International Journal of Biological Macromolecules 78 (2015) 202–208

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Review

Runx2: Structure, function, and phosphorylation in osteoblast differentiation S. Vimalraj, B. Arumugam, P.J. Miranda, N. Selvamurugan ∗ Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu -603 203, India

a r t i c l e

i n f o

Article history: Received 21 January 2015 Received in revised form 2 April 2015 Accepted 3 April 2015 Available online 13 April 2015 Keywords: Runx2 Phosphorylation Osteoblast Bone

a b s t r a c t Runx2 is a master transcription factor for osteogenesis. The most important phenomenon that makes this protein a master regulator for osteogenesis is its structural integrity. In response to various stimuli, the domains in Runx2 interact with several proteins and regulate a number of cellular events via posttranslational modifications. Hence, in this review we summarized the structural integrity of Runx2 and its posttranslational modifications, especially the phosphorylation responsible for either stimulation or inhibition of its regulatory role in osteogenesis. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Runx structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of Runx2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Runx2 phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Negative regulation of Runx2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Ubiquitination/proteasomal degradation of Runx2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Runx2 regulation by miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Osteogenesis is tightly regulated by a diverse set of internal and external factors such as hormones, growth factors, transcriptional factors, and signaling pathways that result in the formation of mineralized bone [1,2]. These factors target receptors of parathyroid hormone (PTH), 1, 25-dihydroxy vitamin D3, estrogen, and glucocorticoids to regulate their intracellular signaling cascades [3,4]. The molecular mechanism of bone formation involves three major phases: (1) proliferation, (2) extracellular matrix maturation, and (3) mineralization, which are orchestrated by various key

∗ Corresponding author. Tel.: +91 9940632335. E-mail addresses: [email protected], [email protected] (N. Selvamurugan). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.008 0141-8130/© 2015 Elsevier B.V. All rights reserved.

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molecules that regulate this phase transfer. During osteogenesis, the proliferation phase is initially downregulated to induce the maturation process with the help of a multitude of gene expression phenomena. Similar processes are involved for maturation and mineralization. Osteoblast-specific phenotypic markers are differentially expressed during various stages of development. Some of the markers consist of type 1 collagen (COL I), alkaline phosphatase (ALP), and collagenous and non-collagenous bone matrix proteins, including osteocalcin (OC), and osteonectin (ON) [2–8]. Runx2 (runt related gene 2) is among the most important transcription factors necessary for the process of osteogenesis and is responsible for the activation of osteoblast differentiation marker genes. For instance, Runx2 triggers the expression of OC by the mechanism of binding to the cis-acting element (osteoblast specific element 2; OSE2 or Runx binding site) of the OC promoter region [2,6].

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There are several other osteoblast marker genes such as ALP, COL I, bone sialoproteins (BSP), and osteopontin (OPN) having OSE2-like elements which are regulated in a similar manner by Runx2. This mechanism emphasizes the vital position of Runx2 in the study of bone development [2]. The bone mineral density was highly correlated with Runx2 expression irrespective of the gender while the level was the same in both peripheral mesenchymal stem cells (PBMSs) and mesenchymal stem cells (MSCs). However, the peak bone mass (PBM) was reached early in females while the bone mineral density (BMD) was higher in males later in life. These parameters are both strictly correlated with the expression of Runx2 [9]. Further, Runx2 knockout mice showed a lack of bone formation and decreased chondrocyte maturation [10–12]. In this review, we summarize a role for Runx2 in the regulation of bone formation, both with respect to the structural integrity of Runx2 as well as the ability of posttranslational modifications such as phosphorylation of various domains of this protein to positively or negatively modulate its activity towards its target genes.

2. Runx structure Runx belongs to the small transcription factor family and all the genes from the Runx family share a common runt domain. It was named the runt domain after the first identified member of this gene family from Drosophila was named runt [13]. Runx2 is a major regulator of bone development, and a mutation in Runx2 leads to the skeletal malformation syndrome cleidocranial dysplasia (CCD). It is also expressed in pre hypertrophic and hypertrophic chondrocytes, indicating the role of Runx2 in cartilage development as well [2,14]. Runx is capable of forming a heterodimer with the transcriptional co-activator, core binding factor beta (CBF␤)/polyoma enhancer binding protein 2 beta (PEBP2␤) that functionally enables the recognition of the consensus sequence PyGPyGGTPy in any of their target genes [15]. There are three members of the RUNX family, namely RUNX1, -2, and -3. RUNX1/AML1/CBFA2/PEBP2␣B is important for the regulation of hematopoietic cell development [2,16]. Osteogenesis, chondrocyte hypertrophy, vascular invasion of developing skeletons, and metastasis of breast cancer to bone are all tightly regulated by RUNX2/AML3/CBFA1/PEBP2␣A, while neurogenesis and gut development are regulated by RUNX3/AML2/CBFA3/PEBP2␣C [2,17,18]. The Runx gene consists of eight exons and two promoters (P1 and P2) involved in the regulation of its transcriptional levels. Based on the transcriptional mechanism of the two promoter regions, two major isoforms with an amino terminus of either type 1 and 2 are produced. The type 1 isoform (P2) includes a MRIPV pentapeptide at its N-terminus and type 2 isoform (P1) includes MASN/DS at its N-terminus (Fig. 1). In addition, Runx2 possesses several active domains such as the transactivation domains (AD1, 2 and 3), glutamine/alanine rich domain (QA), runt homology domain (RHD), nuclear localization signal (NLS), proline/serine/threonine rich domain (PST), nuclear matrix targeting signal (NMTS), repression domain (RD), and VWRPY region [19–21]. At the protein level of Runx1, the P2 and P1 isoforms are constituted of 453 and 480 amino acids, respectively. Similarly, the Runx2 protein is constituted of 507 and 521 amino acids in the P2 and P1 isoforms, respectively and the Runx3 protein contains 415 and 429 amino acids in the P2 and P1 isoforms, respectively [19,20]. Experimental evidence revealed two highly conserved introns, one within and one downstream of the runt domain, and all vertebrates utilize two alternative promoters. The highly conserved 128 amino acid sequence contributes to most of its functional attributes, specifically DNA binding [13,21]. The function of Runx2 protein is mainly dependent on the sites/domains present in its structure and several identified sites/domains in Runx2 are shown in Fig. 1.

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3. Function of Runx2 Runx2 is known for its diversified function in different tissues. It functions as a regulator in osteoblast differentiation at an early stage, and plays a role in skeletal morphogenesis, tooth development, chondrogenesis, and vasculogenesis. Runx2 also has a role in pathological function such as breast cancer metastasis, and bone abnormality was found to be due to Runx2 mutation in the case of fibrous dysplasia, chondrocyte hypertrophy, and CCD [2,14–18,22–26]. Additionally, Runx2 has significant epigenetic control over the ribosomal RNA [23]. Further, it is known to regulate G1 transition in osteoblast cells [24]. Runx2 acts as a platform protein that can regulate bone specific genes, co-regulatory proteins, and chromatin remodeling factors by multiple interactions. It has been identified that Runx2 is important for mouse adipose stem cells to differentiate into an osteogenic lineage in the presence of an osteoinductive medium [27]. Runx2 upregulates expression of PI3K subunits (p85 and p110␤) and Akt, resulting in enhanced DNA binding ability of Runx2 in immature MSCs, immature osteoblastic cells, and pre-chondrocytes [28]. It was also shown that Osx, a downstream effector of Runx2, coordinates with Runx2 for the enhancement of osteoblast differentiation process [29]. Yet other evidence for Runx2 involvement in bone formation is the binding site of Runx2 in the promoter region of RANKL [30]. On the other hand, there are genes identified such as stat1, twist, and hey1 that form complexes with Runx2 resulting in inhibition of osteoblast differentiation [1,2]. This is a critical phenomenon, as there needs to be a delicate balance between osteoblast differentiation and osteoclast desorption. Interestingly, chemicals such as ethanol increased inorganic phosphate (Pi) mediated extracellular matrix calcification depending upon the dosage used in human vascular smooth muscle cells. It also increased the activity of ALP, and expression of Runx2 and OC [31]. During intramembranous bone development, Runx2 and histone deacetylase 3 (HDAC3) mediate repression of Axin2 to prevent the early closure of the calvarial sutures [32]. Moreover, evidence showed that Runx2 is a positive regulator of chondrocyte differentiation, osteoclast differentiation, vascular invasion, and periosteal bone formation. In cartilaginous condensations, Runx2 was found to complex with Runx3 in order to cooperate for early chondrocyte differentiation [33]. Association of Supt3h with the Runx2-P1 promoter region is required to modulate their activity [23]. Experimental evidence proved that Runx2 is important for the regulation of bone specific genes such as BSP, vascular endothelial growth factor (VEGF), OC, and tissue inhibitors of metalloproteinases (TIMPs) [6,29,33].

4. Runx2 phosphorylation Runx2 activity is regulated at various levels including posttranslational modifications such as acetylation, phosphorylation, sumoylation, and ubiquitination. Since this covers an extensive amount of literature, we focus our discussion on the recent developments in Runx2 phosphorylation and its importance in the regulation of osteogenesis in the following section. Phosphorylation, a posttranslational modification, is an essential biochemical process. It is carried out by kinases that add a phosphate group to the R group of any protein, thus giving them a negative charge [34,35]. On the other hand, phosphatases facilitate the dephosphorylation of phosphorylated proteins. The phosphorylation process is specific to particular serine, threonine, or tyrosine residues in the target protein and generally elicits a conformational change. Upon receiving signals from hormones, the phosphorylation state of any protein could be altered [35]. Runx2 phosphorylation typically occurs within the nucleus and

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Fig. 1. Runx2 structure. The mRNA sequence of Runx2 consists of eight exons present in total length of 227,766 nucleotides with introns. Runx2 gene products consist of two isoforms, i) MASNS transcribed from promoter P1 (encodes all eight exons) and ii) MRIPV transcribed from P2 (encodes exons 2–8). The exons 2 and 6 consist of the CpG-rich islands. The exon 8 possesses nuclear matrix targeting signal (NMTS) and VWRPY domains. Runt homology domain (RHD) encodes within exons 2–4 and nuclear localization signal (NLS) encodes at exon 5. RT1, RT2, and RT3 are the truncated exons of Runx2 that are expressed during cancer and viral infections. The genomic information was obtained from UCSC Genome Browser (http://genome.ucsc.edu/). The position of major phosphorylation sites of amino acids in human Runx2 by various kinases are also represented in the figure [13,19–21,39,43,47,87,108–115].

is generally mediated by ERK/MAPK. Certain serine residues are the usual sites of phosphorylation and this modification could lead to either a positive or a negative regulation of its target genes’ expression. For instance, phosphorylation at the conserved serine residue S104 negatively regulated the heterodimerization with PEBP2␤; otherwise, PEBP2␤ would form a complex with Runx2 to maintain its metabolic stability [36]. As such, the stability of Runx2 is maintained by PEBP2␤, a non-DNA binding protein. PEBP2␤ heterodimerized with Runx2 and indirectly promoted DNA binding by increasing the stability of Runx2 and preventing Runx2 from undergoing ubiquitin-mediated proteasomal degradation. Even though phosphorylation of Runx2 at the serine residues S104 and S451 negatively regulated the heterodimerization, phosphorylation at the S14 site did not have any negative impact [36,37]. The coordinated action of dexamethasone, an osteostimulant, and Runx2 stimulated the expression of OC, BSP genes, ALP activity, and mineral deposition in primary dermal fibroblasts by reducing the phosphorylated serine level of Runx2 on S125 with a simultaneous upregulation of MAPK phosphatase-1 (MKP-1) [38,39]. Aside from this, there are multiple phosphorylation-dependent regulatory mechanisms of Runx2 activity by different pathways and protein–protein interactions such as PTH-induced Runx2/AP-1 and BMP mediated Runx2/Smads interactions. In vivo experiments proved that phosphorylation of Runx2 by p38 MAPK is vital in the process of maintaining bone homeostasis [40]. The MAPK pathway activated Runx2 as a consequence of stimuli from the extracellular matrix, BMPs, FGF2, protein kinase A (PKA), mechanical loading, and hormones such as PTH [41,42]. It was reported that PTH stimulates and transactivates Runx2 via PKA through AD3 [39,43]. The upregulated transcription of the matrix metalloproteinase13 (MMP-13) gene was found to be due to phosphorylation of Runx2 by protein kinase G [44]. The PTH activation of Runx2 through the PKA-dependent pathway, in which the S347 site within the AD3 of Runx2 became phosphorylated, also induced MMP13 transcription [43]. Glycogen synthase kinase 3 beta (GSK-3␤) phosphorylation inactivated Runx2 activity, and the phosphorylation process took place at the S369, S373, S377 sites of Runx2 [45]. In FGF2 regulation of the mouse OC gene in pre-osteoblastic cells, Runx2 phosphorylation by ERK1/2 was found to be important

[46]. ERK1 facilitated Runx2 phosphorylation at S301 and S319; this stimulated osteoblast specific gene expression [47]. An extracellular protein, NELL1, led to Runx2 phosphorylation via the MAPK cascade and this required tyrosine kinase activation of the RAS/MAPK cascade [48]. Mechanical stimulus plays a vital role in the regulation of osteoblast genes. It has been shown that the fluid motion within the canaliculi creates a fluid shear stress (FSS) that causes an anabolic response to the bone. FSS also stimulated the activation of the focal adhesion kinases (FAKs), which are involved as mechanosensors converting mechanical energy to intracellular force. This force regulates pathways such as MAPK and PI3K/protein kinase B (Akt), which are responsible for osteoblastogenesis [49]. In addition, FGF2 activated PKC resulting in an induction of Runx2 expression. In silico analysis of a predicted phosphorylation site at S247 of Runx2 proved it to be the site of activation [50]. It has been reported that yes-associated protein (YAP), a mediator of Src/Yes signaling, interacted with Runx2 and that tyrosine phosphorylation of YAP caused dissociation of the Runx2-YAP complex [51]. Similar to many other proteins, Runx2 controls its target genes during the mitotic phase. Runx2 can be phosphorylated by cdk1/cyclin B complex during mitosis and is involved in postmitotic regulation of its target genes [52]. The cdc2-dependent phosphorylation of Runx2 is involved in cell cycle progression through the G2 phase in osteoblasts [53]. Glucose elicits the phosphorylation of Runx2 and changes its sub-nuclear localization [54]. It has also been reported that both Runx2 DNA binding and endothelial cell cycle progression were facilitated by cdk1mediated Runx2 phosphorylation [54]. Runx2 phosphorylation sites in response to various signals and stimuli, as well as alteration of expression of its target genes are summarized in Table 1. The major phosphorylation sites in human Runx2 are also represented in Fig. 1. 5. Negative regulation of Runx2 5.1. Ubiquitination/proteasomal degradation of Runx2 Ubiquitination of Runx2 by E3 ligases such as Smad ubiquitination regulatory factor 1 (Smurf1) and WWP1 facilitates

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Table 1 Runx2 phosphorylation by various signals/stimuli and its targeted gene expression. Name and position of amino acids

Signals/Stimuli

Function

References

S280, S284, S288 S247

GSK3␤ FGF-2 FGF-2

Decreases Runx2 transcriptional activity Positively regulates Runx2 activity Phosphorylates Runx2 through MAPK pathway to increase OC promoter activity

[45] [50] [46]

S347 S28, S347, T340

PKA PTH

Positively regulates transactivation of Runx2 for MMP-13 Phosphorylates though PKA which enables Runx2 transactivation for MMP-13 expression Phosphorylates Runx2 during osteoblast mitosis Phosphorylates Runx2 through MAPK pathway during osteoblast differentiation

[43] [39]

Negatively regulates BMP-2 induced osteogenesis by phosphorylation of Runx2 Negatively regulates osteoblast differentiation by phosphorylation of Runx2 and dexamethasone decreases the level of Runx2 phosphorylation at S125 S301 and S319 phosphorylation positively regulate osteogenesis Facilitates cell cycle progression from G2 to M phase Induces Runx2 ubiquitination and proteasome degradation Negatively regulates Runx2 heterodimerization with CBF␤ Positively regulates osteoblast differentiation IGF-1 phosphorylates Runx2 by the mediation of MKK1/2 which enhances the process of Runx2-DNA binding Phosphorylates Runx2 by MEK/ERK pathway which leads to increased MMP-13 activity

[86]

Induces phosphorylation of Runx2 by ERK/MAP kinase to increase acetylation and decrease ubiquitination Phosphorylates Runx2 by ERK1/2 which enhances osteogenesis Phosphorylates Runx2 by MAPK/p38 to enhance osteoblast differentiation Phosphorylates Runx2 through Ras/MAPK to positively regulate osteogenesis Positively regulates osteoblast differentiation Phosphorylates Runx2 through ERK to positively regulate osteoblast differentiation

[71]

CDK1/cyclin B kinase NELL1 S104

JNK1

S125

Glucocorticoid

S301, S319, S43, S501 S451 S472 S104, S451 S110

ERK/MAPK CDC2 Cyclin D1 and CDK4 BMP-2 IGF-1 FGF-2

S301

FGF-2 FAK Berberine Strontium TGF␤/BMP2 and MAPK Laminin-5

[52] [48,85]

[38]

[47,87] [53] [68] [36] [36] [87,88] [89]

[90,91] [49,92] [93] [94,95] [90,91,96]

S301, S319

DDR2 Connexin43

Phosphorylates through ERK to promote osteoblast differentiation PKC␦ and Runx2 are phosphorylated by FGF-2 and these events are enhanced by Cx43

[97] [98]

S17, S261, S298

TAK1-MKK3/6-p38 MAPK p38 and ERK MAPKs

Phosphorylates Runx2 which enhances the association with CREB-binding protein Phosphorylation of Osterix and Runx2 regulates their cooperative transcriptional activity Positively regulates bone formation by phosphorylating ERK and Runx2

[40]

DDK2 regulates PTHrP by phosphorylating Runx2 in bone metastasis FGF2 phosphorylates Runx2 through Pin1 to regulate bone formation Phosphorylates Runx2 to enhance its stability Phosphorylates through FAK-JNK signaling to enhance cartilage development Decreases the transcriptional activity of Runx2 Phosphorylation of Runx2 regulates bone diseases Regulates phosphorylation of Runx2 Phosphorylation of Runx2 by MAPK and p38 MAPK to increase expression of osteoblast marker genes

[100] [101] [102] [103]

FGF2 S301/319

DDR2 Pin1 Pin1 Type II collagen Palmitate Glutathione CD44 ODN MT01

[94,95] [99]

[104] [105] [106] [107]

S432

Wip1 miR-138 Glucose

Interaction of Wip1 dephosphorylates Runx2 Decreases the phosphorylation of Runx2 through PDGF pathway Phosphorylates Runx2 to promote cell proliferation

[108] [79] [54]

S203/T205/T207, T227, S474

Akt kinase

Phosphorylation of Runx2 enhances metastasis and invasion

[109]

its degradation [55–57]. Schnurri-3 (Shn3), a zinc-finger adapter protein, interacts with WWP1 to induce Runx2 degradation [58]. The TNF receptor associated factor 4 (TRAF4) acts as a substrate for WWP1, where the PY motif of TRAF4 interacts with a domain in Smurf1 to facilitate the degradation of Runx2 [59]. Hence, overexpression of Smurf1 in osteoblast cells inhibits osteoblast differentiation via inhibition of BMP signaling [60,61]. BMP2 stimulates p300-dependent acetylation of Runx2 resulting in

the inhibition of Smurf1-mediated degradation. HDAC4/5 can deacetylate Runx2, and thus Runx2 is vulnerable to Smurfmediated degradation [62]. Akt positively regulates Runx2 transcriptional activity by regulating ubiquitin-mediated proteasomal degradation of Smurf2 [63]. Tumor necrosis factor (TNF) causes degradation of Runx2 and inhibition of osteoblast differentiation through upregulation of Smurf1 expression levels in osteoblast precursor cells [64,65]. The factors that facilitate this process

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include Smad6, a BMP signaling inhibitor [55], casein kinase 2 interacting protein-1 (CKIP) [66], and PTH [67]. The Cyclin D1 and CDK4 complex phosphorylate Runx2 to target it for ubiquitinmediated proteasomal degradation [68,69]. CHIP (C-terminus of HSC 70 interacting protein) also regulates the process of ubiquitination. It is known to have a feedback mechanism, whose levels are inversely proportional to that of Runx2 [70]. It has been demonstrated that FGF2 greatly decreased Runx2 ubiquitination thus increasing the stability of Runx2. This process is controlled by an ERK-dependent pathway. It is predicted that there could be a relationship between phosphorylation and acetylation of Runx2 by FGF2-stimulated ERK/MAP Kinase that increases Runx2 stability and transactivation potential [71]. 5.2. Runx2 regulation by miRNAs MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules (19–25 nts) that regulate gene expression by targeting mRNAs at the post-transcriptional level. They are well known regulators for several physiological processes including osteoblast differentiation [5,7,22,25,72,73]. The experimentally validated microRNA-target interactions database (http://mirtarbase.mbc. nctu.edu.tw/), miRTarBase, provides the information that miR34c-5p, miR-133a-3p, miR-211-5p, miR-204-5p, miR-135-5p, miR-155-5p, miR-335-5p, miR-203a, miR-497-5p, miR-195-5p, miR-23a-5p, miR-204-5p, miR-433-3p, miR-338-3p, miR-2861, miR-124-3p, miR-30d-5p, miR-30a-5p, miR-505-5p, miR-484, and miR-30b-5p are validated to target Runx2 [74]. Further, miR-320c promotes adipogenic lineage by targeting Runx2 [75], miR-30a targets Runx2 and participates in osteolysis in giant cell tumor of bone [76], miR-103a targets Runx2 [77], while miR-135 and miR-203 targets Runx2 in breast cancer cells [78]. miR-138 inhibits osteoblast differentiation by repressing phosphorylation of FAK, ERK1/2, and Runx2 (79). miR-338-3p, miR-133, miR-204, and miR-34c target Runx2 [80–83]. It has been shown that miR-23a, miR-30c, miR34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, and miR-338 target Runx2; thereby regulating osteoblast and chondrocyte differentiation [84]. 6. Conclusions Runx2 plays a vital and diversified role depending on the cellular origin. It acts as an osteogenic differentiation factor in bone cells. Upon various stimuli, Runx2 interacts with different proteins resulting in positive or negative regulation of its target genes. The posttranslational modifications to Runx2, especially phosphorylation, dictate its functional interactions with other proteins controlling osteoblast differentiation. It is important to note that the list of proteins interacting with Runx2 keeps growing. This will pave the way for additional regulatory pathway analysis, which could facilitate the development of alternate and effective therapies for various bone and bone-related diseases. Acknowledgment This work was supported by a research grant from the Department of Science and Technology, Science and Engineering Research Board (DST-SERB), India (Grant No: SR/SO/HS-181/2013 to N. S.). References [1] G. Chen, C. Deng, Y.P. Li, Int. J. Biol. Sci. 8 (2) (2012) 272–288, http://dx.doi. org/10.7150/ijbs.2929 [2] M. Bruderer, R.G. Richards, M. Alini, M.J. Stoddart, Eur. Cell Mater. 28 (2014) 269–286. [3] J.B. Lian, G.S. Stein, J.L. Stein, A.J. van Wijnen, Vitam. Horm. 55 (1999) 443–509, http://dx.doi.org/10.1016/S0083-6729(08)60941-3

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