Stretch-induced MAPK/AP-1 activity affects the differentiation potential of PDL fibroblasts

The purpose nflhis study was to examine ihe involvement of the immediate-early gene products e-Jun and c-Fos in the response of human periodontal ligament (PDL) fibroblasts lo mechanical stretching. The in-gel kinasc approach and electrophore t immobility-shift assays (EMSAsI were used lo investigate the ability of kinases targeting Jun und Fos to phosphorylate these transcription factors and induce AP-1hinding activity after mechanical stimulation of PDL fibroblasts. PDL fihroblasts isolated from healthy individuals were cultivated in Petriperm dishes (flexible bottom) under previously established conditions ( 1,2). Cultured PDL fibroblasts were subjected to mechanical stretching on a convex template with a weight applied on top and producing a -2.59% degree of streich (1,2). After 7-30 min (immediate-early response), extracts were prepared from stretched and control cultures and their kinase activity against c-Jun and c-Fos (served as immobilized substrates) was tested in an elaborated in-gel kinase assay. Our analysis revealed that specific protein serine/threonine kinases, with apparent molecular masses in the range of well characterized Jun/Fos targeting en/ymes (42-58 kDa), were gradually induced in mechanically-stretched PDL llbroblasts and augmented the basal-level phosphorylation of both substrates. This observation correlated well with increased, c-Jun-containing AP-1-binding activity of extracts obtained from stretched PDL cultures on the promoter/regulatory region of the alkaline phusphatase gene and enhanced activity of the corresponding en/yme in these extracts. Because elevated expression of the alkaline phosphatase gene is linked lo the onset ot the osieoblast phenotype, we pose thai mechanical stretching may lead to genetic reprogramming of PDL fibrohlasts via potentiation of c-Jun and c-Fos.