The expression of a novel, epithelium-specific ets transcription factor is restricted to the most differentiated layers in the epidermis.

Ets proteins have been implicated in the regulation of gene expression during a variety of biological processes, including growth control, differentiation, development and transformation. More than 35 related proteins containing the 'ets domain' have now been found which specifically interact with DNA sequences encompassing the core tetranucleotide GGAA. Although ets responsive genes have been identified in the epidermis, little is known about their distribution and function in this tissue. We have now demonstrated that epidermis and cultured epidermal keratinocytes synthesize numerous ets proteins. The expression of some of these proteins is regulated as a function of differentiation. Among these is a novel ets transcription factor with a dual DNA-binding specificity, which we have called jen. The expression of jen is not only epithelial specific, but it is the only ets protein so far described, and one of the very few transcription factors whose expression is restricted to the most differentiated epidermal layers. We show that two epidermal marker genes whose expression coincides with that of jen are transregulated by this protein in a complex mode which involves interactions with other transcriptional regulators such as Sp1 and AP1.

Structural and biological consequences of increased vimentin expression in simple epithelial cell types.

Cytoskeletal intermediate filaments (IFs) constitute a diverse family of proteins whose members are expressed in tissue-specific patterns. Although vimentin IFs are normally restricted to mesenchyme, a variety of cell types express vimentin alone or together with cell-specific IFs during growth, differentiation, and neoplasia. In this study, we have investigated the influence of increased vimentin expression on the simple epithelial cell phenotype. An expression vector encoding a human vimentin cDNA was transfected into murine HR9 endoderm and F9 embryonal carcinoma cell lines, which serve as models for early extraembryonic epithelial differentiation. Stable clones that expressed varying levels of human vimentin were characterized by human vimentin were characterized by immunofluorescence and biochemical analysis. A relatively high level of vimentin expression in HR9 and differentiated F9 epithelial cells resulted in aberrant vimentin structures with co-collapse of keratin K8/K18 filaments and lowered amounts of keratin protein. In F9 epithelial cells, the desmosomal proteins DP I/II did not appear to localize to cell surface desmosome s but rather but rather co-aggregated with the perturbed IFs. Although overall cell morphology was not dramatically altered, individual nuclei were distorted by excess intracellular vimentin. Furthermore, cell proliferation as well as the cell spreading response time were slowed. Ther appears to be a threshold effect regarding overall vimentin levels as cells that expressed lower amounts of the human vimentin exhibited no obvious structural nor biological effects. Our results demonstrate that wild-type vimentin can act as a "mutant" protein when present at high intracellular levels, inducing a variety of phenotypic changes.

Fate of a headless vimentin protein in stable cell cultures: soluble and cytoskeletal forms.

The non-alpha-helical head domains of cytoskeletal intermediate filaments (IFs) are considered to play an important role in IF assembly and stability. We have investigated the fate of a "headless" mutant vimentin protein in cell types that either lack cytoplasmic IFs or contain preexisting IF networks (keratin and vimentin). Stable clones expressing a transfected headless vimentin cDNA were individually analyzed in order to avoid variabilities introduced by transient transfection and to compare the levels and effects of the mutant vimentin protein more accurately. In cells lacking IFs, the mutant protein existed in a diffuse, soluble form as determined by immunofluorescence and biochemical protein fractionation. In cells possessing vimentin IFs, the headless vimentin was highly dispersed throughout the cytoplasm, including lamellopodia. Expression levels in individual clones were as high as sevenfold greater than the endogenous vimentin component. Although the majority of the headless vimentin was highly soluble, a residual portion of the mutant vimentin colocalized with vimentin filaments and consistently comprised about 25% of the cytoskeletal vimentin network. These results demonstrate that the mutant protein can be stably expressed at relatively high levels without deleterious cellular effects or disruption of endogenous vimentin filaments. The observed specific ratio of mutant to wild-type vimentin (1:3) in the cytoskeleton supports IF in vivo assembly via specific hybrid tetramer formation and, further, that at least three intact head domains are required for competent tetramer formation and IF assembly.