Expression of NF-κB in epidermis and the relationship between NF-κB activation and inhibition of keratinocyte growth

Background: Nuclear factor-κB (NF-κB) is a transcription factor involved in a number of signalling pathways in many cell types. NF-κB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation. Objectives: To evaluate the role of NF-κB in keratinocyte growth in human beings, we examined its expression in keratinocytes both in culture and in situ, and studied the relationship between NF-κB activation and the inhibition of keratinocyte proliferation induced by known modulators of keratinocyte growth. Methods: The expression of subunits of the NF-κB family was examined in human skin, primary cultured keratinocytes and an immortalized keratinocyte line by immunohistochemistry and reverse transcriptase-polymerase chain reaction analysis. NF-κkB activation was examined in keratinocytes treated with various modulating agents by electrophoretic mobility shift assay (for DNA-binding activity) and by immunocytochemistry (nuclear translocation). The proliferative capacity of treated keratinocytes was also examined by ³H-thymidine incorporation, cell cycle analysis, and expression of Ki-67, a nuclear marker for cell proliferation. The involvement of NF-κB was assessed using sodium salicylate, which inhibits NF-κB activation. Results: The NF-κB subunits, p5O, p65, RelB, and c-Rel (but not p52), were detected in keratinocytes and in normal epidermis at mRNA and protein levels. The four subunits were expressed in a cytoplasmic (rather than a nuclear) pattern in both basal and suprabasal keratinocytes. Phorbol myristate acetate (PMA), tumour necrosis factor α, and interferon γ each activated NF-κB and inhibited keratinocyte proliferation. Lipopolysaccharide and dexamethasone did not activate NF-κB and had the least effect on proliferation. Finally, a high concentration of calcium (Ca²⁺) and retinoic acid each failed to activate NF-κB, but were potent inhibitors of keratinocyte proliferation, respectively. PMA-induced cell cycle arrest of keratinocytes was blocked by pretreatment with sodium salicylate. Conclusions: NF-κB is constitutively expressed in a resting state in both human cultured keratinocytes and the epidermis. Activation of NF-κB is required for PMA-induced keratinocyte growth arrest.