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Exposure of human keratinocytes to environmental stress is known to induce changes in the expression, phosphorylation, and subcellular relocalization of the 27 kDa heat shock protein. This study demonstrates that ultraviolet B (280-320 nM) irradiation with physiologic doses induces a dose-dependent phosphorylation of 27 kDa heat shock protein, generating the more acidic 27 kDa heat shock protein B, C, and D isoforms. Ultraviolet B also induces perinuclear cytoplasmic relocation and nuclear translocation of 27 kDa heat shock protein and caused aggregation of cytoplasmic actin filaments into a broad perinuclear distribution. The ultraviolet B-induced phosphorylation is reversible, returning to baseline levels 4 h after exposure, and this coincides with the reversal of ultraviolet B-induced actin reorganization. The ultraviolet B-induced phosphorylation is not affected by the protein kinase C inhibitor, GF 109203X, is partially inhibited by epidermal growth factor receptor tyrosine kinase inhibitor, PD 153035, and is substantially inhibited by the specific p38 mitogen-activated protein kinase inhibitor, SB 203580. In addition, pretreatment of cells with the anti-oxidant N-acetyl cysteine partially inhibits ultraviolet B-and oxidant-induced 27 kDa heat shock protein phosphorylation. The p38 mitogen-activated protein kinase cascade is thus the major transduction pathway for ultraviolet B-induced 27 kDa heat shock protein phosphorylation, and reactive oxygen species generated in response to ultraviolet B also contribute to this phosphorylation. As 27 kDa heat shock protein phosphorylation and relocalization has been associated with increased cell survival after environmental insult, our data suggest that ultraviolet B, in addition to initiating recognized cytotoxic events in keratinocytes, also initiates a signaling pathway that may provide cellular protection against this ubiquitous environmental insult.

Stress proteins, which are found ubiquitously in mammalian cells, appear to be implicated in the regulation of cell growth and protection from environmental insult. Although we previously demonstrated the expression of low-molecular-weight stress protein, HSP 27, in cultured keratinocytes, HSP 27 has not yet been identified in human skin. Using standard immunohistochemistry on routinely processed paraffin sections, we examined specimens of common epidermal lesions and normal skin with a monoclonal antibody to HSP 27. Normal skin from the breast, foreskin, and lower extremity demonstrated strong cytoplasmic staining in the suprabasal epidermis. There was no change in the intensity of staining or cellular localization related to age, body location, or gender. Sections of actinic keratosis, superficial basal cell carcinoma, seborrheic keratosis, and psoriasis also exhibited strong cytoplasmic staining in the suprabasal layers of the epidermis. In contrast, cutaneous squamous cell carcinoma demonstrated only weak cytoplasmic staining throughout the infiltrating tumor. This is of particular interest, since other investigators have reported a loss of HSP 27 expression in oncogenically transformed cells that exhibit a tumorigenic phenotype. To our knowledge, this study provides the first demonstration of HSP 27 expression in human skin.

We have begun to characterize the low molecular weight, 27-kD heat shock or stress protein (HSP27) in normal keratinocytes and in HaCaT, a spontaneously transformed keratinocyte line. The presence and location of HSP27 was determined by indirect immunofluorescence on fixed whole cells and immunoblot analysis of cytosolic, membrane, nuclear, and cytoskeletal cell fractions. HSP27 is localized throughout the cytoplasm of cells at 37 degrees C. After heating at 42 degrees C, there is a rapid (within 10 min) increase in nuclear HSP27. Two-dimensional gel analysis of whole cell HaCaT lysates identified multiple isoforms of HSP27 with different isoelectric points. The function of HSP27 is largely unknown but its presence throughout the cytoplasm of cells at 37 degrees C, its translocation to the nucleus after cellular stress, and the presence of multiple isoforms suggest a biologic role in both stressed and unstressed human keratinocytes.