Elevated constitutive IκB kinase activity and IκB-α phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-α transcription

Madhav N. Devalaraja, Ding Zhi Wang, Dean W. Ballard, Ann Richmond

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Abstract

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

Original languageEnglish (US)
Pages (from-to)1372-1377
Number of pages6
JournalCancer Research
Volume59
Issue number6
StatePublished - Mar 15 1999
Externally publishedYes

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Melanocytes
Melanoma
Phosphotransferases
Phosphorylation
Retinal Pigments
Growth
Epithelial Cells
Proteins
Phospho-Specific Antibodies
Cytokines
CXC Chemokines
Proteasome Inhibitors
Nucleic Acid Regulatory Sequences
Luciferases
Immunoprecipitation
Up-Regulation
Antibodies

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Elevated constitutive IκB kinase activity and IκB-α phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-α transcription. / Devalaraja, Madhav N.; Wang, Ding Zhi; Ballard, Dean W.; Richmond, Ann.

In: Cancer Research, Vol. 59, No. 6, 15.03.1999, p. 1372-1377.

Research output: Contribution to journalArticle

Devalaraja, Madhav N. ; Wang, Ding Zhi ; Ballard, Dean W. ; Richmond, Ann. / Elevated constitutive IκB kinase activity and IκB-α phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-α transcription. In: Cancer Research. 1999 ; Vol. 59, No. 6. pp. 1372-1377.
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abstract = "The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-α, is upregulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF) -κB consensus element in the immediate 5' regulatory region of the MGSA/GRO-α 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 IκB-α degradation in Hs294T cells, which leads to an increased nuclear localization of NF-κB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IκB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IκB-α phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-κB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-α. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IκB-α in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho- specific antibody that specifically recognizes the inhibitory form of IκB that is phosphorylated at Ser-32 reacted with IκB-α in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-α or IKK-β are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-α antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-α promoter-luciferase reporter construct with either the dominant negative IKK-α or the repressors of NF-κB, the IκB-α wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-α transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-κB.",
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AU - Ballard, Dean W.

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N2 - The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-α, is upregulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF) -κB consensus element in the immediate 5' regulatory region of the MGSA/GRO-α 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 IκB-α degradation in Hs294T cells, which leads to an increased nuclear localization of NF-κB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IκB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IκB-α phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-κB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-α. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IκB-α in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho- specific antibody that specifically recognizes the inhibitory form of IκB that is phosphorylated at Ser-32 reacted with IκB-α in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-α or IKK-β are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-α antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-α promoter-luciferase reporter construct with either the dominant negative IKK-α or the repressors of NF-κB, the IκB-α wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-α transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-κB.

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