RABL6A Regulates Schwann Cell Senescence in an RB1-Dependent Manner

Abstract

Schwann cells are normally quiescent, myelinating glia cells of the peripheral nervous system. Their aberrant proliferation and transformation underlie the development of benign tumors (neurofibromas) as well as deadly malignant peripheral nerve sheath tumors (MPNSTs). We discovered a new driver of MPNSTs, an oncogenic GTPase named RABL6A, that functions in part by inhibiting the RB1 tumor suppressor. RB1 is a key mediator of cellular senescence, a permanent withdrawal from the cell cycle that protects against cell immortalization and transformation. Based on the RABL6A-RB1 link in MPNSTs, we explored the hypothesis that RABL6A promotes Schwann cell proliferation and abrogates their senescence by inhibiting RB1. Using sequentially passaged normal human Schwann cells (NHSCs), we found that the induction of replicative senescence was associated with reduced expression of endogenous RABL6A. Silencing RABL6A in low passage NHSCs caused premature stress-induced senescence, which was largely rescued by co-depletion of RB1. Consistent with those findings, Rabl6-deficient MEFs displayed impaired proliferation and accelerated senescence compared to wildtype MEFs. These results demonstrate that RABL6A is required for maintenance of proper Schwann cell proliferation and imply that aberrantly high RABL6A expression may facilitate malignant transformation.

Keywords: MPNST; RABL6A; Schwann cell; retinoblastoma protein (RB1); senescence.

Figure 1

RABL6A expression inversely correlates with markers of cellular senescence in primary NHSCs. (A) Low (Lo) versus high (Hi) passage NHSCs were plated at equal density, and cell number was counted on days 2, 5, and 7 after plating. Lo, P2–4; Hi, P8–10. (B) Representative Western blot shows RABL6A expression decreases with sequential passaging of NHSCs, which inversely correlates with markers of cellular senescence (reduced phosphorylation of RB1 and induction of p16). Med, medium passage cells (P5–7). ImageJ quantification of western data from three of more experiments showing (C) reduced RABL6A expression, (D) reduced phopho-RB1 S807/811, and (E) increased p16 expression in Lo, medium (Med), and Hi passage NHSCs. (F) Left, representative bright field images of β-galactosidase staining, showing an increase in senescent cells upon sequential passaging (Lo, Med and Hi). Right, quantification from three or more experiments, each performed in duplicate. Each dot represents a minimum of 150 cell counts per well. (A) p value determined by a generalized linear model to assess the difference between the curves. Error bars, SD from mean. (C–F) p value, One-way ANOVA with Dunnett’s correction. (**, p < 0.001).

Figure 2

Loss of RABL6A causes premature stress-induced senescence in primary NHSCs. (A) Low passage NHSCs with control or RABL6A knockdown (KD1 and KD2) were plated at equal density and cell number counted on days 2, 4, and 6 after plating. (B) Representative Western blot showing effective RABL6A knockdown in KD1 and KD2 and decreased phospho-RB1 S807/811 compared to CON. Below, quantified relative p-RB1 to total RB1. (C) Representative phospho-RB1 S807/811 (top, red) and p16 (bottom, green) immunofluorescence images shows RABL6A depletion causes reduced phospho-RB1 S807/S811 and induction of p16 compared to control cells. Nuclei stained with DAPI. ImageJ quantification of fluorescent intensity from three or more experiments of (D) phospho-RB1 S807/811 and (E) p16. Each dot in D and E represents an individual cell nuclei. (F) Representative bright field images of β-galactosidase staining, showing RABL6A loss induces senescence in NHSCs. (G) Quantification from three or more experiments of β-galactosidase positive cells after RABL6A knockdown. (A) p value determined by a generalized linear model to assess the difference between the KD1 and KD2 curves compared to CON. Error bars, SD from mean. (D,E,G) p value, One-way ANOVA with Dunnett’s correction. (**, p < 0.001).

Figure 3

RABL6A promotes NHSC proliferation via RB1 inactivation. Control (CON) and RABL6A knockdown (KD1 and KD2) in low passage empty vector (EV) and RB1 knockdown (RB1 KD) cells were plated at equal density, and (A) cell number or (B) percent of cell death in the population (as measured by trypan blue exclusion) was counted days 2, 4, and 6 after plating. (C) Representative Western blot shows efficient knockdown of RABL6A in KD1 and KD2 samples relative to CON. (D) Relative mRNA levels of RB1, demonstrating knockdown of RB1 in RB1 KD samples. (E) Quantification of EdU positivity showing RB1 loss restores proliferation in cells with RABL6A knockdown. (F) Representative bright field images of β-galactosidase staining, showing the senescence caused by RABL6A loss is attenuated with co-depletion of RB1. Below, quantification of β-galactosidase staining from three experiments performed in duplicate. (A,B) p value determined by a generalized linear model to assess the difference between CON KD1 vs. RB1 KD1 and CON KD2 vs. RB1 KD2 (as indicated by brackets). Error bars, SD from mean. (E,F) p value, Two-way ANOVA with Bonferroni correction. (**, p < 0.001).

Figure 4

Rabl6a-deficiency leads to reduced proliferative capacity in mouse embryonic fibroblasts. MEFs were harvested from wildtype (WT) or Rabl6 knockout (KO) mice and analyzed. (A) Growth curves from MEFs plated at equally low density and counted on days 2, 4, and 6 after plating. (B) Flow cytometric analyses of DNA content show a reduction in S phase in asynchronously growing Rabl6a KO MEFs as compared to WT. (C) Representative Western blot of sequentially passaged WT versus Rabl6 KO MEFs showing cells lacking RABL6A display elevated p16, a marker of senescence, earlier than WT. Below, quantified p16 levels from ImageJ densitometry analysis. (D) Representative immunofluorescence images of phospho-RB1 S807/811 (green) in low passage WT and Rabl6a KO cells, showing cells lacking Rabl6 activate RB1 earlier than WT. Nuclei stained with DAPI. Right, ImageJ quantification of fluorescent intensity of p-RB1. Dots represent individual cell nuclei. (E) Representative bright field images of β-galactosidase staining at the same passage number. Right, quantification from three or more experiments of β-galactosidase positive cells, showing an increase in Rabl6a knockout MEFs versus WT. (A) p value determined by a generalized linear model to assess the difference between the curves. Error bars, SD from mean. (B,D,E) p value, One-way ANOVA with Dunnett’s correction. (*, p < 0.05; **, p < 0.001).