Maintenance of human skin in organ culture: role for insulin-like growth factor-1 receptor and epidermal growth factor receptor

Recent studies have shown that adult skin incubated in low-Ca2+ (0.15 mM) medium rapidly degenerates but that normal architecture is maintained when the tissue is incubated in high-Ca2+ medium (1.4 mM Ca2+). To investigate whether the skin cell-produced growth factors insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF) play a role in these events, 2-mm skin punch biopsies were obtained and maintained for 8 to 10 days in a basal medium containing 0.15 mM Ca2+ with and without growth factors, or containing 1.4 mM Ca2+ with and without antibodies to the same growth factors. In parallel experiments, cultured human keratinocytes were incubated for 2 days in the same basal medium in the presence or absence of the same growth factors and antibodies. Consistent with previous reports, organ cultures incubated in the low-Ca2+ (0.15 mM) medium rapidly degenerated. Neither IGF-1 nor EGF prevented the complete degeneration of epidermis and dermis in these organ cultures. Interestingly, the addition of an anti-IGF-1 receptor (IGP-1R) antibody to the organ cultures maintained in high-Ca2+ medium induced changes reminiscent of those seen when the organ cultures were maintained in low-Ca2+ medium, i.e. tissue degeneration. In contrast, antibodies to EGF receptor, used for comparison, only produced focal areas of epidermal necrosis. In vitro, IGF-1 is a known mitogen for keratinocytes. In cultured human keratinocytes, anti-IGF-1R antibody partially inhibited the IGF-1-mediated stimulation of human keratinocyte proliferation without affecting normal spontaneous growth. Additionally, IGF-1R immunolocalized to basal keratinocytes in vivo, exhibited specific binding to IGF-1 in vitro. This indicated a critical role for IGF-1R in both organ cultures ex vivo and cultured cells in vitro. Messenger RNA encoding both IGF-1 and IGF-1R were readily detected by RT-PCR in organ cultures incubated in both low- and high-Ca2+ medium. There were no detectable differences in IGF-1 mRNA in organ cultures growing in the low- or high-Ca2+ medium, but lower levels of IGF-1R mRNA were observed in the organ cultures maintained in low-Ca2+ medium than in those in high Ca(2+)medium. These findings are consistent with homeostatic changes in the tissue grown under different calcium concentrations. IGF-1 mRNA was detected in several skin cell populations in vitro, even though it was undetectable in cultured keratinocytes. Taken together these findings indicate that (1) the IGF-1/IGF-1R loop is critically involved in maintenance of human skin organ cultures ex vivo, and (2) IGF-1, locally produced by skin cells other than keratinocytes, interacts with its receptor, predominantly expressed in basal keratinocytes, to maintain tissue homeostasis.