Visit our Expo - Redox and Inflammation signaling 2012

Session XIV : Transcriptional and translational control Poster XIV, 78
NFAT and bone disease: a possible “decoy” therapeutic approach
Margherita Zennaro, Elisabetta Lambertini, Roberto Gambari, Letizia Penolazzi and
Roberta Piva.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy. Email:
piv@unife.it
Bone is a dynamic tissue, under constant remodelling all life long. This process results from
the balance between the activity of osteoblasts (OBs) and osteoclasts (OCs), that deposit and
resorb bone respectively. Any modification of this delicated equilibrium can affect bone
integrity, leading to patologies such as osteopenic disorders or osteopetrosis. A central role in
the control of OCs functions and in some OBs activities too, was recently described for
Nuclear Factor of Activated T-cells (NFAT) c1. In this study, we suggest a possible
involvment of NFATc1 in the transcriptional regulation of human estrogen receptor alpha
(ER) gene in both kind of cells. It is well known that estrogen influences OBs and OCs
activities, playing a fundamental role in protection of bone mass, as indicated, for example, by
association of osteoporosis and menopause.
We previously demonstrated that a specific oligonucleotide (RA4-3’) used as decoy molecule
can increase ER expression in bone cells, by interfering with the activity of an unidentified
negative transcription factor. Interestingly, RA4-3’ sequence contains a putative binding site
(TGAAAA) for NFAT that is the aim of these investigations. First of all we detected a high
level of NFATc1 expression in OCs and in SaOS-2 osteoblastic cells, treated with PMA and
ionomycin. We then verified the specificities of the interaction between NFAT protein and
our sequence, by using cross competition in electrophoretic mobility shift assay. Preliminary
results indicat that a specific interaction between NFAT and our sequence occurs, probably
mediated by some NFAT co-transcription factors, such as AP-1. Next, we verified NFAT
functionality in OCs and SaOS-2 through the transfection of pNFAT-TA luc cis-reporter
vector, containing three tandem copies of the NFAT-consensus sequence upstream of the
luciferase gene, in combination with RA4-3’. This caused a decrease of Luc activity, due to
the recruitment of NFAT proteins by RA4-3’. In addition, we demonstrated that RA4-3’
selectively induces apoptosis in OCs but not in OBs. In view of a possible therapeutic
application of NFAT decoy, the role of NFAT in this issue was also investigated.
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