Characterization of the human a9 integrin subunit gene: promoter analysis and transcriptional regulation

α9b1 is the most recent addition to the integrin family of membrane-bound receptors and consequently remains the one which is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated at the molecular level, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT recombinant plasmids in primary cultured cells and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1/Sp3, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1/Sp3 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Most of all, addition of tenascin-C (TNC), the ligand of α9β1, to the tissue culture plates prior to cell seeding increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing. Primary cultures of human corneal epithelial cells (HCECs; number of replicates: 8), human skin epithelial cells (HSEC; number of replicates: 4), human corneal fibroblast cells (HCFCs; number of replicates: 3), human skin fibroblast cells (HSFCs; number of replicates: 3), human uveal melanocytes (UVM; number of replicates: 3), and various humal uveal melanoma cell lines (T115, T142 and T143; number of replicates: 2-3) were analyzed by gene profiling on microarrays at low culture passages (between passage 2 to 7). HCECs were also cultured on either BSA (number of replicates: 2) or on tenascin-C (number of replicates: 2) and their transcriptome analyzed by gene profiling.