Elastin peptides signaling relies on neuraminidase-1-dependent lactosylceramide generation

Abstract

The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM(3) level decreases while lactosylceramide (LacCer) content increases consistently with a GM(3)/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM(3) blocking antibody shows that GM(3) is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM(3)/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor.

Figure 1. EBP and lipid rafts are colocalized at the plasma membrane.

EBP and lipid rafts localization were analyzed using confocal microscopy as described in the Methods section. EBP appears as a red staining, lipid rafts as a green staining and the colocalization as a yellow staining. The scale represents 10 µm.

Figure 2. Lipid rafts integrity is required for elastin peptide-induced signal transduction.

A) Fibroblasts were incubated for 30 min with 10 mM MCD then treated with 50 µg kE/ml for 30 min. Cellular extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) and anti-ERK 1/2 Western-blots. B) Densitometric analysis: ***, p<0.001.

Figure 3. Gangliosides are essential for elastin peptide-induced signal transduction.

A) Upper panel: Fibroblasts were incubated for 2 h with [1-³H]-sphingosine. The radioactive staining of glycolipids was realized for 48 h. Glycosphingolipids depletion was achieved by incubating cells for 3 days with 20 µM D-PDMP. After extraction, lipids were resolved by HPTLC and the plates were put in presence of a radiographic film during one month at −80°C, and then revealed. Lower panel: cell survival evaluated using MTT assay after D-PDMP incubation. B) Cells were incubated for 3 days with 20 µM D-PDMP. Fibroblasts were then treated with 50 µg kE/ml for 30 min. Cellular extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) and ERK 1/2 Western-blots. The emergence of a third band in ERK1/2 Western-blots is due to an electrophoretic shift because of its phosphorylation. C) Densitometric analysis: ***, p<0.001.

Figure 4. EBP is involved in the kE-induced GM₃ levels decrease.

A) Fibroblasts were incubated for 2 h with [1-³H]-sphingosine, PBS washed, and radioactive staining of glycolipids was performed for 48 h. Cells were then stimulated with or without 50 µg kE/ml during 5 (dark grey) or 30 min (white). After extraction, lipids were separated by HPTLC and the plates were put in the presence of a radiographic film during one month at −80°C and revealed. GM₃ was identified comparing its migration rate to that of controls. Densitometric analysis: ***, p<0.001. B) The radioactive staining and the stimulation have been performed as in A. Lactose (1 mM), an EBP antagonist, was preincubated for 3 h before stimulation with kE (50 µg/ml, 30 min). After extraction, lipids were separated by HPTLC and the plates were put in presence of a radiographic film during one month at −80°C, and revealed. GM₃ was identified by comparing its migration rate to that of controls. Densitometric analysis: NS, not significant; ***, p<0.001.

Figure 5. EBP is involved in the kE-induced LacCer production.

A) Radioactive staining and stimulation were performed as described in Figure 5 caption. LacCer was identified by comparing its migration rate to that of controls. Densitometric analysis: ***, p<0.001. B) Lactose (1 mM) was preincubated for 3 h before stimulation with 50 µg kE/ml for 30 min. After extraction, lipids were separed by HPTLC and the plates were put in presence of a radiographic film during one month at −80°C, and then revealed. LacCer was identified comparing its migration rate to that of controls. Densitometric analysis: ***, p<0.001.

Figure 6. Neu-1 is involved in GM₃/LacCer conversion.

A) Cellular extracts from untransfected fibroblasts or cells transfected with SNC siRNA or Neu-1 siRNA were analyzed by anti-Neu-1 Western-blot. B) Densitometric analysis: NS, not significant; ***, p<0.001. C) Untransfected fibroblasts (control) and fibroblasts transfected with SNC siRNA or Neu-1 siRNA were stimulated with 50 µg/ml of kE. They were stained with or without an FITC-anti-LacCer antibody (1/25 dilution) then analyzed by flow cytometry. The red line represents the control and the green one corresponds to the stimulation with kE (50 µg/ml). D) Fibroblasts were pre-incubated with D-PDMP (20 µM) for 30 min prior to stimulation with kE (50 µg/ml, 30 min). Cell extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) and anti-ERK 1/2 Western-blots.

Figure 7. GM₃ plays a substrate role for the elastin receptor complex.

A) Fibroblasts were incubated for 30 min with 10 µg/ml anti-GM₃ antibody then treated with 50 µg/ml kE for 30 min. Cell extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) and anti-ERK 1/2 Western-blots. The emergence of a third band in ERK1/2 western-blots is due to an electrophoretic shift because of its phosphorylation. B) Densitometric analysis: **, p<0.01.

Figure 8. LacCer triggers ERK 1/2 activation.

A) Fibroblasts were incubated with different concentrations of LacCer for 30 min. Cell extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) Western-blot. B) Densitometric analysis: **, p<0.01; ***, p<0.001.

Figure 9. Exogenous LacCer penetrates into the plasma membrane.

Fibroblasts were incubated with 12.5 µM BODIPY-LacCer (λ_excitation = 505 nm; λ_emission = 511 nm) for 30 min at 4°C followed by washings with defatted-BSA to remove any fluorescent lipid remaining at the plasma membrane as described previously . Cells were fixed then analyzed by fluorescence imaging. Hoechst 33342: nucleus staining by Hoechst 33342 (λ_excitation = 340 nm; λ_emission = 465 nm); BODIPY-LacCer: LacCer staining; Merge: superposition of nuclear and 12.5 µM BODIPY-LacCer stainings. The scale represents 10 µm.