Abstract
BackgroundWe have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2′-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.Methodology/Principal FindingsStable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.Conclusions/SignificanceThese results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.
Highlights
We have previously shown significant alterations in endogenous levels of both glutamic and gamma-aminobutyric acids in particular brain structures after the extremely traumatic water immersion restraint stress (WIRS) experience in rats [1]
Stable Transfection of Myo6 In our previous study [26], transient overexpression of Myo6 led to a decrease in the size of clustered aggregates composed of proliferating cells during culture with all-trans retinoic acid (ATRA) in pluripotent P19 cells
Amongst 6 clones selected by G418 screening in pluripotent P19 transfectants, Myo6 mRNA was strongly detected in several clones, such as M6#1 and M6#4, upon Reverse transcription-polymerase chain reaction (RT-PCR) analysis (Figure 1A)
Summary
We have previously shown significant alterations in endogenous levels of both glutamic and gamma-aminobutyric acids in particular brain structures after the extremely traumatic water immersion restraint stress (WIRS) experience in rats [1]. WIRS and subsequent flashback experiences lead to drastic but transient decreases in the numbers of both neuronal and astroglial cells derived from neural progenitors, with suppression of proliferation in the DG in mice [7]. The extremely stressful experience induced a rapid but transient increase in the expression of both the mRNA and corresponding protein for the cellular motor molecule Myosin VI (Myo6) in the mouse hippocampus [7]. We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo29-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. We investigated the role of Myo in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques
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