Reconstitution of Runx2/Cbfa1-null cells identifies a requirement for BMP2 signaling through a Runx2 functional domain during osteoblast differentiation.
The Runx2/Cbfa1 transcription factor is a scaffolding protein that promotes osteoblast differentiation; however, the specific Runx2-functional domains required for induction of the osteogenic lineage remain to be identified. We approached this question using a TERT-immortalized cell line derived from calvaria of Runx2-null mice by reconstituting the osteogenic activity with wild-type and deletion mutants of Runx2. The presence or absence of osteogenic media (beta-glycerol phosphate and ascorbic acid) and/or with BMP2 did not stimulate osteoblastic gene expression in the Runx2-null cells. However, cells infected with wild-type Runx2 adenovirus showed a robust temporal increase in the expression of osteoblast marker genes and were competent to respond to BMP2. Early markers (i.e., collagen type-1, alkaline phosphatase) were induced (four- to eightfold) at Days 4 and 8 of culture. Genes representing mature osteoblasts (e.g., Runx2, osteopontin, bone sialoprotein, osteocalcin) were temporally expressed and induced from 18- to 36-fold at Days 8 and 12. Interestingly, TGFbeta and Vitamin D-mediated transcription of osteoblast genes (except for osteopontin) required the presence of Runx2. Runx2 lacking the C-terminal 96 amino acids (Runx2 Delta432) showed a pattern of gene expression similar to wild-type protein, demonstrating the Groucho interaction and part of the activation domain are dispensable for Runx2 osteogenic activity. Upon further deletion of the Runx2 C-terminus containing the nuclear matrix targeting signal and Smad-interacting domain (Delta391), we find none of the osteoblast markers are expressed. Therefore, the Runx2 391-432 domain is essential for execution of the BMP2 osteogenic signal.
Bae
Jong-Sup
JS
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA.
Gutierrez
Soraya
S
Narla
Radhika
R
Pratap
Jitesh
J
Devados
Rajitha
R
van Wijnen
Andre J
AJ
Stein
Janet L
JL
Stein
Gary S
GS
Lian
Jane B
JB
Javed
Amjad
A
D020816:Amino Acid Motifs; D000818:Animals; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D048430:Cell Shape; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014807:Vitamin D / Q000494:pharmacology
D015415:Biomarkers; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D010446:Peptide Fragments; C493716:Runx2 protein, mouse; D016212:Transforming Growth Factor beta; D014807:Vitamin D