Redefining the activity of a bone-specific transcription factor: Novel insights for understanding bone formation


  • Jane B Lian,

    Corresponding author
    1. Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA
    • Address correspondence to: Jane B Lian, PhD, University of Vermont College of Medicine, Department of Biochemistry, 89 Beaumont Avenue, Given E210, Burlington, VT 05405-0068, USA. E-mail:

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  • Jonathan A Gordon,

    1. Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA
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  • Gary S Stein

    1. Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA
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  • This is a Commentary on Takarada et al. (J Bone Miner Res. 2013;28:2064–2069. DOI: 10.1002/jbmr.1945).

Runx2: The “Master” Transcriptional Regulator Essential for Formation of a Calcified Skeleton

The discovery in 1997 by several laboratories described the core-binding factor alpha1 (CBFA1)/Runt-related transcription factor 2 (RUNX2) locus as the genetic basis for cleidocranial dysplasia disorders (CCD).[1-3] This finding, together with characterization of the Runx2-null mouse,[4] opened a new chapter in bone biology. Deletion or functional mutation of Runx2[5] resulted in perinatal lethality and a mouse having a predominately cartilaginous skeleton. Komori[4] had observed that only immature osteoblast precursors (weakly positive for alkaline phosphatase) were present in bone tissue of the Runx2-null mouse. Heterozygotic mice also displayed a phenotype characteristic of human CCD syndromes, demonstrating that a full complement of Runx2 expression was critical for normal bone formation. These striking findings pointed to the first bone-specific transcription factor essential for osteogenesis. The absence of a functional Runx2 gene in mice affected intramembranous ossification in the craniofacial skeleton, as well as endochondral bone formation due to an absence of growth plate calcification and formation of mineralized bone.[6, 7] Early studies also showed that the failure of endochondral ossification in Runx2-deficient mice could be rescued by collagen α1(II)-mediated transgenic expression of Runx2 in chondrocytes.[8]

In the past two decades since the initial discovery of Runx2, a large wealth of knowledge has been gained from mutant mouse models and in vitro molecular studies regarding the functional activities of Runx2. The many studies (reviewed in Jensen and colleagues[9]) have revealed a broad spectrum of regulatory mechanisms establishing Runx2 as a “master” transcriptional regulator. These studies have defined several roles for Runx2 that include: (1) activation and repression of phenotypic genes as osteoblasts progress through stages of differentiation; (2) regulation of genes related to bone resorption and osteoclastic coupling[10, 11]; and (3) mediating the transcriptional and functional activity of osteogenic signaling pathways.[12-14] Although the role of Runx2 in bone is well-characterized, Runx2 has been described in non-osseous tissues including: thymus (the tissue from which Runx2 was first identified),[15, 16] astrocytes,[17] testes,[18] ovary,[19] and kidney.[20] The functions of Runx2 in these tissues are completely unknown and beg the question as to the mechanism for specificity of Runx2 required for osteogenesis that does not occur in non-osseous tissues where Runx2 is expressed. In this issue of the Journal of Bone and Mineral Research, the short report by Takarada and colleagues[21] sets a precedent for future studies that can define the role of Runx2 at specific developmental stages of osteogenesis with different Cre drivers, and even more importantly, in non-osseous tissues, using the important resource of the Runx2flox/flox conditional mouse.

Unexpected, But Not Surprising Findings: Runx2 Exhibits Distinct Activities in Subpopulations of Skeletal Cells

The study by Takarada and colleagues[21] validates the fidelity of a conditional deletion of Runx2 in osteoblasts and chondrocytes and compares the phenotypes to the Runx2-null mice.[4] By crossing Runx2flox/flox mice with transgenic mice expressing Cre recombinase under the control of the truncated (2.3 kb) α1(I)-collagen promoter, Runx2 was deleted only in mature osteoblasts because this promoter is very weakly expressed in early osteoprogenitor cells. Surprisingly (but not totally unexpected, for reasons elaborated in this Commentary), these mice displayed relatively normal intramembranous and endochondral bone formation from embryonic day 15.5 (E15.5) up to 6 weeks of age. In striking contrast, conditional deletion of Runx2 in chondrogenic lineage cells by α1(II)-collagen-Cre–excision, generated a perinatal lethal phenotype due to difficulty in breathing, similar to the observation in the original germline deletions of Runx2.[4] The study is also consistent with previous reports identifying failed endochondral bone formation either by expressing a dominant-negative (DN) Runx2 in chondrocytes;[7] or by conditional inactivation of a fully functional Runx2 in chondrocytes, which resulted in dwarfism and perinatal lethality.[22]

Runx2 has been characterized by several groups as a positive regulator of chondrocyte maturation and vascular invasion.[23-25] To explain the chondrocyte-specific skeletal phenotype, Takarada and colleagues'[21] careful histological evaluation of the bone tissues points to a critical Runx2-dependent regulatory event for endochondral bone formation; ie, the requirement for vascular invasion of the growth plate. It is now well established that Runx2 directly activates the expression of vascular endothelial growth factor (VEGF)[25] and conditional deletion of VEGF in osteoblasts has demonstrated the importance of this molecule not only in the endochondral process of bone formation, but in supporting bone mass in the adult.[26] It was shown that VEGF-deficient mice develop osteopenia resulting from deregulated levels of Runx2 and peroxisome proliferator-activated receptor gamma (Pparg).[26] Together, these studies suggest that a Runx2-VEGF circulatory loop contributes to the expanding knowledge regarding central osteogenic regulators that operate for bone development, growth, and homeostasis.

However, it should be noted that the developmental impairment of endochondral bone formation and postnatal growth is likely to involve far more than blocked vascularization of cartilage. Runx2 regulates other key proteins essential for endochondral ossification; eg, Col10a1.[27, 28] Current knowledge of the growth plate signaling pathways essential for long-bone growth links Runx2 to the Indian hedgehog (IHH)/parathyroid hormone–related protein (PTHrP) negative feedback loop that regulates the pace of chondrocyte maturation and formation of the bone collar. Furthermore, other signaling pathways are modulated by Runx2, including: insulin-like growth factor (IGF), Wingless-related integration site (Wnt), transforming growth factor β (TGFβ), and bone morphogenic protein (BMP), all of which impact long-bone growth.[29-31] Retinoic acid increases Runx2, which stimulates IHH, and in turn IHH/GLI family zinc finger 2 (Gli2) upregulates Runx2, driving endochondral bone formation.[28, 32] In addition, SRY (sex determining region Y)-box 9 (Sox9) regulates maturation of growth plate chondrocytes and Runx2 to prevent premature ossification.[33, 34] Therefore, maintaining the correct biological level of Runx2 in different cell types is important, as revealed by transgenic mouse models overexpressing Runx2. In osteoblasts, osteopenia results from both inhibition of osteoblast maturation and induced receptor activator of NF-κB ligand (RANKL), and in chondrocytes an irregular growth plate develops.[24, 27, 35-37] Most of these findings were discovered using cell culture models or histological examinations of mouse phenotypes. Thus, the Runx2flox/flox mice described by Takarada and colleagues[21] will provide a useful option to further examine molecular mechanisms for Runx2 roles in an in vivo context.

Runx2 is a member of a family of three mammalian Runt-related genes. Each of these genes has essential functions in lineage determination: Runx1 in hematopoiesis, Runx2 in osteoblastogenesis, and Runx3 in nerve and gut development. In the developing embryo, the postnatal skeleton, and adult bone, all three factors are present and frequently demonstrate overlapping expression in distinct populations of skeletal cells. Runx1 is highly enriched in the periosteum and perichondrium where Runx2 is also present. Runx2 and Runx3 overlap with each other in cells of the growth plate, and both proteins support IHH expression.[38-40] Chondrocyte maturation is completely blocked in Runx2–/–/Runx3–/–, but not in Runx2–/– mice.[38] It is not clear if the individual proteins have distinct functions in the prehypertrophic and hypertrophic zones, but they do appear to coordinate early-stage and late-stage chondrocyte hypertrophy. The study from Takarada and colleagues[21] indeed demonstrates that the absence of Runx2 is sufficient for the absence of vascularization and development of the marrow cavity that provides the progenitor cells for resorption of cartilage and formation of primary spongiosa.

The absence of a bone phenotype upon αI(I)-collagen conditional deletion of Runx2 has clarified early studies that were somewhat inconsistent with respect to Runx2 loss-of-function in osteoblasts. Transgenic mice expressing a DNA-binding domain Runx2 mutant under the control of a truncated (1.3 kb) osteocalcin (OG2) promoter exhibited osteopenia in postnatal mice[41]; however, transgenic expression of a mutant Runx2 gene under control of a truncated (2.3 kb) collagen α1 promoter, resulted in an anabolic phenotype with increased trabecular bone in young mice.[42] Although these results are contrasting, they highlight the importance of Runx2 function in a stage-specific context. The study by Takarada and colleagues[21] demonstrates that even though there is a complete loss of function of Runx2 in mature osteoblasts, a normal skeleton is formed, clarifying that Runx2 is required for commitment of progenitor cells to the osteogenic lineage and may be less important for maintenance of mature osteoblastic function. To further appreciate the lack of bone abnormalities in Runx2flox/flox × α1(I)-collagen–Cre mice observed in the Takarada and colleagues[21] study, reviewing several well-characterized properties of Runx2 emphasizes the role of Runx2 in lineage commitment. Runx2 expression is driven by two promoters (P1 and P2) producing a longer transcript with a slightly extended amino terminus (P1 promoter driven) or a shorter form (P2 promoter). Although the expression pattern of these isoforms differ, P1 is considered bone-specific because it increases during osteoblast differentiation, and P2 is more ubiquitously expressed, remaining at a lower steady-state level during embryonic development.[43] However, the functional activity of these isoforms is highly similar in that they both regulate expression of bone-related genes. Mouse models that have specifically deleted the P1 isoform have demonstrated that the P2 isoform is competent to form bone and the mice are viable, although long bone and vertebrae exhibit severe osteopenia and intramembranous bones have phenotypic features of CCD.[44, 45] Studies by Liu and colleagues[45] demonstrated that this phenotype is the result of insufficient mesenchymal cells committed to the osteogenic lineage. Additionally, mice hypomorphic for Runx2 exhibit CCD-like calvarial and clavicle defects, demonstrating that a critical level of cellular Runx2 protein is required for recruitment of an adequate number of progenitor cells into the osteoblast lineage to maintain normal bone mass.[46] Thus, the study by Takarada and colleagues[21] provides additional proof that deletion of Runx2 in mature osteoblasts is not a rate-limiting step to bone formation. Once a mesenchymal progenitor is firmly committed to be an osteoblast, there are many other drivers of osteoblast differentiation and mineralized tissue formation to continue the process. The factors that support the program of osteoblastogenesis are transcriptional regulators, such as Osterix (Osx), β catenin/T-cell factor-1 (Tcf-1)/lymphoid enhancing factor-1 (Lef-1),[14] and homeodomain proteins distal-less homeobox 3 (Dlx3), Dlx5/6,[46] activating transcription factor 4 (ATF4).[48] and Hoxa10.[49]

The discovery that the BMP2-responsive transcription factor Osterix/SP7, which functions downstream of Runx2, was also critically required for formation of a mineralized skeleton, brought to realization that Runx2 functions in regulatory networks is essential for osteogenesis.[50] Osterix-null mice were found to lack a mineralized skeleton even though Runx2 was present in the skeletal tissues.[49] The consensus from several subsequent studies has emerged that Runx2 is required to initiate osteogenesis, which Osterix is essential for mineralized tissue formation.[50] There are now many mouse phenotypes exhibiting partial defects in bone formation and structure due to loss-of-function of proteins that involve either direct interaction with Runx2 or alter Runx2 expression.[9, 13, 29, 30] Thus, Runx2 remains as the first requirement for membranous and endochondral bone growth, followed by many other transcriptional regulators and co-regulatory signals established as needed for bone formation through genetic ablation.[29, 31, 51, 52] Together, these factors, with Runx2, contribute to the entire program of osteoblast differentiation, a finding supported by Takarada and colleagues'[21] study in this issue of the JBMR.

The Spectrum and Magnitude of Known Runx2 Functions—More to Come?

Many in vitro studies demonstrated powerful properties of Runx2 that classify Runx2 and other tissue-specific transcription factors as critical for commitment to a cell lineage. Overexpression of Runx2 induces the osteogenic phenotype in progenitor cells and even in non-osseous cells committed to other phenotypes, myoblasts, adipocytes. A novel discovery first characterized for Runx factors was their epigenetic function through retention on mitotic chromosomes during cell division (reviewed in Zaidi and colleagues[54]). In osteoblasts, upon exit from mitosis, Runx2 was associated with genes that contribute to osteogenesis, eg, VEGF and Smads,[55] reflecting a mechanism for “bookmarking” genes to maintain stability of the bone phenotype during proliferation. Runx2 participates in several epigenetic mechanisms by interacting with chromatin remodeling factors for positive and negative regulation of genes[9, 54] and regulating microRNAs to assure osteogenic lineage differentiation.[55] In addition, Runx2 has been implicated in numerous pathologies, including mammary gland development,[56] basal cell carcinoma,[57] breast,[58] prostate,[59] and metastatic bone disease,[58] as well as other forms of malignant cancers.[60] The mouse models used by Takarada and colleagues[21] will allow for further and novel characterization of Runx2 phenotypes in specific tissues involved in these processes and diseases, moving forward to open new and exciting chapters in Runx biology.


All authors state that they have no conflicts of interest.