Specific residues of RUNX2 are obligatory for formation of BMP2-induced RUNX2-SMAD complex to promote osteoblast differentiation.
BMP2 signaling and RUNX2 regulatory pathways converge for transcriptional control of bone formation in vivo. SMAD proteins are recruited to RUNX2 regulatory complexes via an overlapping nuclear matrix targeting signal/Smad interacting domain sequence (391-432) in Runx2. To establish the contribution of RUNX2-SMAD interaction to osteoblastogenesis, we characterized a number of point mutants. Only a triple mutation of amino acids 426-428 (HTY-AAA) results in loss of RUNX2 interactions with either BMP2- or TGF-beta- responsive SMADs and fails to integrate the BMP2/TGF-beta signal on target gene promoters. In a Runx2 null cell reconstitution assay, the HTY mutant did not activate the program of osteoblast differentiation (alkaline phosphatase, collagen type 1, osteopontin, bone sialoprotein and osteocalcin) in response to BMP2 signaling. Thus, subnuclear targeting function and formation of a RUNX2-SMAD osteogenic complex are functionally inseparable. Taken together, these studies provide direct evidence that RUNX2 is essential for execution and completion of BMP2 signaling for osteoblast differentiation.
Javed
Amjad
A
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Mass., USA. javeda@uab.edu
Afzal
Faiza
F
Bae
Jong-Sup
JS
Gutierrez
Soraya
S
Zaidi
Kaleem
K
Pratap
Jitesh
J
van Wijnen
Andre J
AJ
Stein
Janet L
JL
Stein
Gary S
GS
Lian
Jane B
JB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D050659:Core Binding Factor Alpha 1 Subunit / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016212:Transforming Growth Factor beta / Q000378:metabolism
D000596:Amino Acids; D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D051785:Smad Proteins; D016212:Transforming Growth Factor beta