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First published online 21 November 2007
doi: 10.1242/dev.007120

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Differential transmission of MEKK1 morphogenetic signals by JNK1 and JNK2

¹ Department of Environmental Health and Center of Environmental Genetics, University of Cincinnati Medical Center, 123 E. Shields Street, Cincinnati, OH 45267-0056, USA.
² Department of Central Lab, Southern Medical University, Tonghe, Guangzhou, People's Republic of China.

View larger version (49K): [in this window] [in a new window]   Fig. 1. The MEKK1-MKK4 pathway leads to JNK and c-Jun phosphorylation in the developing eyelid epithelium. (A) Immunofluorescence staining of wild-type (WT), Mekk1+/KD (M1+/KD) and Mekk1KD/KD (M1KD/KD) fetuses at embryonic day 15.5 (E15.5) for p-MKK4 (top panels) and p-MKK7 (bottom panels) (green). Nuclei are stained with DAPI (blue). Scale bars: 50 µm. The images are representative of at least three fetuses of each genotype examined. (B) Mouse embryonic stem (ES) cells were either left untreated or treated with activin B (5 ng/ml) for 10 minutes or infected with adenovirus for WT-MEKK1 for 24 hours, as labeled. The cell lysates were subjected to western blot analyses using antibodies for phosphorylated and total MKK4 and MKK7, and MEKK1, as indicated. The results were quantified by chemiluminescence imaging and the fold induction of p-MKK4 and p-MKK7 by activin B and MEKK1 over the control was calculated. (C) Wild-type and Mkk4-/- ES cells were infected with either control or WT-MEKK1 adenoviruses. At 24 hours post infection, cell lysates were examined by western blotting for phosphorylated and total JNK, MEKK1 and MKK4. (D) Immunofluorescence staining of wild-type (WT), Mekk1+/KD (M1+/KD) and Mekk1KD/KD (M1KD/KD) fetuses at embryonic day 15.5 (E15.5) detected phospho-JNK (p-JNK) (left panels) and phospho-c-Jun (p-c-Jun) (middle panels) (green) in the developing eyelid epithelium (arrowheads). Nuclei were stained with DAPI (blue). White rectangular areas of p-c-Jun staining (middle panels) are also shown at higher magnification (right panels). Dotted lines mark dermis (De), basal epithelial layer (Ep. B), suprabasal epithelial layer (Ep. S) and cornea (Co). Scale bars, 50 µm. At least four fetuses (8 eyes) of each genotype were examined. (E) The fraction of phosphorylation-positive cells over total cell count in suprabasal epithelial layer was calculated and statistically significant differences (P<0.05) from wild-type fetuses are denoted with an asterisk.

View larger version (64K): [in this window] [in a new window]   Fig. 2. Defective embryonic eyelid closure of the Mekk1+/KDJnk1-/- mice and impaired migration of the Mekk1+/KDJnk1-/- keratinocytes. (A) Photographs and histological analyses (H&E) of the eyes in Mekk1 heterozygous Jnk1-null (M1+/KDJ1-/-) and Mekk1 heterozygous Jnk2-null (M1+/KDJ2-/-) mice at E19, postnatal day (P)1 and P21. The M1+/KDJ1-/- mice have impaired developmental eyelid closure and an EOB phenotype. H&E staining of the eye tissue sections show developmental eyelid (arrowheads) and ocular tissue inflammation (asterisk). (B) H&E staining of the developing eye at E16. The Jnk1-null (J1-/-), M1+/KDJ1+/- and M1+/KDJ2-/- fetuses had eyelid epithelium (arrowheads) covered ocular surface, whereas the M1+/KDJ1-/- fetus had opened eyelids and fully exposed ocular surface. Scale bar: 200 µm. (C) Immunofluorescence staining for BrdU (red) and keratins (green) of the developing eyes at E16. Similar levels of keratin 6 (K6) and keratin 10 (K10) expression and BrdU incorporation were detected in the developing eyelid epithelium of M1+/KDJ1-/- and M1+/KDJ2-/- fetuses. (D) An in vitro wound healing assay was performed on mouse primary epidermal keratinocytes isolated from wild-type, M1+/KDJ1-/- and M1+/KDJ2-/- mice, in the presence or absence of 10% fetal bovine serum (serum), individual growth factors, TGF (T) and activin B (Act B), or the JNK inhibitor (SP), as indicated. Photographs were taken immediately and 48 hours after wounding. The remaining wound areas at 48 hours were compared with the original wound area. The results represent the mean ± s.d. of at least four independent experiments. (E) Immunofluorescence staining showed PAI1 expression (green) was abundant in developing eyelid epithelium of the M1+/KDJ2-/-, but was almost undetectable in that of the M1+/KDJ1+/- fetuses. Nuclei were stained with DAPI (blue). Arrowheads indicate the developing eyelid tip. Scale bars: 50 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Reduction of JNK and c-Jun phosphorylation in M1+/KDJ1-/- eyelid epithelium. (A) The M1+/KDJ1-/- and M1+/KDJ2-/- fetuses at E15.5 were examined by immunohistochemistry for the phospho- and total JNK and c-Jun, phospho-EGFR (p-EGFR), phospho-ERK (p-ERK) and phospho-Elk (p-Elk). Levels of only p-JNK and p-c-Jun were significantly reduced in the developing eyelid epithelium of the M1+/KDJ1-/- fetuses. Scale bars: 50 µm. (B) Quantitative summary of the percentage of p-JNK- and p-c-Jun-positive cells in suprabasal epithelial layers of the developing eyelids in various gene knockout fetuses. Data are mean ± s.d. of at least four fetuses of each genotype examined. Only the M1+/KDJ1-/- fetuses showed significant (*P<0.01) reduction in p-JNK and p-c-Jun compared with wild-type fetuses. (C) E15.5 Jnk1-null (J1-/-) and Jnk2-null (J2-/-) fetuses were subjected to X-Gal staining to detect the target gene promoter-driven β-galactosidase expression. X-Gal-positive cells were detected in the entire embryonic body of E15.5 fetuses. Arrowheads indicate the developing eyelid tip.

View larger version (30K): [in this window] [in a new window]   Fig. 4. MEKK1 is required for efficient transmission of the activin B signals to JNK. (A) M1+/KDJ1-/- and M1+/KDJ2-/- keratinocytes were either untreated (c) or treated with TGF (T, 10 ng/ml), TGFβ1 (Tβ, 10 ng/ml) or activin B (Act, 5 ng/ml) for 10 minutes, and cell lysates were analyzed for phosphorylated and total JNK and c-Jun, phospho-ERK and phospho-Smad2 by western blotting. Similar results were obtained from at least two independent experiments. The relative ratio of (B) p-JNK/total JNK and (C) p-c-Jun/total c-Jun in the Jnk1-null (J1-/-) and M1+/KDJ1-/- keratinocytes either untreated (control) or treated with TGFβ and activin B were quantified by chemiluminescence imaging analyses and represented graphically. The levels in untreated cells were used as a standard for quantification. Similar results were obtained from at least three independent experiments.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Differential activation of JNK1 and JNK2 by MEKK1. (A) HEK293 cells were transiently co-transfected with plasmids for active MEKK1, HA-JNK1 and HA-JNK2 as indicated, and the JNKs were purified by immunoprecipitation using anti-HA. Cell lysates and immunoprecipitates were analysed by immunoblotting using anti-MEKK1, anti-p-JNK and anti-HA. The amount of phospho-JNK and total immunoprecipitated HA-JNK was quantified by chemiluminescence imaging and pJNK/JNK ratio is shown graphically. Similar results were obtained from at least three independent experiments. (B) Wild-type, M1+/KD, M1+/KDJ1-/- and M1+/KDJ2-/- MEFs were either uninfected or infected with adenoviruses for HA-tagged kinase active MEKK1 [HA-MEKK1(WT)] or for the kinase-inactive mutant MEKK1 [HA-MEKK1(KM)]. Cell lysates were subjected to immunoprecipitation using a mixture of anti-JNK1 and anti-JNK2 and the immunoprecipitates were analyzed by western blotting using anti-HA, anti-JNK and anti-phospho-JNK antibodies. The experiments were repeated twice and the results were consistent. (C) The results in B were quantified by chemiluminescence imaging analyses and the co-immunoprecipitated MEKK1/JNK is represented graphically.

View larger version (28K): [in this window] [in a new window]   Fig. 6. The G177/S179 residues in the JNK1 are crucial for its activity (A) SABLE analysis revealed a putative structural difference between JNK1 and JNK2 in the variable region. The arrows represent β-strands that are predicted in this region. An extra β-strand is predicted in JNK2, but not in JNK1. Asterisks identify the Thr and Tyr residues as JNK phosphate acceptor sites. The triangles indicate the G177/S179 residues in JNK1, which are divergent from C177/N179 in JNK2. (B) HEK293 cells were transiently transfected with wild-type HA-JNK1 and HA-JNK2, and mutant HA-JNK1 (CTN) and HA-JNK2 (GTS). The JNK proteins were isolated by immunoprecipitation using HA-conjugated beads and subjected to western blotting using anti-HA followed by quantification using chemiluminescence imaging analyses. The precipitates were also used for an in vitro kinase assay with an active MKK4 as the kinase in the presence of [-32P]ATP. JNK phosphorylation was detected by exposure to X-ray film and phosphoimager analyses. The values of the relative 32P-JNK/total JNK of the mutants were compared with that of their wild-type counterparts, which were designated as 1. (C,D) HEK293 cells were transiently co-transfected with active MEKK1, wild-type HA-JNK1 and HA-JNK2, and mutant HA-JNK1 (CTN) and HA-JNK2 (GTS), as indicated. The cell lysates were analyzed for MEKK1 expression by western blot analyses and were used for immunoprecipitation with anti-HA. The immunoprecipitates (C) were analyzed for the phospho- and total-JNK by western blotting and chemiluminescence imaging analyses, and (D) were subjected to in vitro kinase assay (KA) using GST-c-Jun as a substrate in the presence of [-32P]ATP. The GST-c-Jun phosphorylation was detected by exposure to X-ray film and the GST-c-Jun and JNK proteins in the reaction were detected by western blot. The relative p-c-Jun/total c-Jun levels were calculated and are shown in the graph. Consistent results were obtained from at least three independent experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 7. Mekk1, Jnk1 and Jnk2 gene doses are crucial for embryonic eyelid closure. (A) M1+/KDJ1+/-J2+/- triple heterozygous mice have EOB phenotype at postnatal day 1, with a partially exposed ocular surface at E16.5; by contrast, the M1+/+ J1+/- J2+/- mice had closed eyelids at E16.5 and P1. The developing eye at E15.5 shows less phosphorylation of JNK and c-Jun (green) in the eyelid epithelium (arrowheads) of the triple hemizygous compared with the double hemizygous fetuses. (B) Diagram illustrating the signaling pathways in the control of eyelid epithelial morphogenesis. The signal is initiated from the eyelid morphogenetic factor activin B, which leads to the activation of MEKK1, a tissue-specific and rate-limiting determinant for transmitting the activin B signals. MEKK1 activates MKK4, which in turn phosphorylates JNK1 and JNK2. Because of the intrinsic variable sequence difference between the JNK isoforms, JNK1 is more effectively phosphorylated than JNK2. The total phospho-JNK determines the nuclear transcription factor c-Jun phosphorylation and induction of gene expression, such as PAI1. The endpoint of this pathway activation is the induction of epithelial cell migration, and eyelid epithelial morphogenesis and closure.