RESUMO
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a central role in the intracellular signaling of P2X7 nucleotide receptors in neurons and glial cells. Fine spatio-temporal tuning of mitogen-activated protein (MAP) kinases is essential to regulate their biological activity. MAP kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues in MAP kinases. This study focuses on how DUSP, DUSP6/MKP3, a phosphatase specific for ERK1/2 is regulated by the P2X7 nucleotide receptor in cerebellar granule neurons and astrocytes. Stimulation with the specific P2X7 agonist, BzATP, or epidermal growth factor (EGF) (positive control for ERK activation) regulates the levels of DUSP6 in a time dependent manner. Both agonists promote a decline in DUSP6 protein, reaching minimal levels after 30 min yet recovering to basal levels after 1 h. The initial loss of protein occurs through proteasomal degradation, as confirmed in experiments with the proteasome inhibitor, MG-132. Studies carried out with Actinomycin D demonstrated that the enhanced transcription of the Dusp6 gene is responsible for recovering the DUSP6 protein levels. Interestingly, ERK1/2 proteins are involved in the biphasic regulation of the protein phosphatase, being required for both the degradation and the recovery phase. We show that direct Ser197 phosphorylation of DUSP6 by ERK1/2 proteins could be part of the mechanism regulating their cytosolic levels, at least in glial cells. Thus, the ERK1/2 activated by P2X7 receptors exerts positive feedback on these kinase's own activity, promoting the degradation of one of their major inactivators in the cytosolic compartment, DUSP6, both in granule neurons and astrocytes. This feedback loop seems to function as a common universal mechanism to regulate ERK signaling in neural and non-neural cells.
RESUMO
Prostaglandin E2 (PGE2) is an important bioactive lipid that accumulates after tissue damage or inflammation due to the rapid expression of cyclooxygenase 2. PGE2 activates specific G-protein coupled EP receptors and it mediates pro- or anti-inflammatory actions depending on the cell-context. Nucleotides can also be released in these situations and they even contribute to PGE2 production. We previously described the selective impairment of P2Y nucleotide signaling by PGE2 in macrophages and fibroblasts, an effect independent of prostaglandin receptors but that involved protein kinase C (PKC) and protein kinase D (PKD) activation. Considering that macrophages and fibroblasts influence inflammatory responses and tissue remodeling, a similar mechanism involving P2Y signaling could occur in astrocytes in response to neuroinflammation and brain repair. We analyzed here the modulation of cellular responses involving P2Y2/P2Y4 receptors by PGE2 in rat cerebellar astrocytes. We demonstrate that PGE2 inhibits intracellular calcium responses elicited by UTP in individual cells and that inhibiting this P2Y signaling impairs the astrocyte migration elicited by this nucleotide. Activation of EP3 receptors by PGE2 not only impairs the calcium responses but also, the extracellular regulated kinases (ERK) and Akt phosphorylation induced by UTP. However, PGE2 requires epidermal growth factor receptor (EGFR) transactivation in order to dampen P2Y signaling. In addition, these effects of PGE2 also occur in a pro-inflammatory context, as evident in astrocytes stimulated with bacterial lipopolysaccharide (LPS). While we continue to investigate the intracellular mechanisms responsible for the inhibition of UTP responses, the involvement of novel PKC and PKD in cerebellar astrocytes cannot be excluded, kinases that could promote the internalization of P2Y receptors in fibroblasts.
