A 17-residue sequence from the matrix metalloproteinase-9 (MMP-9) hemopexin domain binds α4β1 integrin and inhibits MMP-9-induced functions in chronic lymphocytic leukemia B cells.
We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4β1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4β1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC(50) values of 138 and 279 μm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesis.
Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, Spain.
Estefanía
E
Ugarte-Berzal
Elvira
E
Bailón
Irene
I
Amigo-Jiménez
Cidonia L
CL
Vituri
Mercedes Hernández
MH
del Cerro
María José
MJ
Terol
Juan P
JP
Albar
Germán
G
Rivas
José A
JA
García-Marco
Angeles
A
García-Pardo
D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000912:Antibodies, Neoplasm / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002465:Cell Movement; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D039041:Integrin alpha4beta1 / Q000235:genetics / Q000378:metabolism*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D008875:Middle Aged; D016296:Mutagenesis; D020125:Mutation, Missense; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D015854:Up-Regulation; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
D000912:Antibodies, Neoplasm; D004792:Enzyme Precursors; D039041:Integrin alpha4beta1; D061965:Matrix Metalloproteinase Inhibitors; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011480:Protease Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C120487:pro-matrix metalloproteinase 9; D020780:Matrix Metalloproteinase 9