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First published online 26 October 2004
doi: 10.1242/jcs.01490

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FLICE/caspase-8 activation triggers anoikis induced by ß₁-integrin blockade in human keratinocytes

Alessandra Marconi^1,*, Paola Atzei^1,*, Cristina Panza¹, Chiara Fila¹, Rossana Tiberio², Francesca Truzzi¹, Tina Wachter³, Martin Leverkus³ and Carlo Pincelli^1,

¹ Institute of Dermatology, University of Modena and Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy
² Institute of Dermatology, University of Piemonte Orientale, Novara, Italy
³ Department of Dermatology, University of Würzburg Medical School, Josef-Schneider-Str. 2, 97070 Würzburg, Germany

View larger version (27K): [in a new window]   Fig. 1. PARP cleavage is detected in TA cells, but not in KSC. KSC, young TA and TA cells were separated on type IV collagen, as described in Materials and Methods, lysed at 24 hours and analyzed by western blot, using an anti-PARP antibody. 116 kDa represents the full length and 85 kDa the cleaved form of PARP protein.

View larger version (53K): [in a new window]   Fig. 2. Caspase-8 is activated in young TA and TA cells, but not in KSC. KSC, young TA and TA cells were separated on type IV collagen, as described in Materials and Methods, lysed at 24 hours and analyzed by western blotting, using anti-caspase-8, -9, -6 and -3, showing both the zymogen form and the active protein.

View larger version (31K): [in a new window]   Fig. 3. Anti-ß1-integrin induces anoikis, and not differentiation, in human keratinocytes. A pool of KSC and young TA was suspended in KGM and treated with anti-ß1-integrin antibody. The percentage of cells in SubG1 peak (M1) was analyzed by FACS (A). Cells were stained with anti-cytokeratin 10 (CK10) and anti-transglutaminase 1 (TG) antibodies (see Materials and Methods) (B).

View larger version (34K): [in a new window]   Fig. 4. Caspases are activated earlier in young TA (yTA) than in KSC. Keratinocytes were separated on type IV collagen as described in Materials and Methods and suspended in polypropylene. Cells were then lysed at different time points and analyzed by western blot. Membranes were blotted against anti-Bid, and anti-procaspase-3, -6, -8 and -9 antibodies. ß-actin was used to normalize protein content.

View larger version (52K): [in a new window]   Fig. 5. Caspase activation is accelerated by anti-ß1-integrin neutralizing antibody. Young TA keratinocytes were either suspended in polypropylene or treated with anti-ß1-integrin neutralizing antibody (4 µg/ml). Cells were lysed at different time points and membranes blotted against anti procaspases-3, -6, -8, -9, -10 and Bid antibodies. ß-actin was used to normalize protein content (A). (B) Keratinocytes were treated with anti-ß1-integrin neutralizing antibody, as in A. Cytosolic and mitochondrial fractions were extracted from keratinocytes, as described in Materials and Methods, and blotted against anti-cytochrome c antibody.

View larger version (34K): [in a new window]   Fig. 6. Caspase-8 inhibitor delays caspase activation and protects keratinocytes from anoikis. Keratinocytes were pretreated with caspase-9 inhibitor zLEDH-fmk and treated with anti-ß1-integrin antibody. Cells were lysed at different time points and membranes were blotted against anti-procaspase-9, -8, -10 and -3 and anti-Bid antibodies. ß-actin was used to normalize protein content (A). (B) Keratinocytes were pretreated with caspase-8 inhibitor zIETD-fmk and treated with anti-ß1-integrin antibody. Cells were lysed at different time points and membranes were blotted against anti-procaspase-8, -9 and -3 and anti-Bid antibodies. ß-actin was used to normalize protein content. (C) Keratinocytes were pretreated with caspase-8 inhibitor zIETD-fmk or with caspase-9 inhibitor zLEDH-fmk and either suspended in polypropylene or treated with anti-ß1-integrin antibody. Apoptosis was evaluated by TUNEL staining, as described in Materials and Methods. Approximately 100 cells were evaluated, in randomly selected high-power fields, and the percentage of TUNEL-positive cells was counted. Each experiment was repeated three times. Results are expressed as the mean percentage ± s.d. from three experiments. Student's t test was used for comparison of the means.

View larger version (24K): [in a new window]   Fig. 7. cFLIPL overexpression in keratinocytes potently inhibits TRAIL-mediated apoptosis. Stably transfected keratinocytes were generated by retroviral infection of either PINCO-control vector or PINCO-cFLIPL in HaCaT keratinocytes as described in Materials and Methods. Overexpression of cFLIPL was confirmed using 50 µg protein of total cellular lysates by western blotting and subsequent analysis for cFLIPL. As control, cell lysates were blotted against anti-procaspase-8 antibody (C15) (A). (B) For functional analysis, cells were seeded in 96-well-plates in duplicates and were stimulated with the indicated concentrations of recombinant TRAIL for 16 hours. The percentage of living cells was normalized to mock-stimulated cells as marked on the y-axis. One of three representative experiments is shown.

View larger version (59K): [in a new window]   Fig. 8. Anti-ß1-integrin does not activate Bid, the release of cytochrome c and caspase-9 in c-FLIPL overexpressing keratinocytes. c-FLIPL HaCaT cells and mock infected cells were cultivated in the presence of anti-ß1-integrin or irrelevant IgG antibody. Proteins were extracted from the full cell lysate for caspase-8, caspase-9 and Bid (A), whereas they were extracted from mitochondria and cytosol for cytochrome c (B). Western blot was performed with anti-procaspase-9, anti-procaspase-8, anti-Bid and anti-cytochrome c antibodies. ß-actin was used to normalize protein content.

View larger version (35K): [in a new window]   Fig. 9. Anti-ß1-integrin fails to induce anoikis in c-FLIPL overexpressing keratinocytes. After 16 hours of treatment with anti-ß1 integrin, anoikis was measured by TUNEL. Approximately 100 cells were evaluated, in randomly selected high-power fields, and the percentage of TUNEL positive cells was counted. Each experiment was repeated three times. Results are expressed as the mean percentage ± s.d. from three experiments. Student's t test was used for comparison of the means (A). (B) Keratinocytes were fixed in situ with paraformaldehyde and stained by the TUNEL technique and revealed by converter alkaline phosphatase, according to the manufacturer's instruction.