ERK3 is essential for establishment of epithelial architecture [ERK3 KD vs. TFAP2A KD]

Establishment and maintenance of epithelial architecture are essential for embryonic development and adult physiology. Here, we show that ERK3, a poorly characterized atypical MAPK, regulates epithelial architecture in vertebrates. In Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight junction protein distribution, as well as tight junction barrier function, resulting in epidermal breakdown. Moreover, in human breast epithelial cancer cells, inhibition of ERK3 expression induces thickened epithelia with aberrant adherens and tight junctions. Microarray results suggest an involvement of TFAP2A, a transcription factor important for epithelial gene expression, in ERK3-dependent gene expression changes. TFAP2A knockdown phenocopies ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 is required for full activation of TFAP2A-dependent transcription. Our findings thus reveal that ERK3 regulates epithelial architecture, possibly in cooperation with TFAP2A. We used microarrays to compare the changes in ERK3-dependent gene expression profiles with those in TFAP2A-dependent gene expression profiles in Xenopus laevis embryos. Control MO (80 ng), ERK3 MO1/2 (40 ng each of MO1 and MO2), or TFAP2A MO1/2 (40 ng each of MO1 and MO2) were injected into the animal regions of all blastomeres at the 4-cell stage. The animal caps were dissected from the injected embryos at stage 9, cultured alone, and harvested at stage 15. Two independent replicates were prepared. Total RNA was extracted using TRIzol reagent. The quality of the total RNA was assessed using an Agilent 2100 BioAnalyzer. cDNA synthesis and transcriptional amplification were performed using 250 ng of total RNA with the GeneChip 3’ IVT PLUS Reagent Kit (Affymetrix, #902415). Fragmented and biotin-labeled cDNA targets were hybridized to the GeneChip Xenopus laevis Genome 2.0 Array (Affymetrix) according to the manufacturer's protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned chip images were analyzed with GeneChip Operating Software v.1.4 (GCOS). The probe set signal intensities in the raw data (CEL files) were normalized using a robust multiarray average (RMA) algorithm and Expression Console software.