Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cδ, and p38 mitogen-activated protein kinase (MAPK)

Abstract

Mer tyrosine kinase (MerTK) is an integral membrane protein that is preferentially expressed by phagocytic cells, where it promotes efferocytosis and inhibits inflammatory signaling. Proteolytic cleavage of MerTK at an unidentified site leads to shedding of its soluble ectodomain (soluble MER; sMER), which can inhibit thrombosis in mice and efferocytosis in vitro. Herein, we show that MerTK is cleaved at proline 485 in murine macrophages. Site-directed deletion of 6 amino acids spanning proline 485 rendered MerTK resistant to proteolysis and suppression of efferocytosis by cleavage-inducing stimuli. LPS is a known inducer of MerTK cleavage, and the intracellular signaling pathways required for this action are unknown. LPS/TLR4-mediated generation of sMER required disintegrin and metalloproteinase ADAM17 and was independent of Myd88, instead requiring TRIF adaptor signaling. LPS-induced cleavage was suppressed by deficiency of NADPH oxidase 2 (Nox2) and PKCδ. The addition of the antioxidant N-acetyl cysteine inhibited PKCδ, and silencing of PKCδ inhibited MAPK p38, which was also required. In a mouse model of endotoxemia, we discovered that LPS induced plasma sMER, and this was suppressed by Adam17 deficiency. Thus, a TRIF-mediated pattern recognition receptor signaling cascade requires NADPH oxidase to activate PKCδ and then p38, culminating in ADAM17-mediated proteolysis of MerTK. These findings link innate pattern recognition receptor signaling to proteolytic inactivation of MerTK and generation of sMER and uncover targets to test how MerTK cleavage affects efferocytosis efficiency and inflammation resolution in vivo.

FIGURE 1.

Identification of the proteolytic cleavage site of MerTK. Soluble MER was isolated by immunoprecipitation and SDS-PAGE. Gel-purified bands were subjected to mass spectrometric analysis. LC-MS/MS analysis of three separate proteolytic digests (trypsin, chymotrypsin, and Arg-C) identified only peptides originating from the ectodomain of MERTK (A). The trypsin and chymotrypsin digests identified specific peptides in the near proximity of the putative transmembrane domain (sequence highlighted by bars above and below). In contrast, the Arg-C digest identified a peptide with a nonspecific site Pro⁴⁸⁵-Ser⁴⁸⁶ at the C terminus (B). The Arg-C peptide from the in-gel digest and the synthetic peptide IAA ITK GGI GPF SEP VNI IIP EHS KVD YAP were analyzed side-by-side by targeted LC-MS/MS on a high resolution instrument (LTQ-Orbitrap). The identical retention time, accurate mass at both 4⁺ and 3⁺ charge states (mass accuracy of <3 ppm), and MS/MS spectra of both 4⁺ and 3⁺ ions demonstrate correct identification (C).

FIGURE 2.

Deletion of six amino acids including proline 485 renders MerTK resistant to induced proteolysis and efferocytosis suppression by cleavage stimuli. A, mutation scheme indicating the region of site-directed deletion of residues 483–488 from the MERTK stalk, between the fibronectin-III ectodomain and the TM domain. B, Western blot for cellular MERTK and supernatant sMER after overnight transfection of WT or MERTK^Δ483–488 into HEK-293 cells. Post-transfection, cells were treated with or without PMA (50 nm) for 1 h, and cell supernatants and cell extracts were harvested for analysis. C, efferocytosis of apoptotic cells by HEK293 cells was measured by fluorescent microscopy after transfecting with wild type or mutant MerTK^Δ483–488 cDNA with or without PMA. p < 0.05, as indicated. n.s., not significant. Error bars, S.E.

FIGURE 3.

LPS-induced MerTK cleavage requires TLR4-TRIF signaling independent of Myd88. Primary macrophages were incubated with 10 ng/ml TNFα, 10 ng/ml IFNγ, or 50 ng/ml of LPS for 1 h (A), and cell supernatants were harvested for sMER (SOL) immunoblot. Cell lysates were run for full-length cellular MERTK in parallel, and densitometric analysis (bar graph) of the ratio of sMER/full-length MERTK and sMER/actin-loading control were measured. B, cell surface MerTK was measured by immunoblot after surface biotinylation and capture. Macrophages were treated with LPS for the indicated times followed by biotinylation. Densitometric analysis below was normalized to actin loading control. *, p < 0.05. C, sMER generation was measured by immunoblot post-LPS after silencing TLR4 with RNAi versus scrambled (sc) control. D, sMER generation post-LPS treatment in WT and Myd88-deficient primary macrophages. E, sMER generation requires Trif post-LPS treatment, as determined in Trif^−/− macrophages. F, poly(I:C) (shown in μg/ml) induces solMER. Primary macrophages were treated with the indicated doses of poly(I:C) for 2 h, and cell supernatants and cell extracts were subjected to immunoblot for soluble and full-length MER, respectively. Error bars, S.E.

FIGURE 4.

LPS-mediated MerTK cleavage requires NADPH. A, monolayers of elicited primary peritoneal macrophages were treated with 50 ng/ml LPS on tissue culture plates, and intracellular peroxide accumulation was assayed by DCF fluorescence as described under “Materials and Methods.” Three fields for each sample were quantified and expressed as a percentage of DCF-positive cells. B, Western blot of solMER from primary murine macrophage supernatants after treating cells with 1 mm NAC. NAC (freshly prepared) was preincubated with macrophages for 60 min prior to directly adding LPS. Corresponding cell extracts of full-length MERTK are shown below. C, sMER generation by LPS was measured from Nox2-deficient cells by Western blot. Densitometric analysis of the ratio of sMER (SOL) to full-length MERTK (FULL) is to the right. *, p < 0.05. Error bars, S.E.

FIGURE 5.

PKCδ is required for MerTK cleavage. A, primary macrophages were pretreated for 30 min with 250 nm Go6976 or 250 nm Go6983 in complete medium and subsequently treated with 50 ng/ml LPS. Subsequently, levels of sMER from cell supernatants and levels of full-length (FULL) MERTK from cell extracts were measured by Western blot. B, primary macrophages were incubated with PKCδ siRNA for 48 h, and the top panel exhibits representative knockdown efficiency of two PKCδ siRNAs (δ1 and δ2) by Western blot. In parallel, macrophages were cultured with LPS in the presence of PKCδ siRNA and scrambled (sc) control and sMER measured from supernatants and full-length MERTK from cell extracts by immunoblot. Densitometric analysis is shown to the right after knockdown with both PKCδ siRNAs. C, membrane translocation of PKCδ and phospho-PKCδ (PKCδ-P) post-LPS was determined by immunoblot after isolation of membrane pellets as described under “Materials and Methods.” Membrane translocation was also measured after treatment with NAC (right). M, membranous fraction; C, cytosolic fraction; T, total cellular lysate. Error bars, S.E.

FIGURE 6.

Generation of solMER by LPS requires ADAM17. A, immunoblots of sMER (SOL) and full-length MerTK (full) from Adam17^fl/fl and Adam17^fl/flLysmcre peritoneal macrophages with or without LPS. Macrophages from the indicated genotype were elicited and purified by adherence to tissue culture-treated plates in the presence of L-cell conditioned medium for 2 days. Subsequently, 50 ng/ml LPS in serum-free medium was added where indicated for 2 h, and supernatant was collected and concentrated from all samples for immunoblot of sMER. Parallel immunoblot of cellular lysates for full-length MerTK is indicated below. Where indicated, macrophages were pretreated with Adam10 siRNA. B, representative immunoblot of ADAM10 protein after either scrambled (sc) or siRNA (si) knockdown of ADAM10 immature (I) and mature (M) forms and indicated molecular weights. A, actin loading control.

FIGURE 7.

LPS-induced MerTK cleavage requires p38. A, the effects of p38 inhibition and ERK inhibition were determined on LPS-induced sMER generation. Primary peritoneal macrophages were preincubated with p38 (SB 202190, 10 μm) and ERK (PD98059, 10 μm) MAPK inhibitors for 30 min. Subsequently, 50 ng/ml LPS was added where indicated, and cell supernatant and cell extracts were probed by Western blot for sMER and cellular full-length MerTK, respectively. Densitometric measurement is to the right. *, p < 0.05. B, phosphorylation of p38 (P-38; Thr¹⁸⁰/Tyr¹⁸²) and MKK3 (P-MKK3; MKK3 Ser¹⁸⁹/MKK6 Ser²⁰⁷) after LPS treatment of primary macrophages was assessed by immunoblot of cell extracts. Analysis was also performed in the presence of PKCδ siRNA versus scrambled (sc) control. Densitometric analysis is shown to the right and normalized to actin loading control. Error bars, S.E.

FIGURE 8.

LPS induces sMER generation in vivo. Levels of sMER in murine plasma were determined by ELISA after injection of LPS (black bars) or control saline (gray bars). 8–10-week-old Mertk^KD, Adam17^fl/fl, and Adam17^fl/flLysmcre mice were injected with 100 μg of LPS or saline into the peritoneum, and plasma was harvested 3 h later from the left ventricle of the heart. Systemic plasma sMER (A) and plasma TNFα (B) were measured by ELISA as described under “Materials and Methods.” Each bar represents the mean of at least four animals per strain. *, p < 0.05. ND, not detected. Error bars, S.E.