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Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Rolón-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN, Eaton MJ, Harrison JK, Kucheryavykh LY - PLoS ONE (2015)

Bottom Line: Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells.A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration.Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

No MeSH data available.


Related in: MedlinePlus

siRNA knock-down and/or pharmacological blockade of Pyk2 by PF-562,271 eliminates the stimulatory effect of microglia on glioma cell migration.Data obtained from standard migration assays for control glioma cells and cells transfected with siRNA against Pyk2 with and without additional application of PF-562,271 in the presence and absence of microglia in the lower compartment. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant difference from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.
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pone.0131059.g007: siRNA knock-down and/or pharmacological blockade of Pyk2 by PF-562,271 eliminates the stimulatory effect of microglia on glioma cell migration.Data obtained from standard migration assays for control glioma cells and cells transfected with siRNA against Pyk2 with and without additional application of PF-562,271 in the presence and absence of microglia in the lower compartment. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant difference from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.

Mentions: As an independent measure of microglial activation of Pyk2 signaling in glioma cells and its role in glioma cell migration, we used siRNA targeting Pyk2 to selectively knock-down Pyk2 in glioma cells. Three days after transfection with siRNA, levels of Pyk2 were reduced by 60% in glioma cells (S5 Fig). The results obtained using siRNA knock-down of Pyk2 in glioma cells were identical to those obtained using 16 nM PF-562,271 to block Pyk2 (Fig 7). In addition, simultaneous knock-down of Pyk2 using siRNA together with application of 16nM PF-562,271 did not produce further reduction of migration induced by microglia compared to Pyk2 knock-down alone. Taken together the experiments shown in Figs 6 and 7 indicate that Pyk2, but not FAK, is the major signaling pathway involved in microglial stimulation of glioma cell migration.


Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Rolón-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN, Eaton MJ, Harrison JK, Kucheryavykh LY - PLoS ONE (2015)

siRNA knock-down and/or pharmacological blockade of Pyk2 by PF-562,271 eliminates the stimulatory effect of microglia on glioma cell migration.Data obtained from standard migration assays for control glioma cells and cells transfected with siRNA against Pyk2 with and without additional application of PF-562,271 in the presence and absence of microglia in the lower compartment. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant difference from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476590&req=5

pone.0131059.g007: siRNA knock-down and/or pharmacological blockade of Pyk2 by PF-562,271 eliminates the stimulatory effect of microglia on glioma cell migration.Data obtained from standard migration assays for control glioma cells and cells transfected with siRNA against Pyk2 with and without additional application of PF-562,271 in the presence and absence of microglia in the lower compartment. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant difference from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.
Mentions: As an independent measure of microglial activation of Pyk2 signaling in glioma cells and its role in glioma cell migration, we used siRNA targeting Pyk2 to selectively knock-down Pyk2 in glioma cells. Three days after transfection with siRNA, levels of Pyk2 were reduced by 60% in glioma cells (S5 Fig). The results obtained using siRNA knock-down of Pyk2 in glioma cells were identical to those obtained using 16 nM PF-562,271 to block Pyk2 (Fig 7). In addition, simultaneous knock-down of Pyk2 using siRNA together with application of 16nM PF-562,271 did not produce further reduction of migration induced by microglia compared to Pyk2 knock-down alone. Taken together the experiments shown in Figs 6 and 7 indicate that Pyk2, but not FAK, is the major signaling pathway involved in microglial stimulation of glioma cell migration.

Bottom Line: Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells.A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration.Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

No MeSH data available.


Related in: MedlinePlus