Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia.

The p42/p44 mitogen-activated protein kinase (MAPK) cascade includes Ras, Raf, Mek, and Erk MAPK. To determine the effect of a full knockout at a single level of this signaling pathway in mammals, and to investigate functional redundancy between Mek1 and Mek2, we disrupted these genes in murine and human epidermis. Loss of either protein alone produced no phenotype, whereas combined Mek1/2 deletion in development or adulthood abolished Erk1/2 phosphorylation and led to hypoproliferation, apoptosis, skin barrier defects, and death. Conversely, a single copy of either allele was sufficient for normal development. Combined Mek1/2 loss also abolished Raf-induced hyperproliferation. Human tissue deficient in either Mek isoform was normal, whereas loss of both proteins led to hypoplasia, which was rescued by active Erk2 expression. These data indicate that Mek1/2 are functionally redundant in the epidermis, where they act as a linear relay in the MAPK pathway to mediate development and homeostasis.

Mek1 alters epidermal growth and differentiation.

The highly homologous kinases, Mek1 and Mek2, act downstream of Ras and Raf to activate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In epidermis, Ras and Raf promote hyperplasia; however, they act on multiple Mek-independent effectors, and the extent to which Meks can mediate these effects is unknown. To address this, we expressed inducible Meks in transgenic murine and human epidermis. Both Mek1 and Mek2 triggered ERK phosphorylation. Only Mek1, however, recapitulated Ras/Raf effects in increasing proliferation and integrin expression while suppressing differentiation, which are impacts characteristic of epidermal neoplasia. Furthermore, a kinase-dead Mek1 mutant incapable of phosphorylating ERK proteins retained ability to mediate Mek1-driven epidermal proliferation. Mek1 is thus sufficient to promote the proliferative epithelial phenotype in a manner independent of intact kinase function.

Effects of active MEK1 expression in vivo.

Cell transformation is often a result of constitutive activation of genes in signaling pathways that regulate cell proliferation and differentiation. Indeed, the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway is constitutively activated in a large number of cancers. The extent to which a single-gene mutation can alter cell fate, however, remains questionable. In vitro studies have addressed this issue, but organs are comprised of multiple cell types, and in vitro models often poorly approximate these interactions. In response to these limitations, cell-type specific mouse models have been generated as a means to examine the effect of altering a single element of the MAPK pathway in vivo. This review summarizes data from transgenic murine and human tissue models expressing constitutive active forms of MEK1.

Gene transfer via reversible plasmid condensation with cysteine-flanked, internally spaced arginine-rich peptides.

Nonviral gene transfer offers biosafety, stability, and expense advantages over viruses; however, it has suffered from poor efficiency. Because arginine-rich peptides facilitate uptake of macromolecules such as proteins, liposomes, and iron nanoparticles, we explored their potential in enhancing plasmid DNA delivery. In their unmodified form, known protein transduction sequences, including hepta-arginine and Tat(47-57), failed to support effective gene delivery. However, by flanking a core of consecutive arginines with amino- and carboxy-terminal cysteines in vitro gene transfer was observed. Furthermore, interspersing arginines with glycine and histidine residues achieved reversible plasmid condensation and dramatically increased transfection levels in a variety of cell types. Unlike most available cationic homopolymers that function only in vitro, these new peptides also increased gene expression in both murine and human tissue in vivo. Thus, cysteine-flanked, internally spaced arginine-rich (CFIS-R) peptides represent a new approach to efficient nonviral plasmid delivery using rationally designed protein transduction domains.

Erk1/2 MAP kinases are required for epidermal G2/M progression.

Erk1/2 mitogen-activated protein kinases (MAPKs) are often hyperactivated in human cancers, where they affect multiple processes, including proliferation. However, the effects of Erk1/2 loss in normal epithelial tissue, the setting of most extracellular signal-regulated kinase (Erk)-associated neoplasms, are unknown. In epidermis, loss of Erk1 or Erk2 individually has no effect, whereas simultaneous Erk1/2 depletion inhibits cell division, demonstrating that these MAPKs are necessary for normal tissue self-renewal. Growth inhibition caused by Erk1/2 loss is rescued by reintroducing Erk2, but not by activating Erk effectors that promote G1 cell cycle progression. Unlike fibroblasts, in which Erk1/2 loss decreases cyclin D1 expression and induces G1/S arrest, Erk1/2 loss in epithelial cells reduces cyclin B1 and c-Fos expression and induces G2/M arrest while disrupting a gene regulatory network centered on cyclin B1-Cdc2. Thus, the cell cycle stages at which Erk1/2 activity is required vary by cell type, with Erk1/2 functioning in epithelial cells to enable progression through G2/M.

Selective role for Mek1 but not Mek2 in the induction of epidermal neoplasia.

The Ras/Raf/Mek/Erk mitogen-activated protein kinase pathway regulates fundamental processes in normal and malignant cells, including proliferation, differentiation, and cell survival. Mutations in this pathway have been associated with carcinogenesis and developmental disorders, making Mek1 and Mek2 prime therapeutic targets. In this study, we examined the requirement for Mek1 and Mek2 in skin neoplasia using the two-step 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) skin carcinogenesis model. Mice lacking epidermal Mek1 protein develop fewer papillomas than both wild-type and Mek2-null mice following DMBA/TPA treatment. Mek1 knockout mice had smaller papillomas, delayed tumor onset, and half the tumor burden of wild-type mice. Loss of one Mek1 allele, however, did not affect tumor development, indicating that one Mek1 allele is sufficient for normal papilloma formation. No difference in TPA-induced hyperproliferation, inflammation, or Erk activation was observed between wild-type, conditional Mek1 knockout, and Mek2-null mice, indicating that Mek1 findings were not due to a general failure of these processes. These data show that Mek1 is important for skin tumor development and that Mek2 cannot compensate for the loss of Mek1 function in this setting.

Modeling inducible human tissue neoplasia identifies an extracellular matrix interaction network involved in cancer progression.

To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in three-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends on specific extracellular matrix-interacting network hubs. Blockade of one such hub, the beta1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression.