Ectodomain shedding of TNF-α is enhanced by nardilysin via activation of ADAM proteases
Section snippets
Materials and methods
Plasmids. Expression plasmids for human NRDc (pcDNA3.1-hNRDc-V5), the enzymatically inactive mutant of human NRDc (pcDNA3.1-hNRDc E235A-V5), human TACE (pME18S-hTACE), and human ADAM10 (pME18S-hADAM10) were described previously [15], [16]. The human cDNA for full-length TNF-α was cloned into pME18S-FLAG to generate pME18S-hTNF-α-FLAG. The human cDNA for NRDc was cloned into a lentivirus vector, pLenti6 (Invitrogen), to generate pLenti6-hNRDc.
Antibodies and reagents. The antibodies against V5
NRDc and TACE synergistically enhances ectodomain shedding of TNF-α
To examine the effect of NRDc on ectodomain shedding of TNF-α, the transmembrane precursor of human TNF-α was transiently introduced into COS7 cells with NRDc and/or TACE. The amount of the soluble form of TNF-α (sTNF-α) released in the conditioned medium was detected by ELISA. As expected, TACE expression in COS7 cells significantly enhanced the release of TNF-α into the conditioned medium. Addition of NRDc expression dramatically and time-dependently increased the release of TNF-α, although
Acknowledgments
We thank N. Nishimoto and A. Fukumoto for excellent technical assistance and Drs. R. A. Black, K. Ono, and K. Matsumoto for providing materials. Financial support for this study was provided by Research Grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18590809, 18790305, 19041035). This work was also supported by the Takeda Science Foundation, Nagao Memorial Fund and Daiichi Sankyo Sponsored Research Program. The authors declare that they have no
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These authors equally contributed to this work.