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. 2006 Mar 14;45(10):3325-36.
doi: 10.1021/bi0515927.

Chondrocytes utilize a cholesterol-dependent lipid translocator to externalize phosphatidylserine

Monika Damek-Poprawa  1 , Ellis Golub, Linda Otis, Gerald Harrison, Christine Phillips, Kathleen Boesze-Battaglia
Affiliations

Chondrocytes utilize a cholesterol-dependent lipid translocator to externalize phosphatidylserine

Monika Damek-Poprawa et al. Biochemistry. .

Abstract

During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine- (PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol- (GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain.

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Figures

FIGURE 1
FIGURE 1
Lipid profiles of chondrocyte and MV detergent resistant membranes. Cholesterol and sphingomyelin content of (A) chondrocyte membranes and (B) MV was determined as described in Materials and Methods in both detergent-soluble (solid black bars) and DRM fractions (white bars). Phospholipid profile of (C) chondrocyte membranes and (D) MV was determined as described in Materials and Methods in both detergent-soluble (solid black bars) and DRM fractions (white bars). Briefly, lipids were extracted from detergent-soluble and -insoluble fractions, the extract was dried down and resuspended, and phospholipids were separated using HPLC-ELSD or sequential 1D thin-layer chromatography (TLC) on silica gel G. All lipid analyses are an average of four individual analyses from three independent preparations.
FIGURE 2
FIGURE 2
Assessment of chondrocyte maturation. (A) Real-time RT-PCR of collagen X expression. N1511 cells were plated at a high density of 1.7 × 106 cells per well and induced with 50 ng/mL recombinant human BMP-2 and 1 × 10−6 M insulin (solid black bars). The cells in complete media constituted the control (solid gray bars). At the given time points RNA was extracted from cells and analyzed by real-time RT-PCR. The graph shows the mean values ± SD, expressed in mRNA arbitrary units. BMP/Ins-treated cells expressed higher levels of collagen X mRNA than corresponding controls as indicated by an * (P < 0.001 from the Tukey test). On day 10 collagen X expression in BMP/Ins-treated cells significantly increased as compared to days 3 and 7, as shown by a # (P < 0.001 from the Tukey test). Collagen X expression in control cells was the highest on day 7 as compared to days 3, 5, and 10 (**, P < 0.001 from the Tukey test). (B) Surface ALPase staining. Confluent N1511 cells were treated with BMP-2 and insulin, at the days indicated, and cells were immunostained for ALPase surface expression as described in Materials and Methods. Representative data are shown from three different experiments with similar results. (C) ALPase activity in cultured chondrocytes. Confluent N1511 cells were treated with BMP-2 and insulin, at the days indicated, and ALPase activity was measured as described in Materials and Methods. The results are an average of three individual inductions, with ALPase activity measured in duplicate.
FIGURE 3
FIGURE 3
Phospholipid PLSCR1 expression in N1511 cells during chondrocyte differentiation. (A) Real-time RT-PCR of PLSCR1 expression in N1511 cells. N1511 cells were plated at a high density. After 24 h (day 0) the cells were induced with 50 ng/mL recombinant human BMP-2 (R&D Systems), 1 × 10−6 M bovine insulin (Life Technologies, Inc.), and 50 μg/mL L-ascorbic acid phosphate (Wako) in media containing 0.3% FCS. The cells in complete media constituted the control. At the given time points RNA was extracted from cells and analyzed by real-time RT-PCR. The graph shows the mean values ± SD, expressed in mRNA arbitrary units. BMP/insulin-treated cells show higher levels of PLSCR1 expression than control cells as indicated by an *, with a P < 0.05 compared with corresponding untreated cells. (B) PLSCR1 localization in maturing chondrocytes. N1511 cells were treated with anti-PLSCR1 Ab-1 as described in Materials and Methods and assessed using laser confocal microscopy. (C) Expression of PLSCR1 protein in N1511 cells. Confluent N1511 cells were treated with BMP-2 and insulin, at the days indicated cells were isolated, and lysates were prepared in 35 mM OG. (A) The cell lysate was subjected to reducing electrophoresis, immunoblotted, and probed with 1 μg of anti-PLSCR1 antibody/mL of BLOTTO. (B) For actin protein loading controls, membrane was stripped and reprobed with a 1:250 dilution of anti-actin antibody (Santa Cruz Biotechnology).
FIGURE 4
FIGURE 4
Chondrocyte APLT and scramblase activities. N1511 cells isolated at the indicated times were labeled with either NBD-PS (APLT activity) or NBD-PC (scramblase), stained with propidium iodide, and analyzed for transporter activity by flow cytometry as described in Materials and Methods. (A) APLT and scramblase activity at day 5. Cytometry histograms are representative of data from three different experiments with similar results; 15000 cells were analyzed for each sample. (B) APLT and scramblase activity in maturing chondrcoytes. The percent change in mean channel fluorescence of NBD-PC, solid bars (scramblase activity), and NBD-PS, open bars (APLT activity), is indicated as chondrocytes mature. This percent change was calculated as the fluorescence change between BMP/Ins-treated and control cells as a function of BMP/Ins fluorescence for both APLT and scramblase activity. Mean fluorescence values were obtained by gating on viable (PI negative) cells. Routinely, cell viability was between 90% and 95%. (C) Externalization of PS in maturing chondrocytes. N1511 cells isolated at the indicated time points and labeled with acyl-chain-labeled NBD-PS were treated with 1 M sodium dithionite, and the change in fluorescence was recorded over 3 min. The solid line represents PS accessible to quenching in untreated control cells, and bars represent PS accessible to quenching in BMP/Ins-treated cells. The results are an average of three independent measurements of PS externalization. Error bars represent ±SD, with n = 3.
FIGURE 5
FIGURE 5
Colocalization of PLSCR1 with GM1. N1511 cells were treated with CTb conjugated to AlexaFluor 594 and patched with anti-CTb antibody. The cells were then treated with anti-PLSCR1 Ab-1 as described in Materials and Methods and assessed using laser confocal microscopy. Images of AlexaFluor 488 fluorescence (panels A, D, G, and J) and AlexaFluor 594 fluorescence (panels B, E, H, and K) are shown as well as merged images (panels C, F, I, and L) showing both FITC (green) and AlexaFluor 594 (red) fluorescence along with DAPI stained. In controls, panel M, N1511 cells were treated with CTb and a 50:50 mix of AlexaFluor 488 and AlexaFluor 594 secondary antibodies and co-stained with DAPI. Results are representative of three experiments.
FIGURE 6
FIGURE 6
Effect of MβCD on lipid translocator activity. N1511 cells isolated at the indicated times were labeled with either NBD-PS or NBD-PC to access APLT or scramblase activity, respectively. These cells were subsequently depleted of membrane cholesterol, with a short (10 min) incubation with 10 mM MβCD, stained with propidium iodide, and analyzed for transporter activity by flow cytometry as described in Materials and Methods. (A) Cytometry histograms are representative of data from three different experiments with similar results; 15000 cells were analyzed for each sample. (B) The percent change in mean channel fluorescence (MCF) values for scramblase activity (solid bars) and APLT activity (open bars) upon cholesterol depletion with MβCD is indicated. These mean fluorescence values were obtained by gating on viable (propidium iodide negative) cells. The cells used in these experiments are the same as those used in the studies shown in Figure 4 (activity prior to cholesterol depletion). Parallel experiments verified that MβCD did not adversely affect cell viability as determined by propidium iodide exclusion. Routinely, cell viability was approximately 90%. Data were analyzed using a one-way ANOVA analysis as described in Materials and Methods.
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