BMP2 Commitment to the Osteogenic Lineage Involves Activation of Runx2 by DLX3 and a Homeodomain Transcriptional Network

Abstract

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation.Skeletal development is a complex process whereby multiple signaling pathways converge to induce formation of bone structures. The BMP2/4/7 4 members of the transforming growth factor-␤ superfamily of signaling polypeptides are essential for commitment and differentiation of mesenchymal cells to the osteoblast lineage for bone tissue formation. The BMP signal is transduced through the binding to their heterodimeric type I and type II serine/threonine kinase receptors with formation of activated Smad complexes that are translocated to the nucleus to regulate target genes (1). BMP2 target genes include a cohort of transcription factors with specialized activities for induction of cell type-specific lineages. Among the BMP2 target genes are transcription factors that have essential roles in skeletal development, including several homeodomain proteins, the bonerelated runt homology domain factor RUNX2 (CBFA1/AML3), and an SP1 family member, OSTERIX (2-4). BMP/transforming growth factor-␤ intracellular Smads will also form co-regulatory complexes with transcription factors to regulate target genes. The potent osteogenic effects of BMPs are mediated in part by formation of RUNX2-SMAD complexes for phenotypic gene induc...

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

“…Mid-and late-stage osteogenic markers including OPN, bone sialoprotein (BSP), and OCN are temporal successors to RUNX2 and OSX expression after rhBMP-2 stimulation [63]. The complex temporal expression profiles of OPN, BSP, and OCN have been elucidated [15,16,23,25,26,30,53,59,66]; therefore, a coordinated, temporal RNAi treatment cycle may enhance the duration and intensity of gene silencing and prevent HA deposition in vitro. Consequently, the evolution of RNAi therapeutics must match RNAi targets to their expression profiles.…”

A 4 bp deletion mutation in DLX3 enhances osteoblastic differentiation and bone formation in vitro

“…[1] BMP-2 activates DLX3 transcription in hair follicle cells [2] and in combination with Smad can induce DLX3 transcription in keratinocytes. [3] DLX3 expression induced by bone morphogenetic protein (BMP) regulates tissue specific gene expression in developing embryonic ectoderm, [4,5] suggesting important roles of DLX3 in developing tissues modulated by the BMP signaling pathway. DLX3 is also an essential factor for normal placental development in mice.…”

Dlx genes and the maintenance of bone homeostasis and skeletal integrity

“…8,9,18,19 The fact that Dlx3 plays an important role in regulating osteoblast activity is further supported by the analysis of Dlx3-deleted bone marrow stroma cells (BMSCs), which also shows increased osteoblast differentiation and bone-forming activity associated with increased gene expression of Runx2 and Dlx5. The notion that DLX3 acts as a negative regulator of osteoblastogenesis by reducing Runx2, Sp7, Dlx5 and Dlx6 gene expression is further supported by ChIP analysis on BMSCs, which shows that DLX3 binds to the promoters of Sp7, Dlx5 and Dlx6 and Runx2, directly modulating their activity (see also Hassan et al 12 ).…”

“…8,9 In particular, mutations in DLX3 result in Tricho-DentoOsseous syndrome, an autosomal dominant ectodermal dysplasia characterized by curly kinky hair, enamel hypoplasia, taurodontism and increased bone mineral density (BMD) in intramembranous and endochondral bones. 10 The importance of DLX3 in bone is also supported by its capacity to regulate directly in vitro critical determinants of bone differentiation such as osteocalcin (Ocn), Runx2 and osteoactivin, [11][12][13] and by the observation that overexpression of DLX3 in osteoprogenitors stimulates transcription of osteogenic markers.…”

“…In vitro studies have shown that Dlx3 and Dlx5 associate with the Ocn promoter at the onset of transcriptional activation, concomitant with Runx2 occupancy, and, while Dlx3 occupancy on the Ocn promoter decreases from osteoblast maturation to mineralization, Dlx5 occupancy is maximal during the mineralization stage. 12 Isaac et al 15 demonstrate that Dlx3 deletion in calvaria osteoblasts results in increased occupancy of Dlx5 and increased and premature occupancy of Runx2 on regulatory elements on the Ocn promoter. Thus, Dlx3 deletion could directly affect the network of molecular switches and promote osteoblastic differentiation and bone-forming activity via an enhancement of the occupancy of bone-activator TFs such as Dlx5 and Runx2.…”