Neurotrophin-3 increases the DNA-binding activities of several transcription factors in a mouse osteoblastic cell line

In the mouse osteoblastic cell line MC3T3-E1, the signaling responses of several DNA-binding proteins induced by the treatment of neurotrophin-3 were examined using electrophoretic mobility shift assay. Neurotrophin-3 increased binding activities in nuclear extracts of MC3T3-E1 cells to TPA-responsive element (TRE), cyclic AMP-responsive element (CRE) and serum-responsive element (SRE), but not binding activity in the nuclear extracts to c-Myc binding DNA element. Competition experiments revealed that the binding activity to TRE in the nuclear extracts of neurotrophin-3-treated MC3T3-E1 cells was entirely inhibited by the both unlabeled TRE and CRE probes. On the other hand, the binding activity to CRE was abolished by the unlabeled CRE probe but not by the same amount of unlabeled TRE probe. Moreover, immunodepletion/supershift assay using antibodies directed to Fos, Jun and CREB proteins, showed that the binding activities to TRE and CRE in the nuclear extracts were derived in part from these proteins.