Transient Receptor Potential Canonical 3 (TRPC3) Mediates Thrombin-Induced Astrocyte Activation and Upregulates Its Own Expression in Cortical Astrocytes

Abstract

Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca 2ϩ -permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca 2ϩ influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca 2ϩ influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH 2 -terminal kinase, and nuclear factor-B signaling and required de novo protein synthesis. The Ca 2ϩ signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca 2ϩ signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca 2ϩ -signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH 2 , a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own expression.

Gq signaling causes glomerular injury by activating TRPC6

“…As shown in Figure 7H, Rcan1 levels were markedly increased in WT Akita mice compared with levels detected in controls. Rcan1 was also increased ≈3-fold in Gq Akita mice compared with controls, ing on TRPC3 expression (38). TRPC5 levels were not significantly different in the experimental groups.…”

Transient Receptor Potential Canonical 3 Inhibitor Pyr3 Improves Outcomes and Attenuates Astrogliosis After Intracerebral Hemorrhage in Mice

“…11 These results imply that thrombin leaked from ruptured blood vessels can activate astrocytes through TRPC3 channels in vivo. This hypothesis is strongly supported by our present results, demonstrating that the selective TRPC3 inhibitor Pyr3 suppressed reactive astrogliosis and ameliorated functional recovery in mouse ICH model.…”

“…11 Although systemic administration of argatroban, a direct thrombin inhibitor, significantly reduced brain edema after ICH, 12 the effect of TRPC3 inhibition, per se, has not yet been clarified in an in vivo model of ICH.…”

Inversion of neurovascular coupling by subarachnoid blood depends on large-conductance Ca 2+ -activated K + (BK) channels

“…Alternatively, subarachnoid blood may act indirectly by inducing inflammation (57) or reactive astrogliosis (58). Thrombin, for example, has been shown to induce reactive astrogliosis (59). The impact of astrogliosis on astrocyte Ca 2+ signaling and neurovascular coupling is not known, although it has been associated with brain pathologies, including brain cancers, Alzheimer's disease, and stroke (60).…”