American Society of Hematology

Figure 3

$Figure 3. Extrinsic induction of apoptosis induces ADAM17-mediated IL6R shedding. (A) HepG2 cells were transfected with expression plasmids encoding the human IL6R or a control marker protein (EGFP). Cells were treated (6 hours) with cycloheximide (CHX, 100 μM), agonistic anti-Fas antibody CH-11 (αFas, 500 ng/mL), or a combination of cycloheximide and αFas. Cell lysates were prepared and Western blot analysis for PKCδ and IL6R was performed. (B) HepG2 cells were transfected as described in panel A and treated with CHX (100 μM) or CHX (100 μM, 6 hours) in combination with PMA (100 nM, 2 hours) or αFas (500 ng/mL, 6 hours). After stimulation, the sIL6R was immunoprecipitated from supernatants and lysates were prepared from the cell pellets. IL6R and sIL6R were subsequently monitored by Western blot analysis. (C) Apoptosis was induced in the transfected HepG2 cells with CHX alone or in combination with CH-11. The metalloprotease inhibitors TAPI (100 μM), GI254023X (3 μM), or GW280264X (3 μM) were added as indicated 30 minutes prior to stimulation. Western blot analysis of immunprecipitated sIL6R and cleaved PARP (c-PARP) was performed. A vertical line has been inserted to indicate where a gel lane was cut. This gel came from one experiment but irrelevant lanes have been cut out. (D) HepG2 cells were cotreated for 0 hour, 2 hours, 4 hours, and 6 hours with CHX and αFas. ADAM17 was immunoprecipitated from the cell lysates by addition of an anti-ADAM17 polyclonal antibody (3 μg) before detection by Western blot. Release of sIL6R was monitored as before. A17-pro indicates proform of ADAM17; A17-mature, mature form of ADAM17. (E) Western blot of membrane fractions of HepG2 cells expressing p409 empty vector (lane 1) or a dominant-negative ADAM17 mutant that lacks the pro- and catalytic domain (DN-ADAM17, lane 2). (F) HepG2 cells were transiently transfected with IL6R cDNA together with empty p409 vector or p409-DN-ADAM17 plasmid. After 48 hours, cells were treated with CHX (100 μM, 6 hours) in presence of DMSO (vehicle control, 6 hours) to monitor baseline shedding, PMA (100 nM, 2 hours) or αFas for 6 hours. The sIL6R protein in the culture media was quantified using the sIL6R-specific ELISA. The fold increase of the sIL6R is expressed as a percentage of baseline shedding (set to 100%). Baseline shedding of IL6R/mock-transfected cells was 5549.07 (± 1626.3) pg/mL and of IL6R/DN-TACE 3068.84 (± 744) pg/mL. Values represent the mean (± SD) from 3 independent experiments (*P ≤ .001 and **P ≤ .01).$

Extrinsic induction of apoptosis induces ADAM17-mediated IL6R shedding. (A) HepG2 cells were transfected with expression plasmids encoding the human IL6R or a control marker protein (EGFP). Cells were treated (6 hours) with cycloheximide (CHX, 100 μM), agonistic anti-Fas antibody CH-11 (αFas, 500 ng/mL), or a combination of cycloheximide and αFas. Cell lysates were prepared and Western blot analysis for PKCδ and IL6R was performed. (B) HepG2 cells were transfected as described in panel A and treated with CHX (100 μM) or CHX (100 μM, 6 hours) in combination with PMA (100 nM, 2 hours) or αFas (500 ng/mL, 6 hours). After stimulation, the sIL6R was immunoprecipitated from supernatants and lysates were prepared from the cell pellets. IL6R and sIL6R were subsequently monitored by Western blot analysis. (C) Apoptosis was induced in the transfected HepG2 cells with CHX alone or in combination with CH-11. The metalloprotease inhibitors TAPI (100 μM), GI254023X (3 μM), or GW280264X (3 μM) were added as indicated 30 minutes prior to stimulation. Western blot analysis of immunprecipitated sIL6R and cleaved PARP (c-PARP) was performed. A vertical line has been inserted to indicate where a gel lane was cut. This gel came from one experiment but irrelevant lanes have been cut out. (D) HepG2 cells were cotreated for 0 hour, 2 hours, 4 hours, and 6 hours with CHX and αFas. ADAM17 was immunoprecipitated from the cell lysates by addition of an anti-ADAM17 polyclonal antibody (3 μg) before detection by Western blot. Release of sIL6R was monitored as before. A17-pro indicates proform of ADAM17; A17-mature, mature form of ADAM17. (E) Western blot of membrane fractions of HepG2 cells expressing p409 empty vector (lane 1) or a dominant-negative ADAM17 mutant that lacks the pro- and catalytic domain (DN-ADAM17, lane 2). (F) HepG2 cells were transiently transfected with IL6R cDNA together with empty p409 vector or p409-DN-ADAM17 plasmid. After 48 hours, cells were treated with CHX (100 μM, 6 hours) in presence of DMSO (vehicle control, 6 hours) to monitor baseline shedding, PMA (100 nM, 2 hours) or αFas for 6 hours. The sIL6R protein in the culture media was quantified using the sIL6R-specific ELISA. The fold increase of the sIL6R is expressed as a percentage of baseline shedding (set to 100%). Baseline shedding of IL6R/mock-transfected cells was 5549.07 (± 1626.3) pg/mL and of IL6R/DN-TACE 3068.84 (± 744) pg/mL. Values represent the mean (± SD) from 3 independent experiments (*P ≤ .001 and **P ≤ .01).

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