Abstract 1675: Inhibition of Nuclear Factor of Activated T-Cells Signaling Reduces Osteopontin Expression in Vascular Smooth Muscle
Osteopontin (OPN) is a multifunctional protein that can exist as an immobilized extracellular matrix molecule and as a cytokine in body fluids. Its expression increases in response to stress and tissue injury in several cell types including vascular smooth muscle cells. OPN is upregulated in atherosclerotic plaques, neointimal thickenings after balloon angioplasty and in arteries from diabetic rats and patients. The enhanced expression of OPN is not only a marker of vascular disease but it is likely to promote the development of atherosclerotic plaques and restenotic lesions. Thus, mapping the signaling pathway leading to changes in OPN expression may reveal novel pharmacological targets for prevention of vascular disease. OPN expression seems to require the concerted action of several transcription factors. The identity of many of these factors has been revealed in osteoclasts and in cancer cells, but it remains poorly defined in the vasculature. Here we show that the Ca2+/calcineurin-dependent transcription factor Nuclear Factor of Activated T-cells (NFAT) is involved in the regulation of OPN expression in arterial smooth muscle. Using immunofluorescence confocal microscopy and western blotting, we show a time-dependent induction of OPN expression upon culture of intact mouse aorta. OPN was not detected in freshly dissected arteries. Stimulation of arteries during culture with UTP, which is a potent activator of NFAT in this tissue, significantly enhanced OPN expression. Inhibition of NFAT nuclear translocation and NFAT-dependent transcriptional activity, as assessed in arteries from NFAT-luciferase reporter mice, prevented this UTP-mediated upregulation of OPN. We searched the mouse promoter region of OPN and identified multiple potential NFAT binding elements. Using electrophoretic mobility shift assay, interaction of NFAT with these regions was demonstrated in vitro and largely reduced by the inclusion of an antibody directed against NFATc3. Interestingly, even though all four NFAT isoforms (NFATc1-c4) are expressed in mouse aorta, only NFATc3 increases upon organ culture. Our results suggest that OPN expression is regulated in an NFAT-dependent manner in native arteries.