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

Background: It is widely accepted that mechanical loading is necessary to c onstruct the architecture of bone and to maintain bone mass. However, the m olecular mechanisms whereby bone cells respond to mechanical stimuli remain elusive. The mitogen-activated protein kinase (MAPK) signaling cascades ar e known to play a crucial role in the immediate osteoblast response to a va riety 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 facto rs. To identify whether the MAPKs are involved in the mechanotransduction p rocess and to determine the effect on downstream transcription factors, we stimulated human periodontal ligament (hPDL) osteoblast-like cells by mecha nical stretching by employing an established in vitro model of continuous s tretch application. Materials and Methods: Whole-cell extracts were prepared from cultivated hP DL cells that were exposed to short-term, continuous mechanical stretch. In -gel kinase assays were used to assess their kinase activity towards the im mediate-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 previous ly 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 oste oblastic differentiation) were employed to evaluate their specific binding capacity. Results: Selective members of the MAPK family were rapidly induced by stret ching, as manifested by their ability to enhance phosphorylation of their c ognate 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 pe rformed with the relevant sequence from the L/B/K ALP promoter. Conclusions: In as much as AP-1 is instrumental in regulating genes activat ed at the onset of osteoblast differentiation, such as the ALP gene, we pos e that an interplay of distinct MAPKs targeting AP-1 components may dictate the osteogenic response of hDL cells to mechanical stimulation.