Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells

Background: It is widely accepted that mechanical loading is necessary to construct the architecture of bone and to maintain bone mass. However, the molecular mechanisms whereby bone cells respond to mechanical stimuli remain elusive. The mitogen-activated protein kinase (MAPK) signaling cascades are known to play a crucial role in the immediate osteoblast response to a variety of bone-active agents. An important component of this response occurs at the transcriptional level and is executed by downstream phosphorylation substrates, most notably a number of signal-responsive transcription factors. To identify whether the MAPKs are involved in the mechanotransduction process and to determine the effect on downstream transcription factors, we stimulated human periodontal ligament (hPDL) osteoblast-like cells by mechanical stretching by employing an established in vitro model of continuous stretch application. Materials and Methods: Whole-cell extracts were prepared from cultivated hPDL cells that were exposed to short-term, continuous mechanical stretch. In-gel kinase assays were used to assess their kinase activity towards the immediate-early gene products c-Jun and c-Fos [constituents of the activator protein-1 (AP-1) transcription factor]. Electrophoretic mobility-shift and southwestern experiments utilizing a DNA sequence that contained a previously undefined atypical AP-l-binding site in the promoter of the human liver/bone/kidney alkaline phosphatase (L/B/K ALP) gene tan early marker for osteoblastic differentiation) were employed to evaluate their specific binding capacity. Results: Selective members of the MAPK family were rapidly induced by stretching, as manifested by their ability to enhance phosphorylation of their cognate substrates c-Jun and, to a lesser extent, c-Fos in the in-gel kinase assay. This induction was accompanied by markedly increased, phospho-c-Jun-containing AP-l-binding activity, as determined by the binding analyses performed with the relevant sequence from the L/B/K ALP promoter. Conclusions: In as much as AP-1 is instrumental in regulating genes activated at the onset of osteoblast differentiation, such as the ALP gene, we pose that an interplay of distinct MAPKs targeting AP-1 components may dictate the osteogenic response of hDL cells to mechanical stimulation.