Elevated constitutive IkappaB kinase activity and IkappaB-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 up-regulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF)-kappaB consensus element in the immediate 5' regulatory region of the MGSA/GRO-alpha gene at -78 bp. Although the cytokine-inducible mechanisms for transcription of this gene are fairly well delineated, the mechanisms involved in its basal up-regulation of transcription in Hs294T melanoma cells are poorly understood. Recently, we demonstrated an increased rate of IkappaB-alpha degradation in Hs294T cells, which leads to an increased nuclear localization of NF-kappaB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IkappaB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IkappaB-alpha phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-kappaB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-alpha. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IkappaB-alpha in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho-specific antibody that specifically recognizes the inhibitory form of IkappaB that is phosphorylated at Ser-32 reacted with IkappaB-alpha in Hs294T cell, but not in unstimulated 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 with IKK-alpha antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-alpha promoter-luciferase reporter construct with either the dominant negative IKK-alpha or the repressors of NF-kappaB, the IkappaB-alpha wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-alpha transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-kappaB.