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, 23 (26), 8949-54

The Neurofibromatosis 1 Gene Product Neurofibromin Regulates Pituitary Adenylate Cyclase-Activating Polypeptide-Mediated Signaling in Astrocytes

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The Neurofibromatosis 1 Gene Product Neurofibromin Regulates Pituitary Adenylate Cyclase-Activating Polypeptide-Mediated Signaling in Astrocytes

Biplab Dasgupta et al. J Neurosci.

Abstract

Individuals with the neurofibromatosis 1 (NF1)-inherited tumor predisposition syndrome develop low-grade astrocytomas. The NF1 tumor suppressor gene product neurofibromin exhibits GTPase-activating activity (GAP) toward RAS, such that loss of neurofibromin expression leads to high levels of activated RAS and increased cell proliferation. Previous work has demonstrated that Nf1 inactivation in astrocytes leads to increased cell proliferation in vitro and in vivo, accompanied by increased RAS pathway activation. Studies on Nf1 mutant Drosophila have suggested that neurofibromin might also regulate cAMP signaling. Because intracellular cAMP levels have profound effects on astrocyte growth control, we sought to determine the contribution of neurofibromin to astrocyte cAMP regulation. In this report, we demonstrate that Nf1 inactivation in astrocytes results in reduced cAMP generation in response to PACAP and attenuated calcium influx and Rap1 activation. Based on the differential effects of forskolin and dibutyryl-cAMP on Nf1-/- astrocytes, neurofibromin likely functions at the level of adenylyl cyclase activation. Last, the reintroduction of a fragment of neurofibromin containing residues sufficient for restoring RAS-GAP function in Nf1-/- cells resulted in only partial restoration of neurofibromin-mediated cAMP regulation. These results demonstrate that neurofibromin positively influences cAMP generation and activation of cAMP growth regulatory targets in astrocytes and expands the role of the NF1 gene in astrocyte growth regulation.

Figures

Figure 1.
Figure 1.
PACAP-induced cAMP generation is impaired in both Nf1-/- and Nf1+/- astrocytes and is independent of RAS activation. a, Nf1-/- astrocytes exhibit a marked decrease in cAMP generation in response to various concentrations of PACAP. cAMP produced by Nf1+/+ (solid line), Nf1-/- (dotted), and RAS transgenic B8 astrocytes (dashed) are shown. The inset illustrates neurofibromin and PAC-1-R expression in Nf1+/+ and Nf1-/- astrocytes after adenoviral transduction. b, Pretreatment of either Nf1+/+ (white bar) or Nf1-/- astrocytes (black bar) with the RAS farnesyltransferase inhibitor (l-744,832) did not significantly alter PACAP-mediated cAMP generation in Nf1-/- or wild-type astrocytes. The inset demonstrates that l-744,832 treatment resulted in inhibition of MAPK activity (phospho-MAPK) without altering total MAPK (MAPK) expression. c, cAMP generation in response to PACAP stimulation in also impaired in Nf1+/- astrocytes (gray bar) compared with Nf1+/+ (white bar) astrocytes.d,Whereas differences in cAMP levels were observed in Nf1+/+ (white bar) versus Nf1-/- (black bar) astrocytes in response to PACAP treatment, no differences were observed in response to EGF treatment. Each error bar represents mean ± SD. Asterisks denote statistically significant differences (p < 0.004).
Figure 2.
Figure 2.
PACAP activation of Rap1 and induction of calcium influx is impaired in Nf1-/- astrocytes. a, Serum-starved Nf1+/+ and Nf1-/- astrocytes stimulated with PACAP were analyzed for Rap1 activation. The top panel denotes active Rap1 (Rap1-GTP bound to GST-RalGDS), whereas bottom panel demonstrates the total Rap1 in each sample. Significantly reduced Rap1 activation is observed in the Nf1-/- compared with Nf1+/+ astrocytes. b, Serum-starved Nf1+/+ and Nf1-/- astrocytes stimulated with PACAP in presence of 45 Ca2+ demonstrate decreased calcium influx in Nf1-/- (black bar) compared with Nf1+/+ (white bar) astrocytes. Each error bar represents mean ± SD. Asterisks denote statistically significant differences (p = 0.001).
Figure 3.
Figure 3.
Forskolin-stimulated cAMP generation is reduced in Nf1-/- astrocytes, whereas db-cAMP-induced calcium influx and Rap1 activation is similar in both Nf1-/- and Nf1+/+ astrocytes. a, cAMP generation in response to PACAP and forskolin is reduced in Nf1-/- astrocytes (black bars) compared with Nf1+/+ astrocytes (white bar). b, Calcium influx in response to PACAP is reduced in Nf1-/- astrocytes (black bars) compared with Nf1+/+ astrocytes (white bar), but equivalent in response to db-cAMP stimulation. c, Whereas Rap1 activation (Rap1-GTP) in response to PACAP is reduced in Nf1-/- compared with Nf1+/+ astrocytes, equivalent levels of Rap1 activation were observed in response to db-cAMP. Each error bar represents mean ± SD. Asterisks denote statistically significant differences (*p = 0.001; **p = 0.007).
Figure 4.
Figure 4.
Ectopic expression of NF1GRD partially rescues the impaired cAMP response to PACAP in Nf1-/- astrocytes. Whereas reduced cAMP levels were observed in untransduced and vector transduced Nf1-/- (black bars) compared with Nf1+/+ (white bars) astrocytes, a partial restoration of this response was observed in astrocytes expressing the NF1GRD fragment. No effect on cAMP generation was observed in Nf1+/+ astrocytes expressing the NF1GRD fragment. Expression of KT3-tagged NF1GRD in Nf1-/- MSCV-NF1GRD-Pac treated astrocytes is shown in the inset. Each error bar represents mean ± SD. Asterisks denote statistically significant differences (*p = 0.001; **p = 0.005).

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