Runx3 Proteins Research Articles

Transcription Factors Runx1 and Runx3 Suppress Keratin Expression in Undifferentiated Keratinocytes.

The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation.

RUNX3 Inhibits the Invasion and Metastasis of Human Colon Cancer HT‐29 Cells by Upregulating MMP‐2/9

Objective To investigate the effect of Runt-associated transcription factor 3 (RUNX3) on the invasion and metastasis of human colon cancer HT-29 cells and to preliminarily explore the mechanism of its anticancer effect. Methods The RUNX3 plasmid vector was transfected into human colon cancer HT-29 cells by liposome-mediated transfection, while the empty vector and the blank group were used as the control group. After Geneticin (G418) screening, HT-29 cells with stable expression of RUNX3 gene were obtained. The expressions of mRNA and proteins of RUNX3 and metalloproteinases (MMP)-2/9 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cell proliferation was determined by MTT assay. The effect of RUNX3 on invasion and metastasis of HT-29 cells was evaluated by scratch injury assay, Transwell chamber, and Matrigel invasion model. Results RUNX3 was expressed stably in HT-29 cells after transfection. The expressions of RUNX3 mRNA and proteins in the experimental group were significantly higher than those in the blank/empty vector groups. Meanwhile, the expressions of MMP-2/9 mRNA and proteins in the observation group were significantly lower than those in the blank group and the empty vector group. The proliferation and migration ability in the experimental group was significantly lower than blank/empty vector groups from the third day. Transwell chamber experiment and Matrigel invasion assay showed that the number of Transwell cells was decreased significantly than blank/empty vector groups, but no difference was found between the blank group and the empty vector group. Conclusion RUNX3 can inhibit the invasion and metastasis of human colon cancer HT-29 cells, and the mechanism may be related to decreased expression of MMP-2 and MMP-9.

Loss of PIM1 correlates with progression and prognosis of salivary adenoid cystic carcinoma (SACC)

BackgroundIncreasing evidence indicates that PIM1 is a potential prognostic marker and target for cancer treatment but its precise mechanisms of action remain to be determined in salivary adenoid cystic carcinoma (SACC). This study aims to decipher the prognostic and mechanistic role of PIM1 in progression of SACC cells and tumor tissues.MethodsA SACC cell line (ACC-M) was transfected with shRNA plasmids targeting the PIM1 gene. The expression levels of PIM1, RUNX3 and p21 were measured by quantitative real-time PCR and western blot. Subcellular translocalization of RUNX3 and p21 proteins was assessed using immunofluorescence, and cell cycle phase was quantified using flow cytometry. A total of 97 SACC patients were retrospectively analyzed by clinicopathologic characteristics and survival outcomes.ResultsAfter down-regulation of PIM1 in ACC-M cells, RUNX3 and p21 proteins were translocated from cytoplasm to nucleus, with a decrease of p21 expression and increase of G0/G1 phase cells. PIM1 and RUNX3 levels show a distinct covariance. PIM1 is associated with T-status, lymph node involvement, nerve invasion, and distant metastasis in SACC tissues. Patients with low PIM1 level had a better outcome than those with higher PIM1 level.ConclusionsPIM1 is multifunctional in ACC-M cells and it serves as a neoteric therapeutic target and potential prognostic marker for SACC patients.

Core Binding Factor β Plays a Critical Role During Chondrocyte Differentiation.

Core binding factor β (Cbfβ) is a partner protein of Runx family transcription factors with minimally characterized function in cartilage. Here we address the role of Cbfβ in cartilage by generating chondrocyte-specific Cbfβ-deficient mice (Cbfb(Δch/Δch) ) from Cbfb-floxed mice crossed with mice expressing Cre from the Col2a1 promoter. Cbfb(Δch/Δch) mice died soon after birth and exhibited delayed endochondral bone formation, shorter appendicular skeleton length with increased proliferative chondrocytes, and nearly absent hypertrophic chondrocyte zones. Immunohistochemical and quantitative real-time PCR analyses showed that the number and size of proliferative chondrocytes increased and the expression of chondrocyte maturation markers at the growth plates, including Runx2, osterix, and osteopontin, significantly diminished in Cbfb(Δch/Δch) mice compared to wild type mice. With regard to signaling pathways, both PTHrP-Ihh and BMP signaling were compromised in Cbfb(Δch/Δch) mice. Mechanistically, Cbfβ deficiency in chondrocytes caused a decrease of protein levels of Runx transcription factors by accelerating polyubiquitination-mediated proteosomal degradation in vitro. Indeed, Runx2 and Runx3, but not Runx1, decreased in Cbfb(Δch/Δch) mice. Collectively, these findings indicate that Cbfβ plays a critical role for chondrocyte differentiation through stabilizing Runx2 and Runx3 proteins in cartilage.

Correlation between the methylation status of Runx3 gene and positive expression of estrogen receptor in breast cancer

To investigate the relationship between hypermethylation of Runx3 gene promoter and estrogen receptor (ER) and the implications of Runx3 gene promoter hypermethylation in ER positive breast cancer. Western blot and RT-PCR were used to detect the protein and mRNA expression of Runx3 gene in breast cancer cell lines (MCF7 and SKBR3) and normal breast epithelium cell line (MCF10A). Immunohistochemical SP method was used to detect the expression of ER and Runx3 proteins in 113 tissue samples of breast cancer. Moreover, methylation specific PCR was used to detect RUN3 promoter methylation in cell lines MCF7, SKBR3, MCF10A and 113 tissue samples of breast cancer. Of the 3 cell lines, Runx3 protein and mRNA were detectable in MCF10A, but were absent in MCF7 and SKBR3. MCF7 had a high methylation status at Runx3 promoter, in contrast, MCF10A and SKBR3 showed unmethylated RUN3 promoter. Among the 113 cases of breast cancer, 68 cases were ER positive and 45 were negative. The positive rates of Runx3 protein expression in ER positive and negative tumors were 26.5% (18/68) and 66.7% (30/45), respectively (P<0.05). Runx3 promoter hypermethylation was seen in 82.4% (56/68) of ER positive breast cancer cases and 22.2% (10/45) of ER negative ones (P<0.05). Among 68 cases of ER positive cases, Runx3 promoter hypermethylation was positively correlated with the clinical tumor stage (OR=5.84, P<0.05). Runx3 gene promoter hypermethylation is present mainly in the ER positive breast cancers. Testing of Runx3 promoter methylation may provide additional reference for clinical stage and prognosis of breast cancer patients, especially in those with ER positive tumors.

RUNX2: A Master Bone Growth Regulator That May Be Involved in the DNA Damage Response.

RUNX2 is a member of the RUNX family of transcription factors, also containing the RUNX1 and RUNX3 proteins. These factors control the expression of genes essential for proper development in many cell lineages. RUNX2 plays a crucial role in the proliferation and differentiation of osteoblasts, required for bone formation. The cellular level of RUNX2 oscillates in a cell phase-specific manner, reaching a maximum at G2/M in some cells and overexpression of RUNX2 in osteoblasts blocked G1 to S phase progression. Recent studies have shown that RUNX2 may interact with p53 and change the activity of a histone deacetylase. Moreover, RUNX2 may act as an oncogene in cancer transformation, inevitably associated with genomic instability evoked by increased occurrence of DNA damage. We showed that some RUNX2 modifiers changed the sensitivity of differentiating preosteoblasts to DNA damage induced by oxidative stress. All these data suggest the involvement of RUNX2 in cellular DNA damage response (DDR), which is particularly important in osteogenesis as the process of osteoblast differentiation is associated with increasing oxidative stress. However, the mechanism underlying DDR involvement of RUNX2 is unknown. The basic question, whether RUNX2 plays a positive or destructive role in DDR in differentiating cells is still open.

Expression of RUNX3 and β-catenin in the carcinogenesis of sporadic colorectal tubular adenoma

The aim of this study is to investigate the possible roles of runt-related transcription factor 3 (RUNX3) and β-catenin in the carcinogenesis of sporadic colorectal tubular adenomas. The expression of the RUNX3 and β-catenin proteins was evaluated by immunohistochemistry in 23 normal colorectal mucosa (NCM), 81 sporadic colorectal tubular adenomas with different dysplasias (SCTA-D) (mild n=33, moderate n=23, and severe n=25 dysplasia), and 48 sporadic colorectal tubular adenomas with cancerous changes (SCTA-Ca). RUNX3 methylation was assessed by methylation-specific polymerase chain reaction (MSP), combined with laser capture microdissection (LCM), in 17 NCM, 41 SCTA-D (mild n=15, moderate n=12, and severe n=14 dysplasia), and 17 SCTA-Ca tissues. Compared to NCM (82.6 %), RUNX3 in SCTA-D (54.3 %) and SCTA-Ca (27.1 %) was significantly downregulated (P<0.05). In NCM, SCTA-D, and SCTA-Ca, the incidence of positive expression for β-catenin was 13.0, 60.5, and 79.2 %, respectively. A statistically significant difference was observed (P<0.05). RUNX3 levels were markedly higher in adenoma with mild dysplasia (75.8 %) and moderate dysplasia (60.9 %) than in adenoma with severe dysplasia (20.0 %) (both with P<0.05). Likewise, the expression of β-catenin in severe dysplasia adenoma was 84.0 %, which was significantly higher than that in mild dysplasia adenoma (39.4 %). An inverse correlation was found between the protein expression of RUNX3 and β-catenin in SCTA-D and SCTA-Ca (P<0.05). MSP results showed that RUNX3 methylation in NCM, SCTA-D, and SCTA-Ca was 5.9, 17.1, and 41.2 %, respectively, with a statistically significant difference between NCM and SCTA-Ca (P<0.05). However, no significant difference of RUNX3 methylation was observed among different dysplasia groups. RUNX3 and β-catenin play important roles in the carcinogenesis of sporadic colorectal tubular adenomas. In addition, hypermethylation of RUNX3 can downregulate its expression.

Overexpression of RUNX3 Inhibits Malignant Behaviour of Eca109 Cells in Vitro and Vivo

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may play an important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim of this study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpression on biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues using the SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cell line with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence. CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assay to analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice, tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue to be significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinant lentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpression could inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in mice showed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate that underexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.

In vitro nasal epithelial toxicity by cadmium accompanies up-regulation of RUNX3 protein with activation of PI3-kinase/Akt

Nasal epithelium is the first cellular target for inhaled cadmium; however, the specific molecular events that mediate or accompany toxicity are not well studied. Using RPMI-2650 cells as an in vitro nasal epithelial model for the investigation of molecular events of cadmium toxicity, we found that treatment with cadmium acetate induced cell death with the typical morphological changes of apoptosis, such as chromatin condensation and nuclear fragmentation, as well as increased caspase-3/7 activity. This finding was accompanied with concentration-dependent increases of RUNX3, phospho-p53 (p-p53), and phospho-Akt (p-Akt) proteins with concomitant decrease in the Bcl-2 level. However, no significant changes were observed in the levels of Fas ligand and Fas-associated protein with death domain. Cadmium treatment increased the DNA binding activity of p53 toward a p53 consensus binding site. Blockage of RUNX3 expression with siRNA transfection did not affect a cadmium-induced increase in the p-p53 level but suppressed p53 binding activity to DNA. Nuclear co-localization of RUNX3 and p-p53 was enhanced by treatment with cadmium. The nuclear RUNX3 level was suppressed by the pretreatment with LY294002, a PI3-kinase inhibitor, while noticeable changes in the levels of p53 and p-p53 were not observed under the same condition. Cadmium-induced increase in the p53 binding to DNA was partly suppressed by LY294002, while it was enhanced by PD98059, a MEK inhibitor. Collectively, our findings suggest that cadmium induces up-regulation of RUNX3 protein through activation of PI3-kinase/Akt and leads to RPMI-2650 cell death, at least in part, through p53-dependent apoptosis pathway, in which RUNX3 may facilitate p53 binding to DNA.

RUNX3 interactome reveals novel centrosomal targeting of RUNX family of transcription factors

RUNX family proteins are critical regulators of lineage differentiation during development. The high prevalence of RUNX mutation/epigenetic inactivation in human cancer indicates a causative role for dysfunctional RUNX in carcinogenesis. This is supported by well-documented evidence of functional interaction of RUNX with components of major oncogenic or tumor suppressive signaling pathways such as TGFβ and Wnt. Here, we explore the binding partners of RUNX3 proteins to further define the scope of RUNX3 function. Using a mass spectrometry-based approach, we found that RUNX3 binds to centrosomal protein rootletin. This led us to uncover the presence of RUNX proteins at the centrosome. Our findings suggest a potential function for RUNX3 during mitosis.

Roles of VWRPY motif‐mediated gene repression by Runx proteins during T‐cell development

Runx transcription factor family proteins have essential roles during T-cell development by either activating or repressing target genes. For instance, lineage- and stage-specific expression of Cd4 and ThPOK is controlled by a transcriptional silencer embedded in each locus, whose activity requires bindings of Runx complexes. The evolutionarily conserved VWRPY penta-peptide sequences in Runx proteins have been shown to be responsible for repressive function as a platform to recruit Groucho/TLE transcriptional corepressors. However, it remains elusive whether requirement for the VWRPY motif differs among Runx target genes. By examining mice lacking VWRPY motifs in both Runx1 and Runx3 proteins, here, we show a full and partial derepression of Cd4 and ThPOK in CD8-linegae T cells, respectively. Thus, whereas Cd4 silencing completely depends on the VWRPY motif, both VWRPY-dependent and -independent mechanisms operate to repress ThPOK gene. These results indicate that Runx proteins utilize different modes to repress expression of different target genes.

Involvement of Runx3 in the basal transcriptional activation of the mouse angiotensin II type 1 receptor-associated protein gene

We previously cloned a molecule that interacts with angiotensin II type 1 (AT1) receptor to exert an inhibitory function on AT1 receptor signaling that we named ATRAP/Agtrap (for AT1 receptor-associated protein). In the present study we examined the regulation of basal ATRAP gene expression using renal distal convoluted tubule cells. We found that serum starvation upregulated basal expression of ATRAP gene, a response that required de novo mRNA and protein synthesis. Luciferase assay revealed that the proximal promoter region directs transcription and that a putative binding site of runt-related transcription factors (RBE) is important for transcriptional activation. The results of RBE-decoy transfection and endogenous knockdown by small interference RNA showed that the runt-related transcription factor Runx3 is involved in ATRAP gene expression. Chromatin immunoprecipitation assay also supported the binding of Runx3 to the ATRAP promoter in renal distal convoluted tubule cells. Immunohistochemistry demonstrated the expression of Runx3 and ATRAP proteins in the distal convoluted and connecting tubules of the kidney in consecutive sections. Furthermore, the Runx3 immunostaining was decreased together with a concomitant suppression of ATRAP expression in the affected kidney after 7 days of unilateral ureteral obstruction. These findings indicate that Runx3 plays a role in ATRAP gene expression in renal distal tubular cells both in vitro and in vivo.

Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs

Activation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) γ expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-γ, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

Subnuclear targeting of the Runx3 tumor suppressor and its epigenetic association with mitotic chromosomes

Runx proteins are tissue-specific transcriptional scaffolds that organize and assemble regulatory complexes at strategic sites of target gene promoters and at intranuclear foci to govern activation or repression. During interphase, fidelity of intranuclear targeting supports the biological activity of Runx1 and Runx2 proteins. Both factors regulate genes involved in cell cycle control and cell growth (e.g., rRNA genes), as well as lineage commitment. Here, we have examined the subcellular regulatory properties of the third Runx member, the tumor suppressor protein Runx3, during interphase and mitosis. Using in situ cellular and biochemical approaches we delineated a subnuclear targeting signal that directs Runx3 to discrete transcriptional foci that are nuclear matrix associated. Chromatin immunoprecipitation results show that Runx3 occupies rRNA promoters during interphase. We also find that Runx3 remains associated with chromosomes during mitosis and localizes with nucleolar organizing regions (NORs), reflecting an interaction with epigenetic potential. Taken together, our study establishes that common mechanisms control the subnuclear distribution and activities of Runx1, Runx2, and Runx3 proteins to support RNA polymerase I and II mediated gene expression during interphase and mitosis.

Enhancer of Zeste Homologue 2 (EZH2) Down-regulates RUNX3 by Increasing Histone H3 Methylation

Overexpression of enhancer of zeste homologue 2 (EZH2) occurs in various malignancies and is associated with a poor prognosis, especially because of increased cancer cell proliferation. In this study we found an inverse correlation between EZH2 and RUNX3 gene expression in five cancer cell lines, i.e. gastric, breast, prostate, colon, and pancreatic cancer cell lines. Chromatin immunoprecipitation assay showed an association between EZH2 bound to the RUNX3 gene promoter, and trimethylated histone H3 at lysine 27, and HDAC1 (histone deacetylase 1) bound to the RUNX3 gene promoter in cancer cells. RNA interference-mediated knockdown of EZH2 resulted in a decrease in H3K27 trimethylation and unbound HDAC1 and an increase in expression of the RUNX3 gene. Restoration of RUNX3 expression was not associated with any change in DNA methylation status in the RUNX3 promoter region. RUNX3 was repressed by histone deacetylation and hypermethylation of a CpG island in the promoter region and restored by trichostatin A or/and 5-aza-2′-deoxycytidine. Immunofluorescence staining confirmed restoration of expression of the RUNX3 protein after knockdown of EZH2 and its restoration resulted in decreased cell proliferation. In vivo, an inverse relationship between expression of the EZH2 and RUNX3 proteins was observed at the individual cell level in gastric cancer patients in the absence of DNA methylation in the RUNX3 promoter region. The results showed that RUNX3 is a target for repression by EZH2 and indicated an underlying mechanism of the functional role of EZH2 overexpression on cancer cell proliferation.

Pim-1 kinase phosphorylates RUNX family transcription factors and enhances their activity

BackgroundThe pim family genes encode oncogenic serine/threonine kinases which in hematopoietic cells have been implicated in cytokine-dependent signaling as well as in lymphomagenesis, especially in cooperation with other oncogenes such as myc, bcl-2 or Runx family genes. The Runx genes encode α-subunits of heterodimeric transcription factors which regulate cell proliferation and differentiation in various tissues during development and which can become leukemogenic upon aberrant expression.ResultsHere we have identified novel protein-protein interactions between the Pim-1 kinase and the RUNX family transcription factors. Using the yeast two-hybrid system, we were able to show that the C-terminal part of human RUNX3 associates with Pim-1. This result was confirmed in cell culture, where full-length murine Runx1 and Runx3 both coprecipitated and colocalized with Pim-1. Furthermore, catalytically active Pim-1 kinase was able to phosphorylate Runx1 and Runx3 proteins and enhance the transactivation activity of Runx1 in a dose-dependent fashion.ConclusionAltogether, our results suggest that mammalian RUNX family transcription factors are novel binding partners and substrates for the Pim-1 kinase, which may be able to regulate their activities during normal hematopoiesis as well as in leukemogenesis.

Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators.

The AML/RUNX family of transcription factors plays important roles in hematopoiesis, neurogenesis, bone development, and segmentation in vertebrate embryos. The aim of this study was to isolate runt-related genes in a genetically and embryologically exploitable system, the zebrafish, and characterize their function during hematopoietic development. Two runt-related genes were isolated by degenerate PCR and standard library screening, and a radiation hybrid panel, T51 RH, was used to resolve their chromosomal localization. In situ hybridization demonstrated their expression whereas their transcriptional activity was assessed using an AML1-responsive reporter gene in the MLA 144 T-cell line. We isolated the zebrafish runxa and runxb cDNAs, which encode proteins highly homologous to the human and murine Runx1 (AML1) and Runx3 (AML2) proteins. In contrast to a recent report, we detected runxa expression in both hematopoietic and neural tissues of the developing zebrafish. runxa transcripts first appear during segmentation in bilateral mesodermal cells that coexpress one of the earliest blood and endothelial cell markers, scl/tal-1. By 24 hours postfertilization (hpf), runxa transcripts are seen in the ventral wall of the dorsal aorta. Hematopoietic runxa expression is lost in cloche mutants, which are defective in blood and endothelial cell formation. runxb transcripts are seen in nonhematopoietic domains. Both Runxa and Runxb transactivate an AML1-responsive human promoter in hematopoietic cells. Genomic localization studies demonstrate that runxa is located on linkage group 1 (LG1), and the runxb gene is located on LG13. Our gene expression analysis strongly suggests that both the functional and spatial aorta-gonad-mesonephros (AGM) region has been conserved throughout evolution. Our runxa spatiotemporal expression data shed light on the role of vertebrate Runx1/AML1 in primitive vs definitive hematopoietic development.

Analysis of Type-I and Type-II RUNX2 Protein Expression in Osteoblasts

Runt-related transcription factor-2 (RUNX2) is expressed as two isoforms (type-I and type-II) differing only in their amino terminal sequences. The amino terminus of type-I contains MRIPV instead of MASNSLFSAVTPCQQSFFW in type-II. Although type-II mRNA has been considered osteoblast specific, the RUNX2 protein isoforms expressed in osteoblasts have not yet been identified. Using antisera generated against the two different amino terminal sequences of type-I and type-II RUNX2, we show the expression of both isoforms in cells with the mature osteoblast phenotype (fetal rat calvarial cells, and ROS 17/2.8, SaOS-2 and U2OS osteosarcoma cell lines), but only type-I in partially differentiated osteoblast-like cells (the UMR-106 osteosarcoma cell line). Since UMR-106 cells express both type-I and type-II mRNAs, our results suggest that the translation of type-II mRNA is repressed in these cells. No RUNX1 and RUNX3 proteins are detected in any of the osteoblastic cells tested. The antisera we have generated will be useful for studies relating expression of RUNX2 isoforms to control of osteoblast differentiation.