Structural Coupling of Smad and Runx2 for Execution of the BMP2 Osteogenic Signal

Abstract

Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-␤ (TGF␤) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426 -428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGF␤ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity.Skeletal development and bone formation require coordinated activities of multiple signaling pathways that include bone morphogenetic protein 2 (BMP2) 5 and transforming growth factor-␤ (TGF␤). Transduction of these signals results in the activation of target genes that are essential for bone development. Specific receptor-regulated SMADs (R-SMADs) serve as substrates for the BMP and TGF␤/activin/Nodal receptors. SMAD-1, -2, -3, and -5 transduce, whereas Smad-4 serves as a common partner for all R-SMADs to provide the DNA binding property (1, 2). The structural and functional domains of SMAD proteins are well characterized with binding sites for SMAD ubiquitination-related factor (SMURF) ubiquitin ligases, and phosphorylation sites for several classes of protein kinases (3). The MH2 domain mediates interactions with transcriptional activators and repressors for signal transduction; including co-regulators of skeletal development (4 -7).Several studies suggest that the principle activity of BMP and TGF␤ SMADs for the control of skeletogenesis is mediated by their interaction with RUNX2 (CBFA1/AML3). This runt-related transcription factor is critical for osteogenic lineage commitment and formation of the skeleton (8 -12). Mutations in the human RUNX2 cause cleidocranial dysplasia (13,14). Targeted disruption of Runx2 in mice results in the maturational arrest of osteoblasts and a complete lack of mineralized bone (15-17). The gene regulatory properties of RUNX factors are mediated not only by DNA binding to cognate elements, but also through the formation of selective co-regulatory protein interactions with co-activator and co-repressor proteins (9, 10). The C terminus of RUNX proteins contains a 31-amino acid nuclear matrix targeting si...

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

“…2B). This suggests Osx expression was upregulated through an alternate pathway [12], independent of SMAD protein signaling and Runx2 activation [31].…”

Requirement for Runx Proteins in IgA Class Switching Acting Downstream of TGF-β1 and Retinoic Acid Signaling

“…To examine further details of Runx functions in TGF-b1 signaling, mutant constructs of Runx proteins with a defect in the interaction with Smad or TAZ were used for rescue experiments. RunxHTY mutants, which disrupt Runx-Smad interaction, have previously been shown to lose the ability to integrate BMP signal in differentiating osteoblasts using Runx2-deficient cells (33). Unexpectedly, the RunxHTY mutant can rescue the defect in IgA class switching (Fig.…”

TGF-β Family Signaling in Connective Tissue and Skeletal Diseases

“…R-Smads physically interact with and regulate the transcriptional activity of runt-related transcription factor 2 (Runx2, also known as Cbfa1), a master regulator of mesenchymal differentiation toward the osteoblastic cell lineage (Komori et al 1997;Otto et al 1997;Leboy et al 2001;Miyazono et al 2004;Hecht et al 2007;Javed et al 2008Javed et al , 2009reviewed in Lian et al 2004;Schroeder et al 2005). Runx2 function is repressed by TGF-b and Smad2 and Smad3 signaling, and enhanced by BMPs and Smad1, Smad5, and Smad8 signaling (Cohen 2009;van der Kraan et al 2009).…”