Transcriptional regulation of phagocytosis-induced membrane biogenesis by sterol regulatory element binding proteins

Abstract

In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate approximately 100 lipids at appropriate concentrations. To study the regulation of bilayer assembly in a cell cycle-independent manner, we have exploited the fact that phagocytes replenish membranes expended during particle engulfment in a rapid phase of lipid synthesis. In response to phagocytosis of latex beads, human embryonic kidney 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles. Lipid synthesis was accompanied by increased transcription of several lipogenic proteins, including the low-density lipoprotein receptor, enzymes required for cholesterol synthesis (3-hydroxy-3-methylglutaryl CoA synthase, 3-hydroxy-3-methylglutaryl CoA reductase), and fatty acid synthase. Phagocytosis triggered the proteolytic activation of two lipogenic transcription factors, sterol regulatory element binding protein-1a (SREBP-1a) and SREBP-2. Proteolysis of SREBPs coincided with the appearance of their transcriptionally active N termini in the nucleus and 3-fold activation of an SREBP-specific reporter gene. In previous studies with cultured cells, proteolytic activation of SREBP-1a and SREBP-2 has been observed in response to selective starvation of cells for cholesterol and unsaturated fatty acids. However, under the current conditions, SREBP-1a and SREBP-2 are induced without lipid deprivation. SREBP activation is inhibited by high levels of the SREBP-interacting proteins Insig1 or the cytosolic domain of SREBP cleavage-activating protein. Upon overexpression of these proteins, phagocytosis-induced transcription and lipid synthesis were blocked. These results identify SREBPs as essential regulators of membrane biogenesis and provide a useful system for further studies on membrane homeostasis.

Fig. 1.

Phagocytosis induces membrane lipid synthesis in human embryonic kidney 293 cells. On day 0, 293 cells were set up in medium A on coverslips in 24-well plates at 10⁵ cells per well (A and B) or in 96-well plates at 5 × 10⁴ cells per well (C and D). (A and B) On day 2, cells were switched to medium D with 50 μg per well of amine-coated latex beads, incubated for 6 h in the absence (A) or presence (B) of 1 μM latrunculin A, washed, fixed, and analyzed by microscopy. (Bar, 2 μm.) (C) On day 2, cells were incubated for 4 h in medium D plus or minus beads (100 μg per well). Where indicated, 1 μM latrunculin A was added 0.5 h before phagocytosis. Lipids were determined as described in Materials and Methods and corrected for protein concentration. Data indicate the concentrations of cholesterol and phospholipids expressed as % increase with respect to controls. (D) On day 2, cells were incubated for 4 h in medium D plus the indicated concentration of beads. Data are plotted as above. Lipid concentrations in control samples were 69.2 ± 2.7 nmol cholesterol per mg protein and 218.6 ± 3.6 nmol phospholipids per mg protein. Error bars indicate range (n = 2).

Fig. 2.

Phagocytosis induces transcription of key genes required for lipid synthesis and uptake. On day 0, 293 cells were set up in medium A at 5 × 10⁴ cells per well. (A) On day 2, cells were switched to medium D, incubated for 0.5 h plus or minus latex beads (100 μg per well), washed, and incubated for the indicated time. Messenger RNAs were analyzed by quantitative real-time PCR. Data were normalized to the mRNA levels of TATA box binding protein and are expressed as fold change with respect to controls. (B) On day 1, 293 cells were transfected in medium A with 30 ng per well of phRL-CMV (expressing sea pansy luciferase from a CMV promoter) plus 30 ng per well of pFAS-Luc (pFAS), pLDLR-Luc (pLDLR) or pHMGCS-Luc (pHMGCS). On day 2, cells were switched to medium D and incubated for 0.5 h plus or minus beads (100 μg per well), washed, chased for 8 h in medium D, and then analyzed for luciferase activities. Data indicate firefly luciferase corrected for sea pansy luciferase and are expressed as fold change with respect to samples incubated without beads. (C) On day 1, 293 cells were transfected with phRL-CMV plus pFAS-Luc, pHMGCS-Luc, or pLDLR-Luc. On day 2, cells were incubated in medium D for 0.5 h plus or minus latex beads (100 μg per well). Where indicated, wells received 1 μM latrunculin A (Lat. A) 0.5 h before phagocytosis. Cells were washed and chased in the absence of latrunculin A for 6 h in medium D. The three panels represent separate experiments. Luciferase activities are plotted as above. Error bars indicate SD (n = 3).

Fig. 3.

Phagocytosis induces cleavage of SREBPs. (A) On day 0, 293 cells were set up in medium A at 5 × 10⁴ cells per well. On day 2, cells were incubated for 4 h in medium C supplemented with 25 μg/ml ALLN (Calbiochem) and the following: 10% LPDS and 5 μM compactin (lane 1); 10% LPDS, 5 μM compactin, and 1% (wt/vol) hydroxypropyl β-cyclodextrin (lane 2); 10% LPDS, 5 μM compactin, and 1 μg/ml 25HC (lane 3); or 10% FBS plus the indicated concentration of beads (lanes 4–8). Total cell extracts (100 μg of protein per lane) were subjected to SDS/8% PAGE, transferred to nitrocellulose, and blotted with anti-SREBP-1 (IgG-2A4) as described (25). P and M denote the precursor and mature nuclear forms of SREBP-1, respectively. (B–D) On day 0, 293 cells were set up on coverslips in 24-well plates in medium A at 1 × 10⁵ cells per well. On day 1, cells were incubated in medium D for 0.5 h plus (C and D) or minus (B)50 μg per well of beads. Cells were then washed and incubated in medium D for 6 h. ALLN (25 μg/ml) was added during the last 4 h. Cells were stained with anti-SREBP-1 and fluorescently labeled anti-mouse IgG. (B and D) Fluorescence micrographs. (C) Bright field image. C and D show the same cell. (Bars, 2 μm.)

Fig. 4.

Phagocytosis-induced activation of an SREBP-specific reporter gene. On day 0, 293 cells were set up in medium A at 5 × 10⁴ cells per well. On day 1, cells were transfected with 30 ng per well of phRL-CMV plus 30 ng of pSRE-Luc for 16 h. (A) After transfection, cells were switched to medium D and incubated for 0.5 h with the indicated concentrations of latex beads. Cells were washed, chased in medium D for 5 h, lysed, and analyzed for luciferase activities. Data indicate sea pansy luciferase (RLuc) or firefly luciferase corrected for sea pansy luciferase (Fluc/RLuc) and are expressed as fold change. (B) After transfection, 293 cells were switched to medium D/0.4% (vol/vol) DMSO with or without 1 μM latrunculin A for 0.5 h. Cells then received 100 μg per well of latex beads without change of medium. After 0.5 h, cells were washed and chased in medium D for the indicated time. Data are plotted as in Fig. 2B. (C) After transfection, cells were incubated for 6 h in medium C supplemented with 10% FBS or LPDS plus or minus 1 μM latrunculin A. Cells were then lysed and analyzed for luciferase activities. (D) After transfection cells were incubated in the absence or presence of latex beads (100 μg per well) for 0.5 h. Cells were washed (time 0), switched to medium D for 5 h, and analyzed for luciferase activities. During the 5-h period, 1 μg/ml 25HC was added at the indicated time. Error bars indicate SD (n = 3).

Fig. 5.

SREBPs are required for phagocytosis-induced transcription. 293 cells were set up on day 0 in medium A at 5 × 10⁴ cells per well. (A) On day 1, cells were transfected with 30 ng per well of phRL-CMV plus 30 ng per well of pLDLR-Luc (WT) or pLDLR-Luc/mutSRE. (mutSRE). After 16 h, cells were incubated for 0.5 h in medium D plus or minus latex beads (100 μg per well). Cells were washed and chased for 8 h. Data indicate firefly luciferase corrected for sea pansy luciferase and are expressed as fold change. (B) On day 1, cells were transfected with 20 ng per well of phRL-CMV plus 20 ng per well of pFAS-Luc. In addition, wells received 20 ng of either pcDNA3 empty vector (Control), pCMV-Insig1 (Insig1), pCMV-P450-TM/BP2(555–1141) expressing dominant negative SREBP-2 (DN-BP), or pCMV-P450-TM/SCAP (731–1276) expressing dominant negative SCAP (DN-SCAP). After 16 h, cells were incubated with beads, chased and analyzed as above. Error bars indicate SD (n = 3).

Fig. 6.

SREBPs are required for phagocytosis-induced lipid synthesis. (A) On day 0, HD1A immortalized mouse macrophages were set up in medium D at 2 × 10⁴ cells per well. On day 1, cells were infected with adenovirus encoding EGFP, dominant negative SCAP (DN-SCAP) or Insig1 (Insig1) at a MOI of 250. On day 3, cells were incubated plus or minus 100 μg per well of latex beads for 8 h. Cells were then harvested to determine lipid and protein concentrations. Data indicate the concentrations of cholesterol (black bars) and phospholipids (white bars) expressed as percent increase with respect to samples incubated without beads. Lipid concentrations in control samples were 237.7 ± 5.7 nmol of cholesterol per mg of protein and 758.2 ± 4.1 nmol of phospholipids per mg of protein. Error bars indicate SD (n = 2). (B) On day 0, HD1A cells were set up at 3 × 10⁵ cells per well in a 24-well plate. On day 1, cells were infected with adenovirus expressing EGFP, dominant negative SCAP, or Insig1 at a MOI of 250. On day 2, cells received liposomes containing [³H]cholesteryl oleate (1 μCi per 0.5 ml per well; 1 Ci = 37 GBq). On day 3, cells were washed and incubated in fresh medium for 1 h. The dish was then placed on ice, and each well received 50 μg of scintillant yttrium silicate beads for 30 min. Subsequently, the dish was transferred to a scintillation counter at 33°C and scintillation was measured every minute over a period of 3.5 h (28). Data represent the average of triplicate wells. Coefficients of variation ranged from 0.7 to 19.4%, with an average of 9.6%.