Elevated Constitutive IkB Kinase Activity and IkB-a Phosphorylation in Hs294T Melanoma Cells Lead to Increased Basal MGSA/GRO-a Transcription

The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-a, is upregulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokineinducible, functional nuclear factor (NF) -kB consensus element in the immediate 5* regulatory region of the MGSA/GRO-a gene at 278 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 IkB-a degradation in Hs294T cells, which leads to an increased nuclear localization of NF-kB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032–3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IkB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IkB-a phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-kB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-a. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IkB-a in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho-specific antibody that specifically recognizes the inhibitory form of IkB that is phosphorylated at Ser-32 reacted with IkB-a in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-a or IKK-b are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-a antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-a promoter-luciferase reporter construct with either the dominant negative IKK-a or the repressors of NF-kB, the IkB-a wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/ GRO-a transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-kB.