1. EGFR and IL-1 Signaling Synergistically Promote Keratinocyte Antimicrobial Defenses in a Differentiation-Dependent Manner 
Andrew Johnston, Johann E. Gudjonsson, Abhishek Aphale, Andrew M. Guzman, Stefan W. Stoll, James T. Elder 
Journal of Investigative Dermatology 
Volume 131, Issue 2, Pages (January 2011)
DOI: /jid
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2. Figure 1
EGFR ligand expression in skin. Epiregulin (EREG), HB-EGF, TGF-α, amphiregulin (AREG), and epigen (EPGN) RNA transcripts were all more abundant in plaque psoriasis (PP, black) than symptomless psoriasis (PN, gray) or healthy control (NN, open bars) skin, whereas betacellulin (BTC) expression was decreased in PP skin (a). In support of this we found that HB-EGF, TGF-α, and AREG proteins were all substantially elevated in PP versus PN or NN skin (b). Bars represent mean ± SD, n=10 donors for mRNA, n=4 for protein measurements. Statistical significance determined by two-tailed t-test and indicated. *P<0.05, **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
IL-1α and EGFR ligands synergize for the induction of CCL20 and S100A7 but not S100A8 or S100A9 expression by KCs. Postconfluent NHKs were treated with 10ngml−1 IL-1α in combination with 2nM EGF, 2nM HB-EGF, 4nM TGF-α, 2 or 20nM AREG, 2nM BTC, or 2nM EREG. RNA was quantified by quantitative real-time reverse transcriptase PCR normalized to the housekeeping gene RPLP0. (a) DEFB4, CCL20, and (b) S100 family mRNA expression at 4hours of stimulation shows synergism between IL-1α and EGFR ligands for DEFB4, CCL20, and S100A7 but not S100A8 or S100A9 induction. (c) CCL20 (circles) and DEFB4 (squares) have markedly different kinetics with CCL20 mRNA occurring as an early response to IL-1α (closed symbols) or IL-1α +TGF-α (open symbols). (d) The synergism is evident at the protein level as shown by secreted hBD-2 at 24hours of culture measured by ELISA. Open bars, EGFR ligand alone; filled bars, IL-1α+EGFR ligand. Mean±SD, n=3. Statistical significance determined by two-tailed t-test versus control or IL-1α-treated cultures, indicated *P<0.05, **P<0.01, ***P< BTC, betacellulin; HB-EGF, heparin-binding EGF-like growth factor; KC, keratinocyte; NHK, normal human keratinocyte.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Cell density rather than extracellular calcium concentration is crucial to the responses of KCs in vitro. The effect of increased extracellular Ca2+ is only obvious under proliferative conditions (a-d). Subconfluent, proliferating NHKs (a-d), or 4-day postconfluent NHKs (e-h) were starved of growth factors and then treated with 10ngml−1 IL-1α and/or 4nM TGF-α for 24hours. Secreted hBD-2 and CCL20 were assayed by ELISA, S100A7, DEFB4, and CCL20 mRNA quantified by quantitative real-time reverse transcriptase PCR relative to the housekeeping gene RPLP0. Filled bars=0.1mM, open bars=1.4mM Ca2+ indicate mean ± SD, n=3. Statistical significance (two-tailed t-test) indicated ♦P<0.05, ♦♦P<0.01 for low versus high calcium and *P<0.05, **P<0.01, ***P<0.001 for untreated versus IL-1α and IL-1α+TGF-α. Note differences in y axis scaling. KC, keratinocyte; NHK, normal human keratinocyte.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Subconfluent and 4-day postconfluent KCs show differential keratin, IL-1R, and ErbB receptor expression. Postconfluent NHKs express less K5 and K14 mRNA and more K1 and K10 mRNA, characteristic of more differentiated KCs of the skin (a). Subconfluent NHK express higher levels of mRNA for the IL-1 decoy receptor IL-1RII (b). Sub- and postconfluent cells exhibited similar levels of ErbB1 transcript expression, and no detectable ErbB4 expression, but significantly different levels of ErbB2 and ErbB3 (c). Open bars, subconfluent NHKs; filled bars, postconfluent NHKs. Bars indicate mean±SD, n=8. Statistical significance denoted: *P<0.05, **P<0.01, ***P<0.001, two-tailed t-test. KC, keratinocyte; NHK, normal human keratinocyte.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Differential use of signal transduction pathways by (A) sub- and (B) postconfluent KCs in culture. NHKs were grown to the specified confluency, starved of growth factors overnight, and then treated with one of each of the following inhibitors for 1hour before stimulation with 10ngml−1 IL-1α (blue bars) or 10ngml−1 IL-1α+ 4nM TGF-α (red bars) for 24hours. Green bars, where present, represent unstimulated, inhibitor-treated NHKs. Subconfluent NHKs were particularly sensitive to inhibition of NF-κB (parthenolide) and Src family kinases (PD173952) but were largely unaffected by inhibition of EGFR (PD158780) or p38 (SB202190). Bars indicate mean ± SD, n=3 representative of three separate experiments. Statistical significance indicated, *P<0.05, **P<0.01, ***P<0.001, two-tailed t-test versus vehicle control. (c) Sub- and postconfluent NHKs were stimulated with 10ngml−1 IL-1α and/or 4nM TGF-α for 30minutes and assayed for 10 phosphoproteins. Values indicate the concentration of each phosphoprotein in subconfluent (upper value) and postconfluent cells (lower value) and the fold increase (red) or fold decrease (green). Note differences in y axis scaling. Values are representative of two independent experiments. KC, keratinocyte; NHK, normal human keratinocyte.
Journal of Investigative Dermatology  , DOI: ( /jid )
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7. Figure 6
The synergistic effects of IL-1α and TGF-α are evident on reconstituted human epidermal cultures. Cultures were treated for 48hours with 10ngml−1 IL-1a, 4nM TGF-α, or both, and then processed and immunohistochemically stained for hBD-2 (a-d), or loricrin and involucrin (f). Untreated cultures show no hBD-2 expression (a); this is true also of TGF-α treatment that resulted in a thickened epidermal layer (b). IL-1α treatment induced a strong vectorial hBD-2 expression (c), whereas treatment with both IL-1α+TGF-α resulted in altered tissue morphology with enhanced hBD-2 expression (d). The altered epidermal structure was accompanied by decreased loricrin and more extensive involucrin expression in IL-1α+TGF-α-treated cultures (f). To confirm the secretion of hBD-2, culture medium was sampled at 24hours (open bars) and 48hours (filled bars) and hBD-2 was assayed by ELISA (e). Bars indicate mean±SD, n=5. Statistical significance determined by two-tailed t-test IL-1α versus IL-1α+TGF-α, **P<0.01. Scale bar=100μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions