SCAMP4 enhances the senescent cell secretome

Abstract

The senescence-associated secretory phenotype (SASP) is a major trait of senescent cells, but the molecular regulators of SASP factor secretion are poorly understood. Mass spectrometry analysis revealed that secretory carrier membrane protein 4 (SCAMP4) levels were strikingly elevated on the surface of senescent cells compared with proliferating cells. Interestingly, silencing SCAMP4 in senescent fibroblasts reduced the secretion of SASP factors, including interleukin 6 (IL6), IL8, growth differentiation factor 15 (GDF-15), C-X-C motif chemokine ligand 1 (CXCL1), and IL7, while, conversely, SCAMP4 overexpression in proliferating fibroblasts increased SASP factor secretion. Our results indicate that SCAMP4 accumulates on the surface of senescent cells, promotes SASP factor secretion, and critically enhances the SASP phenotype.

Keywords: SCAMP4; cellular senescence; human diploid fibroblasts; senescence-associated secretory phenotype (SASP).

Figure 1.

Identification of SCAMP4 as a cell surface protein selectively elevated in senescent WI-38 fibroblasts. (A) SA-β-gal analysis of proliferating (P; PDL 20) and senescent (S; PDL 50) WI-38 human diploid fibroblasts. (B) ELISA measurement of secreted IL6, IL8, GDF-15, CCL2, and IL1B in conditioned media from proliferating and senescent WI-38 cells, normalized to cell number. (ND) Not detectable. (C–E) Western blot analysis of SCAMP4 levels in whole-cell lysate (WCL) (C), membrane and cytosolic lysates (D), and cell surface (E) from proliferating and senescent WI-38 fibroblasts. Senescence markers SIRT1, p53, p16, and p21; cytosolic marker HSP90; plasma membrane protein markers CAV1 (Caveolin-1) and EGFR; and loading control GAPDH were also assayed. (F) Proliferating and senescent WI-38 cells were fixed with 100% methanol, and endogenous SCAMP4 protein was detected by confocal microscopy. Merged signals. (Blue) Nuclei stained with DAPI; (red) SCAMP4. (G) Detached WI-38 (proliferating and senescent) cells were incubated with anti-SCAMP4-biotin or control human IgG-biotin antibodies and then incubated with streptavidin-APC antibodies. SCAMP4-positive cells were analyzed by flow cytometry; mean fluorescence intensity (MFI) of SCAMP4-APC in proliferating and senescent cells was quantified. (H) Steady-state SCAMP4 mRNA levels were measured by reverse transcription followed by quantitative PCR (RT-qPCR) analysis. p16 and p21 mRNAs were included as senescent cell markers, and ACTB mRNA was included as a loading control; mRNA levels were normalized to 18S rRNA levels in each sample; mRNAs in proliferating cells were set as 1. Graphs in B, G, and H represent the means ± SEM from three independent experiments; (**) P-value < 0.01.

Figure 2.

In proliferating cells, SCAMP4 is degraded by the ubiquitin–proteasome pathway. (A) Analysis of polysomes in proliferating (P) and senescent (S) cells. Lysates of proliferating and senescent WI-38 cells were loaded onto 10%–50% linear sucrose gradients, and the relative distributions of SCAMP4 mRNA and ACTB (control) mRNA were calculated after RT-qPCR analysis in each fraction. (B) The stability of the SCAMP4 protein in proliferating and senescent cells. (Left) Western blot analysis of the levels of SCAMP4 and loading control ACTB in cells harvested after treatment with the protein synthesis inhibitor cycloheximide (CHX) for the times shown. (Right) Quantification of SCAMP4 signals normalized to ACTB signals. Data are the means ± SEM from two independent experiments. (C) Proliferating WI-38 fibroblasts (PDLs 22–25) were incubated with either vehicle (ethanol) or 10 µM MG132 for 4 or 6 h. (Top) Western blot analysis of SCAMP4 levels in WI-38 fibroblasts treated with MG132 for 4 or 6 h. (p21) Marker of proteasome inhibition; (ACTB) loading control. (Bottom) ELISA measurement of secreted IL6, IL8, and MIF. (D) Immunoprecipitation using anti-SCAMP4 or rIgG (control) antibodies in proliferating cells with or without MG132 for 3 h. After lysis, ubiquitinated SCAMP4 was detected by Western blot analysis using anti-ubiquitin antibody. ACTB was detected in lysates (Input) used in immunoprecipitation reactions. (**) P-value < 0.01; (*) P-value < 0.05; (N.S.) not significant.

Figure 3.

Silencing SCAMP4 in WI-38 fibroblasts reduces the secretion of SASP factors. (A–D) Seventy-two hours after transfection with control (Ctrl) siRNA or SCAMP4 siRNA, presenescent WI-38 cells (PDLs 39–42) were analyzed. (A) Levels of p16, p21, IL1A, IL1B, IL6, IL8, and ACTB mRNAs, as measured by RT-qPCR analysis and normalized to 18S rRNA levels. (B) Western blot analysis to assess the levels of SCAMP4, p53, p16, SIRT1, p21, IL1B, and ACTB in whole-cell lysates. (C) SA-β-gal-stained cells (the percentage of β-gal positive cells is shown). (D) Cytokine array analysis of the secreted factors (left) and array signal quantification (right). (E) Experimental scheme (left) and Western blot analysis of SCAMP4 levels (right). (F) ELISA to quantify the levels of secreted IL6, IL8, GDF-15, CCL2, CXCL1, CCL7, IL7, angiogenin, and MIF. Graphs in A and F represent the means ± SEM from three independent experiments. (**) P-value < 0.01; (*) P-value < 0.05.

Figure 4.

SCAMP4 promotes secretion of SASP factors. (A–E) Proliferating WI-38 cells were transduced with lentiviral particles to express either Myc tag alone or SCAMP4-Myc. Twenty days later, RT-qPCR analysis was used to measure the steady-state levels of p16, p21, IL1A, IL1B, IL6, IL8, and ACTB mRNAs (normalized to 18S rRNA levels) (A); SA-β-gal activity was assessed (B); Western blot analysis was performed to assess the levels of SCAMP4-Myc (using anti-SCAMP4 antibody or anti-Myc antibody), p53, IL1A, IL1B, p21, p16, SIRT1, and GAPDH (loading control) in whole-cell lysates (C); [³H]-thymidine incorporation was measured (D); and the secretion of IL6, IL8, GDF-15, IL1B, CXCL1, and IL7 was measured by ELISA (E). Graphs in A, D, and E represent the means ± SEM from three independent experiments. (**) P-value < 0.01; (*) P-value < 0.05. (F) Proposed model. In proliferating cells, SCAMP4 is rapidly degraded by the ubiquitin–proteasome degradation system. In senescent cells, SCAMP4 is stable and localizes to the cell surface, in turn promoting the secretion of SASP factors during senescence.