Abstract
Vascular cell adhesion molecule-1 (VCAM-1) gene expression in cytokine-activated cells depends on two kappaB elements. Since VCAM-1 expression appears developmentally regulated and cytokine-inducible in smooth muscle cells (SMCs), we have studied the role of NF-kappaB in differentiated SMC VCAM-1 expression. Confluent SMCs were cultured either in a serum-free medium in order to induce differentiation, or in medium with serum, stimulated or not by tumor necrosis factor alpha (TNF-alpha). The expression of smooth muscle myosin heavy chain, a SMC marker, and VCAM-1 was induced concomitantly in serum-free medium, whereas only VCAM-1 expression was induced by cytokine-treatment. We showed that the p50 and p65 subunits of NF-kappaB were localized in the cytoplasm of differentiating SMCs, whereas they were translocated into the nucleus of TNF-alpha-activated SMCs. Electrophoretic mobility shift assays with VCAM-1 gene kappaB elements failed to detect any induction of DNA-protein complex with nuclear extracts of differentiating SMCs in contrast to the cytokine-activated SMC nuclear extracts. Furthermore, VCAM-1 mRNA induction was inhibited in TNF-alpha-stimulated SMCs, but not in differentiating SMCs, by pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB protein activation. Taken together, these findings suggest that in contrast to TNF-alpha activation, NF-kappaB is not involved in VCAM-1 gene expression during SMC differentiation.
Highlights
Vascular cell adhesion molecule-1 (VCAM-1)1 is a member of the immunoglobulin superfamily first identified as an adhesion protein expressed on cytokine-activated endothelial cells [1,2,3]
Flow cytometry and RT-PCR analysis showed that smooth muscle cells (SMCs) maintained in medium with serum had a low level of VCAM-1 protein and mRNA (Table I and Fig. 2)
These results showed that VCAM-1 expression was increased in two conditions, either by cytokine treatment in SMCs expressing low levels of smooth muscle myosin heavy chain (sm-MHC), or by medium switch inducing redifferentiation characterized by a high level of sm-MHC expression
Summary
Vascular cell adhesion molecule-1 (VCAM-1) is a member of the immunoglobulin superfamily first identified as an adhesion protein expressed on cytokine-activated endothelial cells [1,2,3]. Studies on the activity of the VCAM-1 gene promoter have shown that in endothelial cells, TNF-␣ induction of VCAM-1 is dependent on two adjacent B sites located at positions Ϫ77 and Ϫ63 relative to the single transcriptional start site [13, 14] These B elements are important for VCAM-1 induction in the embryonic carcinoma cell line P19 stimulated to differentiate along a neural pathway by treatment with retinoic acid [6]. Addition of the antioxidant pyrrolidine dithiocarbamate (PDTC), inhibitor of NF-B/Rel activity [23, 24], did not modify VCAM-1 mRNA induction during SMC differentiation, in contrast with the strong inhibition of TNF-␣-induced expression of VCAM-1 These results indicate that NF-B/Rel proteins are not involved in VCAM-1 gene expression during SMC differentiation
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