Abstract
Using a macrophage cell line that constitutively expresses a human apolipoprotein E (apoE) cDNA, we have investigated the post-translational metabolism of endogenously produced apoE. Inhibition of lysosomal or cysteine proteases led to significant inhibition of apoE degradation but did not increase apoE secretion, indicating that cellular degradation is not limiting for apoE secretion in macrophages. Treatment of macrophages with inhibitors of proteoglycan synthesis (4-methylumbelliferyl-beta-D-xyloside) or sulfation (sodium chlorate) enhanced the release of apoE from cells and significantly attenuated the increase in secretion produced by incubation with phosphatidylcholine vesicles (PV). These observations suggested that a significant fraction of the apoE retained by cells (and released by incubation with PV) was associated with proteoglycans. Treatment of cells with exogenous heparinase led to a greater than 4-fold increase in apoE secretion and similarly attenuated the response to PV, suggesting that apoE was trapped in an extracellular proteoglycan matrix. This conclusion was confirmed in studies showing that PV could enhance the release of apoE from cells during an incubation at 4 degrees C, but this enhanced release was abolished in proteoglycan-depleted cells. Incubation with lactoferrin at 4 or 37 degrees C produced a similar decrement in cellular apoE, again indicating the existence of a cell surface pool of apoE. Pulse-chase studies showed that the apoE trapped in the proteoglycan matrix was susceptible to rapid cellular degradation such that net synthesis of apoE (secreted plus cell-associated) was increased significantly in proteoglycan-depleted cells compared with control cells as early as 45 min during a chase period.
Highlights
Sorting between a degradative or secretory pathway [1]
In view of the above considerations and our previous observations regarding the effect of phosphatidylcholine vesicles (PV) or HDL3 on macrophage apolipoprotein E (apoE) secretion and degradation, we investigated a potential role for extracellular matrix in modulating the secretion and degradation of endogenously produced apoE in macrophages
Our previous observation that addition of PV or HDL3 could preserve a substantial amount of newly synthesized apoE from degradation and enhance its secretion [1] led us to ask which of these effects was primary; i.e. was apoE preserved from degradation because its secretion was enhanced, or was secretion limited by competing cellular degradative pathways? The data in Tables I–III address this question
Summary
Materials—4-Methylumbelliferyl--D-xyloside (DX), sodium chlorate, ALLN, monensin, chloroquine, ammonium chloride, and lactoferrin were obtained from Sigma. Cell Culture—J774 cells were stably transfected to express a wild type human apoE cDNA as described previously in detail [1, 13]. Cells were grown in 10% fetal bovine serum and Dulbecco’s modified Eagle’s medium until the start of the described experimental incubations. Vesicles were dialyzed overnight against methoninine-free Dulbecco’s modified Eagle’s medium plus 1% penicillin/streptomycin and filtered through a 0.45-m membrane prior to use. At the end of the chase period apoE secreted into the medium, and that remaining associated with the cell, were quantitatively immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously in detail [1]. Autoradiographs of sodium dodecyl sulfate-polyacrylamide electrophoresis gels were scanned using an LKB XL Ultrascan laser densitometer and results expressed as fold change over control. For the purposes of our analyses, “secreted” apoE is considered to be that recovered from the medium, and “cell” apoE is considered to be that recovered from lysis of the cell layer
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