Integrin-associated protein stimulates alpha2beta1-dependent chemotaxis via Gi-mediated inhibition of adenylate cyclase and extracellular-regulated kinases - PubMed (original) (raw)

Integrin-associated protein stimulates alpha2beta1-dependent chemotaxis via Gi-mediated inhibition of adenylate cyclase and extracellular-regulated kinases

X Q Wang et al. J Cell Biol. 1999.

Abstract

Integrin-associated protein (IAP/CD47) augments the function of alpha2beta1 integrin in smooth muscle cells (SMC), resulting in enhanced chemotaxis toward soluble collagen (Wang, X-Q., and W.A. Frazier. 1998. Mol. Biol. Cell. 9:865). IAP-deficient SMC derived from IAP(-/-) animals did not migrate in response to 4N1K (KRFYVVMWKK), a peptide agonist of IAP derived from the COOH-terminal domain of thrombospondin-1 (TSP1). When normal SMC were preincubated with 4N1K or an anti-alpha2beta1 function-stimulating antibody, cell migration to soluble collagen was significantly enhanced. 4N1K-induced chemotaxis was blocked by treatment of SMC with pertussis toxin indicating that IAP acts through Gi. In agreement with this, 4N1K evoked a rapid decrease in cAMP levels which was intensified in the presence of collagen, and forskolin and 8-Br-cAMP both inhibited SMC migration stimulated via IAP. 4N1K strongly inhibited extracellular regulated kinase (ERK) activation in SMC attaching to collagen and reduced basal ERK activity in suspended SMC. Pertussis toxin treatment of SMC significantly activated ERK, suggesting that an inhibitory input was alleviated. Inhibition of ERK activity by (a) the MAP kinase kinase (MEK) inhibitor, PD98059, (b) antisense oligonucleotide depletion of ERK, and (c) expression of mitogen-activated protein (MAP) kinase phosphatase-1 in SMC all led to increased migration to collagen, 4N1K, or 4N1K plus collagen. Thus, IAP stimulates alpha2beta1 integrin-mediated SMC migration via Gi-mediated inhibition of ERK activity and suppression of cyclic AMP levels. Both of these signaling pathways could directly modulate the state of the integrin as well as impact downstream components of the cell motility apparatus.

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Figures

Figure 1

IAP is necessary for stimulation of chemotaxis by 4N1K. SMC from both IAP+/+ and IAP−/− mice were tested for chemotactic migration when stimulated with 5 μg/ml collagen (Col), 100 μM 4N1K, or 100 μM 4N1K plus 5 μg/ml collagen-I (4N1K+Col). SMC were allowed to migrate through gelatin-coated filters toward attractants placed in the bottom wells of the micro-Boyden chamber. After staining, cells were counted in five high power fields in each of triplicate wells. Data is expressed as the percentage increased migration relative to control wells with no attractant to control for random motility. Error bars indicate standard deviation.

Figure 2

Activation of α2β1 results in enhanced chemotaxis. Human SMC were pretreated with 100 μM 4N1K, 4NGG, or 1:400 diluted anti-α2β1 antibodies for 30 min at 37°C before being added to the wells. 5 μg/ml of collagen-I was tested as chemoattractant. α2β1-1 was a function stimulating antibody, and α2β1-2 was a function blocking antibody. Data are expressed as cells counted per high power ±SD.

Figure 3

Adhesion of human SMC to collagen and gelatin. Human SMC were harvested as for chemotaxis assays and plated in 96-well plates coated with either collagen or gelatin at the indicated concentrations. After incubation and washing, adherent cells were quantified using a colorimetric assay as described (Wang and Frazier 1998).

Figure 4

Pertussis toxin inhibits chemotaxis. SMC were treated with pertussis toxin (PTX, 50 ng/ml) overnight in serum-free medium, and harvested with EDTA/trypsin. Cells were resuspended in MEM with 0.1% BSA and 50 ng/ml of pertussis toxin, and added directly to the chemotaxis chamber. Chemoattractants were as described in Fig. 1. Data are expressed as in Fig. 2.

Figure 5

4N1K decreases intracellular cAMP levels and pertussis toxin prevents the decrease in cAMP. (A) SMC were incubated in suspension with 4N1K (100 μM), 4NGG (100 μM), soluble collagen-I (5 μg/ml), or 4N1K plus collagen-I for the indicated times at 37°C. The cAMP assay was as described in Materials and Methods. (B) Cells were treated with pertussis toxin (50 ng/ml) overnight and then incubated with 4N1K (100 μM), or 4N1K plus collagen-I (5 μg/ml) for 40 min in suspension. Data are picomoles of cAMP ±SD.

Figure 5

Figure 6

Forskolin and 8-Br-cAMP inhibit SMC chemotaxis. SMC were incubated with forskolin (FK, 24 μM) or 8-Br-cAMP (100 μM) for 15 min before being added to the chemotaxis chambers. Data are cell number ±SD.

Figure 7

4N1K inhibits MAP kinase activation. (A) SMC were plated on gelatin- (100 μg/ml) coated dishes in the presence of 4N1K or 4NGG (100 μM) for the indicated times, and lysed in RIPA buffer. Cell lysates were subjected to immunoprecipitation with anti-ERK antibody. Kinase assays were performed as described in Materials and Methods. Data are MAP kinase activity ±SD. (B) SMC harvested as above were suspended in buffered serum free culture medium and treated indicated for 20 min when they were harvested and processed for ERK activity assay as above. Treatments were: control, no additions; 4NGG and 4N1K (100 μM); Col, collagen at 5 μg/ml; 4N1K+Col, 100 μM and 5 μg/ml; α2β1, function-stimulating anti-α2β1 mAb was used at a 1:50 dilution of the commercial preparation; mIgG, cross-linking anti–mouse IgG was at 20 μg/ml; α2β1+mIgG, both at the stated concentrations.

Figure 7

Figure 8

Pertussis toxin stimulates MAP kinase in SMC. SMC were treated with pertussis toxin (PTX) at the concentrations indicated overnight, and lysed directly in RIPA buffer. Cell lysates were subjected to immunoprecipitation using anti-ERK antibody. Kinase assays were performed as described in Materials and Methods. Data are MAP kinase activity ±SD.

Figure 9

Effect of PD 98059 on SMC chemotaxis. SMC were incubated with PD98059 (PD, 50 nM) for 30 min at 37°C before being added to the chemotaxis chambers. Chemoattractants were as described above. Data are cell number ±SD.

Figure 10

Anti-sense downregulation of ERK enhances SMC chemotaxis. (A) Cell migration assay. Cell migration was tested after ERK was depleted with anti-sense ERK oligonucleotide (AS). Data are expressed as the percentage of increased cell migration above that of the scrambled oligonucleotide-treated cells (SC). (B) Western blotting. The same cells tested in the migration assay in A were lysed in RIPA buffer, and after SDS PAGE, blots were probed with anti-ERK antibody. Lane AS, treated with anti-sense oligonucleotide; lane SC, treated with control oligonucleotide; lane C, no treatment. Blots were stripped and probed with an anti-actin antibody as a loading control.

Figure 10

Figure 11

Expression of MKP-1 in SMC enhances chemotaxis. (A). Western blotting. After transfection and recovery, cells were plated on 100 μg/ml gelatin-coated dishes for the indicated times after which cells were lysed in RIPA buffer, processed for SDS PAGE, and blotted. The activation of MAP kinase was detected through Western blotting with anti-activated MAP kinase antibody. S, cells kept in suspension; Control, untransfected cells attaching to collagen; MT, cells transfected with inactive mutant MKP1; WT, cells transfected with wild-type MKP1; VT, vector control transfectants. PMA, cells in suspension treated with 100 ng/ml PMA for 30 min. (B) Chemotaxis assay. The same cells were tested in the chemotaxis assay as above. Data are expressed as the percentage of increased cell migration over no stimulus.

Figure 11

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Integrin-associated protein stimulates alpha2beta1-dependent chemotaxis via Gi-mediated inhibition of adenylate cyclase and extracellular-regulated kinases - PubMed (original) (raw)