title: Extracellular-signal-related kinase 1/2 is responsible for inhibition of osteogenesis in three-dimensional cultured MC3T3-E1 cells.
abstract: We have previously demonstrated that osteogenic differentiation is inhibited and angiogenic expression is enhanced in murine preosteoblasts (MC3T3-E1) cultured on three-dimensional (3D) poly-L-lactide-co-glycolide (PLGA) scaffolds when compared to two-dimensional (2D) PLGA films. In the present work we investigated the role of the extracellular signal-related kinase 1/2 (ERK1/2) pathway in modulating osteogenic and angiogenic differentiation in 2D and 3D systems made of two distinct biomaterials-type I collagen and PLGA. The addition of a third dimension, regardless of biomaterials, substantially increased ERK1/2 activation as demonstrated by an increase in phosphorylated ERK1/2. Western blot analysis showed significant increases in phosphorylation of ERK1/2 in cells grown in 3D versus 2D cultures at day 4 (5- and 7.7-fold increases 3D vs. 2D in collagens and PLGA, respectively) and day 7 (4.7- and 4.6-fold increases 3D vs. 2D in collagen and PLGA, respectively). Using an ERK-specific inhibitor, PD 98059, we established a correlation between ERK activation and inhibited osteogenic differentiation. Inhibition of ERK activation in 3D cultures significantly enhanced osteogenic differentiation. It in fact restored osteogenic differentiation to a level equal to that of 2D cultured cells. Inhibition of ERK1/2, however, showed little effect on angiogenic gene expression, indicating that two distinct mechanisms are involved in osteogenic and angiogenic differentiation. Taken together, these results allow us to report a mechanistic model in MC3T3-E1 cells in which distinct activation of ERK1/2 in 3D culture has an inhibitory effect on osteogenic differentiation.
authors.affiliation: Plastic Surgery Laboratory, Veteran Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073, USA.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Im
authors.forename: Erwin A
authors.initials: EA
authors.lastname: Kruger
authors.forename: Weibiao R
authors.initials: WR
authors.lastname: Huang
authors.forename: Gregory
authors.initials: G
authors.lastname: Sayer
authors.forename: George H
authors.initials: GH
authors.lastname: Rudkin
authors.forename: Dean T
authors.initials: DT
authors.lastname: Yamaguchi
authors.forename: Reza
authors.initials: R
authors.lastname: Jarrahy
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Miller
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D005419:Flavonoids; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000469:Alkaline Phosphatase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one