Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1).

Vascular cell adhesion molecule-1 (VCAM-1) was first identified as a protein that appears on the surface of endothelial cells after exposure to inflammatory cytokines. Through interaction with its integrin counter receptor VLA-4, VCAM-1 mediates cell-cell interactions important for immune function. We have cloned and begun characterization of the promoter for the VCAM-1 gene. In a series of transfection assays into human umbilical vein endothelial cells (HUVECs), we find that silencers between positions -1.641 kilobases and -288 base pairs restrict promoter activity, and that treatment with tumor necrosis factor-alpha overcomes this inhibition and activates the promoter through two NF kappa B sites located at positions -77 and -63 base pairs of the VCAM-1 gene. This responsiveness appears cell-specific since constructs containing the VCAM-1 NF kappa B sites are not responsive to tumor necrosis factor alpha in the T-cell line Jurkat. The two VCAM-1 NF kappa B sites, which differ slightly in their sequence, form distinct complexes in gel retardation assays, suggesting that they interact with different NF kappa B-site binding proteins. The distribution of these proteins could then control activity of the NF kappa B sites. We conclude that the pattern of VCAM-1 expression in HUVECs is controlled by a combination of these silencers and NF kappa B sites.     

A functional composite cis-element for NF kappa b and RBJ kappa in the rat pregnancy-specific glycoprotein gene.

The rat pregnancy-specific glycoprotein gene rnCGM3 is primarily expressed in the placenta. Previously, three DNase I footprinting sites (FPI, FPII, and FPIII) were identified in the rnCGM3 promoter region, a yeast one-hybrid screen was performed to identify the nuclear factors binding to the FPIII (5'-GCCTGGGAAAAAACTC-3') element, and RBPJ kappa, a downstream effector of the Notch signaling pathway, was identified as one of the FPIII-binding factors. In the present study, the NF kappa B member p65 was identified as another FPIII-binding factor. Electrophoretic mobility shift assays showed that NF kappa B members, including p50 and p65, bound to the FPIII site. The core binding sequence in the FPIII element for p50 and p65 is GGGAAA, which overlaps with that for RBPJ kappa. Competition exists between p50 and RBPJ kappa for binding to the FPIII element. Transient expression analyses revealed that p65 significantly stimulated the expression of a reporter gene directed by the NF kappa B core sequence in the FPIII element. However, RBPJ kappa could block this stimulation. These results suggest that the regulation of rnCGM3 expression involves both NF kappa B and RBPJ kappa, and they are mutually exclusive in the FPIII element.