Systemic overexpression of TNFα-converting enzyme does not lead to enhanced shedding activity in vivo

Abstract

TNFα-converting enzyme (TACE/ADAM17) is a membrane-bound proteolytic enzyme with a diverse set of target molecules. Most importantly, TACE is indispensable for the release and activation of pro-TNFα and the ligands for epidermal growth factor receptor in vivo. Previous studies suggested that the overproduction of TACE is causally related to the pathogenesis of inflammatory diseases and cancers. To test this hypothesis, we generated a transgenic line in which the transcription of exogenous Tace is driven by a CAG promoter. The Tace-transgenic mice were viable and exhibited no overt defects, and the quantitative RT-PCR and Western blot analyses confirmed that the transgenically introduced Tace gene was highly expressed in all of the tissues examined. The Tace-transgenic mice were further crossed with Tace⁻/⁺ mice to abrogate the endogenous TACE expression, and the Tace-transgenic mice lacking endogenous Tace gene were also viable without any apparent defects. Furthermore, there was no difference in the serum TNFα levels after lipopolysaccharide injection between the transgenic mice and control littermates. These observations indicate that TACE activity is not necessarily dependent on transcriptional regulation and that excess TACE does not necessarily result in aberrant proteolytic activity in vivo.

Figure 1. The systemic overexpression of TACE causes no overt defects.

(A) A schematic of the transgene construct. pA, polyadenylation signal sequence. (B) Gross morphology of the 8-week-old control (Ctrl) and Tace-Tg (Tg) mice. (C) Quantitative RT-PCR analysis of Tace expression in the liver, lung, skin, spleen, bone marrow cells (BM), and thymus from 8-week-old control (Ctrl) and Tace-Tg (Tg) mice. The expression level of Tace in each organ of the control mice is set to 1. Bars, S.D. *p<0.05. **p<0.005. (D) Western blot analysis using anti-HA, anti-TACE, and anti-β-Actin antibodies. (E) Hematoxylin and eosin-stained sections of the spleen, liver, and tibia from 8-week-old control, Tace-Tg, and Tace-tg/Tace^−/− (Tg/Tace^−/−) mice.

Figure 2. An increase in the mature TACE in Tace-Tg mEFs does not significantly affect the shedding properties of TGFα.

(A) Quantitative RT-PCR analysis of Tace expression in the control (Ctrl) and Tace-Tg (Tg)-derived mEFs. (B, C) Cell surface molecules in mEFs (B) derived from control (Ctrl), Tace-Tg (Tg), and Tace^−/− (−/−) mice, and splenocytes (C) derived from control (Ctrl) and Tace-Tg (Tg) were biotin-labeled and analyzed by Western blotting before (Whole lysate) and after (Cell-surface) affinity precipitation with neutravidin beads. The membranes were reprobed with anti-β-Actin antibody and anti-ADAM10 antibody to serve as loading controls for total protein from the whole cell lysate and the cell-surface protein, respectively. Black arrowheads, pro-form. White arrowheads, mature form. Please note that there are small amount of biotinylated pro-form TACE and ADAM10 in panel (C) that were labeled through leakage of the biotin reagent during the procedure. (D) Evaluation of TGFα shedding in the control (Ctrl), Tace-Tg (Tg), and Tace^−/− mEFs by a colorimetric assay. Bars, S.D. *p<0.05. ns, not significant.

Figure 3. No apparent change in the serum levels of TACE substrates was observed in Tace-Tg mice.

(A) The serum levels of TNFα, TNFR1, TNFR2, and CD62L were analyzed by ELISA in control and Tace-Tg (Tg) mice. The sera were collected under unchallenged conditions (−) or 3 h after the intraperitoneal injection of LPS and D-galactosamine. (B) Survival curve of the control (Ctrl) and Tace-Tg (Tg) mice treated with LPS and D-galactosamine (n = 18). (C) Quantitative RT-PCR of Timp3 in the liver, lung, skin, spleen, bone marrow cells (BM), and thymus collected from the control (Ctrl) and Tace-Tg mice. Bars, S.D. ns, not significant.