Decrease of Cholesterol in Mouse Melanoma Causes Secretion of Lysosomal Enzymes

Abstract

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.

Mevalonate Pyrophosphate Decarboxylase is Predominantly Located in the Cytosol of both B16 and B16F10 Cells in Mouse Melanoma Treated with Lovastatin

“…4C) and a previous report 22) that the cholesterol content in B16 and B16F10 treated with 10% LDS containing 10 µM lovastatin at 24 hr was significantly decreased. Therefore, it was suggested that the transport of MPD to peroxisomes from cytosol in mouse melanoma was not responsible for the significant decrease of cholesterol contents in cells.…”

“…We previously reported that MPD is predominantly located in the cytosol of B16F10 and is not located in peroxisomes, and the level of MPD (3-fold) in B16F10 was markedly increased and the level of cholesterol content in B16F10 was significantly decreased by treatment with 10% LDS containing 10 µM lovastatin for 24 hr; 22) however, it was not confirmed whether MPD is predominantly located in the cytosol of B16F10 treated with lovastatin, so we examined the subcellular distribution of MPD using B16F10 treated with or without lovastatin. As shown in Fig.…”

“…Also, we previously reported that the cholesterol content in B16F10 significantly decreased, when the amount of MPD was maximally increased by lovastatin. 22) Therefore, the increase in the level of MPD in cells was measured to examine the marked decrease of the cholesterol level in cells or the maximum effect by lovastatin. Figure 3 shows the typical increase Crude extracts were prepared from B16 incubated for 24 hr with 10% FBS or 10% LDS containing 10 µM lovastatin.…”

Reduction of Mevalonate Pyrophosphate Decarboxylase in Mouse Melanoma Cells Treated with δ-Tocotrienol Is Not Associated with Reduction of Cholesterol Content or Release of Lysosomes and Melanosomes

“…We previously reported that the exocytosis of mature enzymes from lysosomes into the medium was caused by a lovastatin-and/or LDS-induced de-crease in the cholesterol content in B16F10 mouse melanoma cells. 13) We suggested that lysosomal exocytosis promotes the rapid repair of wounds in plasma membranes ruptured by a loss of cholesterol, as cholesterol is a major constituent of the plasma membrane. Therefore, it was suggested that not only lysosomes but also lysosome-related melanosomes were released by the decrease in cholesterol.…”

“…12) We previously reported that the exocytosis of mature enzymes from lysosomes into the medium was caused by a lovastatin (HMG-CoA reductase inhibitor)-and/or lipoprotein-deficient serum (LDS)-induced decrease in the cholesterol content in B16F10 mouse melanoma cells. 13) Melanosomes contain several lysosomal enzymes such as β-glucuronidase, 14,15) suggesting that melanosomes are lysosome-related organelles. 16) Therefore, it was suggested that the release of not only lysosomes but also melanosomes was caused by the decrease in cholesterol content.…”