Abstract
Maintenance of the cells of the vessel wall in a quiescent state is an important aspect of normal vascular physiology. Transcriptional repressors are widely believed to regulate this process, yet the exact factors involved and the mechanism of repression are not known. Here, we report that the POU domain transcription factor Oct-1 represses the expression of E-selectin and vascular cell adhesion molecule (VCAM-1), two cytokine-inducible, NF-kappaB-dependent endothelial-leukocyte adhesion molecules that participate in the leukocyte recruitment phase of the inflammatory response. Co-transfection and microinjection studies demonstrate that Oct-1 blocks tumor necrosis factor alpha-stimulated E-selectin and VCAM-1 expression. Gene expression arrays indicate that control of tumor necrosis factor alpha-induced, NF-kappaB-dependent gene expression by Oct-1 is promoter-specific. A DNA-binding mutant of Oct-1 represses NF-kappaB-dependent reporter gene expression. Biochemically, Oct-1 interacts with p65, suggesting that Oct-1 is involved in the regulation of NF-kappaB transactivation function. NF-kappaB-dependent gene expression is more pronounced in Oct-1-deficient than in wild-type murine embryonic fibroblasts, and reintroduction of human Oct-1 abolishes these differences. Finally, the cytokine interleukin-6 induces Oct-1 gene expression, providing a biologically relevant means by which NF-kappaB-dependent gene expression can be selectively reverted by Oct-1 to quiescent levels.
Highlights
Oct-1 Represses Authentic E-selectin and VCAM-1 Gene Expression in Cytokine-activated Endothelial Cells—To determine whether this apparent repression by Oct-1 occurs in vivo on endogenous promoters in a true chromatin environment, we developed a single cell were co-injected with an expression construct for p65
The results showed that E-selectin and VCAM-1 gene expression increases with TNF␣ treatment (Fig. 1D, left) and is significantly blocked in both instances by Oct-1 (Fig. 1D, middle)
In an effort to further clarify how Oct-1 decreases the expression of E-selectin and VCAM-1, we examined whether a direct physical interaction between Oct-1 and DNA was required
Summary
Reagents and Antibodies—Recombinant human TNF␣ and IL-6 were purchased from R&D Systems and used at 10 and 100 ng/ml, respectively. The COS-7 cells were transiently transfected with 1 g of Ϫ578 E-selectin promoter-CAT or VCAM-1 promoter-CAT and 100 ng of a human cytomegalovirus-p65 expression vector by a modified calcium phosphate method. After 24 h of incubation, cells were harvested in reporter lysis buffer (Promega), and CAT activity was determined as previously described [6, 30] and normalized to -galactosidase activity. Rat-1 fibroblasts were seeded on acid-washed glass coverslips at subconfluent density and maintained in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 2 mM GlutaMax, 500 nM methotrexate, 1% penicillin/streptomycin, and 10% fetal bovine serum. GST Pull-down Assays—GST fusion constructs containing various fragments of p65 and Oct-1 were generated by PCR amplification and cloned into the pGEX-2TA vector (Amersham Biosciences). After 24 h, total RNA was isolated from the microinjected cells, and RT-PCR analysis was performed
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