by Reuben Kapur, Ryan Cooper, Lei Zhang, and David A. Williams Cross-talk between α4β1/α5β1 and c-Kit results in opposing effect on growth and survival of hematopoietic cells via the activation of focal adhesion kinase, mitogen-activated protein kinase, and Akt signaling pathways by Reuben Kapur, Ryan Cooper, Lei Zhang, and David A. Williams Blood Volume 97(7):1975-1981 April 1, 2001 ©2001 by American Society of Hematology

Recombinant human FN peptides Recombinant human FN peptides.(A) A schematic representation of FN peptides. Recombinant human FN peptides.(A) A schematic representation of FN peptides. FN is made up of series of type I, II, and III repeats. Regions of FN with cell-binding activity are shown as the RGD-containing cell-binding domain (CELL), which is recognized by integrin α5β1, the nonintegrin-dependent high-affinity heparin-binding site (HEP), and the alternatively spliced non–type III connecting segment (III CS) that is recognized by integrin α4β1 (CS-1). The recognition sites for integrin α4β1, α5β1, as well as the heparin-binding site on FN are indicated by an arrow. (B) Expression of integrins and c-Kit on G1E-ER2 cells. G1E-ER2 cells were stained with anti–c-Kit-PE and analyzed by flow cytometry. The thin line indicates the level of background staining observed with appropriate isotype control antibody. The thick line indicates the level of c-Kit expression. (C) G1E-ER2 cells were stained with anti–α4β1-FITC and analyzed by flow cytometry. The thick line indicates the level of α4β1 expression. (D) G1E-ER2 cells were stained with anti–α5β1-PE and analyzed by flow cytometry. The thick line indicates the level of α5β1 expression. Reuben Kapur et al. Blood 2001;97:1975-1981 ©2001 by American Society of Hematology

Effects of adhesion.Comparison of the effects of adhesion to FN peptides on proliferation of G1E-ER2 cells (A) and primary EPCs (B). Effects of adhesion.Comparison of the effects of adhesion to FN peptides on proliferation of G1E-ER2 cells (A) and primary EPCs (B). Cells were cultured on FN-peptide–coated dishes mediating adhesion via both α4β1 and α5β1(CH296) or α4β1 (H296) or α5β1 (CH271) in the presence of SCF for 48 hours. Proliferation was measured by thymidine incorporation assay. Bars denote the mean thymidine incorporation (cpm ± SEM) of 6 different experiments performed in replicates of 6 (A), and one of the 2 representative experiments performed in replicates of 4 (B). Asterisk indicates P < .05 α4β1 (H296) and α4β1 and α5β1 (CH296) versus α5β1 (CH271). Reuben Kapur et al. Blood 2001;97:1975-1981 ©2001 by American Society of Hematology

FAK and MAPK (ERKs) activation after integrin-mediated adhesion FAK and MAPK (ERKs) activation after integrin-mediated adhesion.Factor-starved G1E-ER2 cells were cultured on FN peptides in the presence of SCF and analyzed at the indicated time points. FAK and MAPK (ERKs) activation after integrin-mediated adhesion.Factor-starved G1E-ER2 cells were cultured on FN peptides in the presence of SCF and analyzed at the indicated time points. (A) Cell lysates were collected and subjected to Western blot analysis with an anti-FAK antibody. Bottom panels indicate the position of phosphorylated (pFAK) and unphosphorylated (FAK) FAK. Upper panels (bars) demonstrate the relative phosphorylation of FAK; 48 hours taken as 100. Bars denote the mean relative phosphorylation (± SEM) of 3 different experiments. Asterisk indicates P < .05 α5β1 (CH271) versus α4β1 (H296). (B,C) Factor-starved G1E-ER2 cells were left unstimulated or cultured on BSA (B) or on indicated FN peptides mediating adhesion via α4β1 (H296) and α5β1 (CH271) (C) in the presence of SCF. Cell lysates were collected and subjected to Western blot analysis with a rabbit antiphospho-ERK antibody that specifically detects phosphorylated T202 and Y204. Bottom panels show total Erk in each lane. The positions of the phosphorylated ERK-1 (pErk-1) and ERK-2 (pErk2) are indicated. Upper panels (bars) demonstrate the relative phosphorylation of ERK-1 and ERK-2 at residues T202 and Y204. Data are presented relative to the phosphorylation of ERK-1 and ERK-2 after 10 minutes (B) (with the level at 10 minutes taken as 100) and 120 minutes (C) (with the level at 120 minutes taken as 100). Bars denote the mean relative phosphorylation (± SEM) of 3 different experiments. Asterisk indicates P < .05 α5β1(CH271) versus α4β1 (H296). (D) G1E-ER2 erythroid progenitors were cultured on BSA or on FN peptides H296 or CH271 in the presence of SCF and the MEK inhibitor (PD98059). Proliferation was measured by thymidine incorporation assay. Bars denote the inhibition in proliferation (± SEM) of 3 independent experiments performed in replicates of 6. Asterisk indicatesP < .05 α4β1 (H296) versus α5β1 (CH271) and BSA; and double asterisks indicate P < .05 α4β1 (H296) and α5β1 (CH271) versus BSA. Reuben Kapur et al. Blood 2001;97:1975-1981 ©2001 by American Society of Hematology

Ligation via α4β1 induces cell death in G1E-ER2 cells Ligation via α4β1 induces cell death in G1E-ER2 cells.G1E-ER2 erythroid progenitors were cultured on FN peptides mediating adhesion to α4β1 (H296) or α5β1 (CH271) or both α4β1 and α5β1(CH296) in the absence (A) or presence (B) of SCF for 48 hours. Ligation via α4β1 induces cell death in G1E-ER2 cells.G1E-ER2 erythroid progenitors were cultured on FN peptides mediating adhesion to α4β1 (H296) or α5β1 (CH271) or both α4β1 and α5β1(CH296) in the absence (A) or presence (B) of SCF for 48 hours. Cell death was quantitated by performing annexin V and PI staining as described in “Materials and methods.” Bars denote the percentage of total cell death (± SD) of 2 independent experiments performed in replicates of 3. Asterisk indicates P < .05 α4β1 (H296) and α4β1 and α5β1(CH296) versus α5β1 (CH271). Reuben Kapur et al. Blood 2001;97:1975-1981 ©2001 by American Society of Hematology

Inhibition of Akt activation via α4β1 enhances cell death in G1E-ER2 Inhibition of Akt activation via α4β1 enhances cell death in G1E-ER2.(A) Reduced activation of Akt at Ser473 via α4β1 (H296) adhesion. Inhibition of Akt activation via α4β1 enhances cell death in G1E-ER2.(A) Reduced activation of Akt at Ser473 via α4β1 (H296) adhesion. Factor-starved G1E-ER2 cells were left unstimulated or cultured on FN peptides mediating adhesion via α4β1 (H296) or α5β1 (CH271) in the presence of SCF for various time points. Subsequently, at various times, cell lysates were collected and subjected to Western blot analysis with a rabbit antiphospho-Akt antibody that specifically detects phosphorylated S473. Bottom panel shows total Akt in each lane. The position of the activated Akt (pAkt) is indicated. Upper panel (bars) quantitatively demonstrates the relative phosphorylation of Akt. Data are presented relative to the phosphorylation of Akt after 120 minutes (taken as 100). Bars denote the mean relative phosphorylation (± SEM) of at least 3 independent experiments. Asterisk indicatesP < .05 α4β1 (H296) versus α5β1 (CH271). (B) G1E-ER2 cells were cultured on BSA or FN peptide CH271 or H296 in the presence of SCF and the PI-3K/Akt inhibitor (wortmannin) for 48 hours. Cell death was quantitated by performing annexin and PI staining as described in “Materials and methods.” Bars denote the percentage of total cell death (± SD) of 2 independent experiments performed in replicates of 3. Asterisk indicates P < .05 BSA, α5β1 (CH271), α4β1 (H296) (wortmannin) versus BSA, α5β1 (CH271), α4β1 (H296) (no inhibitor). (C,D) G1E-ER2 cells were cultured on FN peptides in the presence of SCF and analyzed at the indicated time points. Cell lysates were collected and subjected to Western blot analysis with an anti–Bcl-2 antibody. The position of Bcl-2 and Bcl-xL is indicated. Reuben Kapur et al. Blood 2001;97:1975-1981 ©2001 by American Society of Hematology