Platelet‐derived growth factor‐B gene delivery sustains gingival fibroblast signal transduction

Abstract

Platelet-derived growth factor-BB is a potent mediator of tooth-supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet-derived growth factor-BB application is its short half-life in vivo. Gene therapy has shown strong promise for the long-term delivery of platelet-derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet-derived growth factor-B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad-PDGF-B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell-surface proteins associated with cellular signaling. HGFs from human subjects were treated by adenoviral PDGF-B, PDGF-1308 (a dominant negative mutant of PDGF) and recombinant human platelet-derived growth factor-BB, and then incubated in serum-free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF-B was measured by RT-PCR and Western blot. Cell proliferation was evaluated by [methyl-3H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF-B gene transfer. The expression of alpha and beta PDGFR and Akt were measured by Western blot analysis. Sustained in vitro expression of PDGF-B in HGFs by Ad-PDGF-B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF-BB and Ad-PDGF-1308, Ad-PDGF-B maintained cell growth in serum-free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF-B also prolonged downregulation of the growth arrest specific gene (gas) PDGF alpha R. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long-term cell signaling effect as a result of the over-expression of Ad-PDGF-B, compared with pulse recombinant human platelet-derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad-PDGF-B, for at least up to 96 h. These findings further demonstrate that gene delivery of PDGF-B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet-derived growth factor-BB protein. These data on platelet-derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.