SP1 mediated transcriptional regulation of the ADAMTS-8 gene

ADAMTSs are secreted into the extracellular matrix and degrade structural proteins of the matrix and contain special thrombospondin 1 motifs. As a metalloproteinase, ADAMTS-8 plays an important role in invasion and metastasis processes such as tumour growth and regulation of growth factors. It has been observed that ADAMTS-8 low expression in colorectal cancer cells inhibits cell proliferation, induces apoptosis and suppresses invasion and migration in HCT116, HT-29 models. SP1 is a DNA binding site- recognising protein that regulates mammalian and viral genes. Containing three zinc fingers responsible for increasing transcription in its active site, SP1 binds to GC-rich DNA sequences. SP1 plays an important role in CRC development and progression. In this study, the regulation of ADAMTS-8, which is a suppressor gene in colorectal cancer, by the colorectal cancer regulator SP1 was investigated. For this purpose, promoter fragments of five different lengths were cloned into luciferase vector to determine the transcriptional activity of the promoter region of ADAMTS-8 gene. TS-8[-854/+323] promoter fragment showed the highest activity when basal activities were compared with transient transfection studies in SW480 cells. Bioinformatic analyses revealed that ADAMTS-8 gene contains many SP1 binding sites. Co-transfection studies revealed that SP1 decreased ADAMTS-8 promoter activity especially in TS-8[-854/+323] promoter fragment. ChIP analysis was performed to determine whether SP1 binds functionally to the promoter. Binding to ADAMTS-8(-353/-234), ADAMTS-8(-87/+38) and ADAMTS-8(+188/+312) regions was confirmed. Subsequently, the effect of CEBP, USF and SP1 overexpression on ADAMTS-8 protein level in SW480 model was determined by western blot. SP1 was found to reduce ADAMTS-8 protein expression by almost half. The effect of SP1 overexpression on ADAMTS-8 mRNA level in SW480 and SAOS-2 cell line selected as a side model was determined by qRT-PCR. It was determined that SP1 ovarian expression decreased ADAMTS-8 mRNA level in both SW480 and SAOS-2 model.