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All-trans Retinoic Acid-Induced Hyaluronan Production and Hyperplasia Are Partly Mediated by EGFR Signaling in Epidermal Keratinocytes  Sanna M. Pasonen-Seppänen,

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Presentation on theme: "All-trans Retinoic Acid-Induced Hyaluronan Production and Hyperplasia Are Partly Mediated by EGFR Signaling in Epidermal Keratinocytes  Sanna M. Pasonen-Seppänen,"— Presentation transcript:

1 All-trans Retinoic Acid-Induced Hyaluronan Production and Hyperplasia Are Partly Mediated by EGFR Signaling in Epidermal Keratinocytes  Sanna M. Pasonen-Seppänen, Edward V. Maytin, Kari J. Törrönen, Juha M.T. Hyttinen, Vincent C. Hascall, Donald K. MacCallum, Anne H. Kultti, Tiina A. Jokela, Markku I. Tammi, Raija H. Tammi  Journal of Investigative Dermatology  Volume 128, Issue 4, Pages (April 2008) DOI: /sj.jid Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Hyaluronan and CD44 distribution in organotypic REK cultures. Two-week-old organotypic cultures were fixed, processed for histology, and stained for hyaluronan and CD44 as described in Materials and Methods. (a, b) Controls were treated with the DMSO vehicle. (c, d) All-trans RA induced epidermal hyperplasia, and increased the expression of hyaluronan and CD44. In addition to the pericellular localization, hyaluronan was found intracellularly in all-trans RA-treated cultures (arrows in (c)). (d) Arrowheads indicate CD44 shedding to collagen matrix. Inhibitors reversed the RA-induced accumulation of hyaluronan and CD44 staining (e, f=RA+AG1478 [EGFR inhibitor]; g, h=RA+UO126 [MEK inhibitor]; i, j=RA+GM6001 MMP inhibitor]. (i, j) Epidermal hyperplasia is also reduced, particularly by GM6001. Bar=40μm. (k) The thickness of two-week-old cultures treated with all-trans RA with or without the inhibitors was analyzed as described in Materials and Methods. The data represent means±SD of six experiments. *P<0.05, **P<0.01, ***P<0.001, one-way analysis of variance. Tukey's post hoc test. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 The concentration and molecular mass distribution of hyaluronan. Total hyaluronan contents (epidermis, collagen substrata and media) were measured using the ELSA assay. (a) All-trans RA and the inhibitors were present during the entire culture period (10 days). The results are expressed as percentage of the untreated control cultures (means±SEM; *P<0.05, **P<0.01 one-way analysis of variance). (b) The size distribution of newly synthesized 3H-hyaluronan secreted into the medium during 24hours incubations with all-trans RA with or without the inhibitors was analyzed with gel filtration on a 1 × 30cm column of Sephacryl S The positions of the void volume, V0 (the first hyaluronan positive fraction), and the total volume, Vt, are indicated. (c–e) Organotypic REK cultures were metabolically labeled with [3H]glucosamine and [35S]sulfate for 24hours, and the newly synthesized (c) hyaluronan, (d) chondroitin sulfate, and (e) heparan sulfate were analyzed. The data represent the amounts synthesized during 24hours, expressed as means and ranges of two separate experiments. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 The expression of Has2, Has3, HB-EGF, and CD44. Organotypic REK cultures were treated with all-trans RA with or without the inhibitors (AG1478, UO126, and GM6001) for 1–8hours, total RNA was isolated and reverse transcribed, and analyzed by (a) semi-quantitative RT-PCR (1–8hours treatment) and (b) real-time PCR (4hours treatment). The results are shown as means±SEM of three separate experiments (a).*P<0.05, one-way analysis of variance, Tukey's post hoc test. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 The effects of neutralizing anti-HB-EGF on hyaluronan production and epidermal hyperplasia. (a, b) Organotypic cultures were treated with all-trans RA (0.1μM) with or without the anti-HB-EGF (4μgml−1) for 10 days. The total hyaluronan concentration was measured using the ELSA assay. (a) The results are expressed as percentage of the untreated control cultures (means±range of two cultures). (b) For histology, organotypic cultures were embedded in paraffin and stained with hematoxylin and eosin. Bar=40μm. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 The effect of all-trans RA and inhibitors on protein phosphorylation. Two-week-old organotypic cultures were treated with all-trans RA (0.1μM) with or without the inhibitors for (a) 30minutes, (b) 2hours (EGF 20ngml−1 served as positive control), and (c) 4hours. In (d), organotypic REK cultures were treated with cycloheximide (10μgml−1) and all-trans RA with or without AG1478 for 4hours. Cell lysates were analyzed by western blotting with antibodies for the signaling proteins as indicated in the panels. The molecular mass of each band (in kDa) is shown at the left-hand side. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 The expression of epidermal differentiation markers. (a, b) Two-week-old organotypic cultures were subjected to (b) all-trans RA for 10 days, and stained for keratin 10 as described in Materials and Methods. (a) Control cultures express keratin 10 in suprabasal cells, while its expression is strongly diminished by (b) all-trans RA. (c) Keratinocyte differentiation was estimated with filaggrin and keratin 10 antibodies in western blotting. The bands at 215 and 42kDa show profilaggrin and mature filaggrin, respectively. The figure represents one of two separate experiments with equivalent results. Bar=30μm. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Schematic presentation of the alternatives for all-trans RA-induced signaling leading to epidermal hyperplasia and enhanced hyaluronan production in organotypic REK cultures. All-trans RA can regulate the expression of genes either via nuclear receptors (RXR-RAR) or via a rapid nongenomic mechanism involving, for example, EGFR-ERK activation. All-trans RA possibly activates EGFR via HB-EGF and/or through other cytoplasmic or membrane-associated mechanisms. This causes ERK1/2 activation and downstream activation of transcription factors (e.g. STAT3), which regulate the expression of genes involved in epidermal hyperplasia and hyaluronan synthesis. Has2 is also regulated via nuclear receptors, since it has a functional RARE in its promoter. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

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