Thrombin‐induced reversal of astrocyte stellation is mediated by activation of protein kinase C β‐1

Abstract

Exogenous or endogenous injuries of the central nervous system trigger astrogliosis characterized by proliferation of astrocytes and changes in their morphology from stellate to flat polygonal. Astrocytes in culture are very sensitive to thrombin, a serine protease, which through its proteolytically activated receptor (PAR-1) induces proliferation and morphological changes comparable to astrogliosis. Evaluation of the thrombin signal-transduction pathway in the reversal of astrocyte stellation might help to understand astrogliosis. For this purpose, primary cultured murine cortical astrocytes were treated with H7, a proteinkinase inhibitor, and thrombin, which resulted in an inhibition of stellation reversal. Treatments with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, mimicked the action of thrombin. Subsequently, direct assay of astrocyte PKC activity after thrombin or PMA treatment demonstrated involvement of PKC in thrombin signaling associated with shape change. Western blotting showed that PKC isoform β-1 was involved in this pathway, while PKC A was only weakly activated and PKC β-2 was not activated by thrombin. PKC β-1 translocation was also elicited by a thrombin-receptor active peptide (SFLLRN), demonstrating the involvement of PAR-1 in this process. PKC δ and ε were located constitutively in the membrane fraction in stellate astrocytes. Isoforms γ, η, θ, and ζ were absent from astrocytes. These results suggest that astrogliosis in vivo might be regulated by modulating the activity of thrombin, PAR-1, or specific PKC isoforms.

Thrombin downregulates muscle acetylcholine receptors via an IP3 signaling pathway by activating its G‐protein‐coupled protease‐activated receptor‐1

“…This may explain the discrepancy with the results of Davenport (Davenport et al, 2000) who did not observe such an effect since these authors used only low thrombin concentrations. It is well known that thrombin efficient concentrations depend on the cell-type studied and range from picomolar concentration for reversal of astrocyte stellation (Beecher et al, 1994;Pindon et al, 1998) to nanomolar concentration for tumor cell apoptosis (Zain et al, 2000) and micromolar concentrations for neuronal apoptosis (Donovan et al, 1997). Under our conditions, thrombin did not induce myotube apoptosis as shown by TUNEL analysis and annexin V binding (data not shown).…”

Up-Regulation of Proteinase-Activated Receptor 1 Expression in Astrocytes During HIV Encephalitis

“…We demonstrate that PAR-1 expression in astrocytoma cells is up-regulated by thrombin, but the signaling pathways remain to be elucidated. PAR-1 and thrombin protein expression was predominantly detected on reactive astrocytes in HIVE brains, suggesting a potential important autocrine loop leading to immune activation, calcium signaling (77), and astrogliosis (14). PAR-1 immunoreactivity was also observed on neurons, endothelial cells, and infiltrating monocytic cells, although to a much lesser extent.…”

Activation of Protease-Activated Receptor-1 Triggers Astrogliosis after Brain Injury

“…Furthermore, thrombin seems to induce microglial activation by induction of DNA synthesis as well as production of nitric oxide and tumor necrosis factor-␣ (Ryu et al, 2000;Suo et al, 2002). Many groups have also demonstrated that PAR-1 activation stimulates astrocytes to transform from their normally stellate shape into a polygonal morphology (Beecher et al, 1994;Grabham and Cunningham, 1995;Suidan et al, 1997;Pindon et al, 1998). In addition, thrombin also induces endothelin-1 and nerve growth factor secretion from astrocytes (Ehrenreich et al, 1993;Neveu et al, 1993;Debeir et al, 1996).…”