Macropinocytosis and caveola-dependent endocytosis are major cell uptake pathways for the lysyl oxidase propeptide

Abstract

Introduction: The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of recombinant rat lysyl oxidase propeptide (rLOX-PP) have been identified , rLOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for rLOX-PP is PI3Kdependent macropinocytosis in PWR-IE , PC3, SCC9 and MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that rLOX-PP buffering capacity recovers PI3K-dependent macropinocytosis in low pH endosomes and facilitates rLOX-PP endosomal escape into the cytoplasm enabling its observed interactions with cytoplasmic targets and ultimately its nuclear uptake. rLOX-PP-Atto565 uptake. MC3T3-1E , PWR-lE, DU145 , PC3, SCC9, Cal27, MDA-MB-23l and MDA-kb2 cell cultures were, respectively incubated with rLOX-PP-Atto565 as a function of time and subjected to confocal microscopy and flow cytometry in the presence or absence of uptake inhibitors. To determine optimum time points for analysis of intact rLOX-PP uptake , we determined the stability of rLOX-PP as a function of time. For this purpose , we generated double-labeled rLOX-PP by sequential labeling rLOX-PP with Atto565 followed by a quencher tag QSY® 9 resulting in a non-fluorescent intact rLOX-PP that would become fluorescent after hydrolysis resulting from separation of the quencher and Atto565. To quench extracellular Atto565 fluorescence, N-[epsilon]- (carboxymethyl)-lysine-bovine serum albumin (CML-BSA) was labeled with QSY® 9 to generate a high plasma membrane affinity, Forster resonance energy transfer (FRET) quencher. To measure endosomal pH change before and after rLOX-PP-Atto565 uptake by cells, a pH sensor , Lysosensor Yellow/Blue dextran 10,000 MW, co-localized with rLOX-PP-Atto565. Fluorescence intensity of punctate images was analyzed using Image J and the fluorescent intensity ratio of 4 70 nm to 525 nm was calculated. All uptake studies were performed in live cells except rLOX-PP-Atto565 and F-actin co-localization studies. Results: Data demonstrated that rLOX-PP-Atto565 enters cells primarily by macropinocytosis in all cell lines. Unlike other cell lines tested, rLOX-PPAtto565 uptake in DU145 cells may occur by a PI3K-independent form of macropinocytosis. Additional LOX-PP uptake pathways also occurred to varying degrees in other cell lines. Our inhibition experiments with 1.5 [mu]M Filipin III in PWR-lE, PC3, SCC9 cells suggest that a caveolae-mediated uptake pathway for rLOXPP-Atto565 occurs. While the major rLOX-PPAtto565 uptake pathway in PC3 cells is by macropinocytosis, these cells also employ a dynamin-and clathrin dependent uptake mechanism for rLOX-PP-Atto565 internalization. In summary, the major uptake pathway for labeled rLOX-PP appears to be primarily PI3Kv dependent macropinocytosis. Secondary pathways appeared to be dynamin- and caveola dependent for labeled rLOX-PP uptake by PWR-3, PC3, SCC9, MDA-MB-231 cell lines. Additionally, it appeared that the elevation of the cationic charge of labeled rLOX-PP mayhelp to drive labeled rLOX-PP into low pH environments. Conclusions: The knowledge that rLOX-PP-Atto565 can be taken up by cells via different pathways and that these pathways vary may provide opportunities to target cells in a specific environment that utilize a relatively rare uptake pathways. Both macropinocytosis- and caveola dependent uptake pathways are favorable for escape from endosomes to the cytoplasm and escape from lysosomal degradation. Furthermore, the endosomal alkalinizing effect of rLOX-PP demonstrated may restore Rae I or PI3K dependent macropinocytosis in cells with a low intracellular pH. This property may increase the uptake of rLOX-PP in tumors compared to normal tissues in vivo. This notion is based on the excess production of lactic acid under hypoxic conditions in tumors resulting in relatively low pH(i).