Abstract
Purpose: :
The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is crucial for eyelid morphogenesis. Loss of MAP3K1 in mice results in defective embryonic eyelid closure and an eye-open at birth (EOB) phenotype. We have previously shown that MAP3K1 regulates eyelid development at two levels. One level is the expression of MAP3K1, which is induced in the developing eyelid epithelial cells. Another level is the activity of MAP3K1, which is required to activate a JNK-c-JUN signaling cascade for eyelid closure. Previous in vitro studies show that RhoA, a member of the Rho GTPase family, may act on the second level, activating MAP3K1-mediated JNK pathways. This idea has never been tested in vivo. The current study uses an in vivo compound and cell type-specific gene knockout system and in vitro molecular biology to investigate the interaction between RhoA and MAP3K1 in eyelid development.
Methods: :
The double transgenic mice containing RhoAfloxconditional allele and Le-cre driver were mated with the Map3k1+/ΔKD mice, in which the Map3k1ΔKD allele contains a bacterial β-galactosidasegene knocked-in into the Map3k1 locus, replacing the exons coding for MAP3K1 kinase domain. As a result, the Map3k1ΔKD allele produces a protein that lacks kinase activity and has its N-terminal domain fused to β-gal. The eyelid development in fetuses was examined at E17.5. MAP3K1 expression in the developing eyelids was evaluated by whole mount X-gal staining. Transient transfection was done using 293 cells with Map3k1-promoter-luc and various mammalian expression vectors for active and dominant negative RhoA and ROCKI and II, a protein kinase downstream of RhoA The cells were treated with colchicine, stimulus known to induce MAP3K1 expression, and luciferase activity was determined.
Results: :
We found that MAP3K1 functionally interacted with RhoA in eyelid development. When RhoA is deleted in the developing eyelid epithelial cells, MAP3K1 became haploinsufficient for eyelid closure. Correspondingly, the RhoA ablation reduced MAP3K1 expression in the developing eyelids. In 293 cells, active RhoA stimulated the exogenous Map3k1-promoter-luc and endogenous MAP3K1 expression, whereas, dominate negative RhoA reduced Map3k1-promoter-luc activity induced by colchicine. Furthermore, ROCK also induced Map3k1-promoter-luc activity and the ROCK inhibitor blocked colchicines induced promoter activity and MAP3K1 expression.
Conclusions: :
In addition to activating the MAP3K1-JNK pathways, RhoA can also crosstalk with MAP3K1 by activating its gene promoter and induces its expression.
Keywords: development • signal transduction