Comparative Expression of NFkappaB Proteins in Melanocytes of Normal Skin vs. Benign Intradermal Naevus and Human Metastatic Melanoma Biopsies

Nuclear factor kappa B (NFkappaB) is an essential regulator of gene transcription for hundreds of genes, including many critically involved in apoptosis. NFkappaB complexes containing cRel generally activate pro-apoptotic genes, while those with RelA activate anti-apoptotic genes. We have previously shown that NFkappaB binding by RelA is constitutively elevated in human metastatic melanoma cultures relative to normal melanocytes. Here we extended our investigation to immunohistochemical analysis of human tissue biopsies. We found that RelA expression is significantly elevated in melanocytes of human naevi and melanomas relative to normal skin, but expression of its inhibitor IkappaB-alpha is significantly lower in metastatic melanomas than in intradermal naevi. Antibodies specific for the nuclear localization signal of RelA also showed significantly increased staining in metastatic melanoma biopsies. Notably, in melanomas and in naevi, we also found that RelA is phosphorylated at serine 529, and this activated form accumulates in the nuclei of melanomas. This suggests that increased expression and phosphorylation of RelA occurs at the stage of the benign naevus, but IkappaB-alpha is able to sequester RelA in the cytoplasm and regulate RelA transcriptional transactivation. We also found that antibodies against cRel show a progressive increase in staining from naevi to melanoma. However, staining for IkappaB-epsilon, which primarily inhibits the nuclear localization of cRel was also progressively increased, and cRel expression was predominantly cytoplasmic in melanomas. These results confirm that the altered expression of RelA found in metastatic melanoma cells in tissue culture is relevant to human tumors and offer new insights into the deregulation of NFkappaB signaling

Endotoxin, but not platelet-activating factor, activates nuclear factor-κB and increases IκBα and IκBβ turnover in enterocytes

Bacterial endotoxin (lipopolysaccharide; LPS) and platelet-activating factor (PAF) are important triggers of bowel inflammation and injury. We have previously shown that LPS activates the transcription factor nuclear factor (NF)-κB in the intestine, which up-regulates many pro-inflammatory genes. This effect partly depends on neutrophils and endogenous PAF. However, whether LPS and PAF directly activate NF-κB in enterocytes remains controversial. In this study, we first investigated the effect of LPS and PAF on NF-κB activation in IEC-6 (a non-transformed rat small intestinal crypt cell line) cells, by electrophoresis mobility shift assay and supershift, and found that LPS, but not PAF, activates NF-κB mostly as p50–p65 heterodimers. The effect was slower than tumour necrosis factor (TNF). Both LPS and TNF induce the expression of the NF-κB-dependent gene inducible nitric oxide synthase (iNOS), which occurs subsequent to NF-κB activation. We then examined the effect of LPS and TNF on the inhibitory molecules IκBα and IκBβ. We found that TNF causes rapid degradation of IκBα and IκBβ. In contrast, LPS did not change the levels of IκBα and IκBβ up to 4 hr (by Western blot). However, in the presence of cycloheximide, there was a slow reduction of IκBα and IκBβ, which disappeared almost completely at 4 hr. These observations suggest that LPS causes slow degradation and synthesis of IκBα and IκBβ and therefore activates NF-κΒ via at least two mechanisms: initially, through an IκB-independent mechanism, and later, via an increased turnover of the inhibitor IκB. NF-κΒ activation precedes the gene expression of iNOS (assayed by reverse transcription–polymerase chain reaction), suggesting that LPS up-regulates iNOS via this transcription factor