|
|
Blood, Vol. 110, Issue 1, 82-90, July 1, 2007
Annexin II expressed by osteoblasts and endothelial cells regulates stem cell adhesion, homing, and engraftment following transplantation
Blood Jung et al.
110: 82
Supplemental materials for: Jung et al, Vol 110, Issue 1, 82-90
The N-terminal portion of AXII regulates the adhesion of KG1a cells to osteosarcoma cell lines
Given that AXII is localized to the endosteal surfaces and that antibodies raised against AXII disrupt the adhesion of HSC to OB, we investigated whether AXII itself acted as an adhesion ligand for KG1a cells. Purified AXII protein (10 µg/ml) was immobilized on 96-well plastic plates; these plates were then incubated with CFDA labeled KG1a cells. Binding of KG1a cells increased with increasing concentrations of AXII (Figure S2A). We also found that AXII substantially reduced the number of KG1a cells that bound to MG-63 and SaOS-2 cells (Figure S2B). To identify which region of AXII is responsible for the adhesion of KG1a cells to OBs, 12-amino-acid-long synthetic peptides were designed to correspond to a variety of regions of AXII. As shown in Figure 2C, each of these synthetic peptides bound KG1a cells significantly better compared to BSA. The peptide that corresponded to N-terminal amino acids 1–12 of AXII bound KG1a cells with the greatest affinity (∼1,500% increase in binding relative to the control). Competition binding assays were used to examine the effects of each synthetic peptide on the adhesion of KG1a cells to immobilized AXII. The peptides that corresponded to N-terminal amino acids 1–12, 13–24, and 199–210 of AXII each inhibited adhesion; the peptide that corresponded to N-terminal amino acids 1–12 produced the highest degree of inhibition of adhesion (Figure 2D). In addition, the same peptide also inhibited the adhesion of KG1a cells to MG-63 and SaOS-2 cells (Figure 2E).
Files in this Data Supplement:
- Figure S1. Cell blot–identified annexin-II (ANXA2) expressed on osteoblasts (OBs) mediates adhesion of KG1a cells (JPG, 28.0 KB)
-
SaOS-2 or MG-63 membrane lysates were run on replicative lanes of native lithium dodecyl sulfate–polyacrylamide gels before being blotted onto polyvinylidene fluoride membranes.1,2 After blocking the membranes with bovine serum albumin (BSA), the membranes were probed with biotin-labeled KGla cells for 2 hours at 37°C. The membranes were then fixed and developed with streptavidin-conjugated horseradish peroxidase and contents of the Vector VIP substrate kit. The figure is presented as the original image (An enhanced figure is provided in the manuscript). Adhesion of the biotin-labeled KG1a cells to proteins in the MG-63 membrane lysates revealed a band at ∼36 and ∼90 kDa.
- Figure S2. The N-Terminal Region of ANXA2 Regulates Adhesion of KG1a Cells to Osteosarcoma Cell Lines (JPG, 42.7 KB)
-
(A) Increasing concentrations of ANXA2 were used to coat 96-well plates. The coated plates were blocked with BSA before CFDA-labeled KG1a cells (1 × 104 cells) were deposited directly onto the plates. Adhesion was quantified after 15 minutes at 4°C. Maximal adhesion was achieved at 50 µg/mL, which was set as 100% (∼ 85% ± 12% of the input cells). (B) KG1a cells were deposited directly onto MG-63 or SaOS-2 monolayers in the presence or absence of 10 µg ANXA2 or denatured ANXA2 (100°C for 10 minutes) for 15 minutes at 4°C. Nonperturbed binding on MG-63 cells constituted 60% ± 15 % and 20% ± 12% on SaOS-2 cells of the 10 000 input KG1a cells. (C) To identify which regions of the ANXA2 protein bind KG1a, synthetic peptides that each comprised 12 amino acids that corresponded to the amino-acid sequence of ANXA2 were used to coat 96-well plates that were then blocked with BSA. Subsequently, CFDA-labeled KG1a cells were deposited directly onto the plates and adhesion was quantified relative to BSA controls, which bound less than 5% of the total input (10 000 cells/well) KG1a cells. (D) Competition assays using the 12-amino-acid-long peptides were used to block adhesion of KG1a cells to ANXA2-coated plates. (E) The synthetic peptide corresponding to the N-terminal 12 amino acids of ANXA2 (p1–12) was used to inhibit KG1a adhesion to MG-63 and SaOS-2 cells. Percent binding was set as the maximum achievable KG1a binding for each of the osteosarcoma cell lines. *P < .05 versus the control.
REFERENCES
1. Seshi,B. Discovery of novel hematopoietic cell adhesion molecules from bone marrow stromal cell membrane protein extracts by a new cell-blotting technique. Blood. 1994;83:2399-2409.
2. Seshi,B. Cell adhesion to proteins separated by lithium dodexyl sulfate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane: a new cell-blotting technique. J Immunol Methods. 1994;176:185-201.
|
|
