Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) integrates developmental signals for eyelid closure Esmond Geha,1, Qinghang Menga,1, Maureen Mongana, Jingcai Wanga,b, Atsushi Takatoric, Yi Zhengd, Alvaro Pugaa, Richard A. Lange,f, and Ying Xiaa,e,2 Departments of aEnvironmental Health and eOphthalmology, University of Cincinnati Medical Center, Cincinnati, OH 45267; bSouthern Medical University, Guangzhou 510515, China; cChiba Cancer Center, Chiba 260-8717, Japan; and dExperimental Hematology and Cancer Biology and fVisual Systems Group, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229 Edited* by Michael Karin, San Diego School of Medicine, University of California, La Jolla, CA, and approved September 16, 2011 (received for review February 10, 2011) Developmental eyelid closure is an evolutionarily conserved consequence of epithelial cell apoptosis at the fusion junction, morphogenetic event requiring proliferation, differentiation, cy- resulting in open eyelids. Mice are normally born with their toskeleton reorganization, and migration of epithelial cells at the eyelid closed, but those impaired in embryonic eyelid closure are tip of the developing eyelid. Many signaling events take place born with eye open-at-birth (EOB) phenotype. during eyelid closure, but how the signals converge to regulate Based on genetic mutations in mice that lead to EOB, it has the morphogenetic process remains an open and intriguing qu- become clear that complex signal transduction processes are estion. Here we show that mitogen-activated protein kinase involved in the regulation of eyelid closure. So far, whether kinase kinase 1 (MAP3K1) highly expressed in the developing eye- embryonic eyelid closure is shown depends on signals derived lid epithelium, forms with c-Jun, a regulatory axis that orches- from WNT, Sonic hedgehog, BMP/Activin, FGF, and EGF (5– trates morphogenesis by integrating two different networks of 8). In addition, eyelid closure requires the participation of eyelid closure signals. A TGF-α/EGFR-RhoA module initiates one a number of intracellular signaling kinases, such as MAP3K1, BIOLOGY of these networks by inducing c-Jun expression which, in a phos- JNK, ROCK, and CDH1, and nuclear transcription factors, such DEVELOPMENTAL phorylation-independent manner, binds to the Map3k1 promoter as c-Jun, Fra-2, FOXL2, SMAD, and GRHL3 (9–17). Molecular and causes an increase in MAP3K1 expression. RhoA knockout in and genetic analyses of the knockout mice have shown that the the ocular surface epithelium disturbs this network by decreasing eyelid closure factors are organized into distinct signal trans- Map3k1 MAP3K1 expression, and causes delayed eyelid closure in duction cascades, but how the different pathways crosstalk to or- hemizygotes. The second network is initiated by the enzymatic chestrate the closure of the eyelid has remained largely unknown. activity of MAP3K1, which phosphorylates and activates a JNK-c- One of the well-characterized eyelid closure pathways is me- Jun module, leading to AP-1 transactivation and induction of its diated through the EGFR. Activation of EGFR is necessary for Pai-1 downstream genes, such as . MAP3K1 inactivation reduces the epithelial cells at the developing eyelid tip to migrate, leading AP-1 activity and PAI-1 expression both in cells and developing to embryonic eyelid closure. Consequently, defective EGFR eyelids. MAP3K1 is therefore the nexus of an intracrine regulatory signaling in mice with genetic knockout of the EGFR itself or its loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c-Jun-AP-1 ligands, TGF-α and HB-EGF, or of factors, such as FGF10, c- pathways in developmental eyelid closure. Jun, and GRHL3 that regulate ligand expression, impairs mi- gration, resulting in the EOB phenotype. Another eyelid-closure he mitogen-activated protein kinases (MAPKs) are activated pathway is mediated by MAP3K1 (9). Specifically, MAP3K1 was Tthrough an evolutionarily conserved three-component signal shown to active the JNK MAPKs, which in turn phosphorylate transduction cascade, composed of a mitogen-activated protein the transcription factor c-Jun in epithelial cells at the developing kinase kinase kinase 1 (MAP3K1), a MAP2K. and a MAPK (1). eyelid tip. Activation of the MAP3K1-JNK-c-Jun cascade pro- In the cascade, the MAP3Ks receive signals from upstream cues motes epithelial cell migration and hence, embryonic eyelid and pass them downstream by phosphorylating and activating the closure (9, 18). As far as embryonic eyelid closure is concerned, MAP2Ks, which in turn activate the MAPKs by phosphorylation. the EGFR and MAP3K1 signaling pathways appear to regulate a The MAPKs can modulate transcription factor activity and gene common cell activity, but the point of convergence of these expression, thereby regulating diverse cellular functions. MAP3K1, pathways has not been identified (3). also known as MEKK1, is a member of the MAP3K superfamily, In the course of previous work, we found that MAP3K1 was highly fi known to have highly cell type-speci c roles. In vivo studies using expressed in the developing eyelid tip epithelial cells before eyelid mice lacking either the full-length protein (MAP3K1-null or closure (9). By investigating the molecular mechanisms that control Map3k1−/− Map3k1ΔKD/ΔKD ) or its kinase domain ( ) have shown MAP3K1 expression, we find that TGF-α/EGFR signals, acting that MAP3K1 is involved in immune system development and through RhoA and ROCK, induce c-Jun and the c-Jun–mediated function, injury repair, vasculature remodeling, and tumor pro- activation of the Map3k1 promoter. Once induced, MAP3K1 is re- gression (2). However, the most obvious function of MAP3K1 is the control of eyelid closure during fetal development (3). Mammalian eye development involves a transient closure and Author contributions: A.P. and Y.X. designed research; E.G., Q.M., M.M., J.W., and A.T. reopening of the eyelid (4). In mice, eyelid development begins performed research; Y.Z. and R.A.L. contributed new reagents/analytic tools; E.G., Q.M., at embryonic day 13 (E13), when the surface ectoderm folds into M.M., J.W., A.T., A.P., and Y.X. analyzed data; and Y.X. wrote the paper. the lid buds. The eyelid buds continue to grow toward the center The authors declare no conflict of interest. of the ocular surface and by E15 to E16.5, the epithelial cells at *This Direct Submission article had a prearranged editor. the tip of the eyelid start to elongate and migrate. Ultimately, the 1E.G. and Q.M. contributed equally to this work. epithelium of upper and lower eyelid fuses to form a closed 2To whom correspondence should be addressed. E-mail: [email protected]. eyelid. Mouse eyelids remain closed between E16.5 and post- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. natal day 12, and thereafter the lid fusion breaks down as a 1073/pnas.1102297108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1102297108 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 quired for activation of the JNK-c-Jun pathway and induce AP-1 responsive genes. Thus, MAP3K1 is the nexus of an intracrine reg- ulatory loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c- Jun-AP-1 pathways in the control of eyelid morphogenesis. Results Activation of the EGF Receptor Leads to MAP3K1 Induction. The ΔKD Map3k1 allele contains a bacterial β-galactosidase gene knocked-in into the Map3k1 locus, replacing the exons coding for the MAP3K1 kinase domain (19). As a result, this allele pro- duces a MAP3K1-ΔKD-β-gal fusion protein that lacks kinase but retains β-galactosidase activity. Using fibroblasts derived from heterozygous and knockout fetuses, we established that β-gal expression in genetically modified fibroblasts can be used as a surrogate to measure endogenous Map3k1 promoter directed gene expression (Fig. S1 A and B). Before eyelid closure, MAP3K1 is highly expressed in the leading edge of the developing eyelid, which consists of clumps of rounded periderm cells covering the elongated epithelium (20) (Fig. S1C). Concomitantly, several eyelid morphogens are also abundantly expressed in the protruding tip of the developing eyelids, raising the possibility that some of the morphogenic signals induce MAP3K1 expression (5, 21, 22). To test this ΔKD/ΔKD possibility, we treated the Map3k1 cells with morphoge- netic factors, including: TGF-α and EGF, which activate EGFR; activin B and TGF-β1, which act through receptors of the TGF-β superfamily; FGF10, which act through a FGF receptor; and retinoic acid, which acts through RXR, and measured β-gal ac- Δ Δ Fig. 1. Induction of MAP3K1 expression in fibroblasts. The Map3k1 KD/ KD tivity (Fig. 1A and Fig. S2A). Although both EGFR ligands, fi α fi β broblasts treated with various agents for 24 h or as indicated were (A) TGF- and EGF, signi cantly increased -gal expression, none examined for the relative β-gal activities based on protein concentration, of the other agents had an effect. Correspondingly, TGF-α and (C and D) analyzed by Western blotting with antibodies for (C)anti–p- caused a clear induction of Map3k1 mRNA in wild-type cells, EGFR and (D) β-gal and β-actin. The intensity of the β-gal was compared with ΔKD/ΔKD paralleled by that of β-gal mRNA in Map3k1 cells, that of β-actin. (B) Total RNA isolated from wild-type (solid bars) and Δ Δ reaching a maximum level at 2 h and diminishing to a baseline Map3k1 KD/ KD fibroblasts (white bars) treated with TGF-α for different level by 6 h (Fig. 1B). In addition, TGF-α increased luciferase times were subjected to real-time RT-PCR for Map3k1 and β-Gal transcripts. Δ expression of pMap3k1-luc, a luciferase reporter driven by a 1.9- The fold-induction was calculated based on cycle differences ( Ct) in com- kb fragment of the Map3k1 promoter (Fig.