Abstract
Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a Gi coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of Gq/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, Gi mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.
Highlights
Calcium imaging was performed by loading cultured mouse hippocampal cells with Fura-2 (Fig. 1A)
Primary cultures of mouse hippocampal cells were exposed to Sonic hedgehog (SHH) (500 pM, 10 ng/ml) in the presence of 1 M tetrodotoxin (TTX), and some of the cells exhibited spontaneous Ca oscillations before the agonist application
Agonist-induced Ca frequency increase was evaluated by subtracting the baseline Ca frequency from that after the drug application in each cell (⌬Frequency). ⌬Frequencies of cells applied with concentrations of SHH or Smoothened agonist (SAG) were compared with that of vehicleapplied control cells (0.1% DMSO; Fig. 1, D and E)
Summary
The high levels of SHH and related proteins in the adult hippocampus together with the inferred roles of SHH signaling in adult neurogenesis and brain injury led us to characterize cellular responses to SHH in cultured hippocampal cells. We found the enhancement of Ca oscillations in astrocytes within several minutes after application of SHH pathway agonists, namely SHH and Smoothened agonist (SAG). This enhancement was blocked by inhibition of Gi and removal of extracellular ATP. Together with other lines of evidence, we propose that the enhancement of Ca oscillations in astrocytes is initiated by the activation of SMO-coupled Gi, which leads to ATP release through ATP-permeable channels. This released ATP enhances Ca oscillations in nearby astrocytes. We observed enhanced Ca oscillations in astrocytes in brain slices; this mechanism may be functional in the in vivo brain as well
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