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A. Takatori, R. Parthasarathy, M. Mongan, Y. Xia; MEKK1–JNK Pathway Regulates Mouse Eyelid Morphogenesis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5630.
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MEK kinase 1 (MEKK1) is a mitogen–activated protein kinase kinase kinase, known as an upstream regulator for the c–Jun NH2–terminal kinases (JNKs). Knocking out Mekk1 in mice results in eye–open at birth (EOB) phenotype that leads to severe eye pathologies. There are two Jnk isoforms, Jnk1 and Jnk2, that are ubiquitously expressed in various tissues. This study is aimed at understanding the distinct role for JNK1 and JNK2 in transmitting the MEKK1 signals during eyelid morphogenesis.
Mekk1–/–, Jnk1–/– and Jnk2–/– mice were backcrossed to C57BL/6 background. Intercrosses were carried out to generate Mekk1+/–Jnk1–/–and Mekk1+/–Jnk2–/– mice. Eyelid development was observed at various developmental stages. The eye sections were analyzed by histology and immunohistochemistry. An in vitro wound healing assay was performed using keratinocytes derived from wild type, Mekk1+/–Jnk1–/– and Mekk1+/–Jnk2–/– mice.
The Mekk1+/–Jnk1–/–, but not Mekk1+/–Jnk2–/–, mice displayed EOB similar to the Mekk1–/– mice. Unlike the Mekk1+/–Jnk2–/– fetuses, which showed a thin layer of eyelid epithelium covering the ocular surface at embryonic day 16.5, the Mekk1+/–Jnk1–/– fetuses lacked eyelid closure and had fully exposed cornea. Immunohistochemistry studies showed that there were similar levels of JNK phosphorylation, but a much reduced c–Jun phosphorylation in the developing eyelid epithelium of the Mekk1+/–Jnk1–/–, compared to that of the wild type and the Mekk1+/–Jnk2–/– fetuses. Concomitantly, the expression of PAI–1 was markely decreased in Mekk1+/–Jnk1–/– fetuses. The phosphorylation of ERK and EGFR remained unaltered in the Mekk1+/–Jnk1–/– fetuses. Considering the crucial role of PAI–1 in epithelial cell migration, we studied the keratinocyte migration using an in vitro wound healing assay. Our results indicated that the Mekk1+/–Jnk1–/– keratinocytes had significantly delayed wound healing comparing to the wild type and Mekk1+/–Jnk2–/– keratinocytes.
We show that JNK1 is more critical than JNK2 in transmitting the MEKK1 signals in the regulation of mouse embryonic eyelid closure. Although the MEKK1 signals seem to activate JNK1 and JNK2 equally well, JNK1 appears to be more efficient in c–Jun phosphorylation and activating AP–1 target PAI–1 expression. In the absence of JNK1, one functional Mekk1 allele is insufficient to cause c–Jun phosphorylation and epithelial cell movement, responsible for defective eyelid morphogenesis of the Mekk1+/–Jnk1–/– fetuses.
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