Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Lin
Z
Z
Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA.
Sugai
J V
JV
Jin
Q
Q
Chandler
L A
LA
Giannobile
W V
WV
D000256:Adenoviridae / Q000235:genetics; D000077214:Becaplermin; D049109:Cell Proliferation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D015536:Down-Regulation / Q000235:genetics; D005347:Fibroblasts / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018014:Gene Transfer Techniques; D005881:Gingiva / Q000166:cytology / Q000502:physiology*; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics; D020574:Proto-Oncogene Proteins c-sis / Q000235:genetics*; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000235:genetics; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014161:Transduction, Genetic
D016895:Culture Media, Serum-Free; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin; D004247:DNA; D010770:Phosphotransferases; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases