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Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX-PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full-length LOX cDNA (pre-pro-LOX), the N-glycosylation null pre-[N/Q]pro-LOX cDNA and the deletion mutant pre-LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N-terminal propeptide sequence. The results show that glycosylation of the LOX-PP is not required for secretion and extracellular processing of pro-LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX-PP for pro-LOX exit from the ER and is the first to highlight the influence of LOX-PP glycosylation on LOX enzyme activity.

Everolimus, a pharmaceutical component of drug-eluting stents, inhibits coronary vessel restenosis, but the antirestenotic mechanisms of action remain unclear. Here, we describe the effects of everolimus on key contributors to vessel restenosis, smooth muscle cell proliferation, and migration. In a dose-dependent fashion, everolimus reduced human coronary artery smooth muscle cell (HCASMC) proliferation without toxicity in a bimodal fashion, with accentuated potency occurring at 10 μM. Everolimus arrested the majority of HCASMCs in G1-phase, whereas it reduced the fraction of cells in S-phase at doses that inhibited DNA synthesis (bromodeoxyuridine incorporation). Consistent with this, Western blotting demonstrated that everolimus reduced activation and expression of G1-phase cell cycle progression factors, including p70S6K and cyclin D, respectively, decreased levels of proliferating cell nuclear antigen, and attenuated growth factor/serum-induced phosphorylation of the cell cycle phase transition intermediate, retinoblastoma protein. Everolimus did not, however, affect HCASMC migration. These observations suggest that everolimus acts as an antiproliferative, but not antimigratory, compound to account for at least some of the clinical efficacy exhibited by this drug as an antirestenotic agent. Moreover, everolimus-induced inhibition of the mammalian target of rapamycin complex 1 and regulation of cyclin-mediated cell cycle progression actions likely account for the antiproliferative effects of this compound on HCASMCs.

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.