The human receptor tyrosine kinase Axl gene - promoter characterization and regulation of constitutive expression by Sp1, Sp3, and CpG methylation

Giridhar Mudduluru , Heike Allgayer (1)

1 Dept. Experimental Surgery,Medical Faculty Mannheim

Axl-receptor-tyrosine-kinase promotes antiapoptosis, mitogenesis, invasion, angiogenesis, and metastasis, and is highly expressed in cancers. However, the transcriptional regulation of this important gene has never been characterized. The present study was initiated to characterize the promoter, first cis-acting elements, and promoter methylation driving expression of Axl. The 2.4kb sequence upstream of the translational start site, and sequential 5'-deletions were cloned and revealed a minimal GC-rich region (-556/+7) to be sufficient for basal Axl-promoter activity in Rko, HCT116, and HeLa cells. Within this minimal region, five Sp-binding sites were identified. Two sites (Sp-a, Sp-b) most proximal to the translation start site were indispensable for Axl-promoter activity, whereas mutation of three additional upstream (Sp-c, Sp-d, Sp-e) motifs was of additional relevance. Gelshift and chromatin-immunoprecipitation identified especially Sp1 and Sp3 bound to all 5 motifs, mutations of all motifs abolishing binding. Mithramycin, inhibiting binding of Sp-factors to GC-rich sites, dramatically reduced Axl-promoter activity, Axl-, Sp1-, and Sp3-expression. In Drosophila Schneider SL2-cells, exogenous expression of Sp1/Sp3 increased Axl-promoter activity. Sp1/Sp3-siRNAs significantly reduced Axl-promoter activity and protein in Rko and HeLa cells. Methylation-bisulfite sequencing detected methylated CpG-sites within three Sp-motifs (Sp-a, -b, -c) and GC-rich flanking sequences, demethylation by 5-aza-2'-deoxycytidine upregulating Axl- and Sp3-expression in low Axl-expressing Colo206f/WiDr, but not high Axl-expressing Rko-cells. These data suggest that Axl gene expression in cancer-cells 1) is constitutively driven by Sp1/Sp3 bound to 5 core-promoter motifs, 2) is restricted by methylation within/around Sp-binding sites. This might enhance understanding and treatment of essential mechanisms associated with cancer and other diseases.

Mots clés: Life Sciences

Giridhar Mudduluru, Heike Allgayer. The human receptor tyrosine kinase Axl gene - promoter characterization and regulation of constitutive expression by Sp1, Sp3, and CpG methylation. Bioscience Reports, 2008, 28 (3), pp.161-176. ⟨10.1042/BSR20080046⟩. ⟨hal-00479299⟩