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a low level of MT-SP1, which was increased after THP-1 differentiation into macrophages by PMA treatment (Figure 4C and D, compare lanes 1 and 2). Treatment of THP-1-derived macrophages with hemin or RBC lysate further increased MT-SP1 expression (Figure 4C-F). Immunofluorescent staining of THP-1-derived macrophages demonstrated membrane and cytoplasmic distribution of MT-SP1 (Figure 4G, green) which was sig- nificantly increased upon treatment with hemin (10 μM) or RBC lysate (Figure 4G). Collectively, hemolysis prod- ucts (tested in this study) and hypoxia (meta-analysis data) demonstrated increased expression of MT-SP1 in human macrophages. Higher levels of MT-SP1 expression might induce local activation and accumulation of circulating MSP1.
MSP1 induces endothelial cell motility and activated downstream RON signaling of ERK and AKT
To assess the impact of MSP1 on the endothelium, we examined its effect on cultured human glomerular
endothelial cell line (HGEC) recently generated in our lab- oratory.22 Treatment with 1 μM MSP1 did not induce pro- liferation of HGEC (Figure 5A). In contrast, MSP1 treat- ment significantly increased motility of HGEC in a 2D wound assay (Figure 5B and C). F-actin plays a central role in the endothelial cell motility and permeability.32 Inflammatory mediators can alter F-actin formation and distribution.32 In confluent HGEC, F-actin formed mostly a cortical rim with few stress fibers (Figure 5D, green). MSP1 stimulated re-organization of F-actin and formation of stress fibers. (Figure 5D, green). Treatment cells with specific RON kinase inhibitor (200 nM; RONi) inhibited MSP1-induced formation of stress fibers and vWF expres- sion (Figure 5D, F-actin and vWF staining). MSP1 treat- ment also increased expression of vWF in HGEC (Figure 5D, red), and this increased expression was inhibited by RONi (Figure 5D). Next, we examined activation of RON kinase signaling in HGEC. Binding of MSP1 to RON leads to phosphorylation of the downstream kinases, ERK and AKT.33 Levels of ERK and AKT phosphorylation in HGEC
AB
P=0.008 P=0.002
CD
EF
G
Figure 4. Hemin, red blood cell (RBC) count and hypoxia treatment increase expres- sion of matriptase MT-SP1 in human THP1-derived macrophages. (A and B) Meta-analysis of membrane type serine protease 1 (MT- SP1) expression in human primary monocyte-derived macrophages using Geo database NCBI (A) Data Set GDS3203 and (B) Data Set GDS2036. For quantification graphs, mean and Standard Deviation (SD) are shown. (C and E) Western blot of MT- SP1 in human THP-1-derived macrophages treated with different concentrations of hemin (C) or RBC lysate (D). β-actin was used for normal-
ization. (D
Quantification of MT-SP1 on Western blot. For quantifica- tion graphs, mean and SD are shown. *P<0.05. Results are representative of three independent experiments. (G) Immunostaining of MT- SP1 in THP-1-derived macrophages treated with hemin (10 nM) or RBC lysate (green). DAPI was used for nuclear staining. Bar size 40 μm. PMA: phorbol 12-myris- tate 13-acetate.
and F)
haematologica | 2018; 103(5)