Novel Insights Into Msk1 Phosphorylation By Mrk Beta In Intracerebral Hemorrhage-Induced Neuronal Apoptosis

Neuronal apoptosis is regarded as one of the most important pathophysiological changes of intracerebral hemorrhagic (ICH) stroke-a major public health problem that leads to high mortality rates and functional dependency. Mitogen-and stress-activated kinase (MSK) 1 is implicated in various biological functions in different cell types, including proliferation, tumorigenesis and responses to stress. Our previous study showed that MSK1 phosphorylation (p-MSK1) is related to the regulation of LPS-induced astrocytic inflammation, and possibly acts as a negative regulator of inflammation. In this study, we identified a specific interaction between MSK1 and MRK beta (MLK-related kinase)-a member of the MAPK pathway-during neuronal apoptosis. In an ICH rat model, western blotting and immunohistochemical analysis revealed that both MRK beta and phosphorylation of MSK1 (p-MSK1 Ser376) were significantly upregulated in cells surrounding the hematoma. Triple-immunofluorescent labeling demonstrated the co-localization of MRK beta and p-MSK1 in neurons, but not astrocytes. Furthermore, MRK beta was partially transported into the nucleus, and interacted with p-MSK1 in hemin-treated neurons. Immunoprecipitation showed that MRK beta and p-MSK1 exhibited an enhanced interaction during the pathophysiology process. Utilizing small interfering RNAs to knockdown MRK beta or MSK1, we verified that MSK1 Ser376 is a phosphorylation site targeted by MRK beta. We also observed that the phosphorylation of NF-kappa B p65 at Ser276 was mediated by the MRK beta-p-MSK1 complex. Furthermore, it was found that the neuronal apoptosis marker, active caspase-3, was co-localized with MRK beta and p-MSK1. In addition, flow cytometry analysis revealed that knockdown of MRK beta or MSK1 specifically resulted in increased neuronal apoptosis, which suggested that the MRK beta-p-MSK1 complex might exert a neuroprotective function against ICH-induced neuronal apoptosis. Taken together, our data suggest that MRK beta translocated into the nucleus and phosphorylated MSK1 to protect neurons via phosphorylation of p65-a subunit of nuclear factor kappa B.