Figure 8.

Inhibition of ErbB signaling in reactive astrocytes did not block inflammatory signaling induced by injury. (a) Similar inflammation induced by stab injury in the cortices of Mlc1-dnEGFR and littermate control mice. Shown are representative images of Iba1 immunostaining in injured cortical regions 3 days post injury. ‘Δ’ represents the injury sites. (b) Quantitative analysis of Iba1⁺ cell densities in injured cortical regions of Mlc1-dnEGFR and littermate control mice. n=3 for each group, paired t-test. (c) Real-time RT-PCR results of indicated cytokines in injured cortical tissues from Mlc1-dnEGFR and littermate control mice 7 days post injury. ***P<0.001; **P<0.01; n=3 for each group. (d) Activities of various signaling proteins induced by ErbB receptor ligands were reduced in primary Mlc1-dnEGFR astrocytes. Primary astrocytes from Mlc1-dnEGFR and control mice were treated with saline (Ctrl), rhEGF (1 μg/ml), or rhNRG1 (100 ng/ml) for 15 min, respectively, and indicated proteins and their phosphorylation levels in cell lysates were examined by WB. (e) Quantitative analyses of the protein phosphorylation levels revealed by WB in astrocytes stimulated by ErbB ligands. Phosphorylation levels of indicated proteins were normalized by its total protein levels. ^#P<0.05; ^##P<0.01; as compared with the treatment with saline. *P<0.05, as compared with control astrocytes with the same treatment. n=3 for each protein, paired t-test. (f) STAT3 activity induced by cytokine CNTF was not reduced in primary Mlc1-dnEGFR astrocytes. Primary astrocytes from Mlc1-dnEGFR or control mice were treated with saline (Ctrl) or CNTF (200 ng/ml) for 30 min and indicated proteins and their phosphorylation levels in cell lysates were examined by WB. (g) Quantitative analyses of the protein phosphorylation levels revealed by WB in astrocytes stimulated by CNTF. Phosphorylation levels of indicated proteins were normalized by its total protein levels. ^##P<0.01; ^###P<0.001; as compared with the treatment with saline. **P<0.01, as compared with control astrocytes with the same treatment. n=3 for each protein, paired t-test. (h) Schematic illustration of a working model for the role of ErbB signaling in the induction of reactive astrogliosis. ErbB activation in quiescent astrocytes initiates reactive astrogliosis via diverse downstream signaling pathways. Src and FAK, the non-receptor tyrosine kinases regulating actin polymerization, are activated by ErbB signaling to prompt hypertrophic remodeling in astrocytes. Other signaling proteins downstream of ErbB receptors, such as STAT3, could be activated through different pathways stimulated by multiple factors in the inflammatory environment. Inhibiting ErbB signaling in reactive astrocytes blocks hypertrophy through a direct inhibition on Src/FAK activities, whereas other signaling proteins such as STAT3 remain active to promote proliferation. Note that microglia could react to factors released from reactive astrocytes or other sources.