Unveiling the Role of Ecto-5'-Nucleotidase/CD73 in Astrocyte Migration by Using Pharmacological Tools

Abstract

CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5'-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,β-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.

Keywords: cell adhesion; ecto-5′-nucleotidase/CD73; migration; reactive astrocytes; scratch wound assay.

FIGURE 1

Validation of primary astrocytes culture model. The contents of CD73 in astrocyte cultures prior to (intact) or after creating the scratch wound (SW) were analyzed at mRNA, protein and functional level. (A) Expression of CD73 gene was determined by rt-PCR. Bars represent mean ± SEM of CD73-mRNA abundance relative to GAPDH, determined in n = 3 separate intact (black bar) and SW (white bar) culture preparation. (B) The abundance of CD73 protein was analyzed by Western blotting, after resolving whole cell lysates proteins on SDS-PAGE and probing the support membrane with the anti-CD73 antibodies (1:1500 in TBST; Cell Signaling, US). The antibodies recognized three protein bands on blots, with molecular weights of ∼69, 61, and 55 kDa. (C) Relative abundance of CD73 protein in each sample was assessed by measuring combined optical density of all bands in each lane by using ImageJ and by expressing the value relative to the optical density of GAPDH band in the same lane. The value obtained for intact culture was arbitrarily defined as 100% and was used as a reference (black bar). The bars represent mean CD73 relative protein abundance (% ± SEM) from n = 3 separate culture preparations. (D) Representative confocal images showing cell morphology and membrane topography of CD73 in confluent astrocyte monolayer (left) and cultures subjected to scratch wound (right). The images were obtained by double immunofluorescence labeling for CD73 (red fluorescence) and GFAP (green fluorescence) and nuclear counterstaining with DAPI (blue fluorescence). Scale bar = 25 μm. (E) The level of 5′-AMP phosphohydrolase activity in whole cell culture (black bars) and SW (white bars) cultures, determined in assay conditions as described in section “Materials and Methods.” Levamisole (100 μM) was used as the alkaline phosphatase inhibitor (striped bars). The bars represent mean activity (nmol Pi/mg/min) ± SEM, from n = 3 separate culture preparations performed in sextuplicate. Significance inside the graphs: ^∗ denotes significance at p < 0.05 in respect to intact culture.

FIGURE 2

Influence of different pharmacological inhibitors of CD73 on kinetics of astrocyte migration in vitro. Astrocytes were grown to confluence in normal FBS and wound was made by scraping the bottom of the dish with a sterile 200-μl pipette tip. The cultures were treated with anti-CD73 antibodies and APCP and the effects on the migration were compared with non-treated SW culture. In culture transfected with siCD73, negative control of the transfection was culture transfected with non-specific siRNA duplex (siCTR). (A) Representative images of defined microscopic fields taken at 0 and 24 after creating the wound in cultures treated with different pharmacological inhibitors of CD73. Digitalized images were captured and analyzed in 4-h intervals during 48 h. Scale bar = 100 μm. (B–D) Kinetic parameters of cell migration, cell front displacement (B), wound closure velocity (C) and wound closure (D) obtained in cultures treated with anti-CD73 antibodies (blue), APCP (green) or transfected with siCD73 (magenta). Bars represent means (±SEM) determined from 6 to 8 microscopic fields captured per each dish in n ≥ 7 independent culture preparations for anti-CD73 antibodies and APCP treatments and n = 3 for siCD73 transfection analysis. Significance inside the graphs: ^∗ denotes significance at p < 0.05 in respect to SW; ^# denotes significance at p < 0.001 in respect to siCTR.

FIGURE 3

Effect of adenosine on astrocyte migration. Effect of adenosine and adenine nucleotides on astrocyte migration in SW culture (A). Effect of adenosine, AMP and ATP on cell motility in the scratch wound assay in cultures treated with APCP (B) and anti-CD73 (C). Bars represent means (±SEM) determined from 6 to 8 microscopic fields captured per each dish in n ≥ 7 independent culture preparations. Significance inside the graph: ^∗ denotes significance at p < 0.05 in respect to SW.

FIGURE 4

Effect of pharmacological inhibitors on cell proliferation. Cell proliferation was determined in astrocytes subjected to scratch wound and treatment with APCP or anti-CD73 in the presence or absence of exogenous adenosine, and in the culture transfected with siCD73 or siCTR and subjected to scratch wound. Cells in SW culture were kept without any treatment. Cell proliferation was determined by fluorescence labeling for Ki67⁺, which labels only dividing nuclei and DAPI⁺, which labels all cell nuclei. (A) Representative images of Ki-67/DAPI staining. The cultures were fixed 24 h after the treatment. (B) The percentage of proliferating cells in total cell number in the same field (Ki-67⁺/DAPI⁺) was counted using ImageJ. Bars present mean percentage of proliferating cells (±SEM), determined in two separate culture preparations, 5–7 frames per each treatment per multiple coverslips. Significance inside the graph: ^∗ denotes significance at p < 0.05 in respect to SW.

FIGURE 5

Expression of CD73 in cultures treated with different pharmacological inhibitors. (A) 5′-AMP phosphohydrolase activity was assayed in whole cells, after subjecting the cultures to scratch wound and treatment with APCP (green), anti-CD73 antibodies (blue) and siCD73 (magenta). Bars represent mean relative phospohydrolase activity (%) ± SEM, from n = 7 independent determinations performed in sextuplicate. ^∗ denotes significance at p < 0.01 in respect to SW activity (1.13 ± 0.18 nmol Pi/mg/min); ^#denotes significance at p < 0.05 in respect to siCTR activity (0.76 ± 0.02 nmol Pi/mg/min). (B) Representative Western blot of whole cell lysates obtained from cultures treated with different pharmacological inhibitors. Blots were probed with anti-CD73 antibodies (1:1500 in TBST; Cell Signaling, US) or anti-GAPDH antibodies. (C) Abundance of CD73 protein relative to GAPDH (%) ± SEM, from n = 3 independent determinations. CD73/GAPDH ratio obtained for SW was arbitrarily defined as 100% and used as reference. Significance level inside the graph: ^∗ denotes significance at p < 0.05 in respect to SW; ^#denotes p < 0.05 in respect to siCTR.

FIGURE 6

Membrane topography of CD73. Confocal images of astrocyte cultures immunostained for CD73 (red fluorescence) and GFAP (green fluorescence) and counterstained for DAPI (blue fluorescence). Micrographs show astrocytes in SW culture and cultures treated with APCP and anti-CD73 antibodies at the wound edge (A–F) and at 1–3 cell-row distance away from the wound edge (G–L). Scale bar at F = 50 μm applicable to all micrographs. (H) Relative immunofluorescence intensity corresponding to CD73 (M) and GFAP (N) quantified using the ImageJ software. The results present mean integrated fluorescence density ± SEM, from two separate culture preparations and total 15 frames per treatment. ^∗ denotes p < 0.05 in respect to SW.

FIGURE 7

CD73 shedding. (A) Detection of soluble CD73 in culture media collected 24 h after the addition of the pharmacological inhibitors. Culture media were analyzed for presence of CD73 by dot blot, using either secondary IgG antibody or another set of anti-CD73 primary and matching secondary antibodies, as described in section “Materials and Methods.” Media from n = 4 separate cultures were used in the analysis. (B) Expression level of ERK1/2 in whole cell lysates obtained from cultures treated with different pharmacological inhibitors, detected by Western blotting using ERK1/2-specific antibodies.