Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

J. Cell Sci. 121 (4): 504-513

Integrin {alpha}9β1 is a receptor for nerve growth factor and other neurotrophins

Izabela Staniszewska1, Ilker K. Sariyer1, Shimon Lecht2, Meghan C. Brown1, Erin M. Walsh1, George P. Tuszynski1, Mahmut Safak1, Philip Lazarovici2, and Cezary Marcinkiewicz1,*

1 Department of Neuroscience, Center for Neurovirology and Cancer Biology, Temple University, School of Medicine, Philadelphia, PA 19122, USA
2 Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, School of Pharmacy, Jerusalem 91120, Israel


Figure 1
View larger version (23K):
[in this window]
[in a new window]

 
Fig. 1. Detection of {alpha}9 integrin subunit in a variety of tissues isolated from adult rat. The isolated tissues were cut into small pieces and lysed. Lysates were separated under reduced conditions by SDS-PAGE (7.5% gel) and electrotransferred into a PVDF membrane. Membrane was incubated with anti-{alpha}9 polyclonal antibody against the cytoplasmic domain of integrin (A) or with IgG isolated from pre-bleed serum of the same rabbit (B). The bands were visualized using a chemiluminescent western detection kit. The molecular mass markers are indicated by arrows. Lane a, lysate of mock-SW480 cells – negative control; lane b, lysate of {alpha}9SW480 cells – positive control; lane c, vena cava; lane d, aorta; lane e, brain; lane f, kidney; lane g, heart; lane h, lung; lane i, liver.

 

Figure 2
View larger version (23K):
[in this window]
[in a new window]

 
Fig. 2. Interaction of {alpha}9β1 integrin with neurotrophins in an adhesion assay. (A) Adhesion of {alpha}9SW480- (circles) and mock-SW480- (triangles) transfected cells to immobilized mNGF (filled symbols) and pro-NGF (open symbols). mNGF or pro-NGF was immobilized in a 96-well microplate in PBS by overnight incubation at 4°C. Cells were labeled with CMFDA and added to the wells, previously blocked by BSA. Incubation was performed at 37°C for 30 minutes in HBSS buffer containing calcium and magnesium. After washing off unbound cells, Triton X-100 was added to the wells and the plate was read using a fluorescence microplate reader. The number of adhered cells was calculated from the standard curve prepared in parallel in the same plate from a known number of cells. (B) Adhesion of {alpha}9SW480 cells (filled bars) and mock-SW480 cells (open bars) to immobilized neurotrophins, growth factors, monoclonal antibody and adhesion molecule. All proteins were immobilized on 96-well plates at a concentration of 10 µg/ml in PBS by overnight incubation at 4°C. Adhesion experiments were performed as described above. (C) Effect of various monoclonal antibodies and snake venom disintegrins on adhesion of {alpha}9SW480 cells to immobilized (10 µg/ml) mNGF. Monoclonal antibodies Y9A2 (anti-{alpha}9β1), Lia1/2 (anti-β1), SAM-1 (anti-{alpha}5) LM609 (anti-{alpha}vβ3), or snake venom disintegrins at concentrations of 10 µg/ml, were preincubated with CMFDA-labeled cells for 15 minutes before adding to the wells for adhesion. Adhesion experiments were performed as described above. (D) Effect of mNGF and VLO5 on expression of LIBS epitope on the β1 subunit of integrin in {alpha}9SW480 cells. Anti-LIBS, anti-human monoclonal antibody (B44), was immobilized in 96-well plates at a concentration of 10 µg/ml in PBS, overnight at 4°C. VLO5 or mNGF at a concentration of 1 µM was preincubated with CMFDA-labeled {alpha}9SW480 cells for 15 minutes before addition to the wells for adhesion. Adhesion experiments were performed as described above. Error bars indicate the standard deviation from three independent experiments.

 

Figure 3
View larger version (8K):
[in this window]
[in a new window]

 
Fig. 3. Binding of mNGF to purified {alpha}9β1 integrin in ELISA assay. {alpha}9β1 integrin isolated from {alpha}9K562 cells was immobilized in a 96-well plate at a concentration of 0.5 µg/ml by overnight incubation at 4°C in PBS. After blocking with 5% non-fat milk, a range of concentrations of hrNGF were added to the wells, which were incubated for 30 minutes at 37°C. The wells were then washed, and bound ligand was detected by adding the primary anti-NGF polyclonal antibody and incubation for 60 minutes at 37°C. After washing, the goat anti-rabbit alkaline-phosphatase (AP)-conjugated IgG was added and incubation was continued for another 60 minutes at 37°C. The color was developed with AP substrate (4-nitrophenylphosphate) and the plate was read using an ELISA plate reader at a 405 nm single wavelength. The specific binding ({blacktriangleup}) was estimated by subtraction of binding of NGF to the blocker ({circ}) from binding of NGF to immobilized integrin (bullet). The error bars represent the standard deviation from three duplicated independent experiments.

 

Figure 4
View larger version (29K):
[in this window]
[in a new window]

 
Fig. 4. Identification of TrkA and p75NTR receptors on SW480 cells. (A) RT-PCR analysis of β-actin, TrkA and p75NTR mRNA; the first lane shows markers of a DNA ladder. (B) Comparison of the intensity of bands from the RT-PCR gel in relation to the intensity of β-actin. Bands that are visualized in A were scanned into Uni-Scan-It software and digitalized to obtain the pixel numbers. The relative presence of mRNA of analyzed receptors was calculated relative to β-actin mRNA. Filled bars, TrkA; open bars, p75NTR. Error bars indicate s.d. from five measurements. (C) Binding of 125I-NGF to three cell lines in the presence or absence of VLO5. VLO5 (1 µM) was added to the cell suspension and experiments were performed as described in Materials and Methods. Filled bars, specific binding of NGF in the absence of VLO5; open bars, in the presence of VLO5. Error bars indicate s.d. from three experiments, each performed in triplicate. (D) Western blot analysis of cell lysates obtained from PC12 (lane a), {alpha}9SW480 (lane b), mock-SW480 (lane c), and SY-SY5Y (lane d) using anti-TrkA polyclonal serum. The bands were visualized using a chemiluminescent western detection kit. The numbers above the bands represent the value of the average pixels, reflecting intensity of the bands, digitalized using Un-Scan-It gel software.

 

Figure 5
View larger version (44K):
[in this window]
[in a new window]

 
Fig. 5. {alpha}9β1 integrin-dependent signaling induced by mNGF. (A) {alpha}9SW480 cells were allowed to adhere to immobilized mNGF (20 µg/ml) or BSA for 30 minutes in the absence or presence of {alpha}9β1 integrin inhibitors, Y9A2 (10 µg/ml) or VLO5 (10 µg/ml). Cell lysates were obtained and equal amounts of protein were separated under reducing conditions by 10% SDS-PAGE. The proteins from the gel were electro-transferred onto a PVDF membrane and incubated with primary anti-phospho-Erk1/2 (Thr202/Tyr204) and anti-Erk1/2 polyclonal antibodies. The bands were visualized using chemiluminescent western detection kit. The numbers above the bands represent the average number of pixels, reflecting intensity of the bands in the presented scans, digitalized using Un-Scan-It gel software. (B) Paxillin phosphorylation in {alpha}9SW480 cells induced following binding to immobilized mNGF. Anti-phospho-paxillin (Tyr31) and anti-paxillin polyclonal antibodies were used. (C) Effect of mNGF on phosphorylation of Erk1/2 in GD10 cells transfected with β1 subunit or hybrid β1/β3 subunit and co-transfected with {alpha}9 integrin subunit. Cells were cultured on the plate and stimulated with or without mNGF (50 ng/ml) for 1 hour. Cells were lysed and detection of phosphor-Erk1/2 and total Erk1/2 was performed as described above. All signaling experiments were repeated at least three times.

 

Figure 6
View larger version (16K):
[in this window]
[in a new window]

 
Fig. 6. Chemotaxis of {alpha}9SW480 cells to mNGF. (A) Chemotaxis was assessed in a Boyden chamber with fluoroblock membranes (8.0 µm). Calcein-labeled cells were added to the upper chamber of the membrane with immobilized collagen IV, and mNGF was added to the lower chamber as a chemoattractant. Random migration is indicated by {blacksquare}; bullet, 2.5 µg/ml; {circ}, 5 µg/ml; {blacktriangleup}, 10 µg/ml; {triangleup}, 20 µg/ml. (B) Inhibition of chemotaxis of {alpha}9SW480 cells to mNGF (5 µg/ml). The measurement of migration was assessed after 4 hours. Random migration is represented by bar a; control migration to mNGF without inhibitors, bar b; inhibition by control mouse IgG (10 µg/ml), c; by Y9A2 (10 µg/ml), d; by disintegrins eristostatin (1 µM), e: and by VLO5 (1 µM), f. Error bars indicate s.d. from three separate experiments. *Significant difference (P<0.001) in relation to the group treated with 5 µg/ml of mNGF alone (b).

 

Figure 7
View larger version (17K):
[in this window]
[in a new window]

 
Fig. 7. Effect of NGF and {alpha}9β1 integrin inhibitors on cell proliferation in BrdU assay. (A) {alpha}9SW480 cells (bullet) or mock-SW480 cells ({circ}) were grown in 96-well plates to 70% confluence and starved for 48 hours in FBS-free medium. Cells were not treated or treated with different concentrations of hrNGF in serum-free medium for 48 hours. BrdU color development assay was performed according to the manufacturer's instruction (Roche). Error bars indicate s.d. from triplicate experiments. (B) Effect of {alpha}9β1 integrin and TrkA inhibitors on mNGF-induced proliferation of {alpha}9SW480 cells. Cells were non-treated (a) or treated with 1 µg/ml of mNGF (b) in the presence of 10 µg/ml control mouse IgG (c) 10 µg/ml Y9A2 (d), 1 µM disintegrins eristostatin (e) and VLO5 (f), 10 µg/ml control rabbit serum (g), 10 µg/ml anti-TrkA polyclonal serum (h), or 50 µg/ml vincristine (i). Error bars indicate s.d. from three separate experiments. *Significant difference (P<0.001) in relation to the group treated with 1 µg/ml of mNGF alone (b).

 

Figure 8
View larger version (15K):
[in this window]
[in a new window]

 
Fig. 8. Chemotaxis of neutrophils to {alpha}9β1 integrin ligands. (A) Chemotaxis was assessed in a Boyden chamber with fluoroblock membranes (3.0 µm). Neutrophils isolated from human blood were labeled with calcein by incubation at 37°C for 30 minutes. Neutrophils were added to the upper chamber, and integrin ligands such as Y9A2 (5 µg/ml), VLO5 (1 µM) and mNGF (5 µg/ml), or 2% FBS were applied to the lower chamber. The plate was incubated at 37°C for 2 hours. Migration level was estimated as described in Fig. 5. Error bars indicate s.d. from three separate experiments. All chemoattracted groups were significantly different in comparison with random migration (P<0.001). (B) Inhibition of chemotaxis of neutrophils to mNGF. Migration was assessed after 4 hours. Random migration is represented by bar a and control migration to mNGF without inhibitors by bar b. Inhibition of chemotaxis of neutrophils to mNGF (5 µg/ml) by control mouse IgG (10 µg/ml), c; Y9A2 (10 µg/ml), d; disintegrins eristostatin (1 µM), e and VLO5 (1 µM), f. Error bars indicate s.d. from three separate experiments. *Significant difference (P<0.001) in relation to the group treated with 5 µg/ml of mNGF alone (b).

 


To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882