Abstract
Purpose::
MEK kinase 1 (MEKK1) is a mitogen-activated protein kinase kinase kinase, known as an upstream regulator for the MKK4/7- c-Jun NH2-terminal kinases (JNKs) pathway. Ablation of the Mekk1 gene in mice results in eye-open at birth (EOB) phenotype that leads to severe eye pathologies; however, ablation of the individual Jnk genes, either Jnk1 or Jnk2, does not affect mouse eye development. This study is aimed at understanding the signaling mechanisms by which MEKK1 and JNK contribute to eyelid development.
Methods::
Mekk1-/-, Jnk1-/- and Jnk2-/- mice were intercrossed to generate compound mutant mice. The E15.5 mouse fetuses were subjected to immunohistochemistry for phospho-JNK, phospho-c-Jun and PAI-1 in the developing eyelid epithelium. Mouse embryonic fibroblasts (MEFs) were prepared from wildtype, Mekk1+/-Jnk1-/- and Mekk1+/-Jnk2-/- fetuses. These MEFs and wild type and Mkk4-/- ES cells were infected with adenoviruses for active MEKK1. The cell lysates were subjected to Western blotting for phosphorylated JNK, MKK4 and MKK7. JNK1 and JNK2 mutants {JNK1(C177/N179) and JNK2(G177/S179)} were generated by site-directed mutagenesis. HEK293 cells were transiently co-transfected with cDNAs for MEKK1, wild type and mutant JNK1 and JNK2; the cell lysates were subjected to Western blotting for phospho-JNK and in vitro kinase assay for JNK kinase activity.
Results::
Mekk1+/-Jnk1-/- and Mekk1+/-Jnk1+/-Jnk2+/- triple hemizygous, but not Mekk1+/-Jnk2-/- mice displayed EOB, suggesting that JNK1 and JNK2 are not equivalent in mediating MEKK1 signal during eyelid development. Defective eyelid closure in Mekk1+/-Jnk1-/- and triple hemizygouts was associated with significant less phosphorylation of JNK and its substrate c-Jun in developing eyelid epithelium, and concurrently reduced expression of PAI-1, a known c-Jun target gene. Expression of active MEKK1 strongly activated MKK4, which was necessary for JNK activation. Interestingly, MEKK1 induced phosphorylation of JNK1 twice as efficiently as JNK2. The phosphorylation efficiency was determined by the 177/179 residues in the variable region of JNK, which were Gly177/Ser179 in JNK1 and Cys177/Asp179 in JNK2. Swapping the 177/179 residues between the JNK isoforms reversed their phosphorylation levels by MEKK1.
Conclusions::
Our studies provide the first genetic evidence for a role of JNK in mouse eyelid development. In developing eyelid epithelium, MEKK1 is a rate limiting factor for the activation of the MKK4-JNK pathway, of which JNK1 and JNK2 each makes unique contributions to transmitting the MEKK1 signals, resulting in gene transcription and embryonic eyelid closure.
Keywords: eyelid • signal transduction • transgenics/knock-outs