Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice

Abstract

The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17(ex/ex) were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17(ex/ex) mice was normal, ADAM17(ex/ex) mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target.

Figure 1.

Targeted generation of ADAM17^ex/ex mice. (A) Scheme for targeting the ADAM17 gene. Open arrowheads denote FLP recombinase sites, and LoxP sites are indicated by closed arrowheads. The altered allele contains an artificial exon starting with an in-frame translational stop codon placed downstream of exon 11. The artificial exon (E11a, yellow) containing the stop codon is flanked by noncanonical splice donor and acceptor sites (see Materials and methods). The black bar indicates the hybridization probe used for Southern blotting. (B) mRNA from liver and brain was analyzed by RT-PCR. (C) ADAM17 and ADAM10 Western blots of skin and heart tissues. (D and E) Shedding of L-selectin from B cells (D) and TNF from spleen cells (E) was analyzed by FACS and ELISA, respectively. (F) Serum levels of sTNF-R_II were measured by ELISA. Data in D–F are shown as mean values ± SD. (G) Formation of milk ducts in mice at the age of 12 wk. Representative pictures from four animals are shown. Bars, 500 µm.

Figure 2.

ADAM17^ex/ex mice are highly susceptible to DSS-induced colitis. (A) Body weight after the onset of treatment with DSS. Numbers of mice per group used are indicated. The experiment was performed three times. (B) Colonoscopy of untreated (top) and treated (bottom) mice. Representative pictures are shown. (C) DSS-treated (4 d) and untreated WT and ADAM17^ex/ex mice were immunostained with anti-ADAM17 (magnification, 100×). Bars, 100 µm. Representative macroscopic pictures of four mice per group are shown. (D) Hematoxylin and eosin (H&E), BrdU, anti–phosho-STAT3, and anti-cyclin D1 staining of colons from mice challenged for 10 d with DSS. Bars: (H&E) 200 µM; (BrdU, p-STAT3, and cyclin D1) 100 µm. Representative microscopic images of three experiments (H&E and BrdU), two experiments (pSTAT3), and one experiment (cyclin D1) of ADAM17^ex/ex, ADAM17^WT/ex, and WT controls (five mice per group) are shown. (E) DSS colitis was induced and plasma FITC-dextran concentrations in ADAM17^WT/WT (n = 7) and ADAM17^ex/ex (n = 7) mice 4 h after FITC-dextran administration by oral gavage (60 mg/100 g of body weight) are shown. Data are shown as mean values ± SD. The experiment was performed twice with similar results.

Figure 3.

Intestinal architecture, epithelial proliferation, and gene expression in unchallenged ADAM17^ex/ex mice. (A) H&E staining of paraffin-embedded colon tissue from unchallenged ADAM17^ex/ex mice. Bar, 100 µM. Representative microscopic pictures of eight ADAM17^ex/ex mice are shown. (B) ADAM17^ex/ex (n = 3) and WT (n = 3) mice were injected intraperitoneally with BrdU and stained for BrdU in the distal colon. The arrowheads indicate BrdU-positive nuclei. Bars, 100 µm. The experiment was performed three times with three animals per group. (C) Pathway analysis of microarray data from colon tissue of WT (n = 3) and ADAM17^ex/ex (n = 3) mice. Regulated genes, which are significantly enriched or depleted during biological processes, are displayed. The bar represents the −log(p) of the probability of the enrichment or depletion occurring by chance. Processes that are associated with down-regulated transcripts are shown on the left, and those associated with up-regulated transcripts are shown on the right. The numbers in the bars represent the number of observed regulated transcripts in the category.

Figure 4.

Cellular accumulation of TGF-α and compensatory proliferation induced by recombinant TGF-α. (A) Colonic tissue sections from unchallenged and challenged WT and ADAM17^ex/ex mice (4 d, 2% DSS) were stained for TGF-α. Representative microscopic pictures of three mice per group are shown. Bars, 100 µm. (B) Colitis was induced in WT (n = 5) and ADAM17^ex/ex (n = 9) mice with 2% DSS for 10 d. ADAM17^ex/ex mice were injected daily with recombinant TGF-α (15 µg/mouse; n = 5) or with PBS (n = 4). Representative microscopic images of one experiment with five WT mice, four ADAM17^ex/ex mice treated with PBS, and five ADAM17^ex/ex mice treated with TGF-α are shown. Bars, 100 µm. The experiment was performed twice with similar results. (C) Colitis was induced in WT and ADAM17^ex/ex mice with 2% DSS and the animals were injected daily with the EGF-R ligand EGF (15 µg/mouse; WT, n = 3; ADAM17^ex/ex mice, n = 8) or with PBS, (WT, n = 8; ADAM17^ex/ex mice, n = 9) and body weight was recorded. Data were pooled from two independent experiments.