PI3-kinase-dependent activation of apoptotic machinery occurs on commitment of epidermal keratinocytes to terminal differentiation

Abstract

We have investigated the earliest events in commitment of human epidermal keratinocytes to terminal differentiation. Phosphorylated Akt and caspase activation were detected in cells exiting the basal layer of the epidermis. Activation of Akt by retroviral transduction of primary cultures of human keratinocytes resulted in an increase in abortive clones founded by transit amplifying cells, while inhibition of the upstream kinase, PI3-kinase, inhibited suspension-induced terminal differentiation. Caspase inhibition also blocked differentiation, the primary mediator being caspase 8. Caspase activation was initiated by 2 h in suspension, preceding the onset of expression of the terminal differentiation marker involucrin by several hours. Incubation of suspended cells with fibronectin or inhibition of PI3-kinase prevented caspase induction. At 2 h in suspension, keratinocytes that had become committed to terminal differentiation had increased side scatter, were 7-aminoactinomycin D (7-AAD) positive and annexin V negative; they exhibited loss of mitochondrial membrane potential and increased cardiolipin oxidation, but with no increase in reactive oxygen species. These properties indicate that the onset of terminal differentiation, while regulated by PI3-kinase and caspases, is not a classical apoptotic process.

Figure 1

Phospho-Akt⁴⁷³ labelling of human epidermis. (a-c) Double immunofluorescence labelling with anti-phosphoserine Akt⁴⁷³ (green) and anti-E-cadherin (red), with Hoechst nuclear counterstain (blue). (a-c) show the same section. Arrowheads in (a) mark epidermal-dermal junction. (c) B: basal; S: spinous; G: granular; C: cornified layers. White vertical line indicates viable suprabasal layers that express E-cadherin. (d) Basal layer of human epidermal whole mount labeled with anti- phospho Akt⁴⁷³ (green) and anti-β1 integrin (red). The putative stem cells lie in the integrin-bright clusters; phospho Akt labeled cells are absent from those clusters. (e) To determine the relative labelling intensity of β1 integrins and phospho Akt in an epidermal wholemount a line was drawn across a microscopical field and the red and green fluorescence intensities (y axis, arbitrary units) per unit length (x axis; each division is 200μm) were determined (21, 49). Scale bars: 10μm (a-c), 50μm (d).

Figure 2

Effect of Akt activation on clonal growth of keratinocytes. Cells were transduced with A2AktER (a, b) or myrAktER (c, d) and treated with 100 nM 4-OHT (b, d) or ethanol (vehicle control; a, c). (e) Quantitation of % colony forming efficiency and % abortive colonies.

Figure 3

Inhibition of suspension-induced terminal differentiation with inhibitors of PI3-kinase or caspases. Cells that had been in suspension for 24h (T24) were compared with adherent (ADH) cells (T0; ADH). % involucrin (a, f, k, l) or cornifin (b) positive cells were determined. Cell surface β1 integrin levels were examined (c-e, h-j) and the levels of total and active ERK MAPK (g). Left hand peaks in (c, h) show fluorescence of cells labeled with secondary antibody alone, as a negative control. The effects of the following treatments were examined: 50μM LY294002 (LY), DMSO (solvent control), 100 μg/ml fibronectin (FN), 50 (j) or 100 (f, k, l) μM zVAD-fmk (zVAD), 10μM SB 203580 (SB), 10μM UO126 (UO), 100μM z-VAD-IETD (zIETD) or 100μM z-VAD-LEHD (zLEHD). Error bars show SEM.

Figure 4

Kinetics of caspase activation during terminal differentiation. (a) Caspatag™ labelling of human epidermis. Position of basement membrane is indicated. Scale bar: 10μm. (b-l) Flow cytometry of Caspatag™ labelled keratinocytes that had been freshly harvested (T0) or held in suspension for the number of hours shown. Dead cells were gated out with propidium iodide staining. (m-u) Fluorescence microscopy of cells labelled with Caspatag™ (green), involucrin (red) and Hoechst (blue). Cells were freshly harvested (m-o) or held in suspension for 2h (p-r) or 24h (s-u). Arrows show cells that are strongly labeled with Caspatag™, are involucrin negative and have large, pale nuclei. Note that cells that are less intensely labeled with Caspatag™ are involucrin positive cells with smaller, more intensely stained nuclei. (v) shows total numbers of Caspatag™ positive (strong and intermediate labelling) and involucrin positive cells with time in suspension. The data represent quantitation of the type of flow cytometry data in (b-l) and are combined from three independent experiments ± standard error of the mean.

Figure 5

Effects of PI3-kinase inhibition and fibronectin on caspase activation and involucrin expression. Adherent (T0) keratinocytes were compared with cells that had been incubated in suspension for 4 or 24h in the presence or absence of 100 μg/ml fibronectin (FN), 100μM LY294002 (LY) or DMSO (diluent control). % involucrin positive and Caspatag™ labeled cells are shown ± SEM.

Figure 6

Keratinocytes undergoing suspension induced terminal differentiation do not undergo DNA fragmentation or expose phosphatidylserine. (a, c, e) Flow cytometry of cells labelled with 7AAD and Annexin V. (b, d) Flow cytometry profiles of DNA content. Gates show (left to right) sub-G1, G1, S, G2+M DNA content. (a) MDCK cells induced to undergo apoptosis in suspension; (b, c) adherent keratinocytes induced to undergo apoptosis by treatment with 1 μM staurosporine; (d, e) keratinocytes undergoing terminal differentiation in suspension. In (e) box i denotes cells that are 7AAD and Annexin V negative; box ii denotes Annexin V positive 7AAD negative cells; box iii denotes 7AAD positive, Annexin V negative cells; and box iv denotes double positive cells.

Figure 7

Cytochrome c release and loss of mitochondrial membrane potential during suspension-induced terminal differentiation. (a) Keratinocytes were freshly harvested (0 hr) or held in suspension for 2hr. The total population (All cells) is compared with cells corresponding to areas i (7AAD, Annexin V double negative) and iii (7AAD positive, Annexin V negative) of Figure 6e. (b) Increase in side scatter (SSC) of keratinocytes held in suspension for up to 120 min. (c) The cells with higher SSC at 120 min are 7AAD positive, exhibit loss of cytochrome c and mitochondrial membrane potential (top row), while the cells with lower SCC (bottom row) tend to have lower 7AAD staining with no loss of cytochrome c or mitochondrial membrane potential.