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First published online August 17, 2004
doi: 10.1242/10.1242/jcs.01302

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Transient activation of FOXN1 in keratinocytes induces a transcriptional programme that promotes terminal differentiation: contrasting roles of FOXN1 and Akt

Sam M. Janes^*, Tyler A. Ofstad^*, Douglas H. Campbell, Fiona M. Watt and David M. Prowse^,¶

Keratinocyte Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

View larger version (43K): [in a new window]   Fig. 1. Generation of an inducible form of FOXN1. (A) Schematic diagram showing the fusion protein. (B,C) Immunofluorescence of 3T3 cells expressing FOXN1ER treated with (B) ethanol or (C) 4OHT for 24 hours and stained with an anti-oestrogen receptor antibody (green) and anti ß-tubulin (red). Scale bar: 30 µm. (D) 3T3 cells transduced with pBabepuro (empty vector), pBabepuroFOXN1 or pBabepuroFOXN1ER were transiently transfected with luciferase reporters WT-FP-luc (black bars) or Mut-FP-luc (yellow bars) and treated with 4OHT as shown. The luciferase activity of each reporter was measured in triplicate and the fold induction calculated. Data shown are from a typical experiment.

View larger version (87K): [in a new window]   Fig. 2. The role of FOXN1 in initiation of terminal differentiation. (A-H) Clonogenicity assays. (A, B) Keratinocytes transduced with pBabepuro (empty vector). (C-H) Keratinocytes transduced with pBabepuroFOXN1ER. The plated cells were treated with ethanol (A,C,E) or 4OHT (B,D,H) for 14 days or with 4OHT for 4 hours (F) or 24 hours (G). (I) Differentiation assays were performed by treating FOXN1ER-transduced keratinocytes as shown or suspending uninfected keratinocytes for 24 hours and measuring the percentage of involucrin-positive cells. The means and standard errors are shown from three experiments. (J) Immunoblot of endogenous FOXN1 in keratinocytes held in suspension for the number of hours shown. The blots were probed with antibodies to FOXN1 or, as a loading control, Erk2.

View larger version (40K): [in a new window]   Fig. 3. Validation of the microarray results. (A) Real time PCR analysis of target gene expression. Keratinocytes transduced with empty vector (white bars) or FOXN1ER (grey bars) were treated with ethanol or 4OHT for 24 hours and the relative induction by 4OHT of galectin-7, Gas6, PLCD-1, CRABPI and II, P-cadherin and Akt determined. Results are the mean and standard deviations of three experiments. (B) Immunoblot analysis of target gene expression. Keratinocytes transduced with FOXN1ER were treated with ethanol or 4OHT for 0-48 hours and the induction of galectin-7, CRABPI, CRABPII, Akt, P-cadherin and 14-3-3 determined. The blots were probed with Erk2 antibodies as a loading control.

View larger version (31K): [in a new window]   Fig. 4. The dynamics of Akt activation in cultured human keratinocytes. (A) Adherent keratinocytes that had been starved for 24 hours were treated with 400 ng/ml recombinant Gas6 for 0-40 minutes. Western blots were probed with antibodies to phospho-Akt473 or total Akt. Erk2 was used as a loading control. (B) FOXN1ER transduced keratinocytes were stimulated with 4OHT (100 nM) for 0, 6, 12 or 24 hours and lysates were probed for phospho-Akt473, total Akt or Erk2. (C) Keratinocytes were held in suspension for 0 to 28 hours and total Akt protein was immunoprecipitated. The level of phosphorylated GSK-3ß substrate was analysed by western blotting as a measure of Akt kinase activity. Actin was used as a loading control. (D) Percentage of involucrin-positive cells induced by FOXN1 or suspension culture in the presence or absence of PI 3-kinase inhibitor LY294002 (LY) or Akt inhibitor. The means and standard errors are shown from three experiments.

View larger version (54K): [in a new window]   Fig. 5. Akt activity in reconstituted human epidermis. (A) Western blots probed with anti-ER or anti-phospho-Akt473 antibodies. Cells were transduced with empty retroviral vector (pBabe), myrAktER or A2myrAktER and treated with 4OHT for 24 hours. (B-I) Epidermis reconstituted by keratinocytes transduced with GFP (B,C), FOXN1ER (D,E), myrAktER (F,G) or A2myrAktER (H,I). Sections were subject to immunofluorescent staining using antibodies to the oestrogen receptor (B,D,F,H) or phospho-Akt473 (C,E,G,I) (green). Propidium iodide was used as a nuclear counterstain (red). Scale bar: 50 µm.

View larger version (89K): [in a new window]   Fig. 6. Contrasting effects of FOXN1 and Akt on terminal differentiation. Epidermis was reconstituted by keratinocytes transduced with GFP (A-C), FOXN1ER (D-F), myrAktER (G-I) or A2myrAktER (J-L). (A,D,G,J) Haematoxylin and Eosin staining of paraffin sections. Basal (b), spinous (s), granular (g) and cornified (c) layers are indicated. Sections were stained by immunofluorescence with antibodies to involucrin (B,E,H,K) or loricrin (C,F,I,L) (green). Propidium iodide was used as a nuclear counterstain (red). Scale bar: 50 µm.

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