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E. N. Geh, Y. Xia; Role of the Cytoskeleton in the Regulation of MAP3K1 Expression. Invest. Ophthalmol. Vis. Sci. 2010;51(13):705.
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© ARVO (1962-2015); The Authors (2016-present)
Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is known to be involved in eyelid closure during mouse embryonic development. MAP3K1is highly expressed in the leading edge of the eyelid epithelium just prior to the onset of eyelid closure at embryonic day 15.5 and its ablation in mice results in defective eyelid morphogenesis. We hypothesize that the induction of MAP3K1 expression is a critical determinant for embryonic eyelid closure. The purpose of this study is to investigate the molecular mechanisms that regulate MAP3K1 expression.
Mouse embryonic fibroblasts (MEFs) were isolated from the Map3k1ΔKD fetuses, in which a β-galactosidase gene was knocked in the Map3k1 locus. In these cells, β-gal expression was under the control of the Map3k1 promoter. These cells were used to study the induction of endogenous Map3k1 promoter activity following various treatments by measuring β-galactosidase activity. Total RNA was used for RT-PCR to detect Map3k1 induction at the transcriptional level and whole cell lysates were prepared for Western blotting to detect MAP3K1 protein. A 1.9 kb fragment upstream of the transcription start site (TSS) of mouse Map3k1, encompassing the predicted promoter sequences, was cloned into a promoterless luciferase vector using polymerase chain reaction (PCR). This pMap3k1-luc plasmid was used for transient transfection to study exogenous Map3k1 promoter activities.
Using the Map3k1ΔKD MEFs and measurement of β-galactosidase expression, we have identified that colchicine and nocodazole, microtubule disrupting agents, could stimulate the endogenous Map3k1 promoter by 2.5 fold. This treatment condition also causes an increase in the MAP3K1 protein expression by 2-fold. Using the pMap3k1-luc plasmid, we find that the microtubule disrupting agents also induce the exogenous Map3k1 promoter activity. Interestingly, the exogenous Map3k1 promoter activity was potentiated by over-expressing ROCK, while colchicine induced promoter activity was blocked by treatment with a ROCK inhibitor.
The Map3k1 promoter activity and gene induction is likely stimulated by microtubule disruption through a ROCK-dependent pathway.
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