title: Cell-type-dependent up-regulation of in vitro mineralization after overexpression of the osteoblast-specific transcription factor Runx2/Cbfal.
abstract: Functional expression of the transcriptional activator Runx2/Cbfal is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast-specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast-like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast-specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up-regulated expression in MC3T3-E1 immature osteoblast-like cells. However, Runx2 expression enhanced matrix mineralization in a cell-type-dependent manner. NIH3T3 and IMR-90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2-expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant-dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2-infected C3H10T1/2 cells compared with Runx2-expressing cultures which did not mineralize. Treatment of Runx2-infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast-like MC3T3-E1 cells resulted in acceleration and robust up-regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast-specific activities, in vitro matrix mineralization requires additional tissue-specific cofactors, which supplement Runx2 activity.
authors.affiliation: Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, USA.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Byers
authors.forename: Grace K
authors.initials: GK
authors.lastname: Pavlath
authors.forename: T J
authors.initials: TJ
authors.lastname: Murphy
authors.forename: Gerard
authors.initials: G
authors.lastname: Karsenty
authors.forename: Andrés J
authors.initials: AJ
authors.lastname: García
mesh_terms: D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit; D003907:Dexamethasone / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation; D051379:Mice; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics; D012795:Sialoglycoproteins / Q000235:genetics; D012867:Skin / Q000166:cytology; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D009363:Neoplasm Proteins; D012795:Sialoglycoproteins; D014157:Transcription Factors; D003907:Dexamethasone; D000469:Alkaline Phosphatase