doi: 10.1074/jbc.M111.333039. Epub 2012 Apr 23.
Caveolin-1 and dynamin-2 are essential for removal of the complement C5b-9 complex via endocytosis
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- PMID:22528500
- PMCID: PMC3370175
- DOI: 10.1074/jbc.M111.333039
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Caveolin-1 and dynamin-2 are essential for removal of the complement C5b-9 complex via endocytosis
Oren Moskovich et al. J Biol Chem..
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Abstract
The complement system, an important element of both innate and adaptive immunity, is executing complement-dependent cytotoxicity (CDC) with its C5b-9 protein complex that is assembled on cell surfaces and transmits to the cell death signals. In turn, cells, and in particular cancer cells, protect themselves from CDC in various ways. Thus, cells actively remove the C5b-9 complexes from their plasma membrane by endocytosis. Inhibition of clathrin by transfection with shRNA or of EPS-15 with a dominant negative plasmid had no effect on C5b-9 endocytosis and on cell death. In contrast, inhibition of caveolin-1 (Cav-1) by transfection with an shRNA or a dominant negative plasmid sensitized cells to CDC and inhibited C5b-9 endocytosis. Similarly, both inhibition of dynamin-2 by transfection with a dominant negative plasmid or by treatment with Dynasore reduced C5b-9 endocytosis and enhanced CDC. C5b-9 endocytosis was also disrupted by pretreatment of the cells with methyl-β-cyclodextrin or Filipin III, hence implicating membrane cholesterol in the process. Analyses by confocal microscopy demonstrated co-localization of Cav-1-EGFP with C5b-9 at the plasma membrane, in early endosomes, at the endocytic recycling compartment and in secreted vesicles. Further investigation of the process of C5b-9 removal by exo-vesiculation demonstrated that inhibition of Cav-1 and cholesterol depletion abrogated C5b-9 exo-vesiculation, whereas, over-expression of Cav-1 increased C5b-9 exo-vesiculation. Our results show that Cav-1 and dynamin-2 (but not clathrin) support cell resistance to CDC, probably by facilitating purging of the C5b-9 complexes by endocytosis and exo-vesiculation.
Figures
Inhibition of dynamin abrogates MAC endocytosis. K562 cells transfected with DN-K44A plasmid, Dyn-2-EGFP plasmid or with an empty vector (EGFP) as control were cultured for 48 h (A,B,D) or for 16 h (C). K562 cells were also pretreated with 0, 60, or 80 μm Dynasore for 30 min at 37 °C (D–E). Then, the cells were treated with a sublytic dose of anti-K562 antibodies and then with C9D-NHS supplemented with C9-AF555 (A–D) or C9-AF488 (E) for 10 min at 37 °C. Next, the cells were washed, incubated for 20 min at 37 °C and analyzed under a confocal microscope. Representative cells (four independent experiments) are shown.White arrows in picturesA andB point at C9-AF555 accumulating at the ERC. Inpicture C,arrows point to co-localized C9-AF555 and Dyn-2-EGFP at the cell membrane (white arrows) and at the ERC (white arrowheads). Levels of intracellular C9-AF555 (D) or C9-AF488 (E) were quantified in 50 randomly selected cell images by scanning with the Image J software (A.U.). Results are representative of four independent experiments. *,p < 0.05; **,p < 0.01.
Dynamin protects K562 cells from complement-dependent cytotoxicity. K562 cells were transfected with the K44A plasmid or an empty vector control (EGFP) for 24, 48 or 72 h (A) or for 30 min at 37 °C with Dynasore at different concentrations (B). Then, the cells were subjected to treatment with an anti-K562 antiserum (diluted 1/18 or 1/22) followed by complement for 60 min at 37 °C. Cell lysis was determined by measuring uptake of trypan blue. Results are representative of four independent experiments, *,p < 0.05; **,p < 0.01.
Inhibition of Caveolin-1 abrogates MAC endocytosis.A andB, K562 cells (107) were transfected for 48 h with 20 μg of Cav-1 shRNA (SC) or scrambled shRNA (NS) both mixed with 4 μg of pGFP.C andD, K562 cells (107) were transfected for 48 h with 18 μg of Cav-1-DGI plasmid or an empty vector as control. Transfected cells were treated with a sublytic dose of anti-K562 antibodies and then with C9D-NHS supplemented with C9-AF555 for 10 min at 37 °C. Next, the cells were washed, incubated for 20 min at 37 °C, and analyzed under a confocal microscope.A andC, representative fields are shown (three independent experiments).White arrows point to C9-AF555 accumulating in the ERC. Amount of endocytosed C9-AF555 was quantified in 50 randomly selected GFP-labeled cell images (B) or 50 randomly selected cell images (D) by using the Image J software (A.U.). Results are representative of three independent experiments, *,p < 0.05; **,p < 0.01.
Caveolin-1 protects K562 and Raji cells from complement-dependent cytotoxicity.A andB, K562 cells (107) were transfected with 20 μg of Cav-1-EGFP plasmid or pEGFP as control or with 20 μg of Cav-1 shRNA (SC) or scrambled shRNA (NS) both mixed with 4 μg pGFP.C andD, K562 cells (C) or Raji cells (D) (107) were transfected with 18 μg of Cav-1-DGI or with pcDNA3.1 as control. After 24, 48, 72, or 96 h the cells were treated with anti-K562 antiserum (diluted 1/18 or 1/22) (A–C) or with Rituximab (5 or 30 μg/ml) (D) and then with NHS, for 60 min at 37 °C. Cell lysis was determined by measuring uptake of trypan blue. Results are representative of four independent experiments, *,p < 0.05; **,p < 0.01.
Co-localization of the MAC with Cav-1-EGFP. K562 cells were transfected with Cav-1-EGFP. After 48 h, the cells were treated with a sublytic dose of anti-K562 antibodies and then with C9D-NHS supplemented with C9-AF555 for 10 min at 37 °C. Next, the cells were washed (A) or further incubated for 20 min at 37 °C (B) and analyzed under a confocal microscope. Representative cells of three independent experiments are shown.Arrows point to co-localization of C9-AF555 and Cav-1-EGFP at the cell membrane (white arrows), the early endosome compartment (yellow arrowheads) and at the ERC (white arrowheads).C, an extracellular vesicle containing both Cav-1-EGFP and C9-AF555 is shown.
Caveolin-1 contributes to MAC elimination by exo-vesiculation. K562 cells were transfected with a Cav-1-EGFP or Cav-1-DGI plasmid or with an empty vector as control. After 48 h the cells were treated with a sublytic dose of anti-K562 antiserum and then with NHS for 10 min at 37 °C. The cells were extensively washed with HBSS and incubated at 37 °C for 5 or 10 min. The cell supernatant (sup) was collected and cleared by centrifugation at 5,000 ×g, subjected to SDS-PAGE and analyzed by Western blotting with anti-C9 antibodies.A, representative C9 protein bands in sup.B, results of densitometric analyses of protein bands from three independent experiments.
MAC deposition is inversely correlated with the level of Cav-1-EGFP expression.A andB, K562 cells were transfected with Cav-1-EGFP plasmid or EGFP plasmid as control. After 48 h, the cells were treated with a sublytic dose of anti-K562 antibodies and then with C9D-NHS supplemented with C9-AF555 for 10 min at 37 °C. Next, the cells were washed and analyzed under a confocal microscope. A selected image representing numerous similar images is shown inA andB. White arrows point at 3 cells showing low levels of Cav-1-EGFP and high levels of C9-AF555.C andD, amounts of C9-AF555 and Cav-1-EGFP or EGFP (A.U.) were quantified with the Image J software in 80 randomly selected cell images.C, a significant inverse correlation is shown between the quantities of C9-AF555 and Cav-1-EGFP (R2 = 0.9564;p < 0.05).D, no correlation is seen between the levels of C9-AF555 and EGFP (R2 = 0.0864).
Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD (A,C,D) for 15 min or filipin-III (B) for 30 min at 37 °C. For cholesterol repletion (C), cells were first incubated with MβCD and then washed and treated with 0, 1, 2, or 4 mm cholesterol for 15 min at 37 °C. Cell lysis was activated by treatment with a sublytic dose of anti-K562 antibodies and NHS (or HIS as control) for 60 min at 37 °C.D, cells were first incubated with 0, 3, or 6 mm MβCD, washed and incubated with 200 or 400 units of SLO in DTT or DTT alone (0.4 mm ) as control, for 30 min at 37 °C. Cell lysis was determined by trypan blue uptake. All results are representative of four independent experiments.E, K562 cells were pretreated with 0, 3 or 6 mm of MβCD for 15 min at 37 °C. Then, the cells were treated with a sublytic dose of anti-K562 antibodies and C9D-NHS supplemented with C9-AF488 for 10 min at 37 °C. Next, the cells were washed, incubated for 20 min at 37 °C, and analyzed under a confocal microscope. The amount of intracellular C9-AF488 was quantified with the Zeiss 410 LSM software in 100 randomly selected cell images (Relative fluorescence intensity; R.F.I.). *,p < 0.05; **,p < 0.01; ***,p < 0.001.
Inhibition of clathrin-mediated endocytosis has no effect on complement-dependent cytotoxicity and on MAC endocytosis.A,C, andD, K562 cells were transfected with specific shRNA directed to CHC (SC) or with a nonspecific shRNA (NS).B andE, K562 cells were transfected with a plasmid containing the dominant negative epidermal growth factor receptor substrate 15 (DN-EPS15) or with an empty GFP plasmid.A andB, after 24, 48, or 72 h, the cells were treated with anti-K562 antiserum (diluted 1/18 or 1/22) and then with NHS for 60 min at 37 °C. Cell lysis was determined by measuring uptake of trypan blue.C–E, 72 h (C andD) or 24 h (E) post-transfection the cells were treated with a sublytic dose of anti-K562 antibodies and then with C9D-NHS supplemented with C9-AF555 for 10 min at 37 °C. Next, the cells were washed, incubated for 20 min at 37 °C, and analyzed under a confocal microscope. Representative cells (of four independent experiments) are shown inC;arrows point at a C9-AF555 accumulation in the ERC.D andE, amount of intracellular C9-AF555 was quantified with the Image J software in 50 randomly selected cell images.
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