Andre J. Van Wijnen

Biological Functions of miR-29b Contribute to Positive Regulation of Osteoblast Differentiation

Bone tissue arises from mesenchymal cells induced into the osteoblast lineage by essential transcription factors and signaling cascades. MicroRNAs regulate biological processes by binding to mRNA 3′-untranslated region (UTR) sequences to attenuate protein synthesis. Here we performed microRNA profiling and identified miRs that are up-regulated through stages of osteoblast differentiation. Among these are the miR-29, miR-let-7, and miR-26 families that target many collagens and extracellular matrix proteins. We find that miR-29b supports osteoblast differentiation through several mechanisms. miR-29b decreased and anti-miR-29b increased activity of COL1A1, COL5A3, and COL4A2 3′-UTR sequences in reporter assays, as well as endogenous gene expression. These results support a mechanism for regulating collagen protein accumulation during the mineralization stage when miR-29b reaches peak levels. We propose that this mechanism prevents fibrosis and facilitates mineral deposition. Our studies further demonstrate that miR-29b promotes osteogenesis by directly down-regulating known inhibitors of osteoblast differentiation, HDAC4, TGFβ3, ACVR2A, CTNNBIP1, and DUSP2 proteins through binding to target 3′-UTR sequences in their mRNAs. Thus, miR-29b is a key regulator of development of the osteoblast phenotype by targeting anti-osteogenic factors and modulating bone extracellular matrix proteins.

A microRNA signature for a BMP2-induced osteoblast lineage commitment program

Bone morphogenetic proteins (BMPs) are potent morphogens that activate transcriptional programs for lineage determination. How BMP induction of a phenotype is coordinated with microRNAs (miRNAs) that inhibit biological pathways to control cell differentiation, remains unknown. Here, we show by profiling miRNAs during BMP2 induced osteogenesis of C2C12 mesenchymal cells, that 22 of 25 miRNAs which significantly changed in response to BMP2 are down-regulated. These miRNAs are each predicted to target components of multiple osteogenic pathways. We characterize two representative miRNAs and show that miR-133 directly targets Runx2, an early BMP response gene essential for bone formation, and miR-135 targets Smad5, a key transducer of the BMP2 osteogenic signal, controlled through their 3′UTR sequences. Both miRNAs functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 pathways that synergistically contribute to bone formation. Although miR-133 is known to promote MEF-2-dependent myogenesis, we have identified a second complementary function to inhibit Runx2-mediated osteogenesis. Our key finding is that BMP2 controls bone cell determination by inducing miRNAs that target muscle genes but mainly by down-regulating multiple miRNAs that constitute an osteogenic program, thereby releasing from inhibition pathway components required for cell lineage commitment. Thus, our studies establish a mechanism for BMP morphogens to selectively induce a tissue-specific phenotype and suppress alternative lineages.

Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression

We present an overview of Runx involvement in regulatory mechanisms that are requisite for fidelity of bone cell growth and differentiation, as well as for skeletal homeostasis and the structural and functional integrity of skeletal tissue. Runx-mediated control is addressed from the perspective of support for biological parameters of skeletal gene expression. We review recent findings that are consistent with an active role for Runx proteins as scaffolds for integration, organization and combinatorial assembly of nucleic acids and regulatory factors within the three-dimensional context of nuclear architecture.

Networks and hubs for the transcriptional control of osteoblastogenesis

We present an overview of the concepts of tissue-specific transcriptional control mechanisms essential for development of the bone cell phenotype. BMP2 induced transcription factors constitute a network of activities and molecular switches for bone development and osteoblast differentiation. Among these regulators are Runx2 (Cbfa1/AML3), the principal osteogenic master gene for bone formation, as well as homeodomain proteins and osterix. Runx2 has multiple regulatory activities, including activation or repression of gene expression, and integration of biological signals from developmental cues, such as BMP/TGFβ, Wnt and Src signaling pathways. Runx2 provides a new paradigm for transcriptional control by functioning as a principal scaffolding protein in nuclear microenvironments to control gene expression in response to physiologic signals (growth factors, cytokines and hormones). The protein serves as a hub for the coordination of activities essential for the expansion and differentiation of osteogenic lineage cells through the formation of co-regulatory protein complexes organized in subnuclear domains. Mechanisms by which Runx2 supports commitment to osteogenesis and determines cell fate involve its retention on mitotic chromosomes. Disruption of a unique protein module, the subnuclear targeting signal of Runx2, has profound effects on osteoblast differentiation and metastasis of cancer cells in the bone microenvironment. Runx2 target genes include regulators of cell growth control, components of the bone extracellular matrix, angiogenesis, and signaling proteins for development of the osteoblast phenotype and bone turnover. The specificity of Runx2 regulatory activities provides a basis for novel therapeutic strategies to correct bone disorders.

MicroRNA control of bone formation and homeostasis

MicroRNAs (miRNAs) repress cellular protein levels to provide a sophisticated parameter of gene regulation that coordinates a broad spectrum of biological processes. Bone organogenesis is a complex process involving the differentiation and crosstalk of multiple cell types for formation and remodeling of the skeleton. Inhibition of mRNA translation by miRNAs has emerged as an important regulator of developmental osteogenic signaling pathways, osteoblast growth and differentiation, osteoclast-mediated bone resorption activity and bone homeostasis in the adult skeleton. miRNAs control multiple layers of gene regulation for bone development and postnatal functions, from the initial response of stem/progenitor cells to the structural and metabolic activity of the mature tissue. This Review brings into focus an emerging concept of bone-regulating miRNAs, the evidence for which has been gathered largely from in vivo mouse models and in vitro studies in human and mouse skeletal cell populations. Characterization of miRNAs that operate through tissue-specific transcription factors in osteoblast and osteoclast lineage cells, as well as intricate feedforward and reverse loops, has provided novel insights into the supervision of signaling pathways and regulatory networks controlling normal bone formation and turnover. The current knowledge of miRNAs characteristic of human pathologic disorders of the skeleton is presented with a future goal towards translational studies.

Tyrosine phosphorylation controls Runx2‐mediated subnuclear targeting of YAP to repress transcription

Src/Yes tyrosine kinase signaling contributes to the regulation of bone homeostasis and inhibits osteoblast activity. Here we show that the endogenous Yes‐associated protein (YAP), a mediator of Src/Yes signaling, interacts with the native Runx2 protein, an osteoblast‐related transcription factor, and suppresses Runx2 transcriptional activity in a dose‐dependent manner. Runx2, through its PY motif, recruits YAP to subnuclear domains in situ and to the osteocalcin (OC) gene promoter in vivo. Inhibition of Src/Yes kinase blocks tyrosine phosphorylation of YAP and dissociates endogenous Runx2–YAP complexes. Consequently, recruitment of the YAP co‐repressor to subnuclear domains is abrogated and expression of the endogenous OC gene is induced. Our results suggest that Src/Yes signals are integrated through organization of Runx2–YAP transcriptional complexes at subnuclear sites to attenuate skeletal gene expression.

Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development

Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2ΔC) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2ΔC protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.

The Runx2 Osteogenic Transcription Factor Regulates Matrix Metalloproteinase 9 in Bone Metastatic Cancer Cells and Controls Cell Invasion

ABSTRACT The Runx2 (Cbfa1/AML3) transcription factor and matrix metalloproteinase 9 (MMP9) are key regulators of growth plate maturation and bone formation. The genes for both proteins are characteristic markers of breast and prostate cancer cells that metastasize to bone. Here we experimentally addressed the compelling question of whether Runx2 and MMP are functionally linked. By cDNA expression array analysis, we identified MMP9 as a novel downstream target of Runx2. Like that of MMP13, MMP9 expression is nearly depleted in Runx2 mutant mice. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed the recruitment of Runx2 to the MMP9 promoter. We show by mutational analysis that the Runx2 site mediates transactivation of the MMP9 promoter in osteoblasts (MC3T3-E1) and nonosseous (HeLa) cells. The overexpression of Runx2 by adenovirus delivery in nonmetastatic (MCF-7) and metastatic breast (MDA-MB-231) and prostate (PC3) cancer cell lines significantly increases the endogenous levels of MMP9. The knockdown of Runx2 by RNA interference decreases MMP9 expression, as well as that of other Runx2 target genes, including the genes for MMP13 and vascular endothelial growth factor. Importantly, we have demonstrated using a cell invasion assay that Runx2-regulated MMP9 levels are functionally related to the invasion properties of cancer cells. These results are consistent with Runx2 control of multiple genes that contribute to the metastatic properties of cancer cells and their activity in the bone microenvironment.

Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene

The runt related transcription factor CBFA1 (AML3/PEBP2αA/RUNX2) regulates expression of several bone‐ and cartilage‐related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5′ regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone‐related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt −351 and −92 causes a striking 30‐ to 100‐fold combined decrease in promoter activity. Additionally, 5′ UTR sequences repress reporter gene transcription 2‐ to 3‐fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5′ region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5′ UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation. J. Cell. Physiol. 184:341–350, 2000.

Dlx3 Transcriptional Regulation of Osteoblast Differentiation: Temporal Recruitment of Msx2, Dlx3, and Dlx5 Homeodomain Proteins to Chromatin of the Osteocalcin Gene

ABSTRACT Genetic studies show that Msx2 and Dlx5 homeodomain (HD) proteins support skeletal development, but null mutation of the closely related Dlx3 gene results in early embryonic lethality. Here we find that expression of Dlx3 in the mouse embryo is associated with new bone formation and regulation of osteoblast differentiation. Dlx3 is expressed in osteoblasts, and overexpression of Dlx3 in osteoprogenitor cells promotes, while specific knock-down of Dlx3 by RNA interference inhibits, induction of osteogenic markers. We characterized gene regulation by Dlx3 in relation to that of Msx2 and Dlx5 during osteoblast differentiation. Chromatin immunoprecipitation assays revealed a molecular switch in HD protein association with the bone-specific osteocalcin (OC) gene. The transcriptionally repressed OC gene was occupied by Msx2 in proliferating osteoblasts, while Dlx3, Dlx5, and Runx2 were recruited postproliferatively to initiate transcription. Dlx5 occupancy increased over Dlx3 in mature osteoblasts at the mineralization stage of differentiation, coincident with increased RNA polymerase II occupancy. Dlx3 protein-DNA interactions stimulated OC promoter activity, while Dlx3-Runx2 protein-protein interaction reduced Runx2-mediated transcription. Deletion analysis showed that the Dlx3 interacting domain of Runx2 is from amino acids 376 to 432, which also include the transcriptionally active subnuclear targeting sequence (376 to 432). Thus, we provide cellular and molecular evidence for Dlx3 in regulating osteoprogenitor cell differentiation and for both positive and negative regulation of gene transcription. We propose that multiple HD proteins in osteoblasts constitute a regulatory network that mediates development of the bone phenotype through the sequential association of distinct HD proteins with promoter regulatory elements.