QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 12 September 2006
doi: 10.1242/jcs.03175

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pankow, S. Articles by Werner, S. Search for Related Content PubMed PubMed Citation Articles by Pankow, S. Articles by Werner, S.

Regulation of epidermal homeostasis and repair by phosphoinositide 3-kinase

¹ Institute of Cell Biology, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland
² Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
³ Molecular Oncology, Merck Research Laboratories, Boston, MA 02115, USA

View larger version (85K): [in a new window]   Fig. 1. Expression of p110, p110ß, and p110 in normal and wounded mouse skin. (A) mRNA levels of the PI3K catalytic subunits p110, p110ß, and p110 were assessed by RPA using 10 µg total RNA from murine back skin, tail epidermis, tail dermis and skin-derived primary keratinocytes and fibroblasts or from full-thickness excisional mouse wounds at the indicated time points after injury. Hybridization with a GAPDH-specific riboprobe was performed as a loading control. -p, undigested riboprobes. (B) 30 µg total cellular protein from non-wounded and wounded skin at different time points after injury were analyzed by western blotting for the presence of p110, Ser473-phosphorylated Akt or p85/p55. Coomassie staining of the gel was performed to verify equal loading. High levels of immunoglobulins in early wounds result from invading immune cells. (C) Sections from tail skin and 1- to 14-day full-thickness excisional wounds were stained with an antibody detecting Ser473-phosphorylated Akt. (a) Tail skin showing phosphorylated Akt (red) in the outer root sheath keratinocytes of hair follicles (arrowhead). 1-day wounds harbor high levels of phosphorylated Akt in keratinocytes at the wound rim (arrow, b and higher magnification in c), and in the panniculus carnosus (arrow, b). The eschar (Es) stains non-specifically. (d) Phosphorylated Akt is detected in suprabasal cells of the hyperproliferative epithelium of 5-day wounds (higher magnification in e, arrowheads) but not of 14-day wounds (f). No phosphorylated Akt was detected in normal human epidermis (g), but suprabasal cells were phospho-Akt positive in the hyperproliferative wound epidermis of a 4-day human incisional wound (h, arrow). The basal lamina is indicated by the white dotted line. Nuclei were counterstained with Hoechst 33342 (blue). Bars, 50 m. (D) In situ hybridizations with a digoxygenin-labeled antisense riboprobe (a,b) show p110 mRNA in basal and suprabasal keratinocytes (arrowheads in b) of the normal epidermis and of the hyperproliferative epithelium of 5-day wounds and in dermal fibroblasts (arrows in b). (c) Serial sections incubated with the sense riboprobe revealed only nonspecific background (c). Bars, 100 µm (a); 50 µm (b,c). D, dermis; E, epidermis; Es, eschar; G, granulation tissue; HF, hair follicle, HPE, hyperproliferative epithelium, M, muscle panniculus carnosus.

View larger version (35K): [in a new window]   Fig. 2. Transient upregulation of p110 and p110ß mRNAs during in vitro differentiation of human keratinocytes. (A) Samples of 20 µg total RNA from adult human keratinocytes grown in primary culture under differentiation-promoting conditions were analyzed by northern blotting using cDNA probes specific for p110 and p110ß. ß-actin or involucrin probes were hybridized as a loading control or to monitor differentiation, respectively. (B) Western blot analysis verifies transient upregulation of p110 protein upon differentiation. The blot was re-incubated with antibodies against phosphorylated Akt (Ser473), PTEN, involucrin, keratin 10 (K10), and ß-actin to verify upregulation of differentiation markers (involucrin, keratin 10) or as a loading control (ß-actin).

View larger version (28K): [in a new window]   Fig. 3. Expression of an inducible active form of PI3K in HaCaT keratinocytes. (A) The retroviral vector used for expression of the Myr-p110*-mER fusion protein is shown. IRES, internal ribosomal entry site; iSH2 (p85), inter SH2 domain of the murine p85 protein; L, linker sequence; LTR, long terminal repeat; mER, cDNA encoding the mutated murine estrogen receptor ligand binding domain; mp110*, murine p110 cDNA; Myr, myristoylation signal sequence; neo, neomycin-resistance gene. The position of the riboprobe p110*-p used for RPA is indicated as a black line. (B) Western blotting with 30 µg total cellular protein for detection of endogenous p110 and overexpressed Myr-p110*-mER protein in HaCaT cell clones stably transfected with a retroviral Myr-p110*-mER construct. (C) Western blotting using 30 µg total cellular protein revealed increased amounts of phosphorylated Akt after 4-OHT-treatment of the stable cell clones 1 and 17. The amount of ß-actin in each lane served as a loading control. (D) As shown by western blot analysis of phosphorylated Akt, induction of PI3K signaling by 4-OHT was blocked by simultaneous addition of LY294002. Simultaneous detection of PCNA indicates that cell proliferation is not negatively influenced by LY294002 treatment during this time period. (E) Vector-transfected and Myr-p110*-mER expressing keratinocytes were treated with 4-OHT (+) or solvent (-) as indicated. DNA replication was measured by [3H]thymidine incorporation. Untreated control was arbitrarily set as 1.0. Western blot analysis, which was carried out in parallel, verifies phosphorylation of Akt after 4-OHT-treatment in Myr-p110*-mER cell clones. Error bars indicate s.e.m. Significance was determined with the Student's t-test (two-tailed). **P<0.01; ***P<0.001.

View larger version (33K): [in a new window]   Fig. 4. Validation of PI3K target genes. (A) Western blotting reveals upregulation of RTP801 following PI3K-activation. Unaltered RTP801 levels were seen in vector-transfected control clones. Equal loading was verified by treatment of the membranes with a ß-actin antibody. (B) RNAs isolated from independent Myr-p110*-mER expressing or vector-transfected cell clones were subjected to real-time RT-PCR. Genes encoding -thalassemia/mental retardation syndrome X-linked protein (ATRX), periphilin1 protein (PPHL1), ets-homologous factor 3 protein (EHF) and Cockayne syndrome 1 protein (CKN1) were tested for differential expression in 4-OHT-treated Myr-p110*-mER expressing cell clones. Values are mean ± s.d. The table summarizes the average fold regulation observed by real-time RT-PCR and microarray analysis normalized to endogenous controls and relative to 5 hour solvent controls. Solvent treatment is indicated by -, 4-OHT treatment by +.

View larger version (53K): [in a new window]   Fig. 5. PI3K affects actin reorganization. FITC-coupled phalloidin detected polymerized F-actin filaments (green) in 4-OHT- or solvent-treated Myr-p110*-mER cell clones (clone #17, A,C,E,F) or vector-transfected control clones (B,D). Arrowheads indicate cells with accumulated actin filaments at the cell periphery (C,F). Distribution of paxillin is depicted in red (E,F). Nuclei were counterstained with Hoechst 33342 (blue). Bars, 50 µm.

View larger version (49K): [in a new window]   Fig. 6. PI3K enhances keratinocyte motility. (A) Motility of mitomycin C-treated primary human keratinocytes after 12 hours or 60 hours of treatment with LY294002 in growth medium was monitored by in vitro scratch assay. (B) Immunofluorescence staining of DMSO- and LY294002-treated primary human keratinocytes after scratch wounding using an antibody against phosphorylated Akt. (C) Quantitative analysis of Transwell migration assays using 4-OHT- or solvent-treated Myr-p110*-mER cell clones (left panel) or vector-transfected cells (right panel). Error bars in B and D indicate s.d. Significance was determined by one-way ANOVA with Bonferroni post-correction. *P<0.05; **P<0.01; *** P<0.001. (D) Explant cultures from back skin biopsies of 1-day-old mice were established (a) and treated with DMSO (b), rapamycin (c) or LY294002 (d) in combination with mitomycin C. 10 days after establishment of the culture, the biopsy was removed and the cells, which had grown out from the culture, were stained with hematoxylin and eosin and photographed.

View larger version (45K): [in a new window]   Fig. 7. Activation of the PI3K pathway in HaCaT keratinocytes delays differentiation. (A) Western blotting for detection of involucrin during suspension-culture-induced differentiation of Myr-p110*-mER HaCaT cell clones treated with 4-OHT or solvent, respectively. Western blot detecting phosphorylated Akt (Ser473) indicates successful stimulation of PI3K-signaling by 4-OHT-treatment. Incubation of the membrane with an antibody to ß-actin was used as a loading control. The time of cultivation in suspension is indicated on top of the lanes. (B) Cells grown in serum-free medium over 11 days were harvested after the time points indicated, and the corresponding protein lysates were subjected to western blot analysis for involucrin and phosphorylated Akt (Ser473). Staining of the membrane with an antibody against ß-actin served as a loading control.

View larger version (59K): [in a new window]   Fig. 8. Myr-p110*-mER cell clones form a hyperthickened epithelium in organotypic cultures. (A) hematoxylin and eosin and BrdU stainings of solvent-treated, 4-OHT-treated or untreated organotypic cultures of the two independent Myr-p110*-mER expressing cell clones 1 (a-f) and 17 (g-m) and a vector-transfected cell clone (n-s) are shown. Arrowheads in b indicate disorganized segments of the basal layer, where keratinocytes protrude into the collagen matrix. Arrowheads in e and 1 indicate suprabasal BrdU-positive cells. Bars, 50 µm. (B) Epithelial thickness of the cultures. (C) Bar graph representing the number of BrdU-positive cells per mm basement membrane. Error bars indicate s.e.m. ***P<0.001; ns, not significant.

View larger version (97K): [in a new window]   Fig. 9. Delayed differentiation of keratinocytes expressing Myr-p110*-mER in organotypic cultures. (A) 4-OHT-treated and solvent-treated (control) organotypic cultures of Myr-p110*-mER expressing HaCaT cells were analyzed for phosphorylated Akt (a,b, green), K14 (c,d, green), K10 (e,f, green), involucrin (g,h, blue), loricrin (l,m, green) and 6 integrin (c,d,i,k, red) by immunofluorescence. Arrowheads in d indicate strong K14 expression in the upper suprabasal layers. Nuclei were stained with Hoechst 33342 and are shown in blue in e and f. Note the apical 6 integrin expression in suprabasal cells (k, arrowheads), which is not observed in the controls (i). Counterstaining in l and m was performed with propidium iodide (red). (B) Paraffin sections of 4-OHT-(c,d) or solvent-treated (a,b) Myr-p110*-mER expressing organotypic cultures were subjected to immunofluorescence staining using an antibody recognizing p63. Nuclei are counterstained in a and c with Hoechst 33342. The basement membrane is indicated with a dotted line. Bars, 50 µm.

View larger version (39K): [in a new window]   Fig. 10. PI3K activation induces a hyperthickened epithelium in 3D organotypic cultures. (A) hematoxylin and eosin and BrdU stainings of untreated (nt), solvent-treated or 4-OHT-treated organotypic cultures of the Myr-p110*-mER expressing cell clone 1 are shown. Treatment was initiated in 10-day-old cultures and continued for 15 days. (B) The bar graph represents the number of BrdU-positive cells per mm basement membrane. Error bars indicate s.e.m. *P<0.05; ***P<0.001; ns, not significant. (C) Solvent-treated or 4-OHT-treated organotypic cultures were analyzed by immunofluorescence for the presence of involucrin (a,b, green) and K10 (c,d, blue). Nuclei were counterstained with propidium iodide (red). The basement membrane is indicated as a dotted line. Bars, 50 µm.