No abstract

The regulated dephosphorylation of mitogen-activated protein kinases (MAPKs) plays a key role in determining the magnitude and duration of kinase activation and hence the physiological outcome of signalling. In mammalian cells, an important component of this control is mediated by the differential expression and activities of a family of 10 dual-specificity (Thr/Tyr) MAPK phosphatases (MKPs). These enzymes share a common structure in which MAPK substrate recognition is determined by sequences within an amino-terminal non-catalytic domain whereas MAPK binding often leads to a conformational change within the C-terminal catalytic domain resulting in increased enzyme activity. MKPs can either recognize and inactivate a single class of MAP kinase, as in the specific inactivation of extracellular signal regulated kinase (ERK) by the cytoplasmic phosphatase DUSP6/MKP-3 or can regulate more than one MAPK pathway as illustrated by the ability of DUSP1/MKP-1 to dephosphorylate ERK, c-Jun amino-terminal kinase and p38 in the cell nucleus. These properties, coupled with transcriptional regulation of MKP expression in response to stimuli that activate MAPK signalling, suggest a complex negative regulatory network in which individual MAPK activities can be subject to negative feedback control, but also raise the possibility that signalling through multiple MAPK pathways may be integrated at the level of regulation by MKPs.

We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of %32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation ofa high level ofinduction ofthe enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage.Mammalian cells respond to a wide variety of adverse conditions, both chemical and physical, by inducing the synthesis of a number of stress proteins. The most widely studied of these responses is that induced by heat shock (for review see refs. 1 and 2). Although certain insults other than heat shock itself induce heat shock proteins-for example, treatment of cells with amino acid analogues (3)-others induce stress proteins unrelated to those inducible by heat shock. Both glucose deprivation and anoxic stress are good examples of the latter (4, 5). We have reported (6) the induction of a 32-kDa stress protein in human skin fibroblasts by both near UV radiation (UVA) and the oxidizing agent hydrogen peroxide. Neither of these treatments induced the major heat shock proteins. In addition, we were unable to induce the 32-kDa protein by heat shock in these cells, indicating that the mechanism of induction of this protein is different from that involved in heat shock. Interestingly, a protein of30-35 kDa had been reported (7-10) as inducible by certain chemical treatments, including heavy metal salts and the sulfhydryl reagent sodium arsenite in rodent, avian, and human cells. In all of these cases the heat shock proteins were also induced. A comparison of the 32-kDa proteins induced by UVA, hydrogen peroxide, and sodium arsenite in human skin fibroblasts by partial peptide mapping indicated that they were identical (6).We now describe the isolation ofcDNA clones corresponding to the mRNA species encoding this stress protein and identify the inducible gene as coding for heme oxygenase. We speculate that this enzyme participates in an inducible protective mechanism against oxidative stress induced by UVA and hydrogen peroxide in human skin cells. MATERIALS AND METHODSCell Culture and Treatment with UVA and Chemicals. The normal human skin fibroblast cell line EK4 was derived from a foreskin explant, in this laboratory, and cultured routinely as described (11). Cells were seeded into plastic culture dishes (30-50% confluence) 2-4 days prior to either chemical treatment or UVA irradiation. To treat cells with chemicals, the growth medium was rem...