Elevated constitutive I kappa B kinase activity and I kappa B-alpha phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-alpha transcription

The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-alpha, is upregulated in Hs 294T melanoma cells compared with the normal retinal pigment epithelial (RP E) cells, Previous studies characterized a cytokine-inducible, functional n uclear factor (NF)-kappa B consensus element in the immediate 5' regulatory region of the MGSA/GRO-alpha gene at -78 bp. Although the cytokine-inducib le mechanisms for transcription of this gene are fairly well delineated, th e mechanisms involved in its basal up-regulation of transcription in Hs294T melanoma cells are poorly understood, Recently, we demonstrated an increas ed rate of I kappa B-alpha degradation in Hs294T cells, which leads to an i ncreased nuclear localization of NF-kappa B (R, L. Shattuck-Brandt and A. R ichmond, Cancer Res., 57: 3032-3039, 1997), Here we demonstrate that Hs294T melanoma cells have elevated basal I kappa B kinase (IKK) activity relativ e to RPE cells, causing an increased constitutive I kappa B-alpha phosphory lation and degradation, We also show here that the resultant elevated nucle ar NF-kappa B (p50/p65) in these cells is responsible for the increased bas al transcription of MGSA/GRO-alpha. Pretreatment of Hs294T or RPE cells wit h proteasome inhibitors MG115 or MG132 captures the slower migrating, const itutively phosphorylated form of I kappa B-alpha in Hs294T melanoma cells, but not in RPE cells, In addition, a phospho-specific antibody that specifi cally recognizes the inhibitory form of I kappa B that is phosphorylated at Ser-32 reacted with I kappa B-alpha in Hs294T cell, but nut in unstimulate d RPE cells. Although the basal level of protein expression of IKK-alpha or IKK-beta are the same in both Hs294T and RPE cells, immunoprecipitation wi th IKK-alpha antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGS A/GRO-alpha promoter-luciferase reporter construct with either the dominant negative IKK-alpha or the repressors of NF-kappa B, the I kappa B-alpha wi ld type or mutants lacking the inducible phosphorylation sites, demonstrate s that the increased basal MGSA/ GRO-alpha transcription in the Hs294T cell s is due to the enhanced nuclear activation of NF-kappa B.