| Kallikrein-kinin system is a well-known regulatory system that plays an important part in various physiological and pathological processes of focal cerebral ischemia. Tissue kallikrein (TK) is a critical component in the system, which triggers the course of neuro vascular protection and restoration against ischemia-induced injury via the effects of anti-inflammatory, antioxidant, anti-apoptosis, angiogenesis, and neurogenesis. Our previous work showed that TK could enhance neuron growth, which made it possible for TK to be a therapeutic option for stroke. Focusing on the function of epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK) and flotillin-2, in the present study, we investigated TK-induced neurite outgrowth and its signaling mechanism in cultured human SH-SY5Y cells and mouse primary neurons.Part I Tissue kallikrein activates extracellular regulated protein kinase 1/2 through epidermal growth factor receptorObjective:To explore the signal transduction mechanism of TK on activating EGFR and MAPK.Methods:Human SH-SY5Y cells were cultured in vitro. With or without TK treatment, the phosphorylation of EGFR, extracellular regulated protein kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38) or c-Jun N-terminal kinase (JNK) was detected by western blot. And the intracellular localization of EGFR was observed by immunocytochemistry under a fluorescence microscope. In some experiments, the protein level of EGFR was down-regulated by RNA interference sequences. After knockdown of EGFR, TK-induced ERK1/2, p38 or JNK phosphorylation was evaluated. Also TK-stimulated EGFR phosphorylation was assayed after cells were treated with ERK kinase inhibitor PD98059.Results:(1) SH-SY5Y cells exposed to TK at 0.0625 and 0.125 μM could not activate the phosphorylation of EGFR, while TK at 0.25 to 1.0 μM exhibited a concentration-dependent increase in EGFR phosphorylation. (2) TK facilitated the translocation of EGFR to around the nuclei. (3) TK could trigger the phosphorylation effects of ERK1/2 and p38, which peaked in 5 min and 15 min of TK stimulation respectively, but could not activate JNK phosphorylation. (4) Interestingly, not p38 but ERKl/2 phosphorylation was severely compromised in cells depleted of EGFR. (5) Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation.Conclusion:In cultured SH-SY5Y cells, TK could promote EGFR, ERKl/2 and p38 phosphorylation, and also could facilitate the translocation of EGFR to around the nuclei. Furthermore, TK was able to mediate ERKl/2 pathway via EGFR.Part II Tissue kallikrein activates epidermal growth factor receptor pathway via flotillin-2Objective:To investigate TK signaling pathway through the activation of flotillin-2, EGFR and ERKl/2 in cultured SH-SY5Y cells.Methods:The experiments on blockage of bradykinin B1 receptor, B2 receptor, flotillin-2, EGFR or ERK1/2 were performed by the corresponding inhibitors or RNA interference sequences. TK-induced EGFR or ERK1/2 phosphorylation was evaluated by western blot. Co-immunoprecipitation experiments were carried out for either flotillin-2 or EGFR from unstimulated to TK-stimulated SH-SY5Y cells. With or without TK treatment, the intracellular localization of flotillin-2 or EGFR was observed by immunocytochemistry.Results:(1) SH-SY5Y cells exposed to TK exhibited a significant increase in EGFR or ERK1/2 phosphorylation. (2) With the inhibition of bradykinin B1 receptor or B2 receptor, there was no significant effect on EGFR or ERK1/2 phosphorylation in the TK stimulation. (3) TK-induced EGFR or ERKl/2 phosphorylation was sharply decreased in flotillin-2 knockdown cells. (4) Interestingly, ERKl/2 phosphorylation was severely compromised in cells depleted of EGFR. (5) Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. (6) In the TK-stimulated samples, two bands against EGFR or p-EGFR antibody were detected in the immunoprecipitates that were precipitated with flotillin-2. (7) Flotillin-2 and EGFR were found to reside in the cytoplasm and plasma membrane under normal condition. However, the addition of TK remarkably promoted the two proteins to accumulate around the nuclei, staining colocalized.Conclusion:TK could promote flotillin-2-EGFR-ERK1/2 signaling pathway independent of bradykinin receptors in cultured SH-SY5Y cells. Flotillin-2 and EGFR formed constitutive complex in the TK stimulation. TK treatment was able to facilitate the translocation of flotillin-2-EGFR complex to around the nuclei.Part III Tissue kallikrein promotes neurite outgrowth through flotillin-2, epidermal growth factor receptor and extracellular regulated protein kinase 1/2Objective:To demonstrate that TK could promote neurite outgrowth through flotillin-2, EGFR and ERKl/2.Methods:Mouse primary cortical neurons were cultured in vitro. In some experiments, neuron bradykinin B1 receptor, B2 receptor, flotillin-2, EGFR or ERK1/2 was blocked by the corresponding inhibitors or RNA interference sequences. Unstimulated or TK-stimulated neurons were stained with microtubule associated protein-2 antibody and observed by immunocytochemistry. Neurite outgrowth analysis, including the number of processes and mean process length, was evaluated by using Image J software.Results:(1) TK treatment could promote the number of processes and mean process length in primary neurons. (2) With the blockage of bradykinin B1 receptor or B2 receptor, there was no significant effect on neurite outgrowth in the TK stimulation. (3) TK-induced neurite outgrowth was decreased in flotillin-2 knockdown neurons. (4) Interestingly, TK-mediated neurite outgrowth was severely compromised in neurons inhibition of EGFR. (5) Moreover, impairment of ERK1/2 also seemed to be restricted to TK-stimulated neurite outgrowth.Conclusion:TK could promote neurite outgrowth independent of bradykinin receptors in cultured primary neurons. TK treatment was able to mediate neurite outgrowth through flotillin-2, EGFR and ERKl/2. |
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