Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells.
Runx2/Cbfa1 has been identified as a "master gene" controlling osteoblast differentiation. However, its role in inducing the osteoblast phenotype has been characterized primarily in rodent systems. Thus, we examined Runx2/Cbfa1 messenger RNA, protein, and activity levels during osteoblastic differentiation of human bone marrow stromal (BMSC) cells. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of alkaline phosphatase and osteocalcin mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype by these cells (hMS2-15). Type II Runx2/Cbfa1 messenger RNA was found to be constitutively expressed in hMS2-15 cells and not altered during differentiation; there was no detectable expression of the type I Runx2/Cbfa1 transcript. Interestingly, despite the absence of any change in Runx2/Cbfa1 messenger RNA levels during osteoblastic differentiation of these cells, the activity of Runx2/Cbfa1, as assessed by binding to the osteoblast-specific cis-acting element 2 (OSE2), increased markedly at all time-points examined, with the highest activity level seen at day 7. Similar results were observed in primary cultures of less differentiated human marrow-derived mesenchymal stem cells. Immunoprecipitation and Western blot analysis revealed that whereas there was no increase in Runx2/Cbfa1 protein levels with differentiation in hMS2-15 cells, there was an increase in Runx2/Cbfa1 phosphorylation. Thus, in contrast to rodent systems where osteoblast differentiation is associated with increased synthesis of Runx2/Cbfa1, we find that in human BMSC, osteoblastic differentiation is associated primarily with increases in Runx2/Cbfa1 activity, without a change in messenger RNA or protein levels. Our findings also show that the increase in Runx2/Cbfa1 activity occurs through a posttranslational mechanism involving phosphorylation of key residues.
Shui
Chaoxiang
C
Endocrine Research Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Spelsberg
Thomas C
TC
Riggs
B Lawrence
BL
Khosla
Sundeep
S
D000469:Alkaline Phosphatase / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*
D050659:Core Binding Factor Alpha 1 Subunit; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D000469:Alkaline Phosphatase