Mer tyrosine kinase (MerTK) promotes macrophage survival following exposure to oxidative stress

Abstract

The MerTK plays several important roles in normal macrophage physiology, including regulation of cytokine secretion and clearance of apoptotic cells. Mer signaling in other cell types, including malignant cells that ectopically overexpress the RTK, leads to downstream prosurvival pathway activation. We explored the hypothesis that Mer has a prosurvival role in macrophages exposed to oxidative stress. H(2)O(2) treatment of peritoneal exudate murine macrophages and J774 cells rapidly stimulated Mer phosphorylation in a concentration-dependent manner. Mer phosphorylation was dependent on the ligand Gas6, as treatment with warfarin or MerFc (a fusion protein of the extracellular domain of Mer and the Fc portion of human Ig), inhibitors of Gas6 activity, blocked H(2)O(2)-mediated activation of Mer. Antiapoptotic signals including pAkt and pErk 1/2 were increased dramatically (threefold and 4.5-fold, respectively) in WT Mer-positive macrophages compared with Mer KO macrophages stimulated with H(2)O(2). In a consistent manner, Mer expression led to decreased cleavage of proapoptotic indicators PARP and Caspase-3. Furthermore, Mer provided up to twofold enhanced cellular survival to primary macrophages exposed to H(2)O(2). These data represent the first report of Mer activation in response to oxidative stress and demonstrate the ability of Mer RTK to promote macrophage survival in disease states that involve an oxidative stress environment.

Figure 1.

MerTK activation is induced by H₂O₂ in a dose-dependent manner. (A and B) J774 mouse macrophage cells (A) or primary peritoneal murine macrophages (B) were serum-starved and stimulated with the indicated concentrations of H₂O₂ for 10 min. Whole cell lysates were immunoblotted with an anti-pMer (p-Mer) antibody or an anti-Mer (Mer) antibody. Actin (43 kD) was used as a loading control. (C) WT or Mer KO peritoneal exudate macrophages were treated with 400 μM H₂O₂ for 10 min, and cell lysates were immunoblotted with pMer antibody or total Mer antibody. Actin (43 kD) was used as a loading control. The data shown are representative of three independent experiments.

Figure 2.

H₂O₂ activation of Mer is a Gas6-dependent process. (A) Serum-starved J774 cells were untreated (Untx; Lane 1) or stimulated with 800 μM H₂O₂ (Lane 2) or 200 nM Gas6 for 10 min (Lane 3). Alternatively, starved cells were pretreated with 200 nM MerFc and then stimulated with H₂O₂ or Gas6 (Lanes 4 and 5). Whole cell lysates were immunoblotted with an anti-pMer antibody or an anti-Mer antibody. Actin (43 kD) was used as a loading control. (B) Serum-starved J774 cells were treated with 1 μM warfarin for 44 h prior to stimulation with 800 μM H₂O₂. Whole cell lysates were immunoprecipitated with anti-mouse Mer antibody and immunoblotted with anti-pMer antibody or anti-Mer antibody. (C) J774 cells were untreated or stimulated with 800 μM H₂O₂ as indicated. Conditioned media were collected at the indicated time-points, and an ELISA assay was performed for the presence of Gas6. The data shown are representative of three independent experiments.

Figure 3.

H₂O₂-induced Mer phosphorylation leads to activation of downstream antiapoptotic pathways. (A) Serum-starved WT and KO peritoneal exudate mouse macrophages were treated with 400 μM H₂O₂ for 10 min. Whole cell lysates were evaluated by immunoblot with antibodies for total and pAkt, as well as total and pErk 1/2. pAkt (B) and pErk 1/2 (C) were quantified by densitometry. To reflect relative phosphorylation, the IDV of the phospho band was divided by the IDV of the total protein band, and WT H₂O₂ was normalized to 100% for each experiment. Data shown are the mean ± sem from three independent experiments.

Figure 4.

Mer decreases PARP and Caspase-3 cleavage in response to H₂O₂ stimulation. WT and Mer KO peritoneal exudate mouse macrophages were harvested, serum-starved for 2 h, and treated with 400 μM H₂O₂ for 3 h. Whole cell lysates were immunoblotted with antibodies for PARP and Caspase-3, which detect unprocessed and cleaved forms. The data shown are representative of three independent experiments.

Figure 5.

Mer signaling provides a functional survival advantage for macrophages exposed to H₂O₂. WT and Mer KO peritoneal exudate mouse macrophages were harvested and serum-starved for 2 h. Prior to stimulation with 400 μM H₂O₂, macrophages were incubated in serum-free media, with or without 200 nM MerFc or RetFc for 2 h as indicated. Cultures were treated with H₂O₂ for an additional 3 h and then stained with trypan blue and counted in triplicate to determine cell viability. Survival of WT-untreated cells was normalized to 100% for each experiment. Data shown are the mean ± sem from three independent experiments.