Hyun-Mo Ryoo

Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor β1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation

The bone morphogenetic protein (BMP)‐2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non‐osseous cells in vitro. The runt domain‐related protein Cbfa1/PEBP2αA/AML‐3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP‐2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP‐2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP‐2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone‐related homeodomain protein Msx‐2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF‐β1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP‐2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP‐2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP‐2 requires sequences in the proximal promoter (up to nt ‐365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfa1 may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP‐2 appear to be necessary for complete expression of the osteoblast phenotype. J. Cell. Biochem. 73:114–125, 1999.

Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells.

Bone formation involves several tightly regulated gene expression patterns of bone‐related proteins. To determine the expression patterns of bone‐related proteins during the MC3T3‐E1 osteoblast‐like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone‐related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4–10), bone matrix formation/maturation (days 10–16), and mineralization stages (days 16 –30). During the proliferation period (days 4–10), the expression of cell‐cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10–16), type I collagen expression and biosynthesis, fibronectin, TGF‐β1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF‐β1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16–30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone‐related proteins are temporally regulated during the MC3T3‐E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone‐related proteins in relation to the different stages during the osteoblast differentiation.

Bone Morphogenetic Protein-2-induced Alkaline Phosphatase Expression Is Stimulated by Dlx5 and Repressed by Msx2

Alkaline phosphatase (ALP) is a widely accepted bone marker. Its expression is stimulated by bone morphogenetic protein (BMP)-2 treatment, the activation of BMP receptors and R-Smads, and the expression of Dlx5 and Runx2. However, how BMP-2 induces ALP expression is not clearly understood. We dissected the murine ALP promoter and found within it a Dlx5-binding cis-acting element by electrophoretic mobility shift assays and site-directed mutagenesis of the element. Dlx5 and the product of its target gene, Runx2, stimulated ALP promoter activity in an additive manner. However, because Dlx5 continued to stimulate ALP expression in Runx2–/– cells, the ALP stimulatory activity of Dlx5 is independent of Runx2. We also found that overexpression of Msx2 suppressed the mRNA level and enzyme activity of ALP that were induced by BMP-2 stimulation, and suppressed the Dlx5-stimulated ALP promoter activity by competing with Dlx5 for the cis-acting element in the ALP promoter. Moreover, Msx2 levels are constitutively high in C2C12 myogenic cells but decrease over time after BMP-2 treatment. This may explain why BMP-2 treatment of these cells results in immediate Dlx5 expression yet ALP expression commences only 1–2 days later. In other words, Msx2 in high levels counteracts initially the transcriptional activity of Dlx5 in low levels until a threshold Dlx5:Msx2 ratio is reached to the levels that allow the ALP stimulatory activity of Dlx5 to prevail. Thus, Dlx5 transactivates ALP expression, directly by binding to its cognate response element and/or indirectly by stimulating Runx2 expression, and Msx2 counteracts the direct transactivation of Dlx5.

Runx2 Regulates FGF2-Induced Bmp2 Expression During Cranial Bone Development

Calvarial bone is formed by the intramembranous bone‐forming process, which involves many signaling molecules. The constitutive activation of the fibroblast growth factor (FGF) signaling pathway accelerates osteoblast differentiation and results in premature cranial suture closure. Bone morphogenetic protein (BMP) signaling pathways, which involve the downstream transcription factors Dlx5 and Msx2, are also involved in the bone‐forming processes. However, the relationships between these two main signaling cascades are still unclear. We found that FGF2 treatment of developing bone fronts stimulated Bmp2 gene expression but that BMP2 treatment could not induce Fgf2 expression. Moreover, the disruption of the Runx2 gene completely eliminated the expression of Bmp2 and its downstream genes Dlx5 and Msx2 in the developing primordium of bone, while the expression of Fgf2 was maintained. In addition, cultured Runx2−/− cells expressed very low baseline levels of Bmp2 that were up‐regulated by transfection with a Runx2‐expressing plasmid. These levels in turn were markedly elevated by FGF2 treatment. FGF2 treatment also strongly enhanced the Bmp2 expression in MC3T3‐E1 cells, whose endogenous Runx2 gene is intact and which express Bmp2 at low baseline levels as well. These results indicate that Runx2 is an important mediator of the expression of Bmp2 in response to FGF stimulation in cranial bone development. Developmental Dynamics 233:115–121, 2005.

Differential expression patterns of Runx2 isoforms in cranial suture morphogenesis.

Runx2 (previously known as Cbfa1/Pebp2αA/AML3), a key transcription factor in osteoblast differentiation, has at least two different isoforms using alternative promoters, which suggests that the isoforms might be expressed differentially. Haploinsufficiency of the Runx2 gene is associated with cleidocranial dysplasia (CCD), the main phenotype of which is inadequate development of calvaria. In spite of the biological relevance, Runx2 gene expression patterns in developing calvaria has not been explored previously, and toward this aim we developed three probes: pRunx2, which comprises the common coding sequence of Runx2 and hybridizes with all isoforms; pPebp2αA, which specifically hybridizes with the isoform transcribed with the proximal promoter; and pOsf2, which hybridizes with the isoform transcribed with the distal promoter. These probes were hybridized with tissue sections of mouse calvaria taken at various time points in development. Runx2 expression was localized to the critical area of cranial suture closure, being found in parietal bones, osteogenic fronts, and sutural mesenchyme. Pebp2αA and Osf2 showed tissue‐specific expression patterns. The sites of Pebp2αA expression were almost identical to that of pRunx2 hybridization but expression was most intense in the sutural mesenchyme, where undifferentiated mesenchymal cells reside. The Osf2 isoform was strongly expressed in the osteogenic fronts, as well as in developing parietal bones, where osteopontin (OP) and osteocalcin (OC) also were expressed. However, in contrast to Pebp2αA, Osf2 expression did not occur in sutural mesenchyme. Pebp2αA also was expressed prominently in primordial cartilage that is found under the sutural mesenchyme and is not destined to be mineralized. Thus, Osf2 isoforms contribute to events later in osteoblast differentiation whereas the Pebp2αA isoform participates in a wide variety of cellular activities ranging from early stages of osteoblast differentiation to the final differentiation of osteoblasts.

Erk pathway and activator protein 1 play crucial roles in FGF2-stimulated premature cranial suture closure

Cranial sutures are an important growth center of the cranial bones, and the suture space must be maintained to permit the cranial adjustments needed to accommodate brain growth. Craniosynostosis, characterized by premature suture closure, mainly results from mutations that generate constitutively active fibroblast growth factor (FGF) receptors. FGF signaling, thus, is responsible for the pathogenesis of craniosynostosis. Even though FGF activates many different signaling pathways, the one involved in premature suture closure has not been defined. We observed that placing FGF2‐soaked bead on the osteogenic fronts of cultured mouse calvaria accelerates cranial suture closure and strongly induces the expression of osteopontin, an early marker of differentiated osteoblasts. FGF2 treatment also induced fos and jun mRNAs and later increased the nuclear levels of activator protein 1 (AP1). FGF2 stimulates the expression of osteopontin by inducing expression of AP1, which then binds to its response element in the osteopontin promoter. Blocking of the Erk pathway by PD98059 suppressed the AP1 and osteopontin expression stimulated by FGF2. Coincidently, blocking of the Erk pathway also significantly retarded FGF2‐accelerated cranial suture closure. Thus, the Erk pathway mediates FGF/FGF receptor–stimulated cranial suture closure, probably by stimulating synthesis of AP1 that then stimulates the differentiation of osteoblasts. Developmental Dynamics 227:335–346, 2003.

Effect of Rehmannia glutinosa Libosch extracts on bone metabolism

BACKGROUND Rehmannia glutinosa Libosch extracts (RGX) were investigated to determine if they play roles in bone metabolism. METHODS The effects on osteoblasts were determined by measuring (1) cell proliferation, (2) alkaline phosphatase (ALP) activity, (3) mRNA expression of bone-related proteins, (4) transcriptional activity of Runx2, and (5) osteoprotegerin (OPG) secretion. The effects on the osteoclasts were investigated by measuring (1) tartrate-resistant acid phosphatase-positive [TRAP(+)] multinucleated cell (MNC) formation and (2) resorption areas after culturing osteoclast precursors. Bone mineral density (BMD) measurements and histological observations on rats were also carried out. RESULTS RGX treatment showed a significant increase in both the proliferation and ALP activity of osteoblasts. RGX increased the expression of the bone-related genes. OPG secretion was markedly increased after RGX treatment. In addition, RGX treatment decreased the number of TRAP(+) MNCs and the resorption areas. In vivo studies using ovariectomy-induced osteoporotic rats revealed that RGX alleviated the decrease in the trabecular BMD, and increased the cortical bone thickness and trabeculation of the bone marrow spaces. CONCLUSIONS RGX stimulates the proliferation and activities of osteoblasts, while inhibiting the generation and resorptive activities of osteoclasts. It also shows preventive effects on osteoporotic bone loss induced by an ovariectomy. Although the active substances have not yet been identified, it is believed that the RGX seems to contain active components that have a potential to enhance the bone metabolism in osteoporosis.

Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by haploinsufficiency of the RUNX2 gene. In this study, we analyzed by direct sequencing RUNX2 mutations from eleven CCD patients. Four of seven mutations were novel: two nonsense mutations resulted in a translational stop at codon 50 (Q50X) and 112 (E112X); a missense mutation converted arginine to glycine at codon 131 (R131G); and an exon 1 splice donor site mutation (donor splice site GT/AT, IVS1 + 1G > A) at exon 1–intron junction resulted in the deletion of QA stretch contained in exon 1 of RUNX2. We focused on the functional analysis of the IVS1 + 1G > A mutation. A full‐length cDNA of this mutation was cloned (RUNX2Δe1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Δe1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Δe1 is similar to wild‐type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Δe1, assessed by in situ immunofluorescent staining, was observed with partial retention in both the nucleus and cytoplasm. This finding is in contrast to RUNX2 wild‐type, which is detected exclusively in the nucleus. DNA binding activity was also compromised by the RUNX2Δe1 in gel shift assay. Finally, RUNX2Δe1 blocked transactivation of the osteocalcin gene determined by transient transfection assay. Our findings demonstrate for the first time that the CCD phenotype can be caused by a splice site mutation, which results in the deletion of N‐terminus amino acids containing the QA stretch in RUNX2 that contains a previously unidentified second nuclear localization signal (NLS). We postulate that the QA sequence unique to RUNX2 contributes to a competent structure of RUNX2 that is required for nuclear localization, DNA binding, and transactivation function. J. Cell. Physiol. 207: 114–122, 2006.

Okadaic acid stimulates osteopontin expression through de novo induction of AP-1

Osteopontin, a major non‐collagenous bone matrix protein, is strikingly upregulated in various tissues under certain pathologic conditions, including cancer. However, the mechanism of upregulation of the osteopontin gene in tumor cells remains unclear. Okadaic acid, a strong non‐phorbol ester tumor promoter, is known to stimulate the expression of osteopontin. The aim of the present study was to understand the mechanism by which okadaic acid regulates osteopontin gene expression. Okadaic acid stimulated osteopontin mRNA expression in several cell lines within 3 h, and the increase in osteopontin mRNA was sustained for 24 h. New protein synthesis was required for the okadaic acid‐elicited increase in osteopontin mRNA expression. A serial promoter deletion study showed that the okadaic acid‐response element is located between positions −265 and −73, a sequence that includes the Runx2, Ets‐1, and AP‐1 binding sequences. Okadaic acid increased the mRNA expression of AP‐1 components but not of Runx2 or Ets‐1. Site‐directed mutagenesis and electrophoretic mobility shift assays confirmed that protein binding of the AP‐1 consensus sequence is necessary for the okadaic acid‐mediated osteopontin gene upregulation. These results indicate that de novo induction of the oncoprotein AP‐1 is required for okadaic acid‐stimulated osteopontin gene upregulation. J. Cell. Biochem. 87: 93–102, 2002.

Establishment and characterization of a stable cell line to evaluate cellular Runx2 activity

Runx2 is an essential transcription factor for osteoblast differentiation from early commitment step to final differentiation. Based on its crucial role in osteoblast differentiation, the transcriptional activity of Runx2 protein implies more valuable information for osteoblast differentiation than any other parameters, such as Runx2 mRNA or protein level. Thus, a sensitive, specific, and consistent method to determine the Runx2 transcriptional activity has long been expected. Here we suggest a stable cell line that carries 6xOSE2‐Luciferase reporter vector would be a good evaluation system to determine biological Runx2 transcriptional activity. The proliferation rate, cell shape, and the myogenic differentiation potential of the cloned cell line were similar to those of parental premyoblastic C2C12 cells. The cells specifically responded to Runx2 modulating agent such as FGF2. The stable cell line responded 5–6 folds more sensitively than the transiently transfected cells with Runx2. Though overexpression of any Runx gene stimulated the luciferase activity, Runx2 enhanced the reporter activity the highest. Collectively, the 6xOSE2‐luc stable cells would be a good biological evaluation system to assess the activity of extracellular Runx2 modulating stimulations as well as the signal transduction pathways involved in the stimulations.