Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling

Abstract

Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.

Keywords: ADAM10; endotoxin; epithelium; inflammation; lung; protein degradation.

FIGURE 1.

Proteolysis of SIRPα in monocytes. a, THP-1 cells were seeded in serum-free medium for 2 h and treated with CHX and either vehicle, leupeptin, or MG132. Cells were harvested at various times post-CHX treatment and probed using a SIRPα COOH-terminal antibody. b, THP-1 cells seeded in serum-free medium for 2 h followed by treatment with CHX. Full-length SIRPα (top) and a cleavage product, SIRPc+m (middle), are shown after extended exposure. c, THP-1 cells seeded in serum-free medium for 2 h and treated with CHX 30 min after exposure to either vehicle, leupeptin (Leu), or MG132 (MG), and immunoblots were probed with antibodies as indicated. d, THP-1 cells were incubated with MG132, and total cellular lysates, membrane (Mem) and cytosolic (Cyto) proteins were isolated from cells and probed for SIRPα. e, THP-1 cells were incubated for 4 h with vehicle or PMA at the indicated concentrations with or without MG132 to stabilize the cleaved fragment. Cell lysates were harvested and processed for immunoblotting as indicated. f, RAW264.7 macrophages were incubated with PMA for 4 h with or without inclusion of MG132 and probed using a SIRPα COOH-terminal antibody. Each panel is representative of two to three experiments.

FIGURE 2.

Inflammatory mediators trigger SIRPα proteolysis in monocytes. a, THP-1 cells were pretreated with DMSO (vehicle (Veh)) or Compound 2 (Comp 2). At 30 min post-inhibitor treatment, cells were treated with LPS for various times prior to harvest. In the lower panels, MG132 (MG) was added 30 min post-treatment. b, THP-1 cells were incubated with PMA, TNFα (100 ng/ml), or LPS for 4 h. MG132 was added 30 min post-treatment. Cell lysates were harvested and processed for immunoblotting as indicated. Right, densitometry of stabilized SIRPc+m after treatment with vehicle, PMA, TNF, and LPS (n = 7 (PMA), n = 2 (TNFα), and n = 7 (LPS); data are mean ± S.D. (error bars)). **, p < 0.01 versus control (Con).

FIGURE 3.

Identification of a juxtamembrane region of SIRPα proteolysis. a, schematic of FLAG domains replacing amino acid residues in the SIRPα extracellular membrane-proximal region. Trans, transmembrane domain; Wt, wild-type SIRPα. b, diagram depicting the association of the FLAG domain with both SIRPc+m and the extracellularly released NH₂-terminal fragment. c, HeLa cells were transfected with a WT SIRPα-expressing plasmid. At 24 h post-transfection, medium was replaced with serum-free medium, and cells were incubated with vehicle, PMA, or MG132 (MG) for 6 h as indicated. d, HeLa cells were transfected with the various FLAG-containing SIRPα constructs shown in a. At 24 h post-transfection, the cells were incubated with PMA and MG132 for 6 h. SIRPα proteolysis was examined by immunoblotting using a SIRPα COOH-terminal antibody (top and third panels) or an antibody against the FLAG domain (second and bottom panels). FL, full-length SIRPα. e, HeLa cells were transfected with a plasmid encoding the FLAG site 3 construct representing release of the NH₂-terminal domain (a). At 24 h post-transfection, medium was replaced with serum-free medium, and cells were incubated with PMA and MG132 for 4 h and examined by immunoblotting using a SIRPα COOH-terminal antibody (second and third panels) and an antibody against the FLAG domain (top panel) to detect the cleavage and release of SIRPα. f, HeLa cells were transfected with various FLAG constructs as indicated and treated as described in d. Both cell lysates and concentrated cell medium were analyzed by immunoblotting using a SIRPα COOH-terminal antibody (top and fourth panels) or a FLAG targeted antibody (second, third, and fifth panels). g, HeLa cells were transfected with plasmids expressing WT SIRPα or a FLAG site 1.5 SIRPα mutant and examined for expression at 24 h post-transfection. Green, SIRPα; blue, DAPI. Scale bar, 5 μm. Each panel is representative of two to three experiments. Comp2, Compound 2.

FIGURE 4.

Electrostatic regulation of SIRPα proteolysis. a, schematic of the juxtamembrane region of hSIRP and mSIRP. FLAG site 1.5 in the juxtamembrane region was used as a backbone to generate a variety of charge-dependent SIRPα cut site mutants. A homologous region in the mSIRP was also utilized to examine SIRPα proteolysis. Trans, transmembrane domain. b–e, HeLa cells were transfected with the indicated plasmids. At 24 h post-transfection, cells were incubated with vehicle or PMA to stimulate proteolysis and MG132 (MG) or DAPT for 6 h. b, HeLa cells transfected with plasmids encoding WT, FLAG-D1, FLAG-1.5, FLAG-D2, and FLAG-R. c, HeLa cells transfected with plasmids encoding SIRPα WT, N359D, and N359K and treated with or without PMA or MG132. d, HeLa cells were transfected with plasmids encoding SIRPα WT or point mutants N359A, N359Q, N359R, N359E, and N359F. e, HeLa cells were transfected with plasmids encoding mSIRPα WT or mutant plasmids Q361A, Q361D, or D3 (MQT to DDD). Each panel is representative of two to three experiments.

FIGURE 5.

ADAM10 cleaves SIRPα. a, THP-1 cells were incubated with the serine protease inhibitor PMSF, the broad spectrum MMP inhibitor GM, or a selective MMP2/9 inhibitor (MMP2/9i) at the indicated concentrations followed by PMA treatment in the presence of MG132 (MG). Immunoblots were overexposed in the middle panel to visualize the SIRPα cleaved fragment. b, THP-1 cells were incubated with Compound 2 (Comp 2) or the ADAM10 inhibitor GI at the indicated concentrations and MG132 to stabilize SIRPc+m followed 30 min later by LPS. Immunoblots were overexposed in the middle panel to visualize the cleaved fragment. c, HeLa cells were transfected with hSIRP and mSIRP pretreated with vehicle (Veh), GM, or GI. Cells were then treated with PMA and DAPT. Each panel in a–c is representative of two to three experiments. d and e, HeLa or THP-1 cells were transfected with control DsiRNA (Dsi Con) or DsiRNA against ADAM10 (Dsi A10). d, cleaved SIRPα was visualized (top panel) in THP-1 cells treated with LPS and MG132 at 72 h post-DsiRNA transfection. Right, densitometry of full-length (FL) SIRPα after treatment with DsiRNA is shown (n = 4 experiments, mean ± S.D. (error bars)). *, p < 0.05 versus control (Con). e, at 24 h post-DsiRNA transfection, HeLa cells were transfected with the SIRPα FLAG site 3 construct to visualize cleaved extracellular SIRP. At 72 h post-DsiRNA transfection, cells were treated with PMA to stimulate proteolysis in the presence of MG132. Densitometry of the NH₂-terminal released FLAG-tagged domain after treatment with non-targeted control DsiRNA (Dsi Con) and DsiRNA (Dsi ADAM10 1–3) against three separate ADAM10 targets is shown (n = 6; three independent experiments; mean ± S.D. (error bars)). **, p < 0.01 versus control (Con) DsiRNA.

FIGURE 6.

SIRPc+m undergoes proteolysis by γ-secretase. a, THP-1 cells were treated with PMA to stimulate proteolysis and then incubated with the indicated concentrations of the proteasome inhibitors MG132 (MG) or bortezomib (BTZ) at 30 min post-treatment. Cells were harvested at 4 h post-PMA treatment for immunoblotting using the indicated antibodies. b, THP-1 cells were treated with LPS for 30 min and then treated with the calpain inhibitor PD150606 (PD) at the indicated concentrations, and cells were harvested 4 h post-LPS treatment for immunoblotting as indicated. c, THP-1 cells were treated with LPS for 30 min and then DAPT at the indicated concentrations and harvested 4 h post-LPS treatment. d, HeLa cells were transfected with a WT SIRPα-expressing plasmid. At 24 h post-transfection, cells were incubated with or without DAPT, lactacystin (Lact), or vehicle control. At 30 min post-DAPT and lactacystin treatment, cells were incubated with PMA or vehicle for 4 h. (* represents a γ-secretase-sensitive cleavage product termed SIRPcyto-C). e, SIRPα mutant plasmids were expressed in HeLa cells, and whole cell lysates (WCL) or cellular fractions were analyzed for proteolysis by immunoblotting 24 h after transfection. The SIRPc+m fragment encodes the putative ADAM10-cleaved SIRPα fragment (amino acids 359–504), whereasSIRPcyto-C encodes the potential γ-secretase-cleaved COOH-terminal domain of SIRPα (amino acids 398–504) followed by a V5 tag. f, HeLa cells were transfected with a plasmid expressing the putative SIRPc+m domain. At 24 h post-transfection, cells were incubated with vehicle, DAPT, or lactacystin. Cells were harvested 4 h post-DAPT/lactacystin treatment. g, THP-1 cells were pretreated with bortezomib and/or DAPT. 30 min later, cells were treated with PMA for 4 h. h, RAW264.7 macrophages were pretreated with lactacystin for 30 min prior to incubation with PMA with or without DAPT. Each panel is representative of two to three experiments. Mem, membrane; Cyto, cytosol; Sig, signal sequence; c+m, cytoplasmic + membrane domain.

FIGURE 7.

SIRPα is expressed and proteolytically regulated in human lung epithelia. a and b, lysates from normal human bronchial epithelium (NHBE) derived from three separate subjects (1, 2, and 3), the alveolar type II-like cell line A549, and the human bronchiolar epithelial cell line BEAS-2B were analyzed for SIRPα expression by immunoblotting. In b, human bronchial epithelial cells were probed for SIRPc+m. Patient sample 3 exhibited a cleaved fragment at the same mobility on gels as SIRPc+m when probed with a SIRPα COOH-terminal antibody. c, BEAS-2B cells were pretreated with GM or vehicle. At 30 min post-GM treatment, DAPT was added, and 30 min post-DAPT treatment, cells were incubated with PMA or vehicle for 4 h. d, BEAS-2B cells were incubated with vehicle, DAPT, or bortezomib (BZ) as indicated. At 30 min post-DAPT and bortezomib treatment, cells were incubated with PMA or vehicle for 4 h. e, BEAS-2B cells were incubated with DAPT for 30 min prior to incubation with PA103 (multiplicity of infection, 10) for 8 h (n = 3). f, THP-1 cells were preincubated with DAPT and infected with or without PA103 for 8 h as described above (n = 2). g, BEAS-2B were preincubated with GM, GI, and MMP2/9 inhibitor (2/9i) followed by incubation with PA103. Densitometry of the ratio of cleaved fragment versus full-length SIRPα is shown on the right (control (Con), n = 4; GI, n = 3; control (Con) PA103, n = 5; GI PA103, n = 5; four individual experiments; mean ± S.D. (error bars)). *, p < 0.05 versus uninfected control; #, p < 0.05 versus control PA103). h, BEAS-2B were transfected with control or ADAM10 (A10)-targeted DsiRNA for 48 h followed by incubation with PA103 (multiplicity of infection, 10). Densitometry of the ratio of cleaved fragment versus full-length SIRPα (DsiRNA control (DsiCon), n = 3; ADAM10-targeted DsiRNA (DsiA10), n = 3; three individual experiments; mean ± S.D. (error bars)). Densitometry is shown on the right. *, p < 0.05 versus DsiRNA control. The inset shows levels of ADAM10 protein after DsiRNA transfection.

FIGURE 8.

SIRPα proteolysis modulates inflammatory signaling. a, HeLa cells were incubated with TNFα (10 ng/ml) for the indicated times. Cells were harvested and probed for pIKKα/β and total IKKα. b, HeLa cells were transfected with WT SIRPα and treated with TNFα for 6 h to measure SIRPα proteolysis. c, WT SIRPα- and FLAG-1.5-transfected HeLa cells were treated TNFα in the presence or absence of GI. d, HeLa cells were transfected with an empty vector (EV), WT SIRPα, protease-resistant SIRPα (FLAG-1.5), or the proteolysis mutant SIRPc+m or SIRPcyto-C (Cyto-C). At 48 h post-transfection, cells were incubated with TNFα for 15 min. e, HeLa cells were transfected with an empty vector (EV), WT SIRPα, protease-resistant SIRPα mutants (FLAG-1.5 and FLAG-D2), and the enhanced proteolysis mutant (FLAG-R). Cells were treated as described above. f, HeLa cells were transfected with an empty vector (EV), protease-resistant SIRPα (FLAG-1.5), or the proteolysis mutant SIRPc+m (C+M) and treated with TNFα for 4 and 8 h. Cells were then harvested and immunoblotted for pSTAT1 and total STAT1. g, HeLa cells were transfected with empty vector, WT SIRPα, protease-resistant SIRPα (FLAG-1.5), or proteolysis mutant SIRPc+m. At 48 h post-transfection, cells were incubated with TNFα for 6 h, harvested, and treated as above. h, THP-1 cells were preincubated with GI followed by treatment with LPS for the indicated times. Cells were harvested and probed for pSTAT1 and total STAT1. i, BEAS-2B cells were preincubated with GI followed by incubation with PA103 (multiplicity of infection, 20) for the indicated times. Cells were harvested and probed for pSTAT1 and total STAT1. Each panel is representative of two to three experiments. Veh, vehicle.

FIGURE 9.

Regulatory model for immunomodulation by SIRPα proteolysis. Following exposure to some inflammatory mediators (TNFα, LPS, and PA103), ADAM10 is activated (1). An initial cleavage of SIRPα occurs at the membrane by ADAM10, releasing an NH₂-terminal COOH-terminal fragment (2) (SIRPc+m) that is preserved by DAPT. The SIRPc+m fragment is subsequently cleaved by γ-secretase (3), resulting in release of the COOH-terminal domain (SIRPcyto-C) into the cytosol. Decreased full-length SIRPα and the accumulation of cleaved SIRPα trigger phosphorylation of IKKα/β, suggesting activation of NF-κB, and an increase in STAT1 phosphorylation (4). Treatment with the ADAM10 inhibitor GI254023X, ADAM10 depletion, or cellular expression of the proteolysis-resistant SIRPα FLAG-1.5 protects against cleavage, leading to decreased phosphorylation of IKKα/β and STAT1 (5).