Alterations in NF-kB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-kB

Stratified epithelium contains a mitotically active basal layer of cells that cease proliferating, then migrate outwards and undergo terminal differentiation. The control of this process, which is abnormal in cutaneous neoplasia and inflammation, is not well understood. In normal epidermis, NF-kB proteins were found to exist in the cytoplasm of basal cells and then to localize in the nuclei of suprabasal cells, suggesting a role for NF-kB in the switch from proliferation to growth arrest and differentiation. Functional blockade of NF-kB by expressing dominant-negative NF-kB inhibitory proteins in transgenic murine and human epidermis produced hyperplastic epithelium in vivo. Consistent with this, application of a pharmacologic inhibitor of NF-kB to intact skin induced epidermal hyperplasia. In contrast, overexpression of active p50 and p65 NF-kB subunits in transgenic epithelium produced hypoplasia and growth inhibition. These data suggest that spatially restricted NF-kB activation occurs in stratified epithelium and indicate that NF-kB activation in this tissue, in contrast to its role in other settings, is important for cellular growth inhibition. NF-kB gene regulatory proteins are activated in a range of conditions involving cellular stress and injury (1–3). Stratified epithelial tissues must respond to such frequent environmental stresses while maintaining a precise balance between cellular proliferation and cell loss via desquamation. In stratified epithelium, proliferative basal cells adherent to the underlying basement membrane undergo cell cycle arrest associated with outward migration and activation of terminal differentiation genes (4, 5). Abnormalities in this process disrupt epithelial homeostasis and are characteristic of cutaneous neoplasms as well as a wide array of inflammatory skin diseases. The gene regulatory transcription factors mediating control of epithelial growth and differentiation are not fully known. A number of such transcription regulators, however, may influence keratinocyte gene expression (6), including AP-1 (7–9), c-myc (10), AP-2 (11), LEF-1 (12), steroid family receptors, retinoid receptors, (13–16), and homeodomain proteins (17) such as Skin1ayi (18), Oct6 (19) and distal-less 3 (20). Although there is evidence for activation of each of these gene regulatory factors in specific settings in epithelium, the mechanisms linking gene regulation to cellular growth control have not been clearly elucidated. NF-kByRel proteins are potent inducible gene regulatory factors expressed in a wide array of tissues. NF-kB subunits function as dimeric DNA-binding transcription factors with mammalian family members that include RelA(p65), RelB, c-Rel, p50, and p52, with Dif and Dorsal identified as homologs in Drosophila (1, 2). Studies of NF-kB in lymphoid tissues have revealed potent effects in stimulating proliferation, preventing apoptosis, activating the immune response, and triggering cellular stress response genes. NF-kB activity is controlled at a number of levels, prominent among these being the regulation of its transition from an inactive preexisting cytoplasmic form to an active nuclear protein. IkB family proteins mediate this inhibitory cytoplasmic anchoring effect and functional mammalian members include IkBa, IkBb, p105yIkBg, p100yIkBd, and IkB«, with Cactus an IkB family member identified in Drosophila. NF-kB activation involves phosphorylation and degradation of IkB proteins (21–23), a process that releases NF-kB dimers to translocate to the nucleus and alter expression of target genes (1, 2). The role of NF-kB in epithelium is not well studied. For some time it has been known that NF-kB, consistent with its role as a primary transcription factor, is rapidly activated after UV injury in many cell types, and recent work has confirmed the rapid induction of NF-kB DNA-binding activity in nuclear extracts prepared from cutaneous tissue irradiated with UV light (24). The expression pattern of NF-kB and its effects on global cellular processes that have been characterized in the immune system, however, have not yet been defined in epithelium. We wished to investigate whether NF-kB—as a prototype primary transcription factor controlling important cell fate decisions—may be important in stratified epithelium, a tissue possessing an array of rapidly inducible genetic programs mediating response to external injury, growth, and differentiation. Here we report that NF-kB undergoes a translocation from cytoplasm to nucleus in a pattern that coincides spatially with the differentiation-associated cell cycle arrest in stratified epithelium. To examine a functional role for NF-kB in this setting, we generated murine and human epidermis transgenic for proteins activating or inhibiting NF-kB function. The results of these studies indicate that NF-kB function in stratified epithelium, in contrast to its role in other tissues, is important for growth arrest.