Signal transduction pathways mediating the effect of adrenomedullin on osteoblast survival

Abstract

Adrenomedullin (ADM) plays an important role in the regulation of osteoblastic cells through both a proliferative and an anti-apoptotic effects. The present study investigated mechanisms involved in the effect of ADM on survival. We report that ADM can act in osteoblasts both through a non-transcriptional action, by phosphorylation of different kinases and components, and through a transcriptional effect by activation of CREB. So, we observed by Western blot analysis, modifications in the downstream targets of ERK, the pro-apoptotic protein Bad, which is inactivated by increase in Ser155 phosphorylation, and the transcription factor CREB, which is activated by phosphorylation at Ser133. CREB activation was confirmed by a CRE-dependent gene transcription assay and an immunocytochemical study. This increase in CREB phosphorylation could lead to its enhanced transcriptional activity, as indicated by the induced expression of the proliferation marker, PCNA. Moreover, ADM could also activate the tyrosine kinase Src and the PI3-Kinase, both of which are implicated in survival. The use of specific pharmacological inhibitors allowed to establish that ADM could activate a signaling cascade involving Src, MEK, ERK, p90RSK, and that the effect of ADM, in particular on the CREB protein, greatly depends on the regulatory control of interfering signaling pathways. Moreover, as Wnt signaling plays an important role in the control of osteoblast apoptosis, we explored a major component of this pathway, protein GSK3β. ADM-induced inactivation of GSK3β by phosphorylation at Ser9, highly suggests that ADM could also exert its survival effect in osteoblast via components of the Wnt pathway.