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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02411

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Matrix cell adhesion activation by non-adhesion proteins

¹ Cell Biology Section, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
² The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229, USA
³ SWITCH Biotech AG, 82061 Neuried, Germany

View larger version (36K): [in a new window]   Fig. 1. Proteins at concentrations that promote similar levels of cell adhesion [human platelet osteonectin (10 µg ml–1), osteopontin (4 µg ml–1), fibronectin (1.25 µg ml–1) and laminin (2 µg ml–1)] were coated on 96-well plates and cell adhesion assays were performed as described. Results [mean% binding compared with control, no treatment, (white bars) ± s.d.] are shown for MDA-231 breast carcinoma cells; similar results were obtained with PC-3 prostate carcinoma cells. (A) Anti-integrin blocking antibodies (black bars) 5, v, 5 + v, ß1 and vß3 inhibited MDA-231 cell binding to human platelet osteonectin and fibronectin while a non-blocking 5 antibody (striped bar) did not. MDA-231 cell attachment is inhibited to laminin by 6 and ß1 anti-integrin antibodies and to osteopontin by 3, 6, v, ß1, vß3 and vß5 anti-integrin antibodies. (B) RGD peptide inhibits tumor cell adhesion to human platelet osteonectin at lower concentrations than to fibronectin or osteopontin. RGE was used as the control peptide. *P<0.05, **P<0.01, significant differences were determined in Dunnett's multiple comparison post-test, comparing no treatment (0, white bars) with treatment (peptide or integrin, black bars).

View larger version (21K): [in a new window]   Fig. 2. Bovine bone osteonectin (B.OSN) `activates' the cell adhesion activity of various extracellular matrix proteins. Attachment was assessed as described in Fig. 1. Matrix proteins + B.OSN (4 µg ml–1) were mixed together in solution before binding to the plate. These concentrations (µg ml–1) of matrix proteins [fibronectin (0.1, FN), collagen I (0.005, Col I), laminin (0.4, LM) and vitronectin (0.1, VN)] did not promote or promote a very low level of cell adhesion. Concentrations (µg ml–1) of human platelet osteonectin (10, H.OSN) and fibronectin (1.25, FN) that do promote cell adhesion were used as controls. Results (mean adhesion ± s.d.) are shown for MDA-231 breast carcinoma cells; similar results were obtained with PC-3 prostate carcinoma cells. **P<0.01 and ***P<0.001 significant difference between matrix protein alone (white bars) and matrix protein + B.OSN (black bars), between H.OSN and FN (1.25 µg ml–1) and between B.OSN + FN (0.1 µg ml–1) and FN (1.25 µg ml–1) was determined by Bonferroni's multiple comparison post-test.

View larger version (27K): [in a new window]   Fig. 3. Human platelet osteonectin (H.OSN) contains 1% fibronectin while bovine bone osteonectin (B.OSN) does not contain fibronectin. Serial diluted fibronectin (FN), 500 ng of H.OSN, B.OSN and B.OSN with 3.3 ng FN, was subjected to electrophoresis on 4–12% SDS-PAGE and analyzed by immunoblotting with anti-FN antibody. The amount of osteonectin (500 ng) corresponds to the amount of human platelet osteonectin that promotes cell adhesion. B.OSN was `spiked' with FN as a control. The amount of FN in the H.OSN was quantitated as described and determined to be 5.9 ng, approximately 1% of the H.OSN. This is a representative experiment from three separate experiments.

View larger version (18K): [in a new window]   Fig. 4. Fibronectin rescues cell attachment to human platelet osteonectin after fibronectin (FN) is immunoprecipitated from the human platelet osteonectin (OSN). (A) Fibronectin was immunoprecipitated from human platelet osteonectin (OSN+IP) and cell attachment was assessed as described in Fig. 1. After addition of fibronectin back to the fibronectin-depleted human platelet osteonectin (OSN+IP+FN), cell attachment was restored. The fibronectin added (0.1 µg ml–1) was 1% of the amount of human platelet osteonectin (10 µg ml–1) as determined in Fig. 3 to be the amount of fibronectin present in human platelet osteonectin. Immunoprecipitation with control IgG (OSN+IP con IgG) did not alter the level of cell attachment to human platelet osteonectin. Results (mean adhesion ± standard deviation) are shown for MDA-231 breast carcinoma cells. ***P<0.0001 significant difference from OSN tested by Bonferonni's multiple comparison post-test. (B) ELISA results for levels of fibronectin and osteonectin for mixtures used in A. This assay was not linear after absorbance of 0.4 OD and therefore this assay was used to determine the presence or absence of proteins. Minus sign (–) <0.1 OD, one plus sign (+) 0.1-0.4 OD, two plus signs (++) >0.4 OD; OD was measured at 450 nm. Each sample was measured in triplicate. Data are representative of three assays.

View larger version (25K): [in a new window]   Fig. 5. Effect of varying extracellular matrix protein and osteonectin levels on matrix adhesion `activation'. (A) Effect of varying the amount of osteonectin. A constant amount of fibronectin (0.1 µg ml–1) was coated on the plate together with the indicated amount of osteonectin and attachment was assessed as described in Fig. 1. (B) Effect of varying the amount of matrix protein. A constant amount of osteonectin (4 µg ml–1) was coated on the plate together with the indicated amounts of extracellular matrix protein and cell adhesion was assessed as described in Fig. 1. Results (mean adhesion ± s.d.) are shown for MDA-231 breast carcinoma cells. Approximately 50% of the total cells adhere to wells coated with 0.2 µg ml–1 fibronectin + osteonectin representing 2.8 OD. ***P<0.001 and *P<0.05 significant difference between protein alone and protein + OSN was determined by Bonferroni's multiple comparison post-test.

View larger version (47K): [in a new window]   Fig. 6. Integral distribution plots for van Holde-Weischet G(s) analysis. Circles: fibronectin; squares: osteonectin:fibronectin 1:5; triangles: osteonectin:fibronectin 1:1; diamonds: osteonectin:fibronectin 2:1.

View larger version (28K): [in a new window]   Fig. 7. Effect of bovine serum albumin (BSA) and cytochrome C on `activation' of low levels of fibronectin, laminin, vitronectin and collagen I for cell adhesion. Fibronectin (FN, 0.1 µg ml–1), laminin (LM, 0.4 µg ml–1), vitronectin (VN, 0.1 µg ml–1), collagen I (Col I, 5 ng ml–1), or no protein (none) were mixed with either no protein (None, white bars) or 4 µg ml–1 of bovine bone osteonectin (light gray bars), cytochrome C (dark gray bars), or BSA (black bars). Cell adhesion was then assessed as described. Results (mean adhesion ± s.d.) are shown for MDA-231 breast carcinoma cells. *P<0.05, **P<0.05 and ***P<0.001 significant difference between matrix protein alone and matrix protein + osteonectin, cytochrome C or BSA was determined by Bonferroni's multiple comparison post-test after finding a significant difference P<0.0001 among the groups by two-way analysis of variance (ANOVA).

View larger version (25K): [in a new window]   Fig. 8. Effect of osteonectin and cytochrome C on `activation' of low levels of plasma fibronectin (P.FN), cellular fibronectin (C.FN), or human fibronectin alpha-chymotryptic fragments 40 (40 kDa FN) or 120 kDa (120 kDa FN) for cell adhesion. Fibronectin (0.1 µg ml–1) or no protein (None) was mixed with either no protein (None, white bars) or with 4 µg ml–1 of bovine bone osteonectin (light gray bars) or cytochrome C (dark gray bars). Cell adhesion was then assessed as described. Results (mean adhesion ± s.d.) are shown for MDA-231 breast carcinoma cells. ***P<0.001 significant difference between matrix protein alone and matrix protein + osteonectin or cytochrome C was determined by Bonferroni's multiple comparison post-test after finding a significant difference P<0.0001 among the groups by two-way analysis of variance (ANOVA).

View larger version (26K): [in a new window]   Fig. 9. Schematic model of conformational changes associated with coating adhesion proteins on plastic plates in the presence and absence of non-adhesive proteins.

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