Differential Roles of Insulin Receptor and Insulin-Like Growth Factor-1 Receptor in Differentiation of Murine Skin Keratinocytes  Efrat Wertheimer, Meirav Trebicz, Tora Eldar, Marina Gartsbein, Sharon Nofeh-Moses, Tamar Tennenbaum  Journal of Investigative Dermatology  Volume 115, Issue 1, Pages 24-29 (July 2000) DOI: 10.1046/j.1523-1747.2000.00008.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression levels of the IR and IGFR proteins during keratinocyte differentiation. Primary keratinocytes were isolated and plated as described in Materials and Methods. Proliferating keratinocytes were maintained for 5 d in low Ca2+ medium (0.05 mM) until they reached 80% confluency. Differentiation was then induced by elevating the Ca2+ concentration in the growth medium for 48 h. The Ca2+ concentrations used were 0.05 mM (L) for proliferating keratinocytes; 0.12 mM (M) and 1.0 mM (H) for differentiating keratinocytes. Total protein lysate was extracted from cells and 20 μg were analyzed by western blotting using specific antibodies against IR or IGFR. A representative experiment of six separate experiments is shown. Journal of Investigative Dermatology 2000 115, 24-29DOI: (10.1046/j.1523-1747.2000.00008.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Insulin and IGF-1 binding in proliferating and differentiating keratinocytes. Keratinocytes were cultivated in 24-well plates for 5 d in low Ca2+ medium (0.05 mM). Proliferating keratinocytes were further maintained in 0.05 mM Ca2+ (•) or induced to differentiate for 48 h in 0.12 mM (▪) or 1.0 mM (▴) Ca2+, as described in Figure 1. The 125I-insulin or 125I-IGF-1 binding assay was carried out as described in Materials and Methods. Scatchard analysis was performed. Units of ‘‘Bound’' are shown in fmol per μg protein per min. The experiments were repeated three times, and a representative experiment is shown. Journal of Investigative Dermatology 2000 115, 24-29DOI: (10.1046/j.1523-1747.2000.00008.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Autophosphorylation of IR and IGFR during keratinocyte differentiation. Cells were cultivated as described in Figure 1. Proliferating keratinocytes were further maintained in 0.05 mM Ca2+ (L) or induced to differentiate for 48 h in 0.12 mM (M) or 1.0 mM (H) Ca2+. Insulin (Ins; 10−6 mM) or IGF-1 (IGF; 10−7 mM) were added to the medium for 3 min at room temperature. Control cells (0) were not stimulated. Reactions were terminated by quick washes with cold PBS on ice. (a) Total lysates were immunoprecipitated with anti-phosphotyrosine antibodies and analyzed by western blotting using specific antibodies against IR or IGFR. (b) Cells were stimulated for 3 min with insulin or IGF-1 at the concentrations indicated (0–10−5 M). Membranal fractions were analyzed by western blotting, utilizing anti-phosphotyrosine antibodies. Representative blots of three separate experiments are shown. Arrows indicate the position of IR and IGFR on the blot. Journal of Investigative Dermatology 2000 115, 24-29DOI: (10.1046/j.1523-1747.2000.00008.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Labeling of cell surface receptors by biotinylation. Five day old primary keratinocytes were further maintained for 48 h in medium containing Ca2+ concentrations of 0.05 mM (black columns), 0.12 mM (gray columns), or 1.0 mM (oblique stripes). Thereafter, the cells were surface-labeled by biotinylation, as described in Materials and Methods. Labeled proteins were precipitated on streptavidin-coated beads, separated on a SDS–polyacrylamide gel and receptors were detected by western blotting with antibodies directed against IR or IGFR. Total lysates were run in parallel for estimation of the total expression levels of IR or IGFR in the same samples. Results were quantitated by densitometry, and are presented as the ratio between the biotinylated receptors/total receptor expression. The relative ratio that was calculated for proliferating keratinocytes was set as 1. The experiments were repeated three times, and a representative experiment is shown. Journal of Investigative Dermatology 2000 115, 24-29DOI: (10.1046/j.1523-1747.2000.00008.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 In vitro kinase assays of isolated receptors. Cells were cultivated and differentiated as described in Figure 1. Receptors from cells grown in the presence of 0.05 (L), 0.12 (M), or 1.0 (H) mM Ca2+ were isolated by wheat germ agglutinin (WGA) chromatography, and 20 μg of protein were incubated for 20 min with insulin (Ins; 10−6 M) or IGF-1 (IGF; 10−7 M). Phosphorylation was allowed to proceed for 10 min in the presence of adenosine triphosphate. Thereafter, samples were analyzed by western blotting. Phosphorylated receptors were detected by anti-phosphotyrosine antibodies (αPY). Thereafter, the filters were re-blotted with an anti-IR antibody (αIR), to estimate the total receptor amount in each sample. A representative experiment of three different experiment is shown. Arrows indicate the position of IR and IGFR on the blot. Journal of Investigative Dermatology 2000 115, 24-29DOI: (10.1046/j.1523-1747.2000.00008.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 αβ(IR)–αβ(IGFR)] hybrid formation. Cells were cultivated and differentiated as described in Figure 1. Total protein lysates of proliferating (L, 0.05 mM Ca2+) or differentiating (M, 0.12 mM or H, 1.0 mM Ca2+) cells were subjected to immunoprecipitation by anti-IR antibodies. The samples were analyzed by western blotting and the existence of hybrids was demonstrated by blotting with an anti-IGFR antibody (IGFR) directed against the β subunit of the receptor. Filters were re-blotted with an anti-IR antibody (IR), to demonstrate that equal amounts of IR were precipitated and loaded. A representative blot of four separate experiments is shown. Journal of Investigative Dermatology 2000 115, 24-29DOI: (10.1046/j.1523-1747.2000.00008.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Insulin and IGF-1 regulate keratinocyte differentiation. Skin keratinocytes were let to differentiate for 24–48 h in different Ca2+ concentrations of 0.05 mM (L), 0.12 mM (M), or 1.0 mM (H) in the presence of insulin (Ins; 10−6) or IGF-1 (IGF; 10−7). Hormone was added every 24 h. Control cells were induced to differentiate without the addition of hormone (0). Cytoskeletal fraction was isolated as described in Materials and Methods. The induction of differentiation markers was identified by western blot analysis, using antibodies against keratin 1 (K1) and keratin 10 (K10). Journal of Investigative Dermatology 2000 115, 24-29DOI: (10.1046/j.1523-1747.2000.00008.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions