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Cellular uptake and catabolism of high-density-lipoprotein triacylglycerols in human cultured fibroblasts: degradation block in neutral lipid storage disease.

ABSTRACT: High-density lipoprotein (HDL)-[3H]triolein (i.e. [3H]triolein incorporated into reconstituted HDL) was taken up by cultured fibroblasts through an apparently saturable process, competitively inhibited by non-labelled HDL and independent of the LDL receptor. Using 125I-HDL and HDL-[3H]triolein, binding experiments (at 0 degrees C) followed by a short-time 'chase' at 37 degrees C showed that 125I radioactivity was rapidly released in the culture medium (as trichloroacetic acid-precipitable material), whereas 3H radioactivity remained associated with the cell. The cell-associated HDL-[3H]triolein was rapidly degraded in normal fibroblasts, and the liberated [3H]oleic acid was incorporated into newly biosynthesized phospholipids. In Wolman-disease fibroblasts HDL-[3H]triolein was degraded at a normal rate, and thus independently of the lysosomal compartment. In contrast, the degradation of HDL-[3H]triolein was blocked in fibroblasts from Neutral Lipid Storage Disease (NLSD), similarly to that of endogenously biosynthesized triacylglycerols [Radom, Salvayre, Nègre, Maret and Douste-Blazy (1987) Eur. J. Biochem. 164, 703-708]. Trypsin-treated HDL-[3H]triolein was also taken up by cells and degraded quite similarly to HDL-[3H]triolein. In conclusion, all these data taken together suggest that HDL-[3H]triolein is: (i) associated with the cell through a process independent of intact apolipoprotein (apo) As, thus probably independent of an apoA-receptor-mediated uptake; (ii) internalized by cells, whereas 125I-apoAs are released in the culture medium; (iii) directed to the same non-lysosomal catabolic pool (blocked in NLSD) as for endogenously biosynthesized triacylglycerols.

SUBMITTER: Hilaire N

PROVIDER: S-EPMC1137857 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Project description:In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3' UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal-regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication.

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Cellular uptake and catabolism of high-density-lipoprotein triacylglycerols in human cultured fibroblasts: degradation block in neutral lipid storage disease.

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