The hemopexin domain of matrix metalloproteinase-9 activates cell signaling and promotes migration of schwann cells by binding to low-density lipoprotein receptor-related protein

Abstract

Low-density lipoprotein receptor-related protein (LRP-1) is an endocytic receptor for diverse proteins, including matrix metalloproteinase-9 (MMP-9), and a cell-signaling receptor. In the peripheral nervous system (PNS), LRP-1 is robustly expressed by Schwann cells only after injury. Herein, we demonstrate that MMP-9 activates extracellular-signal-regulated kinase (ERK1/2) and Akt in Schwann cells in culture. MMP-9 also promotes Schwann cell migration. These activities require LRP-1. MMP-9-induced cell signaling and migration were blocked by inhibiting MMP-9-binding to LRP-1 with receptor-associated protein (RAP) or by LRP-1 gene silencing. The effects of MMP-9 on Schwann cell migration also were inhibited by blocking the cell-signaling response. An antibody targeting the hemopexin domain of MMP-9, which mediates the interaction with LRP-1, blocked MMP-9-induced cell signaling and migration. Furthermore, a novel glutathione-S-transferase fusion protein (MMP-9-PEX), which includes only the hemopexin domain of MMP-9, replicated the activities of intact MMP-9, activating Schwann cell signaling and migration by an LRP-1-dependent pathway. Constitutively active MEK1 promoted Schwann cell migration; in these cells, MMP-9-PEX had no further effect, indicating that ERK1/2 activation is sufficient to explain the effects of MMP-9-PEX on Schwann cell migration. Injection of MMP-9-PEX into sciatic nerves, 24 h after crush injury, robustly increased phosphorylation of ERK1/2 and Akt. This response was inhibited by RAP. MMP-9-PEX failed to activate cell signaling in uninjured nerves, consistent with the observation that Schwann cells express LRP-1 at significant levels only after nerve injury. These results establish LRP-1 as a cell-signaling receptor for MMP-9, which may be significant in regulating Schwann cell migration and physiology in PNS injury.

Figure 1.

Immunoblot analysis of pAkt and pERK1/2 after treatment with MMP-9 in LRP-1-inhibited and control cells. A, Primary cultures of Schwann cells treated with MMP-9 (100 nm) for 2 h. B, Primary Schwann cells treated with NRG-1 (0.2 nm), MMP-9 (10 nm) or MMP-2 (10 nm). C, Quantification of pERK1/2 to T-ERK1/2 ratios by densitometry after treatment with MMP-9 or NRG-1 (n = 4 independent experiments), *p < 0.05 compared with SFM. D, Primary Schwann cells were pretreated with GST-RAP (100 nm) 15 min before MMP-9 (10 nm) for 10 min. Erythropoietin (Epo; 4 units/ml) was used as a positive control. E, Primary Schwann cells pretreated with GST-RAP (100 nm) for 15 min before NRG-1 for 10 min. F, Primary Schwann cells were transfected with nontargeting control (NTC) siRNA or LRP-1-specific siRNA. Cells were treated with Epo or MMP-9 for 10 min. Equal amounts of cellular protein (50 μg) were loaded into each lane and subjected to SDS-PAGE and electrotransferred to nitrocellulose for detection with specific antibodies. Total ERK1/2 was used as a loading control. Each blot represents at least two to five independent studies.

Figure 2.

MMP-9 promotes Schwann cell migration that is blocked by the LRP-1 antagonist, GST-RAP. A, Images of primary Schwann cells that penetrated the Transwell membranes to the lower surfaces. The cells were pretreated with GST or GST-RAP (100 nm) for 15 min before treatment with MMP-9 (10 nm) or NRG-1 (0.2 nm). Migration was allowed to proceed for 4 h. B, Quantification of cell migration results. Data are expressed as mean ± SEM (n = 4–6). *p < 0.01 compared with respective vehicle controls.

Figure 3.

LRP-1 gene silencing in Schwann cells blocks basal cell migration and migration in response to MMP-9. A, Images of Schwann cells, transfected with NTC- or LRP-1-specific siRNA, which migrated to the underside surface of Transwell membranes. The cells were treated with MMP-9 (10 nm) or NRG-1 (0.2 nm). Migration was allowed to proceed for 4 h. B, Quantification of cell migration results. Data are expressed as mean ± SEM (n = 6). *p < 0.01 compared with respective vehicle controls. C, Schwann cell death was measured using the Cell Death ELISA in cells transfected with NTC siRNA or LRP-1-specific siRNA, after culturing for 4 h in complete medium (10% FBS), 0.5% FBS-containing medium, or Sato medium. *p < 0.05 compared with complete media.

Figure 4.

MMP-9-induced Schwann cell migration is blocked by inhibitors of MEK1 and PI3K. A, Images of Schwann cells that penetrated to the underside surface of Transwell membranes. The cells were pretreated with PD98059 (50 μm) or LY294002 (20 μm) for 15 min before the addition of MMP-9 (10 nm). Migration was allowed to proceed for 4 h. B, Quantification of cell migration results. Data are expressed as mean ± SEM (n = 4), *p < 0.01 compared with respective vehicle controls.

Figure 5.

Characterization of a GST-fusion protein encompassing the hemopexin domain/LRP-1-binding domain of MMP-9, MMP-9-PEX. A, Schematic diagram of MMP-9. Amino acids 514–704 are contained in GST-MMP-9-PEX. B, Immunoblot analysis of pAkt and pERK1/2 in Schwann cells that were pretreated for 15 min with nonspecific IgG or a monoclonal antibody that specifically recognizes the hemopexin domain in human MMP-9 (anti-MMP-PEX) in SFM and then with MMP-9 or NRG-1. Equal amounts of cellular protein (50 μg) were loaded into each lane and subjected to SDS-PAGE and electrotransferred to nitrocellulose for detection with specific antibodies. Blots represent n = 2. C, Quantification of Schwann cell migration after pretreatment with IgG or anti-MMP-9-PEX and stimulation with MMP-9. Migration was allowed to proceed for 4 h. Data are expressed as mean ± SEM (n = 2), *p < 0.01 compared with respective vehicle controls. D, Purification and characterization of MMP-9-PEX. Aliquots of different fractions (50 μg) were separated by SDS-PAGE and stained with Coomassie Blue. Ind, Induction; Endo, endotoxin. E, Immunoblot analysis of GST-MMP-9-PEX (1 μg). Blots were incubated with either a rabbit polyclonal antibody against GST or with a monoclonal antibody that targets the hemopexin domain of MMP-9.

Figure 6.

Immunoblot analysis of pAkt and pERK1/2 after treatment with MMP-9-PEX in LRP-1-inhibited and control cells. A, Primary Schwann cells treated with NRG-1 (0.2 nm), MMP-9 (10 nm), or MMP-9-PEX (10 nm) for 10 min. B, Primary Schwann cells pretreated with GST-RAP (100 nm) for 15 min before MMP-9-PEX (0–10 nm) for 10 min. C, Primary Schwann cells were transfected with NTC siRNA or LRP-1-specific siRNA. Cells were treated with NRG-1, MMP-9 or MMP-9-PEX for 10 min. All cells were solubilized in RIPA buffer supplemented with sodium orthovanadate and proteinase inhibitors. Equal amounts of cellular protein (50 μg) were loaded into each lane and subjected to SDS-PAGE and electrotransferred to nitrocellulose for detection with specific antibodies. Each blot represents at least three independent studies.

Figure 7.

MMP-9-PEX promotes Schwann cell migration that is blocked by the LRP-1 antagonist, GST-RAP, and by LRP-1 gene silencing. A, Images of Schwann cells which migrated to the underside surface of Transwell membranes. Cells were pretreated with GST or GST-RAP (100 nm) before the addition of MMP-9-PEX (10 nm). Migration was allowed to proceed for 4 h. B, Quantification of cell migration results. Data are expressed as mean ± SEM (n = 6), *p < 0.01 compared with respective vehicle controls. C, Images of Schwann cells transfected with NTC- or LRP-1-specific siRNA, which migrated to the underside surface of Transwell membranes. The cells were treated with MMP-9-PEX or vehicle. Migration was allowed to proceed for 4 h. D, Quantification of cell migration results. Data are expressed as mean ± SEM (n = 6), *p < 0.01 compared with respective vehicle controls.

Figure 8.

MMP-9-PEX-induced Schwann cell migration is blocked by inhibitors of MEK1 and PI3K. A, Images of Schwann cells that penetrated to the underside surface of Transwell membranes. The cells were pretreated with PD98059 (50 μm) or LY294002 (20 μm) for 15 min. MMP-9-PEX (10 nm) was subsequently added. Migration was allowed to proceed for 4 h. B, Quantification of cell migration results. Data are expressed as mean ± SEM (n = 4). *p < 0.01 compared with respective controls. C, MMP-9-PEX promotes Schwann cell migration by activating pERK1/2. Schwann cells were transfected with constitutively active MEK1 (CA-MEK) or empty vector (pEGFP). All cells were cotransfected with pEGFP to express GFP. Vehicle or MMP-9-PEX was added. Cell migration proceeded for 4 h. Migration was quantitated by counting green fluorescing-cells and expressed as a percentage of that observed with control cells that were transfected to express GFP only (mean ± SE, n = 2).

Figure 9.

MMP-9-PEX activates cell signaling selectively in injured sciatic nerve by binding to LRP-1. A, Immunoblot analysis of pAkt and pERK1/2 in uninjured or crush-injured rat sciatic nerves injected with 2 μl of MMP-9-PEX, GST, or MMP-9-PEX + GST-RAP. Nerves were isolated after 15 min. Injured nerves were injected 24 h after crush injury. Equal amounts of nerve protein (30 μg) were loaded into each lane and subjected to SDS-PAGE and electrotransferred to nitrocellulose for detection with specific antibodies. Each blot shows two rats per treatment. β-Actin or total ERK1/2 was used as loading control. B, Quantification of phosphorylated ERK1/2 to total ERK1/2 ratios by densitometry (n = 4–6 rats), *p < 0.01 compared with MMP-9-PEX+GST-RAP. C, Immunofluorescence microscopy for pERK1/2 in crush-injured rat sciatic nerves. The nerves were injected with GST or MMP-9-PEX 24 h after crush injury. Transcardial perfusion was initiated 15 min later. Images are at 100× magnification (scale bar, 100 μm). Note pERK1/2 immunoreactivity (green) in Schwann cell crescents (670× magnification). DAPI (blue) identifies nuclei in the nerve fiber. Images represent n = 4 per treatment.