A role for poly(ADP-ribose) polymerase in the transcriptional regulation of the melanoma growth stimulatory activity (CXCL1) gene expression

The melanoma growth stimulatory activity/growth-regulated protein, CXCL1, i s constitutively expressed at high levels during inflammation and progressi on of melanocytes into malignant melanoma. It has been shown previously tha t CXCL1 overexpression in melanoma cells is due to increased transcription as well as stability of the CXCL1 message. The transcription of CXCL1 is re gulated through several cis-acting elements including Sp1, NF-kappaB, HMGI( Y), and the immediate upstream region(IUR) element (nucleotides -94 to -78) , which lies immediately upstream to the nuclear factor kappaB (NF-kappaB) element. Previously, it has been shown that the IUR is necessary for basal and cytokine-induced transcription of the CXCL1 gene. UV cross-linking and Southwestern blot analyses indicate that the IUR oligonucleotide probe sele ctively binds a 115-kDa protein. In this study, the IUR element has been fu rther characterized. We show here that proximity of the IUR element to the adjacent NF-kappaB element is critical to its function as a positive regula tory element. Using binding site oligonucleotide affinity chromatography, w e have selectively purified the 115-kDa IUR-F. Mass spectrometry/mass spect rometry/matrix-assisted laser desorption ionization/time of flight spectros copy and amino acid analysis as well as microcapillary reverse phase chroma tography electrospray ionization tandem mass spectrometry identified this p rotein as the 114-kDa poly(ADP-ribose) polymerase (PARP1). Furthermore, 3-a minobenzamide, an inhibitor of PARP-specific ADP-ribosylation, inhibits CXC L1 promoter activity and reduces levels of CXCL1 mRNA. The data point to th e possibility that PARP may be a coactivator of CXCL1 transcription.