Mek2 is dispensable for mouse growth and development

Abstract

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.

FIG. 1.

Targeted disruption of the Mek2 gene. (A) Mek2 gene-targeting strategy. Translated Mek2 exons are represented by black boxes. A neo selection marker (stippled box) was inserted between the fourth and sixth Mek2 exons, deleting a 1-kb DNA fragment. The targeting vector contains 3.6 and 2 kb of Mek2 homologous genomic sequences on the 5′ and 3′ sides of the neo insertion, respectively. The herpes simplex virus-thymidine kinase (HSV-TK) selection cassette (hatched box) was added at the 5′ end of the homology. wt, wild type. (B) Southern blot analysis. DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) Mek2 mutant mice digested with BamHI was hybridized with a 5′ probe (BamHI-KpnI targeting vector fragment) and a 3′ probe (EcoRI-BamHI fragment). The 11-kb band represents the wild-type allele, and the 4- and 7-kb bands correspond to the mutant allele hybridized with the 3′ and the 5′ probes, respectively. (C) Western blot analysis of Mek2^−/− mice. Proteins extracted from E11.5 wild-type (+/+), heterozygous (+/−), and homozygous (−/−) Mek2 mutant embryos were analyzed by SDS-PAGE. Duplicate blots were probed with antibodies directed against MEK1 or MEK2.

FIG. 2.

Cell cycle analysis of wild-type and Mek2^−/− MEFs. Confluent cultures of wild-type and Mek2^−/− primary MEFs were serum deprived in 0.1% FBS for 24 h. Reentry into the cell cycle was induced with 20% FBS. Cell samples were collected at 4-h intervals for 24 h by trypsinization, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentages of cells in the G₁, S, and G₂/M phases at each time point are represented. The insets show the percentages of cells in S phase. Representative results from five wild-type and four Mek2^−/− MEF cultures are shown.

FIG. 3.

Time course of serum-stimulated ERK2 phosphorylation in wild-type and Mek2^−/− MEFs. Primary MEFs were serum deprived for 24 h and subsequently stimulated with 20% FBS or 1 ng of EGF/ml for the indicated times (0, 5, 30, 120, 240, and 480 min). (A) Total cell extracts from wild-type and Mek2^−/− MEFs were separated by SDS-PAGE to resolve ERK2 and phospho-ERK2 (p-ERK2), which were revealed by immunoblotting by using an antibody directed against ERK2. (B and C) Quantification with a PhosphorImager of the active form of ERK2 (percentage of phospho-ERK2) in wild-type and Mek2^−/− MEFs when they were stimulated with 20% FBS (B) or 1 ng of EGF/ml (C). The means and standard deviations of the results from five wild-type and four Mek2^−/− MEF cultures are represented.

FIG. 4.

Normal growth of Mek2^−/− mice. Growth was assessed for wild-type (circles), Mek2^+/− (triangles), and Mek2^−/− (squares) female (open symbols) and male (filled symbols) mutant mice. Body weight was determined daily for the first 28 days and then weekly up until the eighth week. Standard deviations of the mean body weights are represented. No statistically significant difference was observed among the various genotypes (wild-type males, n = 30; wild-type females, n = 20; Mek2^+/− males, n = 10; Mek2^+/− females, n = 13; Mek2^−/− males, n = 10; Mek2^−/− females, n = 15).

FIG. 5.

Flow cytometric analysis of CD4 and CD8 expression on T lymphocytes from wild-type and Mek2^−/− lymphoid organs. Thymus, spleen, and lymph node cells from 4- to 6-week-old wild-type and Mek2^−/− mice were stained with the designated antibodies and analyzed by flow cytometry. The percentage of cells in each quadrant is indicated. The results of a representative staining experiment are shown. A summary of all flow cytometry analyses performed on thymocytes is presented in Table 1. No statistically significant differences were observed between wild-type and Mek2^−/− specimens.

FIG. 6.

Mitogen responsiveness of T cells derived from Mek2^−/− mice. (A) Total thymocytes from wild-type and Mek2^−/− mice (2 × 10⁵ cells) were cultured in medium (ctl) or in the presence of IL-2, anti-CD3ɛ, or anti-CD28 alone or in combination in 96-well plates. Cells were stimulated with the indicated concentrations of mitogens, 10 U of IL-2/ml, anti-CD3ɛ (plated at 10 μg/ml), or anti-CD28 (plated at 10 μg/ml) for 72 h. The results shown are the means ± the standard errors of the means of results for nine different experiments done in triplicate. No statistically significant difference was observed. (B) Total T cells from wild-type and Mek2^−/− spleens (2 × 10⁵ cells) were cultured in the presence of various concentrations of anti-CD3ɛ (plated at 0.1 to 10 μg/ml). The results shown are the means ± the standard errors of the means of results from six different experiments done in triplicate. No statistically significant difference was observed. CPM, counts per minute.