1. Analysis of the regulation of human Estrogen Receptor alpha gene expression. The transcriptional regulation of human ERa gene is very complex and only in part known. We are interested in the understanding the role of specific promoter regions of this gene in osteoblasts and osteoclasts Our previous results indicate that: - Runx2 is a regulator of ERa gene expression in osteoblasts: it binds specific cis elements of F promoter of ERa gene, “in vivo”, with opposite effects. - ERa and c-jun, c-fos and ATF-2, but not Fra-2 AP-1 factors interact “in vivo” with specific estrogen-responsive regulatory sequences and AP-1 cis-elements within the F promoter of the human ERa gene in osteoblast-like SaOS-2 cells. This recruitment is oscillatory and is modulated by 17-b-estradiol. This research is based on the employment of EMSA, ChIP, promoter analysis, mutagenesis, quantitative RT-PCR. This study includes also the employment of the “decoy” strategy. The transcription factor (TF) decoy technology involves synthetic double-stranded ODN containing a cis element with high affinity for a target TF and able to bind the TF after transfection into target cells. This attenuates the authentic cis-trans interactions leading to removal of the TF from the endogenous cis elements with subsequent specific modulation of gene expression. Therefore, the decoy approach is a powerful tool to modulate transcription of a target gene and directly study the transcriptional control of a gene of interest. The aim is the improvement of bone matrix deposition and the induction of osteoclast apoptosis. In order to obtain reproducible results we are investigating the better experimental approach in terms of cellular transfection, delivery system and source of primary cells. 2.Wnt signaling and estrogen signaling in bone. The major signaling pathways controlling bone formation and remodeling are those mediated by the following molecules: WNT, Lef1/Tcf, Runx2, AP-1, NFATc1 and Estrogen Receptor alpha. Increasing evidence suggests that all these factors may play a critical role both in osteoblasts and in osteoclasts, acting on different molecular target and associating with themselves. Our field of interest concerns the study of molecular mechanisms supporting the convergence of the above mentioned regulatory signals that may control bone-specific gene expression. The aim of this research is the employment of specific pharmacological molecules or nucleic acids-based drugs able to selectively modulate these pathways in human primary osteoblasts obtained from osteoporotic bones. This research program addresses also the analysis of new compounds present in unexplored plant extracts in terms of their ability to induce osteoclast apoptosis and osteoblast differentiation interfering with Wnt signaling and estrogen signaling. Different plant extracts are tested on primary cultures and modulation of apoptosis, cell proliferation, citotoxicity, EMSA interference, mineralization and osteoblast markers expression are evaluated. 3. Bone tissue regeneration through interaction between human primary osteoblasts and their mesenchymal precursors and biomaterials. The research program on bone tissue regeneration, is addressed to the development of an experimental model based on the combination of biomaterials, bone primary cells and their precursors. The knowledge resulted from molecular study will be employed to improve the performance of the cells combined with different biomaterials. In particular, we are interested in analyzing dental implant surfaces to optimize bone growth at the interface. 4. Analysis of expression and function of Slug gene in human normal osteoblasts and their mesenchymal precursors. In order to identify new potential osteoblast-specific proteins, we are investigating the role of Slug as mediator of Wnt signaling in relation to the expression profile of bone-related genes during human osteoblast differentiation.