Limits...
Slit2 inactivates GSK3β to signal neurite outgrowth inhibition.

Byun J, Kim BT, Kim YT, Jiao Z, Hur EM, Zhou FQ - PLoS ONE (2012)

Bottom Line: Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth.Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons.Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

ABSTRACT
Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

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Inactivation of GSK3β is required for mSlit2-induced inhibition of process extension.(A, B) CAD cells transfected with GSK3β-S9A mutant or EGFP, as a control, were cultured under serum-deprived conditions in the presence or absence of mSlit2, as indicated. Cells were fixed at 72 hr after incubation and immunostained for tubulin to measure the lengths of neurite-like processes. Representative images (A) and quantification of process length (B) are shown. Bar, 200 µm. *** p<0.001. (C) CAD cells were transfected with shGSK3 or treated with 6-bromoindirubin-3′-acetoxime (300 nM), an inhibitor of GSK3 (GSK3i), and cultured for 72 hr under serum-deprived condition. Cells were then fixed and stained for tubulin to measure the lengths of neurite-like processes. *** p<0.001 compared to control.
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pone-0051895-g007: Inactivation of GSK3β is required for mSlit2-induced inhibition of process extension.(A, B) CAD cells transfected with GSK3β-S9A mutant or EGFP, as a control, were cultured under serum-deprived conditions in the presence or absence of mSlit2, as indicated. Cells were fixed at 72 hr after incubation and immunostained for tubulin to measure the lengths of neurite-like processes. Representative images (A) and quantification of process length (B) are shown. Bar, 200 µm. *** p<0.001. (C) CAD cells were transfected with shGSK3 or treated with 6-bromoindirubin-3′-acetoxime (300 nM), an inhibitor of GSK3 (GSK3i), and cultured for 72 hr under serum-deprived condition. Cells were then fixed and stained for tubulin to measure the lengths of neurite-like processes. *** p<0.001 compared to control.

Mentions: We next investigated if the Slit2-induced inhibition of process extension is mediated by inactivation of GSK3β. For this purpose, we transfected CAD cells with a mutant of GSK3β in which the N-terminal serine 9 residue was replaced with alanine (GSK3β-S9A), preventing its inactivation by phosphorylation. Consistent with a previous study [26], ectopic expression of GSK3β-S9A alone resulted in a slight inhibition of processes extension (Figures 7A, 7B). Importantly, Slit2 no longer inhibited process extention in CAD cells when GSK3β-S9A was expressed (Figures 7A, 7B). Furthermore, suppression of GSK3 activity alone - which was achieved either by transfection of the cells with shRNA against GSK3 or treatment with a pharmacological inhibitor of GSK3, 6-bromoindirubin-3′-acetoxime - was sufficient to restrict the extension of neurite-like processes (Figure 7C). Taken together, these results indicate that Slit2 requires the phosphorylation and inactivation of GSK3β to inhibit process extension.


Slit2 inactivates GSK3β to signal neurite outgrowth inhibition.

Byun J, Kim BT, Kim YT, Jiao Z, Hur EM, Zhou FQ - PLoS ONE (2012)

Inactivation of GSK3β is required for mSlit2-induced inhibition of process extension.(A, B) CAD cells transfected with GSK3β-S9A mutant or EGFP, as a control, were cultured under serum-deprived conditions in the presence or absence of mSlit2, as indicated. Cells were fixed at 72 hr after incubation and immunostained for tubulin to measure the lengths of neurite-like processes. Representative images (A) and quantification of process length (B) are shown. Bar, 200 µm. *** p<0.001. (C) CAD cells were transfected with shGSK3 or treated with 6-bromoindirubin-3′-acetoxime (300 nM), an inhibitor of GSK3 (GSK3i), and cultured for 72 hr under serum-deprived condition. Cells were then fixed and stained for tubulin to measure the lengths of neurite-like processes. *** p<0.001 compared to control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526488&req=5

pone-0051895-g007: Inactivation of GSK3β is required for mSlit2-induced inhibition of process extension.(A, B) CAD cells transfected with GSK3β-S9A mutant or EGFP, as a control, were cultured under serum-deprived conditions in the presence or absence of mSlit2, as indicated. Cells were fixed at 72 hr after incubation and immunostained for tubulin to measure the lengths of neurite-like processes. Representative images (A) and quantification of process length (B) are shown. Bar, 200 µm. *** p<0.001. (C) CAD cells were transfected with shGSK3 or treated with 6-bromoindirubin-3′-acetoxime (300 nM), an inhibitor of GSK3 (GSK3i), and cultured for 72 hr under serum-deprived condition. Cells were then fixed and stained for tubulin to measure the lengths of neurite-like processes. *** p<0.001 compared to control.
Mentions: We next investigated if the Slit2-induced inhibition of process extension is mediated by inactivation of GSK3β. For this purpose, we transfected CAD cells with a mutant of GSK3β in which the N-terminal serine 9 residue was replaced with alanine (GSK3β-S9A), preventing its inactivation by phosphorylation. Consistent with a previous study [26], ectopic expression of GSK3β-S9A alone resulted in a slight inhibition of processes extension (Figures 7A, 7B). Importantly, Slit2 no longer inhibited process extention in CAD cells when GSK3β-S9A was expressed (Figures 7A, 7B). Furthermore, suppression of GSK3 activity alone - which was achieved either by transfection of the cells with shRNA against GSK3 or treatment with a pharmacological inhibitor of GSK3, 6-bromoindirubin-3′-acetoxime - was sufficient to restrict the extension of neurite-like processes (Figure 7C). Taken together, these results indicate that Slit2 requires the phosphorylation and inactivation of GSK3β to inhibit process extension.

Bottom Line: Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth.Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons.Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

ABSTRACT
Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

Show MeSH
Related in: MedlinePlus