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The Gs-linked receptor GPR3 inhibits the proliferation of cerebellar granule cells during postnatal development.

Tanaka S, Shaikh IM, Chiocca EA, Saeki Y - PLoS ONE (2009)

Bottom Line: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation.In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression.These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain.

Methodology/principal findings: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 -/- mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit.

Conclusions/significance: These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

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Related in: MedlinePlus

Expression of p27/kip and phosphoCREB expression during postnatal cerebellar development.In panel a, cerebelli from GPR3−/− or wild type mice were harvested at P5, P8, and P14. p27/kip1 immunohistochemistry was detected in IGL (as well as other layers) but was visually more readily apparent in wild-type vs. GPR3−/− mice at all stages. In panel b, pCREB was detected by immunohistochemistry in wild-type and GPR3−/− mice (P12). While pCREB, was detected in the molecular layer (ML) and IGL of widl-type mice, it was not as readily visible in the IGL of the GPR3−/−. In panel c, co-localization of pCREB and p27/kip was determined by double immunofluorescence. Scale bar = 50 µm.
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pone-0005922-g006: Expression of p27/kip and phosphoCREB expression during postnatal cerebellar development.In panel a, cerebelli from GPR3−/− or wild type mice were harvested at P5, P8, and P14. p27/kip1 immunohistochemistry was detected in IGL (as well as other layers) but was visually more readily apparent in wild-type vs. GPR3−/− mice at all stages. In panel b, pCREB was detected by immunohistochemistry in wild-type and GPR3−/− mice (P12). While pCREB, was detected in the molecular layer (ML) and IGL of widl-type mice, it was not as readily visible in the IGL of the GPR3−/−. In panel c, co-localization of pCREB and p27/kip was determined by double immunofluorescence. Scale bar = 50 µm.

Mentions: To provide additional confirmatory evidence for a regulatory relationship between GPR3 and p27/Kip, we sought to determine the expression of p27/Kip1 in wild type or GPR3−/− mice during postnatal cerebellar development (P5, P8 and P14). The expression of p27/Kip1 in wild-type P5 mice was scarce and observed primarily in IGL in P5 cerebellum (figure 6A). However, this expression visibly increased at the P8 and P14 time points in IGL, but also in the inner side of EGL and in migrating GCPs in ML. In contrast, in GPR3−/− mice the expression of p27/Kip1 was visibly reduced in the EGL and IGL of P5, P8 and P14 cerebella compared to that of wild-type mice at the same time points (figure 6a). These data thus indicate that GPR3 gene expression was involved in the regulation of p27/Kip gene expression in the developing postnatal mouse cerebellum.


The Gs-linked receptor GPR3 inhibits the proliferation of cerebellar granule cells during postnatal development.

Tanaka S, Shaikh IM, Chiocca EA, Saeki Y - PLoS ONE (2009)

Expression of p27/kip and phosphoCREB expression during postnatal cerebellar development.In panel a, cerebelli from GPR3−/− or wild type mice were harvested at P5, P8, and P14. p27/kip1 immunohistochemistry was detected in IGL (as well as other layers) but was visually more readily apparent in wild-type vs. GPR3−/− mice at all stages. In panel b, pCREB was detected by immunohistochemistry in wild-type and GPR3−/− mice (P12). While pCREB, was detected in the molecular layer (ML) and IGL of widl-type mice, it was not as readily visible in the IGL of the GPR3−/−. In panel c, co-localization of pCREB and p27/kip was determined by double immunofluorescence. Scale bar = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691605&req=5

pone-0005922-g006: Expression of p27/kip and phosphoCREB expression during postnatal cerebellar development.In panel a, cerebelli from GPR3−/− or wild type mice were harvested at P5, P8, and P14. p27/kip1 immunohistochemistry was detected in IGL (as well as other layers) but was visually more readily apparent in wild-type vs. GPR3−/− mice at all stages. In panel b, pCREB was detected by immunohistochemistry in wild-type and GPR3−/− mice (P12). While pCREB, was detected in the molecular layer (ML) and IGL of widl-type mice, it was not as readily visible in the IGL of the GPR3−/−. In panel c, co-localization of pCREB and p27/kip was determined by double immunofluorescence. Scale bar = 50 µm.
Mentions: To provide additional confirmatory evidence for a regulatory relationship between GPR3 and p27/Kip, we sought to determine the expression of p27/Kip1 in wild type or GPR3−/− mice during postnatal cerebellar development (P5, P8 and P14). The expression of p27/Kip1 in wild-type P5 mice was scarce and observed primarily in IGL in P5 cerebellum (figure 6A). However, this expression visibly increased at the P8 and P14 time points in IGL, but also in the inner side of EGL and in migrating GCPs in ML. In contrast, in GPR3−/− mice the expression of p27/Kip1 was visibly reduced in the EGL and IGL of P5, P8 and P14 cerebella compared to that of wild-type mice at the same time points (figure 6a). These data thus indicate that GPR3 gene expression was involved in the regulation of p27/Kip gene expression in the developing postnatal mouse cerebellum.

Bottom Line: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation.In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression.These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain.

Methodology/principal findings: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 -/- mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit.

Conclusions/significance: These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

Show MeSH
Related in: MedlinePlus