The pathogeneses of toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS), both severe blistering diseases usually associated with drug intake, are not fully elucidated. Histologically, both TEN and SJS are characterized by extensive keratinocyte apoptosis. Previous studies have shown that keratinocyte apoptosis in TEN and SJS was induced by a suicidal interaction between Fas and Fas ligand (FasL), which are both expressed by keratinocytes. However, our preliminary examinations demonstrated that FasL is hardly detected on keratinocytes. We hypothesized that soluble FasL (sFasL) is secreted by peripheral blood mononuclear cells (PBMCs), and this interacts with the Fas expressed on keratinocytes in TEN and SJS. To justify this hypothesis, we investigated whether sFasL secreted by PBMCs could induce the keratinocyte apoptosis in TEN and SJS. Enzyme-linked immunosorbent assay analysis demonstrated that there was no significant sFasL increase in any samples of healthy controls (<40 pg/ml, n = 14) and patients with an ordinary erythema multiforme-type drug eruption (41.5 +/- 3.1 pg/ml, n = 14), whereas high concentrations are detected in all samples of TEN and SJS patients (TEN: 131.5 +/- 57.4 pg/ml, n = 8; SJS: 119.1 +/- 41.0 pg/ml, n = 14) (P < 0.0001). In vitro analysis using cultured keratinocytes revealed that the sera of TEN and SJS patients induced abundant keratinocyte apoptosis compared to erythema multiforme-type drug eruption sera. Furthermore, on stimulation with the causal drug, PBMCs obtained from TEN and SJS patients secreted high levels of sFasL. Taken together, these results indicate that sFasL secreted by PBMCs, not keratinocytes, plays a crucial role in the apoptosis and pathomechanism of TEN and SJS, and that the serum sFasL level may be a good indicator for the early diagnosis of TEN and SJS.

Advanced glycation end products (AGE), nonenzymatically glycated protein derivatives, have been implicated in the development and progression of diabetic angiopathies, including skin dermopathy. Nevertheless, the involvement of AGE in the development and progression of melanoma has not been fully elucidated. In this study we investigated the expression levels of their receptor for AGE (RAGE) in human melanoma and subsequently studied the effects of AGE on melanoma growth and migration. First, RAGE was detected in the cytoplasm of human melanoma cells (G361 and A375). Among the different types of AGE, glyceraldehyde- and glycolaldehyde-derived AGE significantly stimulated the growth and migration of human melanoma cells. Furthermore, tumor formation of melanoma cell xenografts in athymic mice was prevented by treatment with anti-RAGE neutralizing antibodies. In tumor-bearing mice, survival rates were prolonged, and spontaneous pulmonary metastases were inhibited by treatment using anti-RAGE neutralizing antibodies. In addition, all AGE were present in beds of human melanoma tumor, whereas they were barely detected in normal skin. These results suggest that AGE might be involved in the growth and invasion of melanoma through interactions with RAGE and represent promising candidates for assessing the future therapeutic potential of this therapy in treating patients with melanoma.

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, and is involved in the pathogenesis of angiogenic eye disease such as proliferative diabetic retinopathy. However, a functional role for PEDF in tumor growth and angiogenesis remains to be determined. In this study, we have investigated both the in vitro and in vivo growth characteristics of human malignant melanoma G361 cell lines, stably transfected to overexpress human PEDF. Expression levels of PEDF proteins in melanoma cell lines G361 and A375 were comparable with that of human cultured melanocytes, whereas vascular endothelial growth factor levels in two tumor cell lines were much stronger than that in normal melanocytes. Overexpression of PEDF was found to significantly inhibit tumor growth and vessel formation in G361 nude mice xenografts. Furthermore, in vitro proliferation rates of G361 cells were decreased in PEDFtransfected cells. PEDF proteins showed dose-dependent induced growth retardation and apoptotic cell death in nontransfected G361 cells, which were completely prevented by treatment with antibodies against the Fas ligand. Our present study highlights two beneficial effects of PEDF treatment on melanoma growth and expansion; one is the suppression of tumor angiogenesis, and the other is induction of Angiogenesis, a process by which new vascular networks are formed from pre-existing capillaries, is required for tumors to grow, invade, and metastasize.

ABSTRACT

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm 2 ) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm 2 ), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKCpan, PKC␣/␤II, PKC␦ (Thr505), PKC␦ (Ser 643 ), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC␣/␤II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of Cterminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.