This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Devalaraja, M. N. Articles by Richmond, A. Search for Related Content PubMed PubMed Citation Articles by Devalaraja, M. N. Articles by Richmond, A.

Elevated Constitutive IB Kinase Activity and IB- Phosphorylation in Hs294T Melanoma Cells Lead to Increased Basal MGSA/GRO- Transcription¹

Departments of Cell Biology [M. N. D., D. Z. W., A. R.], Microbiology and Immunology [D. W. B.], and Medicine, Division of Dermatology, [A. R.], Vanderbilt University School of Medicine; Veterans Affairs Medical Center [D. Z. W., A. R.]; and Howard Hughes Medical Institute [D. W. B.], Nashville, Tennessee 37212-2637

ABSTRACT

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

This article has been cited by other articles:

				G. Sethi, B. Sung, and B. B. Aggarwal Nuclear Factor-{kappa}B Activation: From Bench to Bedside Experimental Biology and Medicine, January 1, 2008; 233(1): 21 - 31. [Abstract] [Full Text] [PDF]

				D.-S. Son and K. F. Roby Interleukin-1{alpha}-Induced Chemokines in Mouse Granulosa Cells: Impact on Keratinocyte Chemoattractant Chemokine, a CXC Subfamily Mol. Endocrinol., November 1, 2006; 20(11): 2999 - 3013. [Abstract] [Full Text] [PDF]

				H. Rangaswami, A. Bulbule, and G. C. Kundu Nuclear Factor-inducing Kinase Plays a Crucial Role in Osteopontin-induced MAPK/I{kappa}B{alpha} Kinase-dependent Nuclear Factor {kappa}B-mediated Promatrix Metalloproteinase-9 Activation J. Biol. Chem., September 10, 2004; 279(37): 38921 - 38935. [Abstract] [Full Text] [PDF]

				R. D. Sue, J. A. Belperio, M. D. Burdick, L. A. Murray, Y. Y. Xue, M. C. Dy, J. J. Kwon, M. P. Keane, and R. M. Strieter CXCR2 Is Critical to Hyperoxia-Induced Lung Injury J. Immunol., March 15, 2004; 172(6): 3860 - 3868. [Abstract] [Full Text] [PDF]

				X. Bian, A. W. Opipari Jr., A. B. Ratanaproeksa, A. E. Boitano, P. C. Lucas, and V. P. Castle Constitutively Active NFkappa B Is Required for the Survival of S-type Neuroblastoma J. Biol. Chem., October 25, 2002; 277(44): 42144 - 42150. [Abstract] [Full Text] [PDF]

				O. Prakash, Z.-Y. Tang, X. Peng, R. Coleman, J. Gill, G. Farr, and F. Samaniego Tumorigenesis and Aberrant Signaling in Transgenic Mice Expressing the Human Herpesvirus-8 K1 Gene J Natl Cancer Inst, June 19, 2002; 94(12): 926 - 935. [Abstract] [Full Text] [PDF]

				P. Dhawan and A. Richmond A Novel NF-kappa B-inducing Kinase-MAPK Signaling Pathway Up-regulates NF-kappa B Activity in Melanoma Cells J. Biol. Chem., March 1, 2002; 277(10): 7920 - 7928. [Abstract] [Full Text] [PDF]

				A. V. Gasparian, Y. J. Yao, D. Kowalczyk, L. A. Lyakh, A. Karseladze, T. J. Slaga, and I. V. Budunova The role of IKK in constitutive activation of NF-{kappa}B transcription factor in prostate carcinoma cells J. Cell Sci., January 1, 2002; 115(1): 141 - 151. [Abstract] [Full Text] [PDF]

				R. D. Ye Regulation of nuclear factor {kappa}B activation by G-protein-coupled receptors J. Leukoc. Biol., December 1, 2001; 70(6): 839 - 848. [Abstract] [Full Text] [PDF]

				L. W. Shepard, M. Yang, P. Xie, D. D. Browning, T. Voyno-Yasenetskaya, T. Kozasa, and R. D. Ye Constitutive Activation of NF-kappa B and Secretion of Interleukin-8 Induced by the G Protein-coupled Receptor of Kaposi's Sarcoma-associated Herpesvirus Involve Galpha 13 and RhoA J. Biol. Chem., November 30, 2001; 276(49): 45979 - 45987. [Abstract] [Full Text] [PDF]

				J. Yang, J. Luan, Y. Yu, C. Li, R. A. DePinho, L. Chin, and A. Richmond Induction of Melanoma in Murine Macrophage Inflammatory Protein 2 Transgenic Mice Heterozygous for Inhibitor of Kinase/Alternate Reading Frame Cancer Res., November 1, 2001; 61(22): 8150 - 8157. [Abstract] [Full Text] [PDF]

				M. L. Guzman, S. J. Neering, D. Upchurch, B. Grimes, D. S. Howard, D. A. Rizzieri, S. M. Luger, and C. T. Jordan Nuclear factor-{kappa}B is constitutively activated in primitive human acute myelogenous leukemia cells Blood, October 15, 2001; 98(8): 2301 - 2307. [Abstract] [Full Text] [PDF]

				F. Chen, V. Castranova, and X. Shi New Insights into the Role of Nuclear Factor-{kappa}B in Cell Growth Regulation Am. J. Pathol., August 1, 2001; 159(2): 387 - 397. [Abstract] [Full Text]

				J. Yang and A. Richmond Constitutive I{{kappa}}B Kinase Activity Correlates with Nuclear Factor-{{kappa}}B Activation in Human Melanoma Cells Cancer Res., June 1, 2001; 61(12): 4901 - 4909. [Abstract] [Full Text] [PDF]

				R. Romieu-Mourez, E. Landesman-Bollag, D. C. Seldin, A. M. Traish, F. Mercurio, and G. E. Sonenshein Roles of IKK Kinases and Protein Kinase CK2 in Activation of Nuclear Factor-{{kappa}}B in Breast Cancer Cancer Res., May 1, 2001; 61(9): 3810 - 3818. [Abstract] [Full Text]

				M. M. Dikov, T. Oyama, P. Cheng, T. Takahashi, K. Takahashi, T. Sepetavec, B. Edwards, Y. Adachi, S. Nadaf, T. Daniel, et al. Vascular Endothelial Growth Factor Effects on Nuclear Factor-{{kappa}}B Activation in Hematopoietic Progenitor Cells Cancer Res., March 1, 2001; 61(5): 2015 - 2021. [Abstract] [Full Text]

				S. S. Brar, T. P. Kennedy, A. R. Whorton, A. B. Sturrock, T. P. Huecksteadt, A. J. Ghio, and J. R. Hoidal Reactive oxygen species from NAD(P)H:quinone oxidoreductase constitutively activate NF-{kappa}B in malignant melanoma cells Am J Physiol Cell Physiol, March 1, 2001; 280(3): C659 - C676. [Abstract] [Full Text] [PDF]

				X. H. Li, K. M. Murphy, K. T. Palka, R. M. Surabhi, and R. B. Gaynor The Human T-cell Leukemia Virus Type-1 Tax Protein Regulates the Activity of the Ikappa B Kinase Complex J. Biol. Chem., November 26, 1999; 274(48): 34417 - 34424. [Abstract] [Full Text] [PDF]

				V. A. Soldatenkov, A. Dritschilo, Z.'e. Ronai, and S. Y. Fuchs Inhibition of Homologue of Slimb (HOS) Function Sensitizes Human Melanoma Cells for Apoptosis Cancer Res., October 1, 1999; 59(20): 5085 - 5088. [Abstract] [Full Text] [PDF]

				C. Nirodi, S. NagDas, S. P. Gygi, G. Olson, R. Aebersold, and A. Richmond A Role for Poly(ADP-ribose) Polymerase in the Transcriptional Regulation of the Melanoma Growth Stimulatory Activity (CXCL1) Gene Expression J. Biol. Chem., March 16, 2001; 276(12): 9366 - 9374. [Abstract] [Full Text] [PDF]

				C. Nirodi, J. Hart, P. Dhawan, N.-s. Moon, A. Nepveu, and A. Richmond The Role of CDP in the Negative Regulation of CXCL1 Gene Expression J. Biol. Chem., July 6, 2001; 276(28): 26122 - 26131. [Abstract] [Full Text] [PDF]