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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02941

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PLD1 and ERK2 regulate cytosolic lipid droplet formation

Linda Andersson^1,*, Pontus Boström^1,*, Johanna Ericson¹, Mikael Rutberg¹, Björn Magnusson¹, Denis Marchesan¹, Michel Ruiz¹, Lennart Asp¹, Ping Huang², Michael A. Frohman², Jan Borén¹ and Sven-Olof Olofsson^1,

¹ Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden
² Department of Pharmacological Science and the Center for Developmental Genetics, Stony Brook University, Stony Brook, New York 11794, USA

View larger version (48K): [in a new window]   Fig. 1. PLD1, but not PLD2, promotes the formation of lipid droplets in NIH 3T3 cells. (A,B) Effect of transient transfection of NIH 3T3 cells with wild-type PLD1 (PLD1wt), K898R PLD1 (a catalytically inactive mutant) or an empty vector on the area of Oil Red O-stained lipid droplets per cell. (A) One day after the transfection with either PLD1 (left), the catalytically inactive mutant (right) or an empty vector (not shown), the cells were stained with Oil Red O to stain the lipid droplets. Bar, 10 µm. (B) BioPix software was used to calculate the total area of Oil Red O-stained droplets per cell. Values are mean ± s.d. of all cells in 20 randomly selected micrographs from each group (*P<0.001 PLD1wt vs K898R PLD1 and empty vector; Not significant (NS) K898R PLD1 vs empty vector, one-way ANOVA). (C) Accumulation of radioactive triglycerides after incubation with [3H]palmitic acid for the indicated times in NIH 3T3 cells transiently transfected with wild-type PLD1 (triangles) or K898R PLD1 (squares). Values are mean ± s.d. from cells from three different culture dishes. (D) Influence of the transfection with PLD1 on the levels of ADRP in the cell. Cells were transfected as in A and the cell lysate was blotted against antibodies to ADRP. (E) Effect of transient transfection of NIH 3T3 cells with wild-type PLD2 (PLD2 wt), K758R PLD2 (a catalytically inactive mutant) or an empty vector on the area of Oil Red O-stained lipid droplets per cell (values are mean ± s.d. of all cells in 20 randomly selected micrographs from each group; NS). (F) The amount of ADRP as estimated by immunoblotting.

View larger version (42K): [in a new window]   Fig. 2. SiRNA to PLD1 reduces the formation of cytosolic lipid droplets in NIH 3T3 cells. (A) Immunoblots of PLD1 in untransfected cells and of PLD1, GRP78 or ß-actin in cells transfected with PLD1 siRNA or a control siRNA. (B,C) Effect of PLD1 siRNA or a control siRNA on lipid droplet formation in cells treated with oleic acid for 7 hours. Two days after the transfection with the siRNA, the cells were stained with Oil Red O. (B) Micrographs showing staining. Bar, 10 µm. (C) BioPix software was used to calculate the total area of Oil Red O-stained droplets per cell; values are mean ± s.d. of all cells in ten randomly selected micrographs from each group (P<0.001, Mann-Whitney rank sum test). (D) Effect of PLD1 siRNA (triangles) and control siRNA (squares) on the accumulation of radioactive triglycerides in oleic acid-treated NIH 3T3 cells after incubation with [3H]palmitic acid for the indicated times. Values are mean ± s.d. from cells from three culture dishes. (E) Influence of PLD1 siRNA on the amount of ADRP in the cell. Cells were treated with siRNA as in A and the cell lysate was blotted against an antibody to ADRP.

View larger version (13K): [in a new window]   Fig. 3. The insulin-induced increase in the formation of lipid droplets is dependent on PLD1 and ERK2. (A) NIH 3T3 cells were transfected with PLD1 siRNA or control siRNA (as indicated). After 2 days the cells were incubated with 3 nM insulin for 2 hours and the total area of Oil Red O-stained lipid droplets/cell was determined (from all cells in 50 randomly selected micrographs). The total area of Oil Red O-stained lipid droplets/cell in the cells treated with insulin and control siRNA was set to 100% and the recovery of lipid droplets in the other two groups were related to this group (mean ± s.d.; n=3. P=0.002 vs Insulin+PLD1 siRNA and P=0.004 vs Buffer+Control siRNA; NS vs Buffer+Control siRNA; one-way ANOVA). (B) NIH 3T3 were incubated with 25 µM Ste-Mek113 for 1 hours and then with 3 nM insulin together with 25 µM Ste-Mek113 for 2 hours. The total area of Oil Red O-stained lipid droplets/cell were analyzed as in A and the insulin-treated cells were set at 100% (mean ± s.d.; n=5. P=0.036 vs Insulin+Ste-Mek113 and P=0.002 vs control; NS vs control; one-way ANOVA).

View larger version (27K): [in a new window]   Fig. 4. Identification of ERK2 as an activator of lipid droplet formation in the cell-free system. (A) Hydrophobic interaction chromatography (which was the final step in the partial purification of the cytosolic activator for the formation of lipid droplets) on a Resource PheTM column using the Äkta Prime system (see Materials and Methods), starting from rat adipocyte cytosol (200 animals) (Marchesan et al., 2003). (B) The ability of the fractions 1-3 recovered from the Resource PheTM column, to activate the assembly of lipid droplets in the cell-free system (as percentage of the total amount of newly formed triglycerides that are assembled into lipid droplets). (C) Fraction 3, containing the major amount of activity and the least amount of protein (based on the absorption at 280 nm) was collected, concentrated and electrophoresed in a 10% SDS-polyacrylamide gel and stained with Sypro Ruby protein stain. The protein bands (figures on the right-hand side of the gel) were cut out, digested with trypsin and analyzed by MALDI-TOF or, when no hit was obtained, by MS/MS. For the identification of the proteins, see text. The figures on the left-hand side are the molecular mass markers. (D) The ability of recombinant proteins corresponding to ERK2 (0.4 µg/assay) and adiponectin (0.5 µg/assay), or purified citrate lyase (1 U/assay), to activate the assembly of lipid droplets in the cell-free system. Results are given as the percentage of newly formed triglycerides that are assembled into lipid droplets subtracted from the buffer blank (mean ± s.d.; n=3).

View larger version (42K): [in a new window]   Fig. 5. Transfection of ERK2 increases the formation of lipid droplets in intact cells. (A,B) The effect of transfection of ERK2 on the formation of lipid droplets in the cell. Cells were transfected with ERK2 (ERK2) or with a plasmid encoding GFP as control (GFP). After 1 day, the cells were stained with Oil Red O. (A) Micrographs showing staining. Bar, 10 µm. (B) BioPix software was used to calculate the total area of red pixels from Oil Red O-stained droplets per cell in cells transfected with ERK2 or the control plasmid. Values are mean ± s.d. of all cells in 20 randomly selected micrographs from each group (P<0.001, t-test). (C) Transfection of ERK2 increases the total amount of triglycerides in the cell. Cells were treated as in A, harvested and extracted with chloroform:methanol, and the amount of triglycerides in the extract determined and related to the total amount of cell protein (mean ± s.d.; six different pairs of transfected culture dishes; P=0.012, t-test). (D) Transfection with ERK2 increases the amount of ADRP in the cell. Cells, transfected as described above, were harvested and lysed, and blotted using an anti-ADRP antibody. The figure shows the quantification of blots from two different experiments (mean ± range) and one example of a blot. QL, quantum level, i.e. the sum of the grayness of each pixel in the blot.

View larger version (56K): [in a new window]   Fig. 6. Microinjection of ERK2 into the cytosol of NIH 3T3 cells increases the formation of cytosolic lipid droplets. (A) Cells were microinjected with ERK2 (20 ng/µl) and GFP (1 ng/µl), or (as control) GFP alone. The cells were cultured for 2.5 hours and then stained with Oil Red O (no hematoxylin). Bar, 10 µm. (B) BioPix software was used to calculate the total area of red pixels in Oil Red O-stained droplets per cell. Values are mean ± s.d. of 20 microinjected cells from each group (P<0.001, t-test).

View larger version (56K): [in a new window]   Fig. 7. SiRNA to ERK2 causes a reduction in the amount of cytosolic lipid droplets. (A) Immunoblots of ERK2 as well as of GRP78 and GAPDH in cells transfected with ERK2 siRNA, or with control siRNA. The cells were transfected with the constructs 2 days before the experiment. (B,C) Effect of ERK2 siRNA or a control siRNA on lipid droplet formation in cells treated with oleic acid. The cells were transfected 2 days before the experiments and treated with oleic acid for 2 hours before being stained with Oil Red O. (B) Micrographs showing staining. Bar, 10 µm. (C) BioPix software was used to calculate the total area of red pixels in Oil Red O-stained droplets per cell after transfection with ERK2 siRNA or control siRNA. Values are mean ± s.d. of all cells in 20 randomly selected micrographs from each group (P=0.001, t-test). (D) Influence of ERK2 siRNA on the amount of triglycerides in the cell. The cells were treated as in B, and triglycerides were determined as in Fig. 4 (mean ± s.d.; cells from six different pairs of transfected culture dishes; P=0.010, t-test).

View larger version (35K): [in a new window]   Fig. 8. ERK2 is essential for the effect of PLD1 on the formation of lipid droplets, by promoting the phosphorylation of dynein, which increase the association of this protein with lipid droplets. Dynein is essential for the formation of the lipid droplets. (A) NIH 3T3 cells were transfected with PLD1 or GFP as control. After 1 day, the cells were incubated for 2 hours with Ste-Mek113 (25 µM) and stained with Oil Red O. The total area of Oil Red O-stained lipid droplets was determined using BioPix software. The results are given as mean ± s.d. of all cells in 20 randomly selected micrographs (*NS vs control; P<0.001 vs control and P=0.001 vs PLD1 + Ste-Mek113; one-way ANOVA). (B) NIH-3T3 cells were incubated for 24 hours with 360 µM oleic acid, harvested and homogenized. The homogenate was incubated with active ERK2 (0.1 µM for 1 hour at 37°C; ERK2) or buffer (Control) and fractionated with the PhosphoProtein purification kit (`phospho-protein affinity column'). The retained fraction (phosphorylated proteins) was collected and analyzed for the indicated proteins by immunoblotting (anti-dynein and anti-caveolin). (C) NIH 3T3 cells were incubated with oleic acid overnight, homogenized by nitrogen cavitation and subjected to gradient ultracentrifugation. The top fraction containing the lipid droplets was isolated and fractionated with the PhosphoProtein purification kit (`phospho-protein affinity column'). The retained (phosphorylated) proteins and unretained (unphosphorylated) proteins were blotted against antibodies to the dynein intermediate chain. (D) NIH 3T3 cells were transfected with ADRP-HAT and incubated with oleic acid (see B). After 24 hours, the cells were homogenized and the homogenate was incubated with active ERK2 (see B) or buffer. ADRP-HAT was precipitated with talon-Dynabeads (ADRP-HAT precipitate) and analyzed by immunoblot against antibodies to dynein. (E) NIH 3T3 cells were microinjected with a monoclonal antibody directed against dynein (17 cells) or a control immunoglobulin (26 cells) and the effect on the total area of Oil Red O-stained lipid droplets/cell determined. (mean ± s.e.m.; P<0.001, Mann-Whitney rank sum test). (F) NIH 3T3 cells were microinjected with a monoclonal antibody directed against dynein (Anti-dynein: 1 µg/ml) or a control immunoglobulin (Control Ig) and stained with Bodipy. The cells were imaged by confocal microscopy at intervals of 30 seconds over a 5-minute period. BioPix software was used to identify fusions as described previously (Bostrom et al., 2005). Results are mean ± s.e.m. of control immunoglobulins (13 different movies) and anti-dynein (16 different movies) (P<0.001, Mann-Whitney rank sum test).