Abstract
Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.
Highlights
Lysyl oxidase (LOX)3 catalyzes the oxidation of specific lysine residues within extracellular elastin and collagen generating residues of ␣-aminoadipic-␦-semialdehyde within these proteins [1]
It has been documented that the production of hydrogen peroxide accompanying the oxidation of unknown substrate(s) by LOX appeared to be essential for the chemotactic response of cells to this enzyme [19]
by -aminopropionitrile (BAPN)-treated loxϪ/Ϫ mouse embryonic fibroblasts (MEFs) (Kd ϭ 156.3 Ϯ 11.7 pM; Bmax ϭ 2,197 Ϯ 126 molecules/cells) showed affinity and maximal binding similar to control loxϪ/Ϫ MEF (Kd ϭ 196.1 Ϯ 23.4 pM; Bmax ϭ 2,307 Ϯ 115 molecules/cells) (Fig. 9C). These results provide additional evidence suggesting that BAPN does not affect the chemotactic response beyond its inhibitory effect on Inactivation of the LOX Gene Increases the Turnover of Phosphorylated Intermediates of the PDGF Receptor- Signal Transduction Pathway—The PDGFR- signal transduction pathway was analyzed in loxϩ/ϩ and loxϪ/Ϫ MEF as described above for SMC (Fig. 6A)
Summary
We have previously reported that LOX purified from bovine aorta actively induces the migration of peripheral blood monocytes [18] and vascular smooth muscle cells (SMCs) [19] This chemotactic activity of LOX was fully inhibitable by -aminopropionitrile (BAPN), which inactivates catalysis by LOX, or by the presence of catalase in the chemotactic chamber assays [19]. Despite the number of well documented cellular and biological processes dependent on LOX activity, the relevant molecular mechanisms, including the identification of cell protein substrates that might be oxidized by LOX in these instances, has yet to be accomplished We have approached this issue in part by studying the role of LOX in chemotaxis, a basic cellular response underlying several of the biological functions in which LOX is involved. This newly documented priming effect of LOX on chemotaxis is clearly distinguishable from the acute chemotactic effect of this enzyme previously described [19]
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