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Department of Experimental Medicine, University of L’Aquila, via Vetoio, Coppito, 2, 67100 L’Aquila, Italy
¹ European Molecular Biology Laboratories, Heidelberg, Germany
² Dompè S p A, via Campo di Pile, L’Aquila, Italy
³ Department of Medical Physiopathology, University ‘La Sapienza’ of Rome, Policlinico Umberto I, Rome, Italy

(Requests for offprints should be addressed to A Teti; Email: teti{at}univaq.it)

(S Migliaccio and A Teti contributed equally to this work)
(Gianfranco Caselli is now at Rotta Research Laboratorium S p A, Monza, Italy)

The human estrogen receptor (ER ) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ER F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C (PKC ). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in over-confluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, F-promoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ER resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ER signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ER in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.

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