Intact human epidermis resists invasion by pathogenic microbes but the biochemical basis of its resistance is not well understood. Recently, an antimicrobial peptide, human beta-defensin-2, was discovered in inflamed epidermis. We used a recombinant baculovirus/insect cell system to produce human beta-defensin-2 and confirmed that at micromolar concentrations it has a broad spectrum of antimicrobial activity, with the striking exception of Staphylococcus aureus. Immunostaining with a polyclonal antibody to human beta-defensin-2 showed that the expression of human beta-defensin-2 peptide by human keratinocytes required differentiation of the cells (either by increased calcium concentration or by growth and maturation in epidermal organotypic culture) as well as a cytokine or bacterial stimulus. Interleukin-1alpha, interleukin-1beta, or live Pseudomonas aeruginosa proved to be the most effective stimuli whereas other bacteria and cytokines had little or no ability to induce human beta-defensin-2 synthesis. In interleukin-1alpha-stimulated epidermal cultures, human beta-defensin-2 first appeared in the cytoplasm in differentiated suprabasal layers of skin, next in a more peripheral web-like distribution in the upper layers of the epidermis, and then over a few days migrated to the stratum corneum. By semiquantitative Western blot analysis of epidermal lysates, the average concentration of human beta-defensin-2 in stimulated organotypic epidermal culture reached 15--70 microg per gram of tissue, i.e., 3.5-16 microM, well within the range required for antimicrobial activity. Because of the restricted pattern of human beta-defensin-2 distribution in the epidermis, its local concentration must be much higher. Defensins and other antimicrobial peptides of inflamed epidermis are likely to play an important antimicrobial role in host defense against cutaneous pathogens.

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The role of c-Jun NH2-terminal kinase (JNK) signaling cascade in the stress-inducible phosphorylation of heat shock factor 1 (HSF1) was investigated using known agonists and antagonists of JNK. We showed that treatment of HeLa cells with MG132, a proteasome inhibitor and known INK activator, caused the transcriptional activation domain of HSF1 to be targeted and phosphorylated by JNK2 in vivo. Dose-response and time course studies of the effects of heat shock and anisomycin treatment showed a close correlation of the activation of JNK and hyperphosphorylation of HSF1. SB203580 inhibited INK at the 100 microM concentration and significantly reduced the amount of hyperphosphorylated HSF1 upon heat shock or anisomycin treatment. SB203580 and dominant-negative JNK suppress hsp70 promoter-driven reporter gene expression selectively at 45 degrees C but not at 42 degrees C heat stress, suggesting that JNK would be preferentially associated with the protective heat shock response against severe heat stress. The possibility that JNK-mediated phosphorylation of HSF1 may selectively stabilize the HSF1 protein and confers protection to cells under conditions of severe stress is discussed.