The metalloprotease TACE/ADAM17 is required for P2Y-stimulated TGF-α shedding and EGFR transactivation. (A) EC-4 EGFR cells were serum-starved for 4 h and pretreated with vehicle or 20 μM TAPI-2 for 30 min as indicated. Cells were then stimulated with vehicle, 20 ng/ml EGF, or 100 μM ATP for 5 min. Phosphorylated EGFR (top) was detected in cell lysates by immunoblotting followed by stripping and detection of total EGFR (bottom). Representative results of three separate experiments are shown. (B) EC-4 EGFR cells were transiently transfected with 100 nM siRNA targeting TACE or a scrambled control (Scr). Forty-eight hours after transfection, cells were serum-starved and harvested as described above. Phosphorylated EGFR (top) was detected in cell lysates by immunoblotting followed by stripping and detection of total EGFR (middle) and TACE expression (bottom). Representative results of three separate experiments are shown. (C) EC-4 EGFR or EC-2 (Tace^ΔZn/ΔZn) EGFR cells were serum-starved and harvested as previously described. Phosphorylated EGFR (top) was detected in cell lysates by immunoblotting followed by stripping and detection of total EGFR (bottom). Representative results of three separate experiments are shown. (D) EC-4 and EC-2 cells or (E) EC-2 and EC-2 cells stably expressing transfected mouse TACE (bottom) were serum-starved for 4 h and then stimulated for 5 min with vehicle or 100 μM ATP. Media and lysates were harvested and concentrated as described in Materials and Methods, and analyzed for TGF-α content by specific RIA. Data are presented as picograms of TGF-α in conditioned media per milligram of total lysate protein from three separate experiments with at least three replicates. *p < 0.05 compared with the control; ^#p < 0.05 compared with ATP.

ATP-stimulated TGF-α shedding requires calcium and ROS. CHO AP-TGF-α cells were serum-starved for 4 h and pretreated with vehicle (DMSO) or the following concentration of inhibitors for 30 min: 50 μM BAPTA-AM (BAP); 10 μM PP2; 10 μM PP3; 20 nM BIM-I; 10 μM SB202190 (SB2); 10 μM SB203580 (SB3); 20 μM PD98059 (PD); and 5 mM NAC. After stimulation with vehicle or 100 μM ATP for 5 min, media were collected and analyzed for alkaline phosphatase (AP) activity. AP activity is presented relative to the unstimulated control. *p < 0.05 compared with control; ^#p < 0.05 compared with uninhibited stimulation. All others not significant.

ROS mediate ATP-induced TGF-α shedding. (A) CHO AP-TGF-α cells were pretreated with vehicle or 5 mM NAC for 30 min. Cells were then stimulated for 5 min with vehicle or 100 μM ATP. (B) CHO AP-TGF-α cells were pretreated with vehicle or 20 μM TAPI-2 for 30 min. Cells were then stimulated for 5 min with vehicle, 100 μM ATP, or 30 μM hydrogen peroxide (HP). (C) Mutant M2 CHO AP-TGF-α clones, which lack functional endogenous TACE, or TACE M2 CHO AP-TGF-α clones, which stably express mouse TACE (bottom panel), were stimulated for 5 min with vehicle or 30 μM HP. Media for all experiments were collected and analyzed for alkaline phosphatase activity. Results are an average of three separate experiments with at least three replicates. AP activity is presented relative to the unstimulated control. *p < 0.05 compared with control; ^#p < 0.05 compared with uninhibited stimulation.

Mitochondrially derived ROS are required for ATP-induced TGF-α shedding. (A) CHO AP-TGF-α cells were pretreated with DMSO or 1 μM myxothiazol for 30 min. Cells were then stimulated for 5 min with vehicle or 100 μM ATP. (B) CHO AP-TGF-α cells were pretreated with DMSO or 25 μM rotenone for 30 min. Cells were then stimulated for 5 min with vehicle or 100 μM ATP. (C) CHO AP-TGF-α cells were pretreated with DMSO, 5 mM NAC, or 1 μM myxothiazol and 25 μM rotenone for 30 min. Cells were then stimulated for 5 min with vehicle or 100 μM ATP. (D) CHO cells were pretreated with vehicle or 1 μM myxothiazol and 25 μM rotenone for 30 min. During pretreatment, cells were also loaded with 250 nM MitoTracker Red and treated as described in Materials and Methods. Cells were stimulated with vehicle or 100 μM ATP. Boxes represent the 25th, 50th, and 75th quartiles and error bars the minimum and maximum reading and are presented in this manner to more fully display all the data points. These data represent three separate experiments of at least four replicates relative to the unstimulated control. *p < 0.03 compared with the unstimulated control; ^†p < 0.001 compared with the stimulated control. (E) CHO AP-TGF-α cells were serum-starved for 4 h and pretreated with DMSO, 5 mM NAC, 1 μM myxothiazol, 25 μM rotenone, or 1 μM myxothiazol and 25 μM rotenone for 30 min. Cells were then stimulated for 5 min with vehicle or 1 μM A23187. Media for all alkaline phosphatase (AP) assays were collected and analyzed for AP activity. All assays are an average of three separate experiments with at least three replicates and presented relative to the unstimulated control samples. *p < 0.05 compared with control; ^#p < 0.05 compared with uninhibited stimulation.

GPCR stimulation induces EGFR transactivation and TGF-α shedding. Cells were serum-starved for 4 h. Results are representative of three separate experiments. (A) Cells were stimulated with vehicle, 20 ng/ml EGF, or 100 μM ATP for 5 min. Phosphorylated EGFR (top) was detected in cell lysates by immunoblotting followed by stripping and detection of total EGFR (bottom). (B) EC-4 EGFR cells were inhibited with vehicle or 20 μM AG1478 for 30 min and then stimulated with vehicle, 20 ng/ml EGF, or 100 μM ATP for 5 min. Cell lysates were subjected to anti-Shc immunoprecipitation followed by immunoblotting for phosphotyrosine (top, RC-20) and total Shc protein (bottom). (C) EC-4 cells were stimulated for 5 min with vehicle, 100 μM ATP, 100 μM ATPγS, or 100 μM UTP. Media and lysates were harvested and concentrated as described in Materials and Methods and analyzed for TGF-α content by specific RIA. Data are presented as the average amount of TGF-α in conditioned media per milligram of total lysate protein from three separate experiments with at least three replicates. *p < 0.02 compared with the control. (D) Total RNA from EC-4 mouse fibroblast cells (Fb) or mouse jejunum tissue (Jej) was subjected to first strand cDNA synthesis with or without reverse transcriptase (RT) and amplified for 28 cycles to detect the expression of P2Y₂ (top) and P2Y₄ (middle) receptors. β-actin was used a loading control (bottom).

CHO cells demonstrate P2Y₂-TACE-TGF-α shedding. (A) Total RNA from CHO cells was subjected to first strand cDNA synthesis with or without reverse transcriptase (RT) and amplified for 28 cycles to detect the expression of P2Y₂ (top) and P2Y₄ (middle) receptors. β-Actin was used as a loading control (bottom). (B) CHO AP-TGF-α cells were pretreated with vehicle or 20 μM TAPI-2 for 30 min. Cells were then stimulated for 5 min with vehicle, 100 μM ATP, or 100 μM UTP. Media were collected and analyzed for alkaline phosphatase (AP) activity. (C) Mutant M2 CHO AP-TGF-α clones, which lack functional endogenous TACE, or TACE M2 CHO AP-TGF-α clones, which stably express mouse TACE (bottom), were serum-starved for 4 h. Cells were then stimulated for 5 min with vehicle or 100 μM ATP. Media were collected and analyzed for AP activity. AP assay results are an average of three separate experiments with at least three replicates and are presented relative to the unstimulated control. *p < 0.05 compared with control; ^#p < 0.05 compared with uninhibited stimulation.

Calcium regulates ATP-induced, TACE-dependent TGF-α shedding. (A) CHO AP-TGF-α cells were pretreated with vehicle or 50 μM BAPTA-AM for 30 min. Cells were then stimulated for 5 min with vehicle, 100 μM ATP, or 1 μM A23187. (B) Mutant M2 CHO AP-TGF-α clones, which lack functional endogenous TACE, or TACE M2 CHO AP-TGF-α clones, which stably express mouse TACE (bottom panel), were stimulated for 5 min with vehicle or 1 μM A23187. (C) CHO AP-TGF-α cells were pretreated with water or 500 μM EGTA for 30 min. Cells were then stimulated for 5 min with vehicle, 100 μM ATP, or 1 μM A23187. (D) CHO AP-TGF-α cells were pretreated with water or 500 μM EGTA for 30 min. Cells were then stimulated for 5 min with vehicle, 1 μM A23187 or 30 μM hydrogen peroxide (HP). Media for all experiments were collected and analyzed for alkaline phosphatase (AP) activity. Results are an average of three separate experiments with at least three replicates. AP activity is presented relative to the unstimulated control. *p < 0.05 compared with control; ^#p < 0.05 compared with uninhibited stimulation.

ATP-induced TGF-α shedding is independent of NADPH oxidase activity. (A) CHO AP-TGF-α cells were pretreated with DMSO or 1 mM apocynin for 30 min. Cells were then stimulated for 5 min with vehicle or 100 μM ATP. Media were collected and analyzed for alkaline phosphatase (AP) activity. Results are an average of three separate experiments with at least three replicates. AP activity is presented relative to the unstimulated control. Plasma membrane proteins from stimulated CHO (B) and HL-60 (C) cells incubated in cytochrome C reaction buffer were used to measure superoxide production by way of cytochrome C reduction as measured by changes in absorbance at 550 nm in the presence and absence of SOD at the indicated times. Results are an average of three independent experiments. *p < 0.05 compared with control.