Adhesion of monocytes to type I collagen stimulates an APP-dependent proinflammatory signaling response and release of Abeta1-40

Abstract

Background: Amyloid precursor protein (APP) is a ubiquitously expressed cell surface protein reported to be involved in mediating cell-cell or cell-matrix interactions. Prior work has demonstrated that APP co-localizes with beta1 integrin in different cell types.

Methods: In an effort to determine the function of APP on monocytic lineage cells, in particular, the human monocyte cell line, THP-1, was used to assess the role of APP during adhesion to the extracelluar matrix component type I collagen.

Results: Pull-down assays demonstrated that THP-1 adhesion to collagen stimulated a tyrosine kinase-associated signaling response which included subsequent phosphorylation of p38 MAP kinase and increased association of APP with alpha2beta1 integrin, specifically. In addition, cell adhesion was dependent upon APP expression since APP siRNA knockdown attenuated THP-1 adhesion to collagen compared to mock transfected controls. One consequence of the tyrosine kinase-dependent signaling response was increased secretion of interleukin-1beta (IL-1beta) and Abeta1-40 but not the Abeta1-42 fragment of APP. Increased secretion of IL-1beta was dependent upon p38 MAP kinase activity while Abeta1-40 secretion required Src family kinase activity since the specific p38 inhibitor, SB202190, and the Src family kinase inhibitor, PP2, attenuated IL-1beta and Abeta1-40 secretion, respectively.

Conclusions: These data demonstrate that APP is involved in classic integrin-dependent tyrosine kinase-associated adhesion and activation of peripheral monocytic cells. Moreover, divergent APP-dependent signaling is required for increased secretion of both IL-1beta and Abeta1-40 as a component of the adhesion-dependent change in phenotype. This suggests that APP may have a broad role in not only mediating cell-matrix adhesion but also in the function of peripheral immune cells.

Figure 1

Adhesion of monocytes to collagen stimulated a tyrosine kinase-dependent signaling response and a tyrosine kinase-independent decrease in full-length APP protein levels. A) THP-1 cells were either untreated or treated for 30 minutes with PP2 (5 μM) or DMSO vehicle. Cells were plated on tissue culture plastic alone or on type I collagen. B) THP-1 cells were either not transfected or transfected with individual APP siRNA duplexes. At 24 hours post-transfection, non-transfected cells were unstimulated (control) or stimulated by adhesion to collagen, and transfected cells were stimulated by adhesion to collagen. Cells were plated on tissue culture plastic alone or on type I collagen. Cells were lysed at 30 minutes in RIPA buffer, separated by 7% or 10% SDS-PAGE and Western blotted using anti-phosphotyrosine antibody, 4G10, anti-phospho-p38 antibody, anti-p38 antibody (loading control), anti-APP antibody, and anti-ERK2 antibody (loading control). Antibody binding was visualized by chemiluminescence.

Figure 2

Ligation of α2β1 integrin with type I collagen stimulated co-localization with APP. A) THP-1 cells were pre-treated with LPS (25 ng/mL) for 24 hours prior to adhesion to collagen. Cells were plated on tissue culture plastic alone or on type I collagen. Cells were lysed at 30 minutes in 1% Triton X-100 buffer, and either α2 integrin, β1 integrin or APP was immunoprecipitated. Immunoprecipitates were resolved by 7% SDS-PAGE and Western blotted with anti-APP antibody, anti-α1 integrin antibody, anti-α6 integrin antibody, anti-α2 integrin antibody or anti-β1 integrin antibody. B) THP-1 cells were either untreated (c) or treated with LPS (25 ng/mL) for 24 hours. Cells were lysed in RIPA buffer separated by 7% SDS-PAGE and Western blotted using anti-α2 integrin antibody and anti-ERK2 antibody (loading control). C) THP-1 cells were untreated (con) or treated for 24 hours with LPS (1 μg/ml). Cells were plated on coverslips and fixed with 4% paraformaldehyde. Cells were immunostained with and without 1% triton permeabilization with anti-APP antibody. D) THP-1 cells were untreated (c) or treated for 24 hours with LPS (1 μg/ml) or 10 μM Aβ. Cells were lysed in RIPA buffer separated by 7% SDS-PAGE and Western blotted using anti-APP antibody and anti-ERK2 antibody (loading control). Antibody binding was visualized by chemiluminescence.

Figure 3

Collagen stimulates a selective increase in Aβ1-40 that is dependent on Src tyrosine kinase activity. THP-1 cells were either untreated or treated for 30 min with SB202190 (100 nM), PP2 (5 μM), γ-secretase inhibitor (10 μM), β-secretase inhibitor (5 μM) or DMSO vehicle. Cells were plated on tissue culture plastic or collagen for 48 hours. Media was then collected and analyzed by human Aβ1-40 and Aβ1-42 ELISA. Data were analyzed by unpaired ANOVA with Tukey's post-test comparison and are expressed as mean +/- SD (* = p < 0.001 from control, ** = p < 0.001 from collagen). SB202190 and PP2 treated collagen conditions were normalized to percent decrease due to drug only. Values are representative of three independent experiments.

Figure 4

Adhesion of monocytes to collagen stimulates a p38-dependent increase in IL-1β production. THP-1 cells were either untreated or treated for 30 min with SB202190 (100 nM), PP2 (5 μM), γ-secretase inhibitor (10 μM), β-secretase inhibitor (5 μM) or DMSO vehicle. Cells were then plated on tissue culture plastic or collagen for 24 hours. Media were collected and analyzed by human interleukin-1β ELISA. Data were analyzed by unpaired ANOVA with Tukey's post-test comparison and are expressed as mean +/- SD (* = p < 0.001 from control, ** = p < 0.01 from collagen, *** = p < 0.001 from collagen). Values are representative of three independent experiments.

Figure 5

Adhesion of monocytes to collagen is dependent on expression of APP. THP-1 cells were either not transfected or transfected with an individual APP siRNA duplex. A) At 24 hours post-transfection, cells were lysed with RIPA buffer, separated by 7% SDS-PAGE and Western blotted with anti-APP antibody and anti-ERK2 antibody (loading control). B) At 24 hours post-transfection, transfected cells and non-transfected cells were loaded with 5 μg/mL calcein-AM and plated on tissue culture plastic alone, poly-L-lysine, or type I collagen for 30 minutes. Fluorescence from total cell number was read prior to washing the wells. Wells were then washed and fluorescence of adhered cells was determined. Data were analyzed by unpaired ANOVA with Tukey's post-test comparison and are expressed as mean +/- SD (*** = p < 0.001 from control, ** = p < 0.001 from mock transfected cells). Values are representative of three independent experiments.