Amphiregulin Carboxy-Terminal Domain Is Required for Autocrine Keratinocyte Growth  Stefan W. Stoll, Jessica L. Johnson, Yong Li, Laure Rittié, James T. Elder  Journal of Investigative Dermatology  Volume 130, Issue 8, Pages 2031-2040 (August 2010) DOI: 10.1038/jid.2010.98 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Amphiregulin (AREG) expression is strongly inhibited by tetracycline (TET)-induced expression of AREG small hairpin RNA (shRNA). (a) Inducible, lentivirus-mediated gene expression is tightly regulated by TET as assessed by enhanced green fluorescent protein (EGFP) immunofluorescence and immunoblotting. The photographs in the upper panel were taken with identical exposure times. Bar=200μm. The “+” and “-” labels on the immunoblot indicate ±TET. (b) AREG mRNA expression. N-TERT-keratinocytes (KCs) were grown under autocrine conditions ±TET and analyzed by quantitative real-time PCR. Asterisks denote a significant reduction in gene expression relative to untreated controls, P<0.005, n=2 for shEGFP, n=5 for shAREG. (c, d) AREG protein expression. N-TERT KCs were grown under autocrine conditions ±TET for 48hours (48hours pre-TX), followed by incubation for an additional 2–24hours in basal keratinocyte serum-free medium (KSFM)±TET. AREG protein levels were analyzed in radioimmunoprecipitation assay extracts (c) or in KC-conditioned medium (d) by ELISA as described in Materials and Methods. Data are expressed as % of controls, *P<0.02 (c) or P<0.001 (d) versus untreated controls (n=4 for all conditions, except n=2 for shEGFP at 2 and 14hours). Journal of Investigative Dermatology 2010 130, 2031-2040DOI: (10.1038/jid.2010.98) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Epidermal growth factor receptor (EGFR) ligand expression and shedding in normal human keratinocytes (NHKs) and N-TERT keratinocytes (KCs). KCs were grown to 40% confluence and incubated in basal medium for 24hours. Culture supernatants were collected for ELISA and the cells were lysed for quantitative real-time PCR analysis. (a) Relative EGFR-ligand transcript levels were analyzed by quantitative real-time PCR using TaqMan gene-expression assays (Applied Biosystems, Foster City, CA) as previously described (Stoll et al., 2010). Amphiregulin (AREG) mRNA expression was analyzed with three different TaqMan assays (Applied Biosystems; assay #1: HS00155832; assay #2: HS00950668; assay #3: HS00950669). Data are expressed as % of 36B4 transcript levels, mean±SEM, n=3, *P<0.05 versus all other EGFR ligands. (b) KC supernatants conditioned for 24hours were analyzed by ELISA for the presence of shed AREG. Data are expressed as ng of soluble AREG per ml of supernatant. Journal of Investigative Dermatology 2010 130, 2031-2040DOI: (10.1038/jid.2010.98) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Autocrine extracellular signal regulated kinase (ERK) phosphorylation is reduced in the presence of amphiregulin (AREG) small hairpin RNA (shRNA). N-TERT-tetracycline repressor (TR) shAREG or shEGFP was grown under autocrine conditions for 48hours as described in Materials and Methods, followed by treatments as described below, and radioimmunoprecipitation assay lysates were analyzed by ERK and phospho-ERK western blotting. (a) Cells were incubated in fresh M154 medium for 2–4hours ±TET, with or without 5μgml−1 AREG-neutralizing antibodies (Abs) or IgG isotype control or 1μM PD158780, 40μM GM6001, or 100ngml−1 epidermal growth factor (EGF). (b) Cells were incubated in fresh M154 medium ±TET for 30minutes, followed by treatment with EGF (20ngml−1) or AREG (100ngml−1) for 10minutes. Journal of Investigative Dermatology 2010 130, 2031-2040DOI: (10.1038/jid.2010.98) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Amphiregulin (AREG) knockdown strongly inhibits autocrine keratinocyte (KC) growth and promotes KC differentiation. Cell growth assays were performed on N-TERT-tetracycline repressor (TR)-shAREG or N-TERT-TR-shEGFP ±tetracycline (TET), with and without PD158780 (1μM), GM6001 (40μM), or neutralizing antibodies (Abs) against EGFR or its ligands (each at 5μgml−1). (a) Cells were stained with crystal violet and photographed. Each photograph represents duplicate wells. (b) KC growth was evaluated by hemocytometer counting. Data are expressed as cell number, % of untreated controls; mean±SEM, n=2–7, *P<0.05, **P<0.005 versus untreated controls. (c) Morphology of N-TERT-KCs at the end of growth assays. Bar=200μm. (d) Quantitative real-time PCR of various markers of KC differentiation. Data are expressed as % of 36B4 (mean±SEM, n=6, *P<0.05, **P<0.005, ***P<0.0001 versus (-) TET controls, NS=not significant). Journal of Investigative Dermatology 2010 130, 2031-2040DOI: (10.1038/jid.2010.98) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inhibition of autocrine keratinocyte (KC) growth by amphiregulin (AREG) small hairpin RNA (shRNA) cannot be reversed by addition of recombinant EGFR ligands. Cell growth assays were performed using N-TERT-tetracycline repressor (TR)-shAREG cells in the presence or absence of EGFR ligands ± tetracycline (TET) or ±AREG-blocking antibodies (Abs). Cell counts were determined in the presence or absence of TET (a) or AREG blocking Abs (b). Data are expressed as total cell number. Error bars indicate SEM, n=3–8 except epigen (EPGN), n=2. All TET-treated conditions in Figure 5a were significantly reduced (P<0.05) versus (-) TET controls. Asterisks in b indicate significant changes versus controls without anti-AREG or EGF (P<0.05). (c) KC cell morphology at the end of the growth assay. Arrows indicate groups of differentiated cells that were observed in cultures incubated with anti-AREG or TET. Bar=200μm. Journal of Investigative Dermatology 2010 130, 2031-2040DOI: (10.1038/jid.2010.98) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Amphiregulin (AREG) transmembrane (TM) precursor restores keratinocyte (KC) proliferation and prevents KC differentiation in AREG knockdowns. N-TERT-tetracycline repressor (TR)-shAREG were infected with lentivirus constructs encoding the AREG extracellular domain (ECD) or complete AREG (TM) precursor. After selection, cells were used in cell-growth assays. (a) Overview of the protein domain structure of the AREG TM precursor, soluble mature AREG, and AREG expression constructs. The approximate location of the TaqMan gene expression assays (Applied Biosystems) is indicated. The small hairpin RNA (shRNA) targets a sequence in the 3′ untranslated region of the AREG mRNA (nucleotide position 998–1,019 of Genbank # M30704). (b) AREG expression in N-TERT using expression assays specific either for the TM or for both constructs as shown in a (mean±SEM, n=4–7, #P<0.005 versus (-) tetracycline (TET) controls, *P<0.05 and **P<0.005 versus N-TERT-TR-shAREG with and without TET, respectively). (c) Crystal violet staining of growth assays with N-TERT-TR-shEGFP and N-TERT-TR-shAREG cell lines incubated in the presence or absence of TET±epidermal growth factor (EGF) or AREG. (d) KC morphology at the end of the growth assays. Bar=200μm. (e) quantitative real-time PCR analysis of markers of KC differentiation. The data for N-TERT-TR-shAREG are the same as in Figure 4d and are included here for comparison. Error bars indicate mean±SEM, n=4–7, *P<0.05, **P<0.005 versus TET-treated N-TERT-TR-shAREG. Journal of Investigative Dermatology 2010 130, 2031-2040DOI: (10.1038/jid.2010.98) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions