A disintegrin and metalloproteinase 17 regulates TNF and TNFR1 levels in inflammation and liver regeneration in mice

Abstract

A disintegrin and metalloproteinase 17 (ADAM17), or tumor necrosis factor (TNF)-α-converting enzyme, is a key metalloproteinase and physiological convertase for a number of putative targets that play critical roles in cytokine and growth factor signaling. These interdependent pathways are essential components of the signaling network that links liver function with the compensatory growth that occurs during liver regeneration following 2/3 partial hepatectomy (PH) or chemically induced hepatotoxicity. Despite identification of many soluble factors needed for efficient liver regeneration, very little is known about how such ligands are regulated in the liver. To directly study the role of ADAM17 in the liver, we employed two cell-specific ADAM17 knockout (KO) mouse models. Using lipopolysaccharide (LPS) as a robust stimulus for TNF release, we found attenuated levels of circulating TNF in myeloid-specific ADAM17 KO mice (ADAM17 m-KO) and, unexpectedly, in mice with hepatocyte-specific ADAM17 deletion (ADAM17 h-KO), indicating that ADAM17 expression in both cell types plays a role in TNF shedding. After 2/3 PH, induction of TNF, TNFR1, and amphiregulin (AR) was significantly attenuated in ADAM17 h-KO mice, implicating ADAM17 as the primary sheddase for these factors in the liver. Surprisingly, the extent and timing of hepatocyte proliferation were not affected after PH or carbon tetrachloride injection in ADAM17 h-KO or ADAM17 m-KO mice. We conclude that ADAM17 regulates TNF, TNFR1, and AR in the liver, and its expression in both hepatocytes and myeloid cells is important for TNF regulation after LPS injury or 2/3 PH, but is not required for liver regeneration.

Keywords: amphiregulin; lipopolysaccharide; partial hepatectomy; tumor necrosis factor receptor; tumor necrosis factor-α converting enzyme.

Fig. 1.

Tissue-specific inactivation of a disintegrin and metalloproteinase 17 (ADAM17). Data presented are the results of real-time RT-PCR analyses using 18S expression for normalization. A: reduced expression of Adam17 in whole liver RNA from unoperated mice with hepatocyte-specific ADAM17 deletion (ADAM17 h-KO) (open bars) (n = 4–5). B: confirmed knockdown of Adam17 mRNA expression in primary hepatocytes from ADAM17 h-KO mice (n = 4–6). C: Adam17 mRNA levels in CD11b⁺ nonparenchymal cells (NPC) are similar in Cre- (black bars) and ADAM17 h-KO mice (n = 2–4). D: similar expression of Adam17 mRNA in whole liver RNA from Cre- and myeloid-specific ADAM17 KO (ADAM17 m-KO) mice (hatched bars) (n = 5). E: abrogated Adam17 expression in CD11b⁺ NPC cells from ADAM17 m-KO mice (n = 3–4) compared with controls. F: primary hepatocytes isolated from Cre- and ADAM17 m-KO mice express similar levels of Adam17 mRNA (n = 5). Data are presented as fold change using the values from Cre- controls as the denominator and are presented as means ± SE. #P = 0.09 and **P < 0.01. Non-op, unoperated mice.

Fig. 2.

Inhibition of tumor necrosis factor (TNF) and tumor necrosis factor receptor 1 (TNFR1) release after lipopolysaccharide (LPS) injection in ADAM17 m-KO and ADAM17 h-KO mice. Data presented are from samples collected 90 min after injection of saline or LPS (1 μg/g body wt). A: serum levels of TNF, as determined by ELISA, are significantly lower in ADAM17 h-KO and ADAM17 m-KO mice after injection of LPS compared with controls (n = 4–5). B: LPS induction of Tnf mRNA in the liver is similar between Cre-, ADAM17 m-KO, and ADAM17 h-KO mice (black, hatched, and open bars, respectively) (n = 2–5). C: serum levels of TNFR1, as determined by ELISA, are lower in ADAM17 h-KO and ADAM17 m-KO mice after injection of LPS compared with controls (n = 3–4). D: serum levels of IL-6 are significantly lower in ADAM17 h-KO mice, but not ADAM17 m-KO mice, compared with control mice after LPS injection (n = 5). ELISA data are presented as means ± SE. Real-time RT-PCR data are presented as fold change using the values from untreated Cre- controls as the denominator and are presented as means ± SE. *P < 0.05 and **P < 0.01 compared with Cre- group and normalized to 18S expression. ND, not detectable. Cre- represents control Adam17^FlNeo/FlNeo mice.

Fig. 3.

Suppression of cytokine and growth factor induction in the liver after partial hepatectomy (PH) in ADAM17 h-KO mice, determined by specific ELISAs. A: levels of TNF in liver tissue after PH are significantly lower in ADAM17 h-KO mice (open bars) compared with controls (black bars) (n = 3–5). B: levels of TNFR1 are lower in liver tissue from ADAM17 h-KO mice compared with controls after PH (n = 3–5). C: levels of serum IL-6 are similar in ADAM17 h-KO mice and control mice after PH (n = 2–6). D: upregulation of amphiregulin protein is abrogated after PH in ADAM17 h-KO mice compared with controls (n = 3–6). Data are presented as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with Cre- controls; n = 3–5 mice.

Fig. 4.

Liver regeneration after PH is largely unaffected in ADAM17 h-KO mice. A: liver Adam17 expression in Cre- controls is upregulated after PH compared with basal levels (n = 3–5). B: staining for bromodeoxyuridine (BrdU) indicates no effect of hepatic ADAM17 deficiency on DNA synthesis after PH (n = 3–7). C: the overall extent of mitosis after PH is similar in control and ADAM17 h-KO mice, except at the 48-h time point (n = 3–6). D: restoration of liver mass, assessed by liver-to-body weight (BW) ratio, after PH is unaffected by ADAM17 deficiency in hepatocytes (n = 3–6). Data are presented as means ± SE. *P < 0.05 compared with Cre- mice.

Fig. 5.

Induction of liver TNF after PH is abrogated in ADAM17 m-KO mice, but liver regeneration is not affected. A: levels of TNF protein in the liver after PH are significantly lower in ADAM17 h-KO mice compared with controls (n = 3–5). B: liver levels of TNFR1 protein in ADAM17 m-KO mice are not significantly different from controls at baseline or 6 h after PH (n = 3). C and D: DNA synthesis, as determined by BrdU incorporation (n = 4) (C) and mitosis (n = 4–5) (D) are unaffected in ADAM17 h-KO mice compared with Cre- littermates. E: restoration of liver mass, determined by liver-to-body weight ratio, is similar in ADAM17 h-KO mice and Cre- controls (n = 3–6). Data are presented as means ± SE. ***P < 0.001.

Fig. 6.

Liver regeneration after carbon tetrachloride (CCl₄)-induced hepatotoxicity in ADAM17 m-KO mice. A and B: CCl₄-induced release of liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST) is significantly reduced in ADAM17 m-KO mice compared with controls at 48 h (n = 3–4). TACE: tumor necrosis factor-α-converting enzyme. C and D: significant necrosis (traced) is evident in representative images of liver sections from Cre- and ADAM17 h-KO mice 48 h after CCl₄ injection. E: necrotic liver area, as determined using NIH Image J on hematoxylin and eosin (H&E)-stained sections, is similar in ADAM17 m-KO mice and controls (n = 3–6). F: caspase-3 activity in liver tissue is unaffected by ADAM17 inactivation in myeloid cells (n = 3–5). G and H: DNA synthesis and mitosis after CCl₄ injection are unaffected by ADAM17 deficiency in myeloid cells (n = 3–6). Data are presented as means ± SE. ***P < 0.001 compared with Cre- mice. Scale bars = 500 μm.