% RA under occlusion as described in Materials and Methods. Biopsies were taken at the end of the treatment period and stained with hematoxylin and eosin or with an antibody to Ki-67. The number of keratinocytes per unit length of epidermis was assessed in hematoxylin and eosin stained sections using NIH Image software. Values shown represent means and standard errors based on n = 17 separate volunteers for the vehicle and ROL groups and n = 4 for the RA group. ***p <0.01 vs vehicle alone. Scale bar: 100 μm. Journal of Investigative Dermatology 2001 117, 1335-1341DOI: (10.1046/j.0022-202x.2001.01564.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 c-erbB-RTK antagonist inhibits RA-induced hyperplasia in organ-cultured human skin. Human skin was maintained in organ culture for 8 d. At the end of the incubation period, tissue was stained with hematoxylin and eosin. Control skin (KBM + Ca2+); skin treated with 3 µM RA; skin treated with 3 µM RA and 1 µM PD169540; quantitation of results. The number of keratinocytes per unit length of epidermis was assessed in hematoxylin and eosin stained sections using NIH Image software. Values shown represent means and standard errors based on organ cultures from n = 5 different volunteers. *p <0.05 vs control; **p <0.05 vs RA alone. Scale bar: 100 μm. Journal of Investigative Dermatology 2001 117, 1335-1341DOI: (10.1046/j.0022-202x.2001.01564.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Irreversible and reversible c-erbB-RTK antagonists inhibit proliferation of human epidermal keratinocytes in monolayer culture. Keratinocytes were incubated for 2 d in KBM alone or KBM supplemented with 0.5 µg per ml insulin and 2% pituitary extract. Concomitantly the cells were treated with varying concentrations of the two c-erbB-RTK antagonists. At the end of the incubation period, cells were harvested and counted. Journal of Investigative Dermatology 2001 117, 1335-1341DOI: (10.1046/j.0022-202x.2001.01564.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 RA induction of HB-EGF mRNA overcomes high-Ca2+-induced suppression in human skin organ culture. Organ cultures of human skin were incubated for 8 h in KBM containing either 0.1 or 1.4 mM Ca2+ in the presence or absence of 3 µM RA as indicated above the autoradiograms. RNAs were isolated and northern blots were prepared and hybridized with HB-EGF or 36B4 probes as described in Materials and Methods. Journal of Investigative Dermatology 2001 117, 1335-1341DOI: (10.1046/j.0022-202x.2001.01564.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Antibody to HB-EGF inhibits RA-induced hyperplasia in organ-cultured human skin. Human skin was maintained in organ culture for 8 d. At the end of the incubation period, tissue was stained with hematoxylin and eosin. Control skin (KBM + Ca2+); skin treated with 3 µM RA; skin treated with 3 µM RA and 10 µg per ml anti-HB-EGF. Quantitation of results. The number of keratinocytes per unit length of epidermis was assessed in hematoxylin and eosin stained sections using NIH Image software. Values shown represent means and standard errors based on organ cultures from n = 3 different volunteers. Normal goat IgG (10 µg per ml) was ineffective. *p <0.05 vs control; **p <0.05 vs RA alone. Scale bar: 100 μm. Journal of Investigative Dermatology 2001 117, 1335-1341DOI: (10.1046/j.0022-202x.2001.01564.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 c-erbB RTK antagonists and antibody to HB-EGF do not interfere with the ability of RA to induce fibroblast survival and growth under low-Ca2+ conditions in monolayer culture. Fibroblasts were incubated for 2 d in KBM with or without varying concentrations of the two c-erbB-RTK antagonists or anti-HB-EGF monoclonal antibody. At the end of the incubation period, cells were harvested and counted. Journal of Investigative Dermatology 2001 117, 1335-1341DOI: (10.1046/j.0022-202x.2001.01564.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions