Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts

Abstract

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.

Figures

Fig. 1. The Ras binding domain of Raf1 can be used as an immunocytochemical probe for Ras-GTP in situ

NIH-pJ5W-Ha-Ras(61L) cells were grown in 0, 5, or 10 μM dexamethasone for 24 h and then assayed for levels of Ras-GTP using either a biochemical (A) or immunocytochemical (B–I) assay. A, cells treated with 5 μM dexamethasone demonstrated a 0.8–1.2-fold increase in Ras-GTP (range over three experiments; 0.8-fold shown here), while cells treated with 10 μM dexamethasone had a 3.2–3.5-fold increase (3.2-fold shown here). B and C, cells grown in the absence of dexamethasone; D and E, cells grown in 5 μM dexamethasone; F and G, cells grown in 10 μM dexamethasone; H and I, cells grown in 10 μM dexamethasone but probed with GST in place of Raf1-RBD-GST.

Fig. 2. Raf1-RBD-GST recognizes GTP-bound Ha-, K-, and N-Ras

NIH 3T3 cells were transfected with pCGN-hyg constructs encoding HA-tagged Ha-Ras (C and D), K-Ras (E and F), or N-Ras (G and H) or the HA tag alone (A and B) and then grown for 24 h. Cells were then switched to serum-free medium for 48 h, processed for HA immunocytochemistry (A, C, E, and G) and Ras-GTP analysis (B, D, F, and H), and then examined by fluorescence microscopy. Note that cells transfected with all three HA-tagged Ras species show high levels of Raf1-RBD-GST immunofluorescence, compared with cells transfected with HA tag alone. Untransfected NIH 3T3 cells grown in the absence of serum for 48 h demonstrated only background staining when examined by Raf1-RBD-GST immunocytochemistry (I). However, after treatment for 2 min with 20 ng/ml insulin (J), these cells demonstrated increased, punctate immunofluorescence that was localized in patches, apparently at the cell membrane (see “Results”). These data indicate that Raf1-RBD-GST can detect elevated Ras-GTP in situ.

Fig. 3. Schwann cells from Nf1 null mice have constitutively elevated Ras-GTP

Schwann cells from wild type (A) and Nf1−/− mouse embryos (B and C) were grown in serum-free N2 medium supplemented with rhGG2 and forskolin as described previously and then switched to N2 alone for 48 h and processed for Ras-GTP immunocytochemistry. Compared with wild type Schwann cells, Nf1−/− Schwann cells show significantly increased basal levels of Ras-GTP. This basal Ras activity dropped to levels comparable with those of wild type cells in cultures grown in the presence of 1 μM L-744,832 for 48 h. (c).

Fig. 4. Ras-GTP is unaltered in fibroblasts from Nf1 null mice

Fibroblasts from wild type (A–C) or Nf1−/− mice (D and E) were grown in DMEM in the absence of serum for 24 h and then treated with 20 ng/ml insulin for 30 min (B and E) or 1 h (C and F) and examined by Ras-GTP immunocytochemistry. Fibroblasts from these same Nf1−/− embryos demonstrated increased proliferation and collagen deposition (not shown), characteristic features of mouse fibroblasts lacking neurofibromin. Note that Ras-GTP levels were similar in Nf1−/− and wild type fibroblasts and that Ras-GTP levels decreased over a similar time course in both cell populations.

Fig. 5. A subpopulation of Schwann cells, but not fibroblasts, from NF1 patient neurofibromas has constitutively elevated Ras-GTP

A and B, phase-contrast and Ras-GTP staining in normal human Schwann cells. Note that none of the cells from normal nerve express detectable levels of Ras-GTP. Schwann cells (C–F) and fibroblasts (G–J) from a neurofibroma of a 40-year-old male NF1 patient (C, D, G, and H) and a plexiform neurofibroma of a 10-year-old female NF1 patient (E, F, I, and J) were analyzed for Ras-GTP in first passage cultures. C, E, G, and I, phase-contrast images of cells; D, F, H, and J, Ras-GTP immunofluorescence. Tumors were dissociated and split into two cultures: one grown on a poly-L-lysine-coated dish in N2 plus rhGG2 and forskolin (conditions favoring Schwann cell growth and survival) and one grown on an uncoated dish in DMEM plus 10% fetal bovine serum (conditions favoring fibroblast growth and survival). After 1 week, cells were passaged by trypsinization, grown for an additional 2 days, and then processed for S100 protein and Ras-GTP immunocyto-chemistry. Note that Ras-GTP is elevated in Schwann cell cultures but not in fibroblasts. The arrowheads show cells with elevated Ras-GTP, while arrows indicate cells with basal Ras-GTP.