Abstract
ADAM17 is a transmembrane metalloprotease involved in the proteolytic release of the extracellular domain of many cell surface molecules, a process known as ectodomain shedding. Despite its likely participation in tumor progression and its current consideration as a therapeutic target, very little is known about the regulation of the expression of ADAM17. Here we show that long term treatment with epidermal growth factor (EGF) leads to a marked increase in the levels of ADAM17. EGF receptor activation does not affect the levels of the mRNA that encodes for, or the rate of synthesis of, ADAM17 but increases its half-life. The effect of EGF is biologically relevant because it increases the shedding of several substrates of ADAM17, including the desmosomal cadherin Dsg-2. Analysis of protein and mRNA levels in mammary tumor samples shows that in vivo the levels of ADAM17 can also be controlled post-transcriptionally. Finally, we show that both the shed extracellular domains of Dsg-2 and ADAM17 are frequently expressed in tumors, further supporting the participation of the metalloprotease in malignant progression.
Highlights
Removal of the ectodomain of a significant number of membrane-anchored proteins
ADAM17, know as tumor necrosis factor-␣-converting enzyme, participates in the activation of the epidermal growth factor receptor (EGFR) and related receptors [6], which play a crucial role in the development of different tumors of epidermal origin [7]
We did not analyze mRNA levels, we have recently found that ADAM17 protein levels are dramatically augmented in the majority of breast cancer samples analyzed [24]
Summary
Reagents and Antibodies—BB-94 and recombinant EGF were obtained from British Biotech and R&D Systems, respectively. After 24 h, cells were serumstarved for an additional 24 h and treated with 50 ng/ml EGF, 1 M Iressa ( known as ZD1839), 10 M BB-94, or 20 g/ml cycloheximide as indicated for different periods of time. The cells were lysed or washed three times with a quenching solution (phosphate-buffered saline containing 1 mM CaCl2, 0.5 mM MgCl2, and 100 mM glycine) and incubated at 37 °C for different times in the absence or presence of 50 ng/ml EGF and lysed. Metabolic Labeling and ADAM17 Immunoprecipitation— ϳ2 ϫ 104 cells/cm exponentially growing A431 cells were serum-starved and treated with EGF for different times, washed twice with methionine- and cysteine-free medium, and incubated for 30 min in medium supplemented with 1 mCi/ml Tran35S-label (Biolink 2000; Arlington Heights, IL). Precleared extracts were immunoprecipitated overnight at 4 °C with polyclonal anti-ADAM17 antibody, followed by a 1-h incubation at 4 °C with protein A-agarose. Tumor samples were fixed in 10% neutral formalin following the standard procedures and stained with a polyclonal antibody against the cytoplasmic tail of ADAM17
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