J Biol Chem 285(40): 30506-30515

PMID: 20679348

Suppression of ADAM17-mediated Lyn/Akt Pathways Induces Apoptosis of Human Leukemia U937 Cells

Introduction

Protease inhibitors play a critical role in the regulation of several biological processes such as blood coagulation, complement fixation, fibrinolysis, fertilization, and embryogenesis (1). Dysregulation of proteinases leads to many pathophysiological conditions that include cancer, atherosclerosis, and inflammation. In particular, cell surface proteases, including meprin, matrix metalloproteinase, dipeptidyl peptidase IV, and seprase, have been demonstrated to play an important role in facilitating cell invasion into extracellular matrix and may contribute significantly to extracellular matrix degradation by metastatic cancer cells (2). Abundant expression of these enzymes is associated with poor prognosis. Thus, protease inhibitors that repress cell surface proteases may be applicable to cancer therapy. Protease inhibitors are grouped into a number of families, including the Kunitz, Kazal, Serpin, and mucous families (3). Several Kunitz-type protease inhibitors, including bikunin, hepatocyte growth factor activator inhibitor-2, and tissue factor pathway inhibitor-2, are found to suppress tumor invasion and metastasis (4,–7). It was suggested that bikunin and tissue factor pathway inhibitor-2 exerted their biology activities through a cell surface receptor-mediated process (3, 4). Moreover, tissue factor pathway inhibitor-2 elicits pro-apoptotic signaling pathway in the human fibrosarcoma cell line (8).

Snake venoms are complex mixtures of pharmacologically active polypeptide toxins that are believed to have evolved to alter functionally the physiological activities along with predator-prey interaction (9,–11). In addition to enzymes and toxins, snake venom also contains serine protease inhibitors. Several Kunitz/bovine pancreatic trypsin inhibitors from the venom of Viperidae and Elapidae snakes have been isolated and sequenced (12,–15). These snake venom Kunitz-type protease inhibitors have been demonstrated to specifically inhibit the proteolytic activity of trypsin or chymotrypsin. Nevertheless, their physiological roles in the regulatory mechanisms that influence the proteases in coagulation, fibrinolysis, and inflammation have been rarely considered. Three protease inhibitor-like protein genes have been cloned from Bungarus multicinctus genome in our laboratory (16). The deduced protein sequences of protease inhibitor-like proteins are highly homologous with those of Kunitz-type protease inhibitors. However, their biological activities remain elusive. Because soybean Kunitz-type trypsin inhibitor has been found to induce apoptotic death of human leukemia Jurkat cells (17), anti-leukemia activity of B. multicinctus protease inhibitor-like proteins is thus examined. In this study, human leukemia U937 cells were treated with B. multicinctus protease inhibitor like protein-1 (PILP-1). It was found that PILP-1-induced down-regulation of a disintegrin and metalloprotease 17 (ADAM17) led to inactivation of Lyn/Akt pathways. The signaling pathways further triggered apoptosis of U937 cells through the mitochondrion-mediated death pathway. Collectively, our data elucidate a novel ADAM17/Lyn/Akt signaling pathway in maintaining the viability of leukemia cells and suggest a strategy in improving leukemia therapy through suppression of ADAM17 protein expression.

EXPERIMENTAL PROCEDURES

Materials

B. multicinctus PILP-1 was prepared according to our established procedure (16). MTT,² propidium iodide, digitonin, U0126 (MEK1 and MEK2 inhibitor), SB202190 (p38 MAPK inhibitor), and anti-β-actin antibody were obtained from Sigma, and annexin V-FITC/propidium iodide flow cytometry assay kit and rhodamine-123 were purchased from Invitrogen. Gefitinib was purchased from LC Laboratories (Woburn, MA). Anti-ADAM17 (H-300) antibody (specifically recognized pro-ADAM17), anti-Fas (N-18) antibody, and anti-Lyn (SC-15) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p38 MAPK and anti-phospho-p38 MAPK, anti-ERK and anti-phospho-ERK, anti-JNK and anti-phospho-JNK, anti-TNFR2, anti-Akt and anti-phospho-Akt(Ser-473), anti-phospho-Src(Tyr-416), anti-phospho-Lyn (Tyr-507), anti-caspase-9, anti-PARP, anti-Bcl-2, and anti-FasL antibodies were the products of Cell Signaling Technology (Beverly, MA). Anti-caspase-3 antibody, anti-caspase-8 antibody, Ac-DEVD-p-nitroanilide, Ac-LEHD-p-nitroanilide, Z-IETD-fmk (caspase-8 inhibitor), Z-DEVD-fmk (caspase-3 inhibitor), and PP2 (Lyn inhibitor) were purchased from Calbiochem. Anti-cytochrome c and anti-Bid antibodies were the products of Pharmingen. Anti-human TNFR1 antibody and monoclonal anti-human ADAM17-fluorescein were purchased from R & D Systems (Minneapolis, MN) and anti-ADAM17 activation site (ab39163) antibody (specifically recognized mature ADAM17) was obtained from Abcam (Cambridge, MA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Pierce. Cell culture supplies were purchased from Invitrogen Unless otherwise specified, all other reagents were of analytical grade.

Cell Culture

Human acute myelogenous leukemia U937 cells and human chronic myelogenous leukemia K562 cells obtained from ATCC (Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen), 2 mml-glutamine, penicillin (100 units/ml)/streptomycin (100 μg/ml), and 1% sodium pyruvate incubated at 37 °C in an incubator humidified with 95% air and 5% CO₂. Exponentially growing cells (1 × 10^{) were plated in 96-well plates and treated with PILP-1 in serum-free medium. For pharmacological experiments, culture cells were pretreated with 10 μ}m SB202190, 10 μm U0126, 100 μm Z-DEVD-fmk, and 100 μm Z-IETD-fmk before PILP-1 was added.

RNA Preparation and RT-PCR

Total RNA was isolated from untreated control cells or PILP-1-treated cells using the RNeasy minikit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Reverse transcriptase reaction was performed with 2 μg of total RNA using Moloney murine leukemia virus reverse transcriptase (Promega) as recommended by the manufacturer. A reaction without reverse transcriptase was performed in parallel to ensure the absence of genomic DNA contamination. After initial denaturation at 95 °C for 10 min, PCR amplification was performed using GoTaq Flexi DNA polymerase (Promega) followed by 30 cycles at 94 °C for 60 s, 55 °C for 60 s, and 72 °C for 60 s. After a final extension at 72 °C for 5 min, PCR products were resolved on 2% agarose gels and visualized by ethidium bromide transillumination under UV light. Primer sequences were as follows: TNFR2 (forward), 5′-ACATCAGACGTGGTGTGCAA-3′, and TNFR2 (reverse), 5′-CCAACTGGAAGAGCGAAGTC-3′; ADAM17 (forward), 5′-CAGCACAGCTGCCAAGTCATT-3′ and ADAM17 (reverse), 5′-CCAGCATCTGCTAAGTCACTTCC-3′. The PCR yielded PCR products of 323 and 235 bp for TNFR2 and ADAM17, respectively. Each reverse-transcribed mRNA product was internally controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR using primers 5′-GAGTCAACGGATTTGGTCGT-3′ (forward) and 5′-TGTGGTCATGAGTCCTTCCA-3′ (reverse), yielding a 512-bp PCR product. The TNFR2 and ADAM17 reverse transcriptase-PCR products were subsequently confirmed by direct sequencing.

Cloning of Luciferase Reporter Plasmid of ADAM17 Promoter and Luciferase Assay

DNA segment containing nucleotides −938 to +235 of the human ADAM17 gene was amplified by PCR from human genomic DNA. The PCR-amplified genomic DNA was subcloned into the firefly luciferase reporter vector pGL3-basic (Promega) between KpnI and XhoI sites. The nucleotide sequences of constructs were identified by DNA sequencing. The resulting pGL-ADAM17 was used for promoter activity assay, and luciferase assay was performed with the luciferase reporter assay system (Promega).

DNA Transfection

The pCMV-MEK1 (expressed the constitutively active MEK1) and constitutively activated, myristoylated Akt vector were generous gifts from Dr. W. C. Hung (National Sun Yat-Sen University, Taiwan). Human ADAM17 expression plasmid, pME18S-ADAM17, was kindly provided by Dr. E. Nishi (Kyoto University, Japan), and pMX-IRES-GFP-Lyn expression vector was obtained from Dr. N. J. Donato (University of Michigan Comprehensive Cancer Center). LynY507F (gain of function mutant) cDNA was prepared from pMX-IRES-GFP-Lyn expression vector using the PCR method and subcloned into pcDNA3 expression vector. The plasmids were transfected into U937 cells using the pipette-type electroporator (MicroPorator-MP100, Digital Bio Technology Co., Korea).

Co-immunoprecipitation of ADAM17 and Lyn

After treatment with or without 10 μm PILP-1 for 24 h, U937 cells were harvested and washed with cold PBS. The cells were then incubated with lysis buffer (50 mm NaCl, 50 mm Tris-HCl, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mm PMSF, 50 mm NaF, and 1 mm Na₂VO₄) in ice for 10 min. After centrifugation step at 15,000 × g for 20 min, the supernatant was mixed with protein G Plus/protein A-agarose suspension (Calbiochem) and incubated for 1 h at 4 °C (preclearing). After removal of agarose, the cell lysate was incubated with antibodies (anti-ADAM17 or anti-Lyn) overnight at 4 °C on a rotating plate. Then protein G Plus/protein A-agarose suspension (Calbiochem) was added to each sample. Following an additional 2 h of incubation at 4 °C, immunoprecipitates were washed three times with lysis buffer and eluted by SDS-gel loading buffer for SDS-PAGE and Western blot analyses.

RNA Interference

ADAM17 siRNA (catalogue number sc-36604) and negative control siRNA (catalogue number sc-37007) were purchased from Santa Cruz Biotechnology, Inc. For the transfection procedure, cells were grown to 60% confluence, and 100 nm siRNA were transfected using Lipofectamine^{2000 (Invitrogen) according to the manufacturer's instructions.}

Separation of Cytosolic and Mitochondrial Fractions

Following specific treatment, cytosolic and pellet (mitochondrial) fractions were generated using a digitonin-based subcellular fractionation technique (18). Cytochrome c and proteins of the Bcl-2 family were detected by Western blot analysis.

Determination of Soluble TNF-α Receptor 2 (TNFR2) by ELISA

After treatment with PILP-1 for 24 h, the culture media of U937 cells were collected and centrifuged at 12,000 rpm for 10 min, and the clarified supernatants were collected. ELISA for soluble TNFR2 (sTNFR2) was performed according to the manufacturer's protocol (R & D Systems, Inc.). Developed assay plates were read at wavelength 450 nm using a plate reader, and the results were calculated using a standard curve generated each time an assay was performed.

Flow Cytometry Analyses of Cell Surface Expression of ADAM9, ADAM10, and ADAM17

After specific treatment, nonspecific antibody-binding sites were blocked by incubation with PBS containing 0.01% human IgG. One fluorescent parameter flow cytometry was performed by staining cells with monoclonal anti-human ADAM17-fluorescein according to the manufacturer's protocol (R & D Systems). The stained cells were analyzed by a Beckman Coulter Epics XL flow cytometer. For detection of ADAM10 and ADAM9 protein expression on the cell surface, cells were incubated with anti-ADAM9 (AP7437a) (Abgent, San Diego) and anti-ADAM10 (MAB1427) (R & D Systems) antibodies at 4 °C for 30 min. After washing, cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse or anti-rabbit IgG (H+L) (Anaspec, Fremont, CA) and subjected to flow cytometric analysis.

Measurement of ADAM17 Activity

ADAM17 activity was measured according to the manufacturer's protocol (Calbiochem). Its fluorescence-related enzymatic cleavage was monitored at 320 nm excitation and 405 nm emission wavelength using a microplate fluorescence reader.

Statistical Analysis

All data are presented as means ± S.D. Significant differences among the groups were determined using the unpaired Student's t test. A value of p < 0.05 was taken as an indication of statistical significance. All the figures shown in this study were obtained from at least three independent experiments with similar results.

Other Tests

Cell viability assay, annexin V/propidium iodide staining, DNA content analysis, measurement of mitochondrial membrane potential, caspase-3 and -9 activity assay, separation of human peripheral blood mononuclear cells (PBMCs), and Western blot analysis were performed in essentially the same manner as described previously (18). Results of Western blots were quantified by a scanning densitometer. All bands were normalized to β-actin expression, and fold changes in protein expression were determined on the basis of β-actin loading control.

Materials

Cell Culture

RNA Preparation and RT-PCR

Cloning of Luciferase Reporter Plasmid of ADAM17 Promoter and Luciferase Assay

DNA Transfection

Co-immunoprecipitation of ADAM17 and Lyn

RNA Interference

Separation of Cytosolic and Mitochondrial Fractions

Determination of Soluble TNF-α Receptor 2 (TNFR2) by ELISA

Flow Cytometry Analyses of Cell Surface Expression of ADAM9, ADAM10, and ADAM17

Measurement of ADAM17 Activity

Statistical Analysis

Other Tests

Materials

Cell Culture

RNA Preparation and RT-PCR

Cloning of Luciferase Reporter Plasmid of ADAM17 Promoter and Luciferase Assay

DNA Transfection

Co-immunoprecipitation of ADAM17 and Lyn

RNA Interference

Separation of Cytosolic and Mitochondrial Fractions

Determination of Soluble TNF-α Receptor 2 (TNFR2) by ELISA

Flow Cytometry Analyses of Cell Surface Expression of ADAM9, ADAM10, and ADAM17

Measurement of ADAM17 Activity

Statistical Analysis

Other Tests

Materials

Cell Culture

RNA Preparation and RT-PCR

Cloning of Luciferase Reporter Plasmid of ADAM17 Promoter and Luciferase Assay

DNA Transfection

Co-immunoprecipitation of ADAM17 and Lyn

RNA Interference

Separation of Cytosolic and Mitochondrial Fractions

Determination of Soluble TNF-α Receptor 2 (TNFR2) by ELISA

Flow Cytometry Analyses of Cell Surface Expression of ADAM9, ADAM10, and ADAM17

Measurement of ADAM17 Activity

Statistical Analysis

Other Tests

RESULTS

Upon exposure to PILP-1, U937 cells showed a concentration- and time-dependent decrease in cell viability (supplemental Fig. 1A). PILP-1 treatment caused an increase in annexin V staining and induced an increased accumulation of cells in the sub-G₁ phase (supplemental Fig. 1, B and C). Moreover, degradation of procaspase-3, procaspase-8, and PARP (caspase-3 substrate) was noted in PILP-1-treated cells (supplemental Fig. 1D). Pretreatment with caspase-3 inhibitor (Z-DEVD-fmk) and caspases-8 inhibitor (Z-IETD-fmk) restored significantly the viability of PILP-1-treated cells (supplemental Fig. 1E). These data indicated that PILP-1 induced apoptotic death of U937 cells.

Increasing evidence suggests that altered mitochondrial function is linked to apoptosis, and a decreasing mitochondrial transmembrane potential (ΔΨ_m) is associated with mitochondrial dysfunction (19). As shown in supplemental Fig. 2A, the increasing population of U937 cells exhibited the loss of ΔΨ_m after PILP-1 treatment. As seen in supplemental Fig. 2B, a time-dependent release of cytochrome c into cytosol was detected relative to gradual decrease in mitochondrial cytochrome c. Moreover, down-regulation of Bcl-2 and production of t-Bid were notably observed after PILP-1 treatment for 24 h. In the mitochondrial pathway, caspase-8 converts the Bid from the inactive form (22 kDa) to the active form (15 kDa), which is called truncated Bid (tBid). tBid associates with the mitochondria outer membrane, disrupts mitochondrial membrane potential (ΔΨ_m), and releases cytochrome c into the cytoplasm. Apoptotic signals converge on mitochondria to trigger the release of cytochrome c into the cytosol, causing caspase-9 and -3 activation and cell death (20). Consistent with this result, procaspase-9 degradation was noted with PILP-1-treated cells. Moreover, PILP-1 treatment induced an increase in activities of caspase-3 and caspase-9 in U937 cells (supplemental Fig. 2C). Pretreatment with caspase-8 inhibitor abolished the production of active caspase-3 in PILP-1-treated cells, whereas caspase-3 inhibitor was unable to block PILP-1-elicited procaspase-8 degradation (supplemental Fig. 2D). It suggested that caspase-8 was located on the upstream position for procaspase-3 degradation. Moreover, Z-IETD-fmk (caspase-8 inhibitor) abolished PILP-1-induced loss of ΔΨ_m (supplemental Fig. 2A). Taken together, the data indicate that PILP-1-induced apoptosis of U937 cells was mediated via the caspase-8/mitochondrial pathway.

Because MAPKs are common components of the apoptotic program (21), phosphorylation of MAPKs was examined in PILP-1-treated U937 cells. Fig. 1A shows that the level of phospho-p38 MAPK was notably increased after 8 h of PILP-1 treatment, whereas that of phospho-ERK was reduced after PILP-1 treatment. In contrast, JNK was not phosphorylated in PILP-1-treated cells. As shown in Fig. 1B, the viability of PILP-1-treated cells was restored by pretreatment with SB202190 (p38 MAPK inhibitor). Moreover, SB202190 pretreatment abrogated PILP-1-elicited procaspase-8 degradation (Fig. 1B) and mitochondrial depolarization (supplemental Fig. 2A). The data reflect that p38 MAPK activation elicited caspase-8/mitochondrial mediated death pathway in PILP-1-treated U937 cells. Fig. 1C shows that PILP-1 was unable to induce procaspase-8 degradation, p38 MAPK activation, and ERK inactivation in cells transfected with constitutively active MEK1. Moreover, transfection of constitutively active MEK1 restored the viability of PILP-1-treated cells (Fig. 1D). These suggested that ERK inactivation was associated with cytotoxicity of PILP-1.

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FIGURE 1.

PILP-1-elicited ERK inactivation and p38 MAPK activation in U937 cells.A, Western blot analyses of phosphorylated MAPKs. U937 cells were treated with 10 μm PILP-1 for indicated times. B, effect of SB202190 on viability and procaspase-8 degradation in PILP-1-treated cells. U937 cells were pretreated with 10 μm SB202190 for 1 h and then incubated with 10 μm PILP-1 for 24 h. Left panel, cell viability was analyzed by MTT assay. The values represent averages of three independent experiments with triplicate measurements (mean ± S.D., *, p < 0.05). Right panel, Western blot analyses of procaspase-8 degradation in PILP-1-treated U937 cells. Ctrl, control. C, effect of constitutively active MEK1 on p38 MAPK phosphorylation and procaspase-8 degradation in PILP-1-treated cells. pCMV-MEK1-transfected cells were treated with 10 μm PILP-1 for 24 h. D, transfection of constitutively active MEK1 restored viability of PILP-1-treated cells. U937 cells were transfected with control vector or pCMV-MEK1. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Cell viability was analyzed by MTT assay. The values represent averages of three independent experiments with triplicate measurements (mean ± S.D., *, p < 0.05).

Death receptors of the tumor necrosis factor (TNF) family such as Fas and tumor necrosis factor-α receptor 1 (TNFR1) are the best understood death pathways that recruit FADD and procaspase-8 to the receptor (22). Recruitment of procaspase-8 through FADD leads to its auto-cleavage and activation, and in turn it activates effector caspases such as caspase-3 or initial apoptosis through the mitochondrial pathway (23). Because up-regulation of death receptors evokes caspase-8 activation, protein expression of TNFR1, TNFR2, Fas, and FasL in PILP-1-treated cells was thus examined. Fig. 2A shows that, unlike that of Fas, FasL, and TNFR1, protein expression of TNFR2 increased markedly in PILP-1-treated cells. Nevertheless, transcription of TNFR2 mRNA did not significantly change in PILP-1-treated cells as revealed by reverse transcription-PCR amplification (Fig. 2B). ELISA revealed that PILP-1 treatment led to a reduction in detectable soluble TNFR2 (sTNFR2) concentration in culture medium of U937 cells (Fig. 2C). Given that ectodomain shedding of TNFR2 resulted in the production of sTNFR2, our data suggested that PILP-1-induced up-regulation of TNFR2 arose from reduction in TNFR2 shedding. Knockdown of FADD did not significantly affect PILP-1-elicited procaspase-8 degradation and rescue viability of PILP-1-treated cells (Fig. 2D), reflecting that the death receptor-mediated pathway was not involved in PILP-1-induced cell death.

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FIGURE 2.

PILP-1-induced cell death is not mediated through death receptor-mediated pathway.A, Western blot analyses of Fas, FasL, TNFR1, and TNFR2 protein expression in PILP-1-treated cells (top panel). U937 cells were treated 10 μm PILP-1 for the indicated times. Bottom panel, quantification of the expression of TNFR2 from Western blot analyses. Three independent experimental results were analyzed by densitometry. B, detecting the transcription of TNFR2 mRNA using RT-PCR. U937 cells were treated with 10 μm PILP-1 for 24 h. RT-PCR was conducted according to the procedure described under “Experimental Procedures.” Ctrl, control. C, measurement of soluble TNFR2 (sTNFR2) in culture medium using ELISA. After treatment with 10 μm PILP-1 for the indicated times, culture media of U937 cells were collected for determining sTNRF2 concentrations. The data are the means ± S.D. of three independent experiments in triplicate measurements (*, p < 0.05). D, effect of down-regulation of FADD on PILP-1-induced procaspase-8 degradation and cell death. U937 cells were transfected with 100 nm control siRNA or FADD siRNA. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Left panel, Western blot analyses of procaspase-8 degradation. Right panel, effect of PILP-1 on viability of FADD siRNA-transfected cells. Cell viability was determined using MTT assay. The values represent averages of three independent experiments with triplicate measurements (mean ± S.D.).

Given that ADAM17 is involved in the release of the ectodomain of TNFR2 (24), protein expression of ADAM17 in PILP-1-treated cells was examined. Fig. 3A shows that protein expression of pro-ADAM17 and ADAM17 in U937 cells was reduced in response to PILP-1 treatment. ADAM17 has been shown to be synthesized as a zymogen, which is constitutively processed to cell membrane after removal of the prodomain of pro-ADAM17. The increase in the surface expression of ADAM17 up-regulated sheddase activity of ADAM17 (25). Flow cytometry analyses revealed that PILP-1 treatment elicited a decrease in the amount of detectable ADAM17 on the cell surface. Taken together, these results suggested that PILP-1-induced decrease in TNFR2 shedding was mediated through down-regulation of ADAM17. Transcriptional level of ADAM17 mRNA of PILP-1-treated cells was notably lower than that of control untreated cells as evidenced by RT-PCR assay (Fig. 3B). Promoter assay revealed that PILP-1 attenuated the luciferase activity of the ADAM17 promoter in U937 cells (Fig. 3C). SB202190 pretreatment and overexpression of the constitutively active MEK1 abrogated PILP-1-evoked decease in ADAM17 mRNA transcription, reduction in luciferase activity of the ADAM17 promoter, and down-regulation of ADAM17 (Fig. 3, B–E). Consistently, PILP-1-elicited up-regulation of TNFR2 was restored by SB202190 and transfection of constitutively active MEK1 (Fig. 3, D and E). Moreover, SB202190 and overexpression of constitutively active MEK1 abrogated PILP-1-evoked decrease in ADAM17 activity and TNFR2 shedding (Fig. 3F). Taken together, these results suggested that PILP-1-elicited ADAM17 down-regulation was mediated through p38 MAPK activation and ERK inactivation.

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FIGURE 3.

Effect of SB202190 and constitutively active MEK1 on PILP-1-induced ADAM17 down-regulation and TNFR2 shedding.A, protein expression of pro-ADAM17 and ADAM17 in PILP-1-treated cells. U937 cells were treated with 10 μm PILP-1 for the indicated times. Top left panel, Western blot analyses of pro-ADAM17 and ADAM17 protein expression. Bottom left panel, flow cytometry analyses of cell surface ADAM17 protein expression. Right panel, quantification of the expression of pro-ADAM17 and ADAM17 from Western blot analyses. Three independent experimental results were analyzed by densitometry. Ctrl, control. B, effect of SB202190 and constitutively active MEK1 on the transcription of ADAM17 mRNA. Top panel, U937 cells were pretreated with 10 μm SB202190 for 1 h and then incubated with 10 μm PILP-1 for 24 h. Bottom panel, U937 cells were transfected with pCMV-MEK1. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. RT-PCR was conducted according to the procedure described under “Experimental Procedures.” C, PILP-1 treatment elicited increase in transcriptional activity of the ADAM17 promoter. After transfection with indicated plasmid for 24 h, transfected cells were treated with 10 μm PILP-1 for 24 h and then harvested for measuring luciferase activity (mean ± S.D., *, p < 0.05). D, SB202190 suppressed PILP-1-induced ADAM17 down-regulation. U937 cells were pretreated with 10 μm SB202190 for 1 h and then incubated with 10 μm PILP-1 for the indicated times. Left panel, Western blot analyses of pro-ADAM17, ADAM17, and TNFR2 protein expression. Right panel, quantification of the expression of pro-ADAM17, ADAM17, and TNFR2 from Western blot analyses. Three independent experimental results were analyzed by densitometry. E, effect of constitutively active MEK-1 on PILP-1-induced ADAM17 down-regulation. U937 cells were transfected with pCMV-MEK1. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Left panel, Western blot analyses of pro-ADAM17, ADAM17, and TNFR2 protein expression. Right panel, quantification of the expression of pro-ADAM17, ADAM17, and TNFR2 from Western blot analyses. Three independent experimental results were analyzed by densitometry. F, effect of SB202190 (SB) and constitutively active MEK1 on ADAM17 activity and TNFR2 shedding in PILP-1-treated cells. U937 cells was pretreated with 10 μm SB202190 for 1 h and then incubated with 10 μm PILP-1 for 24 h. U937 cells were transfected with pCMV-MEK1. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Left panel, soluble TNFR2 (sTNFR2) in the culture medium was determined by ELISA. Right panel, ADAM17 activity was determined using fluorimetric substrate. The data are the mean ± S.D. of three independent experiments in triplicate measurements (*, p < 0.05).

Given that down-regulation of ADAM17 protein expression induced apoptosis of HeLa cells (26), the causal relationship between ADAM17 down-regulation and cytotoxicity of PILP-1 was investigated. Fig. 4A shows that knockdown of ADAM17 by siRNA reduced viability of U937 cells. Moreover, down-regulation of ADAM17 by siRNA elicited degradation of procaspase-3, -8, and -9 in U937 cells (Fig. 4B). On the other hand, overexpression of ADAM17 attenuated PILP-1-induced cell death (Fig. 4C) and suppressed PILP-1-induced degradation of procaspase-3, -8, and -9 (Fig. 4D). These reflected that PILP-1-induced cell death was associated with down-regulation of ADAM17.

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FIGURE 4.

Effects of ADAM17 down-regulation and ADAM17 overexpression on cell viability and degradation of procaspases in PILP-1-treated cells.A, down-regulation of ADAM17-induced death of U937 cells. U937 cells were transfected with 100 nm control siRNA or ADAM17 siRNA. 24 h post-transfection, cell viability was determined by MTT assay. The values represent averages of three independent experiments with triplicate measurements (mean ± S.D., *, p < 0.05). Ctrl, control. B, Western blot analyses of ADAM17 protein expression and degradation of procaspases in ADAM17 siRNA-transfected cells. U937 cells were transfected with 100 nm control siRNA or ADAM17 siRNA. 24 h post-transfection, the cells were harvested for Western blot analyses. C, overexpression of ADAM17 restored viability of PILP-1-treated cells. U937 cells were transfected with control vector or pME18S-ADAM17. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Cell viability was determined by MTT assay. The values represent averages of three independent experiments with triplicate measurements (mean ± S.D., *, p < 0.05). D, Western blot analyses of degradation of procaspases in pME18S-ADAM17-transfected cells after PILP-1 treatment. U937 cells were transfected with control vector or pME18S-ADAM17. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h.

ADAM17 is demonstrated to be a primary sheddase for multiple EGFR pro-ligands. Activation of EGFR by its ligand, which subsequently activates downstream PI3K/Akt and Ras/MAPK/ERK signaling pathways, is important for regulating cell proliferation and cell survival (27). Fig. 5A shows that PILP-1 inactivated Akt in U937 cells. Knockdown of ADAM17 by siRNA led to a reduction in the level of phospho-Akt (Fig. 5B). The finding that overexpression of ADAM17 abolished PILP-1-induced Akt inactivation (Fig. 5C) again supported a link between ADAM17 protein expression and Akt phosphorylation. Overexpression of constitutively active Akt (CA-AKT) restored the viability of PILP-1-treated cells (Fig. 5D). PILP-1 down-regulated ADAM17 in constitutively activated, myristoylated Akt-transfected cells, but degradation of procaspases was not observed after treatment with PILP-1 (Fig. 5E). This indicated that Akt inactivation evoked activation of caspases. Moreover, overexpression of constitutively active Akt did not alter PILP-1-evoked p38 MAPK activation and ERK inactivation (Fig. 5E), whereas SB202190 pretreatment or overexpression of constitutively active MEK1 abrogated PILP-1-induced Akt inactivation (Fig. 5F). These reflected that ADAM17 down-regulation was located at an upstream position for Akt inactivation in PILP-1-treated cells.

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FIGURE 5.

Effect of PILP-1 and ADAM17 protein expression on Akt phosphorylation in U937 cells.A, PILP-1 induced Akt inactivation. U937 cells were treated with 10 μm PILP-1 for the indicated times. B, effect of ADAM17 down-regulation on Akt phosphorylation in U937 cells. U937 cells were transfected with 100 nm control siRNA or ADAM17 siRNA. 24 h post-transfection, the cells were harvested for Western blot analyses. Ctrl, control. C, effect of ADAM17 overexpression on Akt phosphorylation in PILP-1-treated cells. U937 cells were transfected with control vector or pME18S-ADAM17. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. D, transfection of constitutively active Akt restored viability of PILP-1-treated cells. U937 cells were transfected with control vector or constitutively active Akt (CA-Akt). 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Cell viability was analyzed by MTT assay. The values represent averages of three independent experiments with triplicate measurements (mean ± S.D., *, p < 0.05). E, effect of constitutively active Akt on levels of phospho-ERK and phospho-p38 MAPK, ADAM17 protein expression, and degradation of procaspases in PILP-1-treated cells. U937 cells were transfected with control vector or constitutively active Akt (CA-Akt). 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. F, effect of SB202190 and constitutively active MEK1 on PILP-1-induced Akt inactivation. Top panel, U937 cells were transfected with pCMV-MEK1. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Bottom panel, U937 cells was pretreated with 10 μm SB202190 for 1 h and then incubated with 10 μm PILP-1 for 24 h.

In primary leukemic blast cells for patients with acute myeloid leukemia, the Lyn kinase is found to be constitutively activated (28, 29). Lyn is one member of the Src kinase family and has been reported to be upstream of Akt and ERK1/2 in intracellular cascades of acute myeloid leukemia (29, 30). Moreover, Kang et al. (31) found that interaction between Src and ADAM12 played a role in activating Src tyrosine kinase. The commercially available antibody against the phosphorylated Src Tyr-416 residue recognizes the highly conserved tyrosine phosphorylation site (EDNEpYTAR) in other members of the Src kinase family. Thus, the corresponding tyrosine site at position 396 (the positive regulatory phosphorylation site) of Lyn is recognized by the anti-phospho-Src Tyr-416 antibody. Moreover, Western blot for phosphorylated LynY507 (the negative regulatory phosphorylation site) was also conducted. Fig. 6, A and B, shows that PILP-1 treatment or down-regulation of ADAM17 led to a decrease in the level of pY396-Lyn accompanied with an increase in the level of pY507-Lyn. Overexpression of ADAM17 suppressed PILP-1-evoked changes in the levels of pY396-Lyn and pY507-Lyn (Fig. 6B). These reflected that ADAM17 was associated with activation of Lyn in U937 cells. Inhibition of Lyn activity by PP2 caused a reduction in approximately 50% viability of U937 cells (Fig. 6C). PP2 treatment did not significantly affect PILP-1-induced p38 MAPK activation, ERK inactivation, and down-regulation of ADAM17 (Fig. 6D). Nevertheless, PP2 treatment induced degradation of procaspase-8 and a decrease in the level of phospho-Akt regardless of PILP-1 treatment. Fig. 6, C and E, shows that transfection of pcDNA3-LynY507F restored viability of PILP-1-treated cells and suppressed PILP-1-induced Akt inactivation and procaspase-8 degradation. PILP-1-induced ERK inactivation, p38 MAPK activation, and ADAM17 down-regulation were still noted with pcDNA3-LynY507F-transfected cells. Taken together, these results revealed that ADAM17 down-regulation led to inactivation of the Lyn/Akt pathway in U937 cells. Previous studies found that the EGFR inhibitor (Gefitinib)-induced apoptosis of U937 cells through the Akt pathway (32). Gefitinib treatment reduced the levels of phospho-Akt and phospho-ERK but did not alter phosphorylation of Lyn and p38 MAPK and ADAM17 protein expression (Fig. 6G). It reflected that ADAM17-mediated Lyn/Akt activation was not related to the EGFR pathway. To examine if endogenous ADAM17 binds to Lyn, cell lysates of control untreated cells and PILP-1-treated cells were incubated with anti-ADAM17 or anti-Lyn antibodies, respectively. The protein complexes captured by antibodies were subjected to Western blot analyses. Fig. 6F shows that Lyn was co-immunoprecipitated with ADAM17, suggesting that ADAM17 and Lyn formed protein complexes. Meanwhile, the protein complexes immunoprecipitated by either anti-ADAM17 or anti-Lyn antibodies revealed a reduction in pY396-Lyn and an increase in pY507-Lyn after PILP-1 treatment.

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FIGURE 6.

PILP-1-induced Lyn inactivation.A, Western blot analyses of Lyn phosphorylation in PILP-1-treated cells. U937 cells were treated with 10 μm PILP-1 for the indicated times. B, effect of ADAM17 protein expression on Lyn phosphorylation in U937 cells. Left panel, U937 cells were transfected with 100 nm control (Ctrl) siRNA or ADAM17 siRNA. 24 h post-transfection, the cells were harvested for Western blot analyses. Right panel, effect of ADAM17 overexpression on Lyn phosphorylation in PILP-1-treated cells. U937 cells were transfected with control vector or pME18S-ADAM17. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. C, effect of PP2 (Lyn inhibitor) and overexpression of LynY507F on viability of PILP-1-treated cells. Cells were treated with 10 μm PILP-1, 20 μm PP2, or a combination of 20 μm PP2 and 10 μm PILP-1 for 24 h. Alternatively, U937 cells were transfected with pcDNA3-LynY507F. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Cell viability was analyzed by MTT assay. The values represent averages of three independent experiments with triplicate measurements (mean ± S.D., *, p < 0.05). D, effect of PP2 (Lyn inhibitor) on ERK phosphorylation, p38 MAPK phosphorylation, ADAM17 protein expression, Akt phosphorylation, and degradation of procaspase-8 in U937 cells. Cells were treated with 10 μm PILP-1, 20 μm PP2, or a combination of 20 μm PP2 and 10 μm PILP-1 for 24 h. E, effect of LynY507F overexpression on ERK phosphorylation, p38 MAPK phosphorylation, ADAM17 protein expression, Akt phosphorylation, and degradation of procaspase-8 in PILP-1-treated U937 cells (top panel). U937 cells were transfected with control vector or pcDNA3-LynY507F. 24 h post-transfection, the cells were treated with 10 μm PILP-1 for 24 h. Bottom panel, quantification of the expression of pro-ADAM17 and ADAM17 from Western blot analyses. Three independent experimental results were analyzed by densitometry. F, effect of Gefitinib (EGFR inhibitor) on ERK phosphorylation, p38 MAPK phosphorylation, ADAM17 protein expression, Lyn phosphorylation, and Akt phosphorylation in U937 cells. Cells were treated with 10 μm PILP-1, 20 μm Gefitinib, or a combination of 20 μm Gefitinib and 10 μm PILP-1 for 24 h. F, co-immunoprecipitation of ADAM17 and Lyn using anti-ADAM17 antibody or anti-Lyn antibody. Lysates of untreated control cells or PILP-1-treated cells were incubated with anti-ADAM17 antibody (top panel) or anti-Lyn antibody (bottom panel) followed by incubation with protein G Plus/protein A-agarose. Protein G Plus/protein A-agarose bead-bound immunocomplexes were eluted using SDS gel loading buffer and subjected to Western blot analyses. Control human IgG could not bind with either ADAM17 or Lyn.

The supplemental Fig. 3A shows that PILP-1 treatment induced ADAM17 down-regulation and procaspase-8 degradation in chronic myeloid leukemia K562 cells. Knockdown of ADAM17 by siRNA also elicited a decrease in viability of K562 cells (supplemental Fig. 3B). Moreover, p38 MAPK activation, ERK inactivation, Akt inactivation, and Lyn inactivation were also noted with PILP-1-treated K562 cells (supplemental Fig. 3A). Obviously, PILP-1-induced death of K562 cells and U937 cells was likely mediated through the same pathways. In sharp contrast, PILP-1 did not induce significant changes in protein expression of ADAM17 and the levels of phospho-ERK, phospho-p38 MAPK, pY396-Lyn, and phospho-Akt in PBMC cells (supplemental Fig. 4A). Moreover, marginal reduction in viability of PBMCs was noted after PILP-1 treatment (supplemental Fig. 4B). These data indicated that PILP-1 showed selective cytotoxicity toward U937 and K562 cells.

Given that ADAM17, ADAM9, and ADAM10 share common membrane substrates (33), the effect of PILP-1 on protein expression of ADAM9 and ADAM10 was examined. As shown in supplemental Fig. 5A, protein expression of mature ADAM9 and ADAM10 did not significantly change in PILP-1-treated cells. Flow cytometry analyses also revealed that PILP-1 treatment did not significantly alter the amount of detectable ADAM9 and ADAM10 on the cell surface (supplemental Fig. 5B). To examine if endogenous ADAM9 or ADAM10 binds to Lyn, cell lysates of control untreated cells and PILP-1-treated cells were incubated with anti-Lyn antibodies. The protein complexes captured by antibodies were subjected to Western blot analysis. It was found that Lyn could not form protein complexes with ADAM9 or ADAM10 (supplemental Fig. 5C).

DISCUSSION

ADAM is a gene family of multidomain membrane-anchored proteins, including more than 30 members in various animal species, and has been implicated in pathophysiological conditions (33, 34). ADAM17 is a member of the ADAM family and originally described as being responsible for the proteolytic cleavage of the membrane-anchoring precursor form of TNF-α to generate a soluble form of TNF-α (35). Subsequent studies have shown that ADAM17 is also involved in the shedding of other biologically active proteins, including heparin-binding epidermal growth factor, transforming growth factor β, TNFR1, TNFR2, EGFR, vascular cell adhesion molecule-1, L-selectin, interleukin receptors, Notch, and prion proteins (36, 37). ADAM17 has been shown to be synthesized as a zymogen, which is constitutively processed in the secretory pathway. An increase in the surface expression of ADAM17 up-regulated sheddase activity of ADAM17 (25). Thus, in addition to protein expression, generation of mature ADAM17 from its proform is proved to be related to its sheddase activity (37, 38).

Several reports have focused on the importance of ADAM17 up-regulation in tumor malignancy. In colon carcinoma, the up-regulated expression of ADAM17 induced the activation of EGFR through the shedding of EGFR ligands (39). Tanaka et al. (40) demonstrated an increase in the expression of heparin-binding epidermal growth factor in advanced ovarian cancer and also found that it correlated significantly with the ADAM17 expression in ovarian cancer. Aberrant expression of ADAM17 has also been reported in breast cancer (41), prostate cancer (42), pancreatic ductal adenocarcinoma (43), and oral squamous cell carcinoma (44). Thus, blocking of ADAM17 expression or development of specific ADAM17 inhibitors might have potential for cancer therapy. Although specific ADAM17 inhibitors have been reported previously (45, 46), ADAM17-targeted drugs also lead to the inhibition of several nontarget ADAMs or metalloproteinases (47). Thus, suppression of ADAM17 protein expression may become specific modalities for cancer therapy. Our data show that PILP-1-induced death of leukemia cells is mediated through down-regulation of ADAM17 and subsequent inactivation of Lyn and Akt. Lyn has been reported to be the major Src kinase in acute myeloid leukemia, and its constitutive activation is associated with proliferation of leukemia cells (28, 29). Src family contains a unique N-terminal region, an Src homology 2 (SH2) domain, an SH3 domain, a catalytic (tyrosine kinase) domain, and a short C-terminal tail. The SH3 domain is important for inter- as well as intramolecular interactions that regulate Src catalytic activity, cellular location, and recruitment of protein substrates (31). Noticeably, the cytoplasmic tail of ADAM17 contains an SH3-binding site, which is suggested to potentially activate SH3 domain-containing signaling molecules such as Src and Grb (36). Consistent with the finding that the interaction between ADAM12 and Src elicits activation of Src, our data reveal that ADAM17 is probably involved in Lyn phosphorylation through a similar mechanism. Noticeably, as compared with PBMC cells, leukemia cells, including U937 cells and K562 cells, are susceptible to being death induced by PILP-1. Thus, down-regulation of ADAM17 by PILP-1 may be an adaptable strategy in improving leukemia therapy. Conclusively, PILP-1 treatment suppresses ADAM17 expression through p38 MAPK activation and ERK inactivation-mediated pathways. Down-regulation of ADAM17 leads to inactivation of Lyn/Akt pathways and consequently evokes the caspase-8/mitochondria-mediated death pathway in U937 cells.

Supplementary Material

Supplemental Data:

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From the Institute of Biomedical Sciences, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, National Sun Yat-Sen University, Kaohsiung 804, Taiwan

^{To whom correspondence should be addressed. Fax: 886-7-5250197; E-mail:}wt.ude.usysn.liam@gnahcsl.

Received 2010 Jun 18; Revised 2010 Jul 30

Abstract

Cell surface proteases have been demonstrated to play an important role in facilitating cell invasion into the extracellular matrix and may contribute significantly to extracellular matrix degradation by metastatic cancer cells. Abundant expression of these enzymes is associated with poor prognosis. Thus, protease inhibitors that repress cell surface proteases may be applicable to cancer therapy. Because soybean Kunitz-type trypsin inhibitor has been found to induce apoptotic death of human leukemia Jurkat cells, anti-leukemia activity of Bungarus multicinctus protease inhibitor-like protein-1 (PILP-1) is thus examined. PILP-1 induced apoptosis of human leukemia U937 cells, characteristic of loss of mitochondrial membrane potential, degradation of procaspase-8, and production of t-Bid. FADD down-regulation neither restored viability of PILP-1-treated cells nor attenuated production of active caspase-8 and t-Bid in PILP-1-treated cells, suggesting that the death receptor-mediated pathway was not involved in the cytotoxicity of PILP-1. It was found that PILP-1-evoked p38 MAPK activation and ERK inactivation led to PILP-1-induced cell death and down-regulation of ADAM17. Knockdown of ADAM17 by siRNA induced death of U937 cells and inactivation of Lyn and Akt. Immunoprecipitation suggested that ADAM17 and Lyn form complexes. Overexpression of ADAM17, LynY507F (gain of function), and constitutively active Akt suppressed the cytotoxic effects of PILP-1. PILP-1-elicited inactivation of Lyn and Akt was abrogated in cells with overexpressed ADAM17 or LynY507F. Taken together, our data indicate that ADAM17-mediated activation of Lyn/Akt maintains the viability of U937 cells and that suppression of the pathway is responsible for PILP-1-induced apoptosis.

Keywords: Apoptosis, Metalloprotease, Protease Inhibitor, Signal Transduction, Snake Venom, ADAM17, Akt, Lyn

Abstract

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^{This work was supported by Grant NSC98-2320-B110-002-MY3 from the National Science Council, Taiwan (to L.-S. C.), and a grant from National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center.}

^{The on-line version of this article (available at}http://www.jbc.org) contains supplemental Figs. 1–5.

^{The abbreviations used are:}

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
TNFR: TNF-α receptor
Z: benzyloxycarbonyl
fmk: fluoromethyl ketone
PBMC: peripheral blood mononuclear cell
EGFR: EGF receptor
SH: Src homology.

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