Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with alphavbeta3 integrin that activates PKCalpha and RhoA

Abstract

Clustering of alphavbeta3 integrin after interaction with the RGD-like integrin-binding sequence present in neuronal Thy-1 triggers formation of focal adhesions and stress fibers in astrocytes via RhoA activation. A putative heparin-binding domain is present in Thy-1, raising the possibility that this membrane protein stimulates astrocyte adhesion via engagement of an integrin and the proteoglycan syndecan-4. Indeed, heparin, heparitinase treatment and mutation of the Thy-1 heparin-binding site each inhibited Thy-1-induced RhoA activation, as well as formation of focal adhesions and stress fibers in DI TNC(1) astrocytes. These responses required both syndecan-4 binding and signaling, as evidenced by silencing syndecan-4 expression and by overexpressing a syndecan-4 mutant lacking the intracellular domain, respectively. Furthermore, lack of RhoA activation and astrocyte responses in the presence of a PKC inhibitor or a dominant-negative form of PKCalpha implicated PKCalpha and RhoA activation in these events. Therefore, combined interaction of the astrocyte alphavbeta3-integrin-syndecan-4 receptor pair with Thy-1, promotes adhesion to the underlying matrix via PKCalpha- and RhoA-dependent pathways. Importantly, signaling events triggered by such receptor cooperation are shown here to be the consequence of cell-cell rather than cell-matrix interactions. These observations are likely to be of widespread biological relevance because Thy-1-integrin binding is reportedly relevant to melanoma invasion, monocyte transmigration through endothelial cells and host defense mechanisms.

Fig. 1.

Heparin and heparitinase treatments inhibit Thy-1-stimulated responses in astrocytes. (A) DI TNC₁ cells were serum starved for approximately 16 hours and then stimulated for 2 minutes with 3% fetal bovine serum (FBS, positive control) or 15 minutes with Thy-1-Fc-beads that had been previously treated with heparin (50-400 μg/ml) or left untreated for 30 minutes at 4°C. The cells were then lysed and affinity-precipitated active RhoA or total RhoA from whole cell lysates were visualized by immunoblotting with anti-RhoA monoclonal antibody. A representative western blot is shown. The values indicated were averaged from three independent experiments and indicate the fold increase in RhoA activity normalized to total protein. (B) DI TNC₁ astrocytes were treated with 0.5 mU of heparitinase (+Hase) for 3 hours at 37°C in serum-free medium, or left untreated (–Hase) for the same amount of time in serum-free medium. The cells were then stimulated for 10 minutes with Thy-1(RLD)-Fc-beads. Non-stimulated cells (NS) were used as controls. After the different treatments, cells were washed, fixed and permeabilized. Focal adhesions (FA) were stained with an anti-paxillin monoclonal antibody followed by Alexa Fluor 488 anti-mouse IgG (green). Stress fibers (SFs) were stained with Rhodamine-conjugated phalloidin (red) and were visualized by confocal microscopy. Scale bar: 15 μm. White squares indicate the areas shown at a higher magnification in the top row. (C) The average number of FAs per cell ± s.e.m. (white bars), as well as the average area per FA ± s.e.m. (black bars), obtained after treating the cells with medium (NS), Thy-1(RLD)-Fc-beads or Thy-1(RLE)-Fc-beads, were determined for at least 50 cells in each experimental condition using the ImageJ program and the function `Analyze particles'. The values shown were obtained by averaging data from at least three independent experiments. Non-parametric Mann-Whitney analysis was used to compare the data. Significant differences are indicated between NS and stimulated cells treated (+Hase) and non-treated (–Hase) with heparitinase (^*P<0.05). Significant differences are indicated in the average number of FAs per cell (white bars) between cells in the presence (+Hase) or the absence (–Hase) of heparitinase treatment (^**P<0.05).

Fig. 2.

Thy-1 stimulates FAK autophosphorylation of cells in suspension. Astrocytes in suspension (2×10⁵ cells) were treated for the indicated period of time with Thy-1(RLD)-Fc complexed to protein A (4:0.4 μg/tube), pre-incubated or not with heparin (200 μg/ml). Then, cells were rinsed with cold PBS and lysed in Laemmli buffer. Total protein extracts from DI TNC₁ astrocytes were separated by SDS-PAGE and analyzed by western blotting with anti-pY397FAK antibodies. Tyrosine phosphorylated FAK levels were quantified by scanning densitometric analysis of western blots and normalized to actin (graph). Phosphorylated Y397FAK levels in astrocytes treated with either Thy-1(RLD)-Fc (white bars) or heparin-treated Thy-1(RLD)-Fc (black bars) at different time points were compared with those in the non-stimulated control (gray bar). Results shown are the means ± s.e.m. of data from three independent experiments. Non-parametric Mann-Whitney analysis was used to compare the data. Statistically significant differences are indicated (^*P<0.05, ^**P<0.01).

Fig. 3.

Mutation of the heparin binding domain of Thy-1 renders the protein unable to bind heparin and to promote focal-adhesion formation. (A) Partial sequences (amino acids 1-45) of rat, mouse and human Thy-1. Underlined amino acid residues are almost identical for all three species. Gray boxes indicate the integrin-binding sequence (RLD) and the putative heparin-binding domain (REKRK) present in Thy-1. Mutations in both integrin- and heparin-binding domains were obtained by the double-PCR technique to obtain four different forms of the secreted Thy-1-Fc molecules by transiently transfecting HEK293T cells: wild-type Thy-1(RLD), integrin-binding-site-mutated Thy-1(RLE), double-mutated Thy-1(RLE)(AEAAA), HBD-mutated Thy-1(RLD)(AEAAA). (B) The aformentioned wild-type and mutated Thy-1-Fc proteins were precipitated from supernatants with protein-A-Sepharose or heparin-Sepharose, and analyzed by western blotting using anti-human IgG (Fc specific)-HRP. Additionally, the supernatant of cells transfected with pEGFP-C1 alone was used as a control. (C) The effect of these proteins on FA formation was tested and evaluated by immunofluorescence as indicated in Fig. 1C. White bars, the number of focal adhesions per cell; black bars, the area of focal adhesions. Values shown are mean ± s.e.m. of data from at least three different experiments. Non-parametric Mann-Whitney analysis was used to compare the data. Statistically significant differences are indicated (^*P<0.05).

Fig. 4.

Silencing of syndecan-4 with siRNA transfections decreases focal-adhesion and stress-fiber formation stimulated by Thy-1. Syndecan-4 silencing was performed by co-transfecting pEGFP-C1 with different siRNA directed against syndecan-4. (A) Expression levels of syndecan-4 and actin mRNA or syndecan-4 and syndecan-2 protein, in either non-transfected cells (Control) or cells transfected with siRNA control (Co siRNA) or different mixes of syndecan-4-specific siRNA, were measured by semi-quantitative RT-PCR or western blots, respectively. Bands obtained for syndecan-4 (S-4) and syndecan-2 (S-2) by western blot were quantified by densitometry and results are presented as average ratio ± s.e.m. between values obtained (S-4/S-2) from three independent experiments. (B) Astrocytes were co-transfected with pEGFP-C1 (Control GFP+) and either control siRNA or siRNA (1+2). After allowing GFP expression, cells were stimulated with Thy-1-Fc-beads for 10 minutes. The graph shows the percentage of GFP-positive cells that were stimulated by Thy-1 under each condition. Cells were scored as stimulated when elongated focal adhesions were present and there was at least a 1.5-fold increase in the number of focal adhesions per cell. Data were obtained from three independent experiments. Values mean ± s.e.m. calculated from counting at least 100 GFP-positive cells per condition.

Fig. 5.

Transfection of astrocytes with a syndecan-4 mutant lacking the cytoplasmic domain decreases Thy-1-stimulated focal-adhesion formation. Cells were transiently co-transfected with EGFP-containing plasmid and either truncated (A,C) or wild-type (B,D) syndecan-4. DI TNC₁ astrocytes were then left unstimulated (NS; A,B) or were stimulated with Thy-1(RLD)-Fc-beads [+Thy-1(RLD); C,D]. Samples were analyzed by immunofluorescence using mouse anti-paxillin antibodies followed by Cy3-labeled anti-mouse IgG. Red and green colors were inverted to improve visualization of focal adhesions; therefore, in images obtained using confocal microscopy, focal adhesions are in green in both non-transfected (no color) and GFP-transfected (red) cells. Scale bar: 25 μm. (E) Quantification of cells with focal adhesions within the transfected cell population in each transfection condition (GFP+) is shown for non-stimulated (gray bars) and Thy-1-stimulated cells (black bars). Cells with focal adhesions were evaluated as indicated in Fig. 4. Values were obtained by evaluating at least 100 GFP-positive cells per condition from three independent experiments. Statistically significant differences are indicated between values obtained for wild-type syndecan-4 with either transfected control (^**P<0.05) or mutated syndecan-4 (^*P<0.05). (F) Transfected DI TNC₁ cells were serum-starved overnight and then stimulated or not (NS) for 15 minutes with Thy-1-Fc-beads [+Thy-1(RLD)]. The cells were then lysed and active RhoA was affinity-precipitated with RBD-GST-beads. RhoA was visualized by immunoblotting as indicated in Fig. 1A. A representative result from three independent experiments is shown.

Fig. 6.

Pharmacological inhibition of PKCα reduces the formation of focal adhesions and stress fibers, as well as RhoA activity in astrocytes. (A) DI TNC₁ cells were treated with 100 nM Gö 6976, left untreated or treated with 0.01% DMSO (vehicle) for 30 minutes at 37°C in serum-free medium and were then either stimulated with Thy-1(RLD)-Fc-beads for 10 minutes or not stimulated (NS). Samples were analyzed by immunofluorescence as described in Fig. 1B. Scale bar: 25 μm. (B) The number of FAs per cell and the average area of FAs were determined as for Fig. 1C. Values shown are mean ± s.e.m. of data from at least three different experiments. Significant differences are indicated (^**P<0.05) between NS cells and cells stimulated with Thy-1(RLD)-Fc-beads, for the indicated conditions (–DMSO, white bars; + DMSO, gray bars). A significant difference (^#P<0.05) is indicated between cells treated or not with Gö 6976 (black bars versus gray bars) in Thy-1(RLD)-Fc-stimulated cells. (C) Astrocytes were serum-starved and then treated with either 10 nM or 100 nM Gö 6976 before stimulating with Thy-1(RLD)-Fc-beads for 15 minutes. Active RhoA levels were determined as described in Fig. 1A. Controls included starved cells and cells stimulated with 3% fetal bovine serum (FBS) for 2 minutes. Results are representative of two independent experiments.

Fig. 7.

The expression of a dominant-negative mutant of PKCα reduces the formation of focal adhesions and stress fibers, as well as RhoA activity in astrocytes. (A) DI TNC₁ cells were transduced with adenoviral vectors for wild type (wt) and dominant-negative (dn)PKCα, and empty vector using MOIs of 0, 300, 600 and 1000. After 24 hours, astrocytes were lysed and samples were analyzed by immunoblotting with anti-PKCα antibodies. Rat brain extract was used as a positive control (+C) for PKCα. Bands of 80 and 45 kDa correspond to full-length and the catalytic region of PKCα, respectively. (B) Astrocytes were transduced with empty or (dn)PKCα adenoviral vectors using a MOI of 1000. After 24 hours, cells were seeded on coverslips and, the next day, stimulated with Thy-1(RLD)-Fc-beads, or left unstimulated (NS). Focal adhesions and stress fibers were visualized by immunofluorescence as detailed in Fig. 1B. Scale bar: 25 μm. (C) Astrocytes were transduced with empty vector (light gray bars), (wt)PKCα (dark gray bars), (dn)PKCα (black bars) as in B or were not transduced (white bars). Cells were then stimulated with Thy-1(RLD)-Fc-beads, treated with TRAIL-R2-Fc-beads (negative control) or not stimulated (NS). For each experimental condition, the number of FAs per cell and the average size of FAs were determined as for Fig. 1C. Values shown are mean ± s.d. of one representative experiment performed in duplicate. (D) Astrocytes were transduced with the different adenoviral vectors described in C and RhoA activity was measured as in Fig. 1A. Results are representative of two independent experiments.