Runx3/AML2/Cbfa3 Regulates Early and Late Chondrocyte Differentiation

Authors


  • The authors state that they have no conflicts of interest.

Abstract

We studied the expression and function of Runx3 during chondrogenesis and chondrocyte maturation. We found that Runx3 is essential for mediating the early stage of endochondral ossification through cooperation with other Runx family members.

Introduction: Runx proteins are spatially and temporally co-expressed during skeletal formation. A cooperative and/or redundant function between these factors was postulated, yet the mechanisms underlying these cooperative effects are unknown.

Materials and Methods: Expression patterns of Runx3 transcripts were assessed during mouse embryonic developments and limb bud—derived mesenchymal cell differentiation into mature chondrocytes by real-time RT-PCR. Runx3 protein distribution was also determined by immunohistochemistry in mouse embryos. Runx3 gain and loss of function was performed through overexpression and siRNA knockdown of Runx3 into the limb bud—derived cell line MLB13MYC clone17, respectively. Co-transfection experiments were performed in clone 17 cells using the Runx1 promoter and Runx3 cDNA. Promoter activity was measured by luciferase reporter assay.

Results: Both Runx3 isoforms are significantly upregulated at the onset of cartilage mineralization and bone formation in E15.5 mice. This upregulation follows that of Sox9 and is concomitant with that of alkaline phosphatase. Furthermore, Runx3 expression remains high during later stages of embryonic development when the levels of osteocalcin are maximal. We determined the expression patterns of Runx3 during chondrogenesis and chondrocyte maturation using mouse limb bud—derived micromass cultures between days 3 and 21. Whereas Runx3 mRNAs are progressively upregulated between days 3 and 14, it is dramatically downregulated at day 21. Markers of chondrocyte maturation alkaline phosphatase and type X collagen are upregulated and maintained throughout the 21 days of culture. Runx3 role in mediating chondrocyte terminal differentiation through gain and loss of function in MLB13MYC clone17 shows that Runx3 regulates both early and late markers of chondrocyte maturation. Finally, Runx3 transcriptionally inhibits Runx1 expression in chondrocytes.

Conclusions: We show a role for Runx3 in mediating stage-specific chondrocyte maturation. Our study clearly suggests that, whereas Runx3 may cooperate with Runx2 to induce chondrocyte terminal differentiation, it inhibits Runx1 expression during late maturation.

INTRODUCTION

The runt-related factor X family of transcription factors (Runx1/AML1/Cbfa2, Runx2/Cbfa1/AML3, and Runx3/Cbfa3/AML2) are phylogenetically conserved between C. elegans, Drosophila, zebrafish, and vertebrates.(1–3) Gene ablation and gain of function experiments established all three proteins as important regulators of cell growth and differentiation during embryonic development.(4–11) Thus, Runx1/AML1 null phenotype results in early embryonic lethality of the homozygous mice, showing Runx1 requirement for definitive hematopoiesis,(4,5) whereas Runx2 knockout results in impaired chondrocyte terminal maturation followed by total absence of mineralized bone.(6–8) More recently, abrogation of the Runx3 gene showed a critical role in central nervous system development,(9) in addition to its anti-oncogenic function in the gastrointestinal tract.(10,11)

The growing evidence that Runx proteins have both independent and redundant functions in various tissues mainly originate from Runx mRNA or protein co-distribution during skeletal development. We have previously shown that Runx1 and Runx2 are co-expressed in various skeletal elements during mouse embryonic development,(12) whereas Levanon et al.(13) showed overlapping expression of Runx1 and Runx3 proteins during cartilage and bone formation. Stricker et al.(14) also showed by in situ hybridization that Runx2 and Runx3 transcripts overlap in immature and mature chondrocytes, suggesting a redundant or cooperative function between both factors in mediating chondrogenesis.(14) Furthermore, co-expression of Runx proteins in both normal and malignant tissues indicates temporal and spatial shared functions between them in multiple cell systems.(15–17) Additionally, Runx genes share structural and organizational features, and all three Runx factors target the same DNA recognition motif (5′PuACCPuCA3′) through their highly conserved runt homology DNA binding domain.(16,17) Finally, Runx family members interact with common co-regulatory proteins, to exert their activating or repressing effects on their respective target genes.(16,18) However, the mechanisms underlying cooperative or independent activities among the Runx family members are yet to be determined during cartilage development.

Our previous studies showed that Runx1 mediates the onset of chondrogenesis and chondrocyte differentiation,(19) whereas Runx2 is responsible for chondrocyte terminal maturation and mineralization.(20,21) Interestingly, although the loss of Runx3 function was not associated with any apparent skeletal phenotype, simultaneous loss of Runx2 and Runx3 functions induced a dramatic delay in cartilage formation in axial and appendicular skeleton compared with Runx2 knockout alone.(22) This reduction in cartilage maturation is attributed to the arrest of chondrocyte differentiation before hypertrophy.(22) Thus, the purpose of this study was to investigate the expression patterns and regulation of Runx3 during mouse embryonic development and mouse limb bud—derived mesenchymal stem cell chondrogenic differentiation. We also preformed gain and loss of function studies to determine if Runx3 is a stage-specific mediator of chondrocyte maturation.

MATERIALS AND METHODS

Animals

For our gene expression studies in whole embryos, CD-1; ICR pregnant mice were purchased (Charles River Laboratories Wilmington, MA, USA) and housed at the University of Rochester animal facility according to state and federal law requirements and experimental protocols approved by the University animal care committee. Whole embryos were removed at E11.5 through E16.5 and at E18.5. Embryos were eviscerated and the head were removed to ensure minimal contribution of soft tissues and central nervous system to our gene expression before total RNA extraction.

Immunohistochemistry

E15.5 and E16.5 embryos were fixed in 10% neutral-buffered formalin for 5 days before Runx3 protein detection by immunohistochemistry as previously described.(19) Embryos embedded in paraffin were sectioned and placed on glass slides. Three-micrometer sections were first deparaffinized in xylene and heated in 10 mM citrate buffer, pH 6.0, for 1 h at 70°C for Runx3 antigen retrieval (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Sections were incubated overnight at 4°C with anti-Runx3 (1:30 dilution). Runx3 protein was detected using goat anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) followed by horseradish peroxide streptavidin (1:250; Zymed, San Francisco, CA, USA) for 30 min treatment at room temperature. Secondary antibody alone was also used as a negative control.

Limb bud cell cultures

Forelimbs were dissected from E11.5 embryos and limb bud—derived mesenchymal stem cells were isolated as previously reported.(23) Briefly, forelimbs were digested in a 10% chick serum along with 1 U/ml of dispase for 3.5 h at 37°C and passed through a 20-μm Nitex nylon mesh filter to remove the ectoderm and cell clumps and generate single cells. Cells were counted and concentrated to 1.0 × 105 cells/10 μl of 50% DMEM/50% F12 media with 10% FBS and antibiotics. Cells were seeded in a 12-well plate at a density of 1.0 × 105 cells/well in a final volume of 10 μl and incubated for 1 h at 37°C to allow adherence. Two milliliters of fresh media was added to each well. Cells were cultured or treated with BMP-2 for varying amounts of time before RNA extraction.

Cell lines

Cells were maintained at 37°C in a humidified incubator in presence of 5% CO2 and cultured in appropriate media supplemented with FBS, antibiotics (500 units/ml of penicillin and 500 μg/ml of streptomycin), and 5% l-glutamine as follows. The mouse C3H10T1/2 embryonic fibroblast cells were cultured in BME supplemented with 10% FBS. The mouse NIH3T3 fibroblast cells and the mouse MLB13MYC clone17 limb bud—derived clonal cells were cultured in DMEM supplemented with 10% FBS. The mouse chondrogenic ATDC5 cells were cultured in DMEM/F-12 supplemented with 5% FBS. The rat calvaria-derived RCJ3.1 C5.18 chondrogenic cells and the mouse calvaria-derived MC3T3-E1 pre-osteoblast cells were cultured in αMEM supplemented with 15% and 10% FBS, respectively. For our BMP-2 treatments of MLB13MYC clone17 cell experiments, cells were seeded into 6-well plates at a density of 1.5 × 105 cells/well and maintained in complete media for up to 6 days. During this period, cells were either treated with 50 ng/ml of BMP-2 or vehicle as control. Media were changed every other day before harvesting RNA or protein.

RNA extraction and real-time RT-PCR

RNA was extracted using Rneasy mini kit (Qiagen, Valencia, CA, USA) according to manufacturer's instruction. One microgram of total RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer's protocol. Freshly reverse transcribed cDNA (0.2 μl) was used for real-time RT-PCR using the fluorescent dye SYBR Green I (Absolute QPCR SYBR Green Mix, ABgene, Rochester, NY, USA) to monitor DNA synthesis using specific primers (Table 1). The PCR was carried out in the RotorGene real-time DNA amplification apparatus (Corbett Research, San Francisco, CA, USA) using the following cycling protocol: a 95°C denaturation step for 15 min followed by 45 cycles of 95°C denaturation (20 s), 57–63°C annealing (30 s), and 72°C extension (30 s). Gene expression was normalized to the housekeeping gene β-actin. PCR products were subjected to a melting curve analysis, and the data were analyzed and quantified with the RotorGene analysis software (Corbett Research).

Table Table 1.. Primers Used for Real-Time RT-PCR Experiments
  1. M, mouse; H, human.

original image

Western blot analysis

Twenty-five to 50 μg of total proteins were separated on 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA). Membranes were incubated overnight at 4°C with a blocking solution containing 5% nonfat dry milk. Membranes were incubated with Anti-AML2 (Runx3; Calbiochem, San Diego, CA, USA) at room temperature for 1 h and further incubated after appropriate washing with a horseradish peroxidase—conjugated secondary antibody at room temperature for 1 h. Anti-β-Actin (Sigma, St Louis, MI, USA) was used as a control for equal protein loading. Immune complexes were detected using SuperSignal West Femto detection reagent (Pierce, Rockford, IL, USA).

Overexpression experiment

MLB13MYC clone 17 cells were seeded at a density of 5.0 × 105 in a 6-cm dish and maintained in medium supplemented with 10% FBS without antibiotics 1 day before transfection. Eight micrograms of the Runx3 isoform starting with the amino acid sequence MRIPV (methionine, arginine, isoleucine, proline, and valine) expression vector were mixed with 20 μl of lipofectamine 2000 according to manufacturer's protocol. The complex of Runx3 CMV-driven cDNA and lipofectamine 2000 transfection agent was added to the cells. Media were replaced after 6-h incubation and cells were cultured for 12, 24, and 48 h before harvesting and total RNA extractions.

RNA interference experiments

The mouse limb bud—derived mesenchymal cells MLB13MYC clone 17(24) were seeded at a density of 1.25 × 105 cells/well into 6-well plates and 7.5 × 105 cells/well into 10-cm plates and incubated in DMEM media supplemented with 10% FBS in the absence of antibiotics. Cells were transfected for 24, 48, and 72 h with 200 pmol small interference RNA (siRNA) oligo of Runx3 (5′-UGGCCAGGUUCAACGACCUUCGAUU-3′) per well in 6-well plates (RNA experiments) or for 24 h with 600 pmol of RNAi oligo in 10-cm plates (Western blot analyses) using the Lipofectamine 2000 transfection reagent according to manufacturer's recommendation (Invitrogen). Block-it Fluorescent (Invitrogen) was used as an unrelated control.

Transfection and luciferase activity assay

For Runx1 promoter experiments, MLB13MYC (clone 17) cells were seeded at 1.0 × 105 cells per well in a 12-well plate. After a 24-h incubation, cells were transfected with 100 ng of pGL3-Runx1 promoter (generously provided by Dr Farrell, Imperial Collagen Faculty of Medicine) and CMV-driven human Runx3 cDNA (0, 125, or 250 ng) incubated for 34 h. pGL3 was used as an empty reporter vector and pRL (Renilla luciferase vector; 100 ng/well) was also co-transfected to standardize for transfection efficiency control. Runx1 promoter activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) and a luminometer (Optocomp 1; MGM Instruments, Hamden, CT, USA).

RESULTS

Runx3 transcripts are regulated throughout mouse embryonic development

All Runx1, 2, and 3 genes encode for at least two protein isoforms starting with the amino acid sequences MASXS or MRIPV. These family members were shown to control various aspects of organogenesis during embryonic development. We studied the expression patterns of Runx3 MASNS and MRIPV isoforms using total RNA isolated from whole mouse embryos from which gut and CNS were excluded. We concomitantly assessed the expression of early and late chondrocyte phenotypic markers Sox9, alkaline phosphatase, and osteocalcin.

Figure 1A shows that the MASNS isoform of Runx3 is significantly upregulated 2-fold at E14.5 in comparison with E11.5. The levels of this isoform later reached a maximal expression at E15.5 (8-fold), to be thereafter downregulated at E18.5. A 3-fold intermediate induction was also observed at E16.5 compared with E11.5 (Fig. 1A). Similarly, the Runx3 MRIPV isoform is dramatically induced at E15.5 by at least 10-fold and continues to be upregulated through E18.5 (Fig. 1B). Together, these results suggest that the overall expression of both Runx3 isoforms seems to reach maximal expression after E14.5 corresponding to the beginning of cartilage terminal maturation and the onset of bone formation during mouse embryonic development. As expected, Sox 9 mRNA levels (Fig. 1C) were upregulated at E13.5 corresponding to the onset of cartilage early matrix formation in the mouse embryo to be thereafter downregulated between E15.5 and E18.5 as the skeleton mineralizes and the cartilage is replaced with bone. The induction of both Runx3 isoforms is observed after that of Sox9. On the other hand, Runx3 upregulation is concomitant to the significant upregulation of alkaline phosphatase (Fig. 1D) and osteocalcin (Fig. 1E) transcripts between E14.5 and E18.5. These results suggest a role for Runx3 during cartilage maturation and bone formation in the mouse embryo.

Figure Figure 1.

Runx3 is differentially expressed during mouse embryogenesis. Whole embryos at various developmental stages were homogenized to extract total RNA. mRNA levels of (A) MASNS and (B) MRIPV isoforms of Runx3, (C) Sox9, (D) alkaline phosphatase, and (E) osteocalcin were measured using real-time RT-PCR. Values are expressed as means ± SE, normalized to β-actin. *Statistical significance from E11.5 levels (p < 0.05). (F) The protein expression of Runx3 was also detected in E15.5 and E16.5 using immunohistochemistry. Runx3 protein was expressed in prehypertrophic chondrocytes (#), the perichondrium (↓), osteoblasts and primary spongiosa (▸), and hematopoetic cells (•) but in the absence of hypertrophic chondrocytes (*) in the limb.

To identify the cellular distribution of Runx3 protein during embryonic development, we performed immunohistochemistry using a Runx3 antibody. Figure 1F shows that Runx3 protein is primarily expressed in prehypertrophic chondrocytes, the perichondrium, and osteoblasts. Runx3 is also expressed in hematopoietic cells and primary spongiosa. However, no Runx3 expression was detected in hypertrophic chondrocytes in the limb of E15.5 and E16.5 embryos.

Runx3 is regulated during chondrocyte differentiation

Previous reports showed Runx3 expression during limb development and endochondral bone formation in vivo.(14,22) However, the mechanisms underlying Runx3 function during chondrogenesis and chondrocyte maturation are not yet characterized. To study a possible role for Runx3 during chondrogenesis, we first examined its expression pattern in primary mouse limb bud—derived cells that spontaneously differentiate into mature chondrocytes when cultured in micromass.(23) Using specific primers to distinguish between both Runx3 isoforms (starting with the amino acid sequences MASNS versus MRIPV), we performed real-time RT-PCR experiments using total RNA extracted from limb bud cells at various time-points. Total endogenous levels of all three Runx family members and early and late chondrocyte phenotypic genes were also assessed using these RNA. Figure 2A shows that the MASNS isoform of Runx3 is upregulated between day 3 and day 7 by 4-fold. This increase is maintained and enhanced at day 10 (8-fold) and is maximal at day14 (15-fold) to be thereafter reduced at day 21. The MRIPV isoform of Runx3 transcripts are high and unchanged between day 3 and day 7 of culture, whereas they are significantly upregulated at day 10 (2.5-fold) compared with day 7 (Fig. 2B). This isoform is dramatically downregulated at day 14 and to a higher extent at day 21, where it is repressed compared with day 3 (3-fold inhibition).

Figure Figure 2.

Runx3 is upregulated during chondrogenesis and chondrocyte differentiation in embryonic mouse limb bud mesenchymal cells. Limb bud cells were plated in micromass for 3–21 days. Total RNA was extracted from the cultures at the indicated time-points (days 3, 7, 10, 14, and 21). mRNA levels of (A) MASNS, (B) MRIPV, and (C) total of Runx3, (F) total Runx1, (I) total Runx2, and (D) chondrogenic phenotypes, type II collagen, (E) Sox9, (G) alkaline phosphatase, and (H) type X collagen were measured by real time RT-PCR. Values are expressed as means ± SE, normalized to β-actin. *Statistical significance from day 3 data (p < 0.05).

Along with Runx3 isoform expression, we also assessed early and late chondrocyte phenotypic gene expression in this cell culture model. Sox9 (Fig. 2E) and type II collagen (Fig. 2D) levels are induced in the early time-points between days 3 and 10 to be downregulated thereafter as the cells become more mature. The markers of chondrocyte maturation and hypertrophy alkaline phosphatase (Fig. 2G) and type X collagen (Fig. 2H) transcripts are progressively and significantly upregulated between days 3 and 21.

To complement these results, total Runx3 gene expression was also assessed using specific primers that do not distinguish between both isoforms. Figure 2C shows that overall expression of Runx3 is progressively upregulated between days 3 and 14 (12-fold induction in comparison with day 3) to be dramatically downregulated at day 21 to basal levels. Runx3 expression overlaps that of Runx1, which is upregulated at day 7 to be downregulated thereafter (Fig. 2F), and that of Runx2, which is only upregulated and maintained during the late time-points between day 10 and day 21 (Fig. 2I). These results suggest a role of Runx3 during both early and late chondrocyte maturation, and further underline its possible redundant function with the other Runx family members.

Both isoforms of Runx3 are induced by BMP-2 in primary limb bud cells

BMP-2 has been previously shown to accelerate chondrocyte differentiation in limb bud cells as evidenced by elevated gene expression of alkaline phosphatase and type X collagen.(19) We studied the effect of BMP-2 on Runx3 isoform transcript levels in this limb bud chondrogenic cell model. Figure 3 shows that BMP-2 is able to upregulate the MASNS isoform of Runx3 after 3 days of culture by ∼2-fold (Fig. 3A) and significantly induces the MRIPV isoform by 2.5- and 2.7-fold at day 7 and day 14, respectively (Fig. 3B). However, BMP-2 continuously repressed the mRNA levels of the marker for early chondrocyte differentiation Sox9 at day 14 by 24.4-fold and reduced its suppression at day 21 by 2.4-fold (Fig. 3C). As expected, treatment with BMP-2 strongly enhanced alkaline phosphatase (Fig. 3D) with a similar pattern to that of the Runx3 gene, whereas BMP-2 continuously upregulated osteocalcin throughout day 21 (Fig. 3E). Together these results show that the induction of Runx3 by BMP-2 is concomitant with that of chondrocyte maturation.

Figure Figure 3.

BMP-2 induces Runx3 gene expression in limb bud mesenchymal cells. Limb bud cells were plated in micromass culture and incubated in the presence or absence of BMP2 (50 ng/ml) for 21 days. Total RNA was extracted from those cells at the indicated time-points (days 3, 7, 14, and 21). mRNA levels of (A) MASNS and (B) MRIPV of Runx3, (C) Sox9, (D) alkaline phosphatase, and (E) osteocalcin were measured by real time RT-PCR. Values, which are normalized to β-actin, are presented as fold induction of BMP-2 over control and expressed as means ± SE. *Statistical significance from controls at each time-point (p < 0.05).

MLB13MYC clone 17 as a substitute for primary limb bud cells

Because Runx3 is expressed at multiple stages of chondrogenesis, we assessed the expression levels of Runx3 proteins by Western blot analysis in various mesenchymal cell lines capable or not to differentiate into mature chondrocytes. Figure 4A shows that the highest Runx3 levels are present in RCJ3.1 C5.18 rat calvarial chondrogenic cells and in MC3T3-E1 mouse pre-osteoblasts. Significant levels of Runx3 are also observed in the mouse chondrogenic cells ATDC5 and MLB13MYC clone 17, whereas very low levels are detected in the mouse embryonic fibroblasts C3H10T1/2, which also possess mesenchymal chondroprogenitor capabilities and the mouse fibroblast cells NIH3T3.

Figure Figure 4.

Endogenous Runx3 protein expression in various cell lines. C3H10T1/2 (mouse embryonic fibroblast cells), NIH3T3 (mouse fibroblast cells), ATDC5 (mouse chondrogenic cells), MLB13MYC clone 17 (mouse limb bud-derived mesenchymal stem cells), RCJ3.1C5.18 (rat calvaria-derived cells), and MC3T3-E1 (mouse osteoblastic cells). (A) Runx3 proteins were measured using Western blot analysis. BMP-2 induces Runx3 gene expression in the limb bud derived cell line, MLB13MYC13 clone17. mRNA levels of (B) Runx3, (C) Runx1, (D) Runx2, (E) type II collagen, (F) alkaline phosphatase, and (G) type X collagen were measured by real-time RT-PCR. Values, which are normalized to β-actin, are expressed as means ± SE. *Statistical significance from controls at each time-point (p < 0.05).

MLB13MYC clone 17 showed intermediate levels of Runx3 compared with other cell lines tested in this study and is a surrogate of limb mesenchymal origin.(24) We therefore characterized these cells in our hands under our culture conditions on BMP-2 treatments for 4 and 6 days. Regulation of Runx3 as well as Runx1, Runx2, and chondrocyte phenotypic genes by BMP-2 in MLB13MYC clone17 was assessed by real time RT-PCR.

Figure 4 shows that, whereas BMP-2 significantly enhanced Runx 2 (Fig. 4D) and Runx3 (Fig. 4B) levels, it inhibited that of Runx1 (Fig. 4C). Furthermore, whereas BMP-2 significantly but mildly upregulated the early chondrogenesis marker type II collagen (Fig. 4E), it dramatically enhanced alkaline phosphatase (Fig. 4F) and type X collagen (Fig. 4G), the later markers of chondrocyte differentiation in these cells. Together, these results show the ability of these cells to exhibit chondrocyte phenotypic features that are enhanced by BMP-2 treatment. Thus, we decided to use the MLB13MYC cells for both gain and loss of Runx3 function experiments.

Runx3 regulates early and late stages of chondrocyte differentiation

Because both isoforms of Runx3 are comparatively expressed and regulated during chondrocyte differentiation, we used the MRIPV isoform of the Runx3 for our gain of function studies. We used the MLB13MYC clone 17 cells to overexpress Runx3 through transient transfection of a CMV-driven human Runx3 cDNA. We chose these cells because they are limb bud—derived immortalized cells and exhibit intermediate endogenous levels of Runx3 compared with the other cell lines as shown in Fig. 4A. Additionally, the use of the same cell line for both our gain and loss of function for comparative purposes makes them preferably suitable for our functional studies. Figure 5A shows that Runx3 transcripts are potently upregulated 12, 24, and 48 h after transfection of these cells in comparison with the controls. These results confirmed the overexpression of Runx3 in these cells. To study the effect of Runx3 gain of function on chondrogenesis and chondrocyte differentiation, we assessed the expression levels of early (type II collagen) and late (alkaline phosphatase and type X collagen) chondrocyte phenotypic genes at the above time-points. Runx3 significantly inhibited type II collagen (Fig. 5 D) mRNA levels at 12 and 24 h, whereas it had no effect at 48 h. However, Runx3 overexpression significantly induced the late marker of chondrocyte maturation type X collagen (Fig. 5F), 24 and 48 h after transfection. Runx3 effect on alkaline phosphatase was, however, not significant (Fig. 5E) in these cells. To evaluate possible cross-regulation between Runx3 and the other Runx members, we assessed the expression levels of Runx1 and Runx2 in these cells. Whereas Runx 3 overexpression slightly but significantly downregulated Runx1 mRNA levels at all time-points (Fig. 5B), it dramatically reduced Runx2 transcripts at 12 and 24 h and to a lesser extent at 48 h (Fig. 5C). Together these results indicate that Runx3 regulates both early and late chondrocyte differentiation but also regulates the other members of the Runx family of transcription factors.

Figure Figure 5.

Overexpression of Runx3 regulates early and late chondrocyte differentiation. For the Runx3 gain of function, MLB13MYC clone 17 cells were tranfected with either CMV-driven Runx3 cDNA vector or pUC19 as a control. After the cells were incubated for 12, 24, or 48 h, total RNA was extracted from the cells. mRNA levels of (A) Runx3, (B) Runx1, (C) Runx2, and chondrogenic phenotypic genes, (D) type II collagen, (E) alkaline phosphatase, and (F) type X collagen were measured using real-time RT-PCR. Values, which are normalized to β-actin, are expressed as means ± SE. *Statistical significance over controls at each time-point (p < 0.05).

We complemented our Runx3 gain of function studies by performing loss of function experiments using siRNA to inhibit Runx3 expression in the MLB13MYC clone 17 cells that express significant endogenous Runx3 (as shown in Fig. 4A). As expected, Runx3 siRNA time-dependently inhibited its gene expression 24, 48, and 72 h after transfection by 65%, 80%, and 81%, respectively (Fig. 6A). Runx3 siRNA also effectively knocked down Runx3 protein by 69% (Fig. 6B). This inhibition of Runx3 expression induced significant upregulation of type II collagen (76%) only 24 h after transfection but not at 48 and 72 h (Fig. 6D). Similarly, Runx3 knockdown also enhanced mRNA levels of alkaline phosphatase (59%) at 48 h, but not at 24 and 72 h (Fig. 6E). Finally, type X collagen was significantly inhibited by Runx3 loss of function at all time points (Fig. 6F). Interestingly, loss of Runx3 function also inhibited Runx2 mRNA levels at 24, 48, and 72 h by 78%, 67%, and 53%, respectively (Fig. 6C). Together our gain and loss of function studies indicate that Runx3 mediates both chondrogenesis and chondrocyte maturation. The mechanisms underlying precise cross regulation and redundant functions between all three Runx proteins seem to be stage specific.

Figure Figure 6.

Runx3 knock-down modulates chondrocyte differentiation. MLB13MYC clone 17 cells (chondrogenic cells), were transfected with Runx3 siRNA oligo (200 pmol) and were incubated for 24, 48, and 72 h. Total RNA was extracted at the indicated time-points. mRNA levels of (A) Runx3, (C) Runx2, and chondrogenic phenotypic genes, (D) type II collagen, (E) alkaline phosphatase, and (F) type X collagen were measured using real-time RT-PCR. (B) Total protein was also isolated from transfected clone 17 cells 24 h after transfection with Runx3 siRNA to measure Runx3 protein levels using Western blot. Values are expressed as means ± SE and are normalized to β-actin. *Statistical significance (p < 0.05) from the siRNA-NS (nonspecific sequences) control group.

Runx3 inhibits Runx1 expression in chondrocytes

The cooperative effects between Runx3 and Runx2 in mediating chondrocyte terminal maturation have been previously reported in vivo.(22) We have also previously reported that Runx1 mediates onset of mesenchymal cell progression toward chondrogenesis. Because our data strongly suggest a dual role for Runx3 in regulating early and late stages of chondrocyte differentiation, we studied possible cooperative or antagonistic effects between Runx3 and Runx1 in chondrocytes. We first examined the regulation of Runx1 by Runx3 through our Runx3 loss of function experiments. Runx3 knockdown significantly enhanced Runx1 mRNA levels by 80% at 24 h, 67% at 48 h, and 73% at 72 h (Fig. 7A). This induction of Runx1 by Runx3 loss of function was further confirmed at the transcriptional level by assessing the effect of Runx3 overexpression on Runx1 promoter activity. Figure 7B shows that overexpressed Runx3 dose-dependently inhibited Runx1 P1 promoter activity. These results indicated that Runx3 both directly and indirectly induces chondrocyte differentiation by regulating Runx1 expression.

Figure Figure 7.

Runx3 negatively alters Runx1 gene expression. (A) MLB13MYC clone 17 cells were transfected with Runx3 siRNA oligo (200 pmol) and incubated for 24, 48, and 72 h. Total RNA was extracted at the indicated time-points. mRNA levels of Runx1 was measured using real-time RT-PCR. Values are expressed as means ± SE, normalized to β-actin. *Statistical significance (p < 0.05) from the siRNA-NS (nonspecific sequences) control group. (B) Clone 17 cells were co-transfected with CMV-driven human Runx3 cDNA (0, 125, or 250 ng) and Runx1 P1 promoter (300 ng) and incubated for 34 h. Runx1 promoter activity was measured using a dual luciferase reporter assay kit. pGL3 (300 ng) was used as a control, and pRL (100 ng) was used as an internal control to normalize for transfection efficiency. Values are expressed as means ± SE. *Statistical significance (p < 0.05) relative to the corresponding pGL3 control. ♦, statistical significance (p < 0.05) relative to the Runx1 basal promoter; •, statistical significance (p < 0.05) relative to the Runx1 promoter co-transfected with 125 ng of CMV-Runx3.

DISCUSSION

Runx family members (Runx1/Runx2/Runx3) have been extensively studied in their individual fields, where Runx1 plays a major role in hematopoiesis,(4,5) Runx2 is essential for skeletal development,(6–8) and Runx3 plays a key role in central nervous system development in addition to its anti-oncogene properties.(9–11) The similarity in the mRNA and protein structure between these family members, as well as their spatial and temporal co-distribution during embryonic and skeletal development, prompted us and others to hypothesize that Runx2 is not the only member of this family that mediates skeletal development. We present compelling evidence that Runx1 and Runx3 cooperatively orchestrate endochondral ossification along with the master gene Runx2.

We have previously shown that Runx1 co-regulates endochondral ossification with Runx2(12) and is essential for the transition of mesenchymal stem cells into a chondrocyte differentiation pathway.(19) In this study, we found that Runx3 transcripts are upregulated during embryonic development using total RNA extracted from whole embryos between E11.5 and E18.5. Previous reports described the spatial expression patterns of the Runx3 protein(13) and transcripts(14) in mouse embryos. The use of whole embryos provides us with the unique temporal expression of Runx3 at stages before (E11.5) and after cartilage is formed (E13.5) in comparison with when bone matrix is mineralized (E15.5—E18.5). This also complements data by Yoshida et al.(22) using the Runx3+/− Lac-Z knockin mice showing that Lac-Z activity reflecting Runx3 gene expression in embryos at E12.5, E13.5, and E15.5 is mainly attributed to skeletal elements including the scapulae, ribs, limbs, pelvic bones, and vertebrae.(22) However, we could not ignore that the expression of Runx3 at the early to late stages of embryos (E10.5—E16.5) is from dorsal root ganglia.(9,13) Our immunohistochemistry experiments show that Runx3 proteins are expressed in both immature and mature chondrocytes. However, we did not detect Runx3 protein in the hypertrophic cell population. Although these results are consistent with the in situ hybridization data published by Striker et al.(14) showing that Runx3 is expressed in the perichondrium and prehypertrophic chondrocytes but is excluded from the hypertrophic chondrocytes in embryonic limbs at E 14.5 and E15.5, Levanon et al.(13) found that Runx3 protein is present in the prehypertrophic and hypertrophic chondrocytes in vertebrae at E15.5. These differences in expression patterns may be caused by temporal differences in the examined skeletal tissues. Runx3 is first detected in the mouse limbs at E12.5, whereas it is only expressed in vertebrae at E13.5.(22)

To recapitulate the cellular and molecular events occurring during cartilage formation of the limb, we performed in vitro experiments using the mouse limb bud—derived chondrogenic cell model.(23) We observe that transcripts for Runx3 are upregulated during chondrocyte maturation as evidenced by a progressively elevated expression level of the type X collagen gene. However, we also observed a subsequent inhibition of Runx3 transcripts in the later time-points of terminal maturation while hypertrophy is not arrested. One would speculate that the concomitant expression of Runx3 and type X collagen may be caused by the heterogeneous maturation stage of cell populations in micromass cultures, This Runx3 expression pattern during chondrocyte differentiation in this model is very similar to that previously described for Runx2.(19) Together, our findings clearly suggest that Runx3 plays a key role during cartilage development in addition to its conventional function as a gastrointestinal cancer suppressor(10,11) and a supporter of neuronal development and survival.(9)

The expression pattern of Runx3 during embryonic development, skeletal formation, and chondrocyte maturation begs for defining the function of Runx3 during cartilage formation despite the lack of apparent skeletal phenotype in the Runx3 KO mice.(22) However, in the manuscript by Levanon et al., it is apparent that Runx3 knockout mice exhibit decreased growth compared with their wildtype littermates, suggesting a possible function of Runx3 in the growth plate.(9,25) For our gain and loss of function studies, we used the immortalized MLB13MYC clone 17 established by Rosen et al., and isolated from E13 mouse limb bud—derived cells.(24) This cell line expresses endogenous type II collagen and expresses high levels of ALP in response to BMP-2. Interestingly, continuous BMP-2 treatment of these cells decreases type II collagen expression and switches them to a more osteoblastic phenotype as evidenced by enhanced BGP and type I collagen expression. Other investigators(26,27) reported that a novel gene named BIG-3 (BMP-2 induced gene 3kb) in MLB13MYC stimulated both chondrocyte and osteoblast differentiation in vitro. Thus, MLBMYC clone 17 is suitable as an in vitro model of studying endochondral ossification. Our characterization of other chondrocyte phenotypic markers in this cell line on BMP-2 treatment shows that these cells express all Runx family members. Furthermore, whereas Runx2 and Runx3 levels are enhanced by BMP-2, the expression of Runx1 is inhibited by this differentiation growth factor. This suggests a progression of these cells to a terminally mature phenotype, as evidenced by enhanced alkaline phosphatase and type X collagen expression possibly through induction of Runx2 and Runx3.

Our loss of function studies using Runx3 knockdown by siRNA (1) enhanced the early marker of chondrocyte maturation type II collagen at the early but not in the later time-points but (2) inhibited the marker of chondrocyte hypertrophy type X collagen concomitantly to that of Runx3 at all time-points. This Runx3 knockdown in our in vitro system is consistent with that seen in vivo using the Runx3 knockout mouse embryos at E15.5,(22) where cartilage formation was slightly delayed in comparison with the wildtype littermates. Yoshida et al.(22) also reported that the dosage of Runx factors is important to mediate early cartilage formation and endochondral ossification. Specifically, Runx2 and Runx3 three-quarter knockout exhibits delayed chondrocyte differentiation, whereas the double knockout mice present complete abrogation of cartilage maturation.(22) Because to date, no gain of function model for Runx3 in skeletal tissues has been described, our gain of function in vitro experiments represent the seminal findings indicating that Runx3 plays a role both in early and late stages of chondrocyte maturation. Surprisingly, forced expression of Runx3 led to a potent repression of Runx2 transcripts in our in vitro model. Loss of Runx3 function also shows an inhibitory effect on Runx2; one could explain these results by an indirect regulation of the overall Runx2 and Runx3 levels necessary to drive terminal maturation. Further studies addressing the Runx protein dosage will shed more light into the molecular mechanisms underlying Runx mediated control of endochondral ossification.

Our previous studies showed cooperative but not redundant function between Runx1 and Runx2,(12,19) yet this study shows a role for Runx3 in both early and late chondrocyte maturation. While studying the mechanism by which Runx3 may regulate early chondrogenesis, we found that Runx3 inhibits Runx1 mRNA levels. Grueter et al.(28) have also assessed the inter-relationship between Runx1 and Runx3 in vivo after forced expression of Runx3 in the thymus using the CD4-enhancer promoter and found that Runx1 protein levels are downregulated in these transgenic mice. The effects of our Runx3 loss of function on Runx1 gene expression were also further supported by our promoter assay in which overexpressed Runx3 dose-dependently inhibited Runx1 promoter activity. These results are consistent with a recent study by Spender et al.(29) showing that Runx3 binds to the Runx1 promoter resulting in inhibition of Runx1 gene expression of in B cells. This regulation of Runx1 by Runx3 is therefore not specific to chondrocytes. Additional in vivo evidence for this regulation of Runx1 by Runx3 was recently provided by Fukushima-Nakase et al.(30) showing that Runx3 C-terminal knockin into the Runx1 locus similarly induced the osteocalcin promoter in comparison with the wildtype Runx1. However, substitution of the Runx1 C terminus with Runx2 did not show any induction of the osteocalcin promoter.(30) These findings indicate that Runx1 can be interchangeable with Runx3 but not with Runx2. Overall, one could envision two distinct mechanisms through which Runx3 may regulates cartilage formation: one being a cooperative effect with Runx1 to induce early chondrogenesis and an antagonistic effect with Runx1 to mediate late chondrocyte maturation, likely in support of Runx2.

We believe Runx family members cooperatively orchestrate endochondral ossification in a spatial and temporal manner. Our previous studies established Runx1 as a mediator of mesenchymal cell commitment toward chondrogenesis and early chondrocyte differentiation,(12,19) whereas Runx2 is necessary for chondrocyte terminal differentiation and cartilage maturation and mineralization.(20,31,32) Here we provide evidence that Runx3 not only cooperatively induces early and late chondrocyte maturation but may also serve as an inhibitor of Runx1 and potentate Runx2 function during endochondral ossification. Further studies will determine the precise combined roles of all three factors during skeletal development.

Acknowledgements

The authors thank Dr Paul J Farrell for providing them with the Runx1 promoter and Ruth Belflower, Arlene Martinez, Krista Canary, and Barbara Stroyer for technical assistance. This work was supported by the National Institutes of Health (NIH RO1 Grant AR052674–01).

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