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Patients with cleidocranial dysplasia (CCD) caused by mutations in RUNX2 have severe dental anomalies, including delayed or absent eruption of permanent teeth. This requires painful and expensive surgical/orthodontic intervention because of the absence of medicine for this condition. Here, we demonstrate that nicotinamide, a vitamin B3 and class III histone deacetylase inhibitor, significantly improves delayed tooth eruption in Runx2 +/- mice, a well-known CCD animal model, through the restoration of decreased osteoclastogenesis. We also found that Csf1 mRNA and protein levels were significantly reduced in Runx2 +/- osteoblasts as compared with wild type whereas RANKL and OPG levels had no significant difference between wild type and Runx2 +/- osteoblasts. The nicotinamide-induced restoration of osteoclastogenesis of bone marrow-derived macrophages in Runx2 +/- mice was due to the increased expression of RUNX2 and CSF1 and increased RANKL/OPG ratio. RUNX2 directly regulated Csf1 mRNA expression via binding to the promoter region of the Csf1 gene. In addition, nicotinamide enhanced the RUNX2 protein level and transacting activity posttranslationally with Sirt2 inhibition. Taken together, our study shows the potential and underlying molecular mechanism of nicotinamide for the treatment of delayed tooth eruption by using the Runx2 +/- murine model, suggesting nicotinamide as a candidate therapeutic drug for dental abnormalities in patients with CCD.

Gain-of-function mutations in fibroblast growth factor receptors (FGFRs) cause congenital skeletal anomalies, including craniosynostosis (CS), which is characterized by the premature closure of craniofacial sutures. Apert syndrome (AS) is one of the severest forms of CS, and the only treatment is surgical expansion of prematurely fused sutures in infants. Previously, we demonstrated that the prolyl isomerase peptidyl-prolyl cis-trans isomerase interacting 1 (PIN1) plays a critical role in mediating FGFR signaling and that Pin1+/- mice exhibit delayed closure of cranial sutures. In this study, using both genetic and pharmacological approaches, we tested whether PIN1 modulation could be used as a therapeutic regimen against AS. In the genetic approach, we crossbred Fgfr2S252W/+, a mouse model of AS, and Pin1+/- mice. Downregulation of Pin1 gene dosage attenuated premature cranial suture closure and other phenotypes of AS in Fgfr2S252W/+ mutant mice. In the pharmacological approach, we intraperitoneally administered juglone, a PIN1 enzyme inhibitor, to pregnant Fgfr2S252W/+ mutant mice and found that this treatment successfully interrupted fetal development of AS phenotypes. Primary cultured osteoblasts from Fgfr2S252W/+ mutant mice expressed high levels of FGFR2 downstream target genes, but this phenotype was attenuated by PIN1 inhibition. Post-translational stabilization and activation of Runt-related transcription factor 2 (RUNX2) in Fgfr2S252W/+ osteoblasts were also attenuated by PIN1 inhibition. Based on these observations, we conclude that PIN1 enzyme activity is important for FGFR2-induced RUNX2 activation and craniofacial suture morphogenesis. Moreover, these findings highlight that juglone or other PIN1 inhibitors represent viable alternatives to surgical intervention for treatment of CS and other hyperostotic diseases.

Background: The standard sunitinib schedule, 4 weeks on, followed by 2 weeks off (4/2 schedule), is associated with troublesome toxicities, and maintenance of adequate sunitinib dosing and drug levels, which are essential for achieving an optimal treatment outcome, is challenging. The objective of this study was to investigate the efficacy and safety of an alternative sunitinib dosing schedule of 2 weeks on and 1 week off (2/1 schedule) compared with the standard sunitinib schedule of 4 weeks on and 2 weeks off (4/2 schedule)., Patients and Methods: In this multicenter, randomized, open-label, phase II trial, treatment-naïve patients with clear-cell type metastatic renal cell carcinoma (mRCC) were randomly assigned to 4/2 or 2/1 schedules after stratification by Memorial Sloan Kettering Cancer Center risk group and the presence or absence of measurable lesions. The primary end point was the 6-month failure-free survival (FFS) rate, determined by intention-to-treat analysis., Results: From November 2007 to February 2014, 76 patients were accrued, and 74 were eligible. FFS rates at 6 months were 44% with the 4/2 schedule (N = 36) and 63% with the 2/1 schedule (N = 38). Neutropenia (all grades, 61% versus 37%; grade 3-4, 28% versus 11%) and fatigue (all grades, 83% versus 58%) were more frequently observed with schedule 4/2. There was a strong tendency toward a lower incidence of stomatitis, hand-foot syndrome, and rash with schedule 2/1. Objective response rates (ORRs) were 47% in schedule 2/1 and 36% in schedule 4/2. With a median follow-up of 30.0 months, the median time to progression (TTP) was 12.1 months in schedule 2/1 and 10.1 months in schedule 4/2., Conclusion: Sunitinib administered with a 2/1 schedule is associated with less toxicity and higher FFS at 6 months than a 4/2 schedule, without compromising the efficacy in terms of ORR and TTP (NCT00570882)., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: This study explored the impact of genetic polymorphisms in cytochrome P450 (CYP) enzymes and transporters on the plasma trough concentration of imatinib mesylate (IM) and clinical response in chronic myeloid leukemia (CML)., Patients and Methods: In total, 82 patients with CML who had been administered 400 mg IM daily for over 6 months were genotyped for 11 single-nucleotide polymorphisms in nine genes (CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP2D6, ABCB1, SLC22A1, SLC22A2 and ABCG2) using blood samples. The trough imatinib concentration and clinical responses were assessed 6 months after the initiation of IM therapy., Results: The CC, CA and AA genotypes in ABCG2 421C>A gave significantly different frequencies for the major molecular response (MMR) (P = 0.02). However, no significant differences were found between the genotypes of the CYP enzymes and transporters identified in this study and the imatinib plasma trough concentrations and clinical response frequencies, except for the correlation of ABCG2 with MMR., Conclusions: The results of the present study may indicate that the ABCG 421C>A genetic polymorphism influences the MMR of imatinib in patients with CML.

Previous studies have shown that histone deacetylase (HDAC) inhibitors stimulate osteoblast differentiation in vitro and bone formation in vivo. However, the effects of HDAC inhibitors on odontoblasts have not been elucidated. Therefore, in this study, we examined the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on odontoblast differentiation using an MDPC23 odontoblast-like cell line. SAHA significantly enhanced matrix mineralization and the expression levels of odontoblast marker genes. SAHA increased the expression levels of nuclear factor I/C (Nfic) and dentin sialophosphoprotein (Dspp). Nfic bound directly to the Dspp promoter and stimulated Dspp transcription. SAHA increased both basal and Nfic-induced Dspp promoter activity. SAHA-induced Dspp promoter activity disappeared when mutations were introduced within the Nfic binding element of the Dspp promoter. Nfic knockdown by siRNA blocked SAHA stimulation of Dspp expression. These results indicate that SAHA enhances odontoblast differentiation and that SAHA increases Dspp expression, at least in part, by increasing the expression level of Nfic.

Previous linkage analysis of an Asian population proposed possible candidate genes for mandibular prognathism, such as Matrilin-1 (cartilage matrix protein). To investigate the association between the single-nucleotide polymorphisms (SNPs) in Matrilin-1 and mandibular prognathism, we investigated three sequence variants (-158 T>C, 7987 G>A, 8572 C>T) in 164 mandibular prognathism patients and 132 control individuals with a normal occlusion. The results showed that the 8572 TT genotypes in Matrilin-1 showed increased risk of mandibular prognathism (OR = 9.28, 95% Cl = 1.19~197.57, P < 0.05), whereas the 7987 AA genotype showed a protective effect for mandibular prognathism (OR = 0.16, 95% Cl = 0.05~0.47, P < 0.05). Genotyping results showed that the Matrilin-1 polymorphism haplotype TGC (ht4; 158T, 7987G, and 8572C alleles) had a pronounced risk effect for mandibular prognathism compared with controls (OR = 5.16, 95% Cl = 2.03~13.93, P < 0.01). The results suggest that polymorphisms in Matrilin-1 could be used as a marker for genetic susceptibility to mandibular prognathism.

Objective: Mutations in the RUNX2 gene, a master regulator of bone formation, have been identified in cleidocranial dysplasia (CCD) patients. CCD is a rare autosomal-dominant disease characterized by the delayed closure of cranial sutures, defects in clavicle formation, and supernumerary teeth. The purposes of this study were to identify genetic causes of two CCD nuclear families and to report their clinical phenotypes., Materials and Methods: We identified two CCD nuclear families and performed mutational analyses to clarify the underlying molecular genetic etiology., Results: Mutational analysis revealed a novel nonsense mutation (c.273T>A, p.L93X) in family 1 and a de novo missense one (c.673C>T, p.R225W) in family 2. Individuals with a nonsense mutation showed maxillary hypoplasia, delayed eruption, multiple supernumerary teeth, and normal stature. In contrast, an individual with a de novo missense mutation in the Runt domain showed only one supernumerary tooth and short stature., Conclusions: Mutational and phenotypic analyses showed that the severity of mutations on the skeletal system may not necessarily correlate with that of the disruption of tooth development.

The present study was conducted to evaluate the efficacy and safety of a combination regimen of docetaxel plus oxaliplatin in patients with advanced gastric cancer. Patients with previously untreated metastatic or recurrent, measurable gastric cancer received intravenous docetaxel 65 mg m(-2) plus oxaliplatin 120 mg m(-2) on day 1 based on a 3-week cycle. Forty-two patients were enrolled in the current study, among whom 39 were assessable for efficacy and all assessable for toxicity. One complete response and 18 partial responses were confirmed, giving an overall response rate of 45.2% (95% confidence interval (CI); 31.7-59.7%). At a median follow-up of 7.7 months, the median time to progression and median overall survival was 5.7 (95% CI; 4.3-7.2) months and 9.9 (95% CI; 7.8-12.0) months, respectively. Grade 3/4 neutropenia occurred in 11 patients (26.1%) and febrile neutropenia was observed in four patients (9.5%). The common non-haematologic toxicity was fatigue (grade 1/2, 61.9%) and nausea (grade 1/2, 47.7%). The combination of docetaxel and oxaliplatin was found to be well tolerated and effective in patients with advanced gastric cancer.

Objective: Disruption of the vascular supply to the bone and subsequent hypoxia has been implicated in the pathogenesis of osteonecrosis (ON) of the femoral head (ONFH). To evaluate the genetic effect of HIF1alpha, a key transcription factor in controlling hypoxia condition, on ONFH, we analyzed HIF1alpha polymorphism and its genetic association with ONFH., Methods: We directly sequenced the HIF1alpha gene in 24 Korean individuals and identified four sequence variants. Four polymorphisms (-2755C>A, +41224T>C, +45319C>T, +51610C>T) were genotyped in ONFH (n=384). ONFH patients were divided into three subgroups based on etiological factors: idiopathic (129 cases), steroid (59 cases) and alcohol (196 cases) ON groups., Results: We found that the allele frequency of -2755C>A and the genotype frequencies of +41224T>C and +51610C>T were significantly associated with idiopathic ONFH in men (P=0.0409, 0.0113, 0.0269, respectively). In addition, haplotype (CTCC) of HIF1alpha was also significantly associated with idiopathic ONFH in men (P=0.017)., Conclusions: We found that HIF1alpha polymorphisms are associated with idiopathic ONFH in men. These results suggest that variations in HIF1alpha may play an important role in the pathogenesis and risk factor for ONFH.

This study intended to evaluate the morphological characteristics of the cranial base and maxillomandibular structures of facial asymmetry in adult patients, so as to determine whether mandibular asymmetry is a result of primary mandibular deformity or if it is influenced by cranial base deformity. Forty-two adult patients with dentofacial deformity were placed into two groups according to the deviation of the chin: Asymmetry group (n = 24) and Non-asymmetry group (n = 18). They were compared with three-dimensional (3D) CT reformatted images using a 3D visualization and analyzing program. The differences between the two groups, the correlation between the cranial base and maxillomandibular asymmetry were evaluated statistically. The degree of cranial base asymmetry in the Asymmetry group was not statistically different from the Non-asymmetry group. The asymmetric condyle position was found to be associated with skull base characteristics. The 3D position of the condyle and cranial base, however, was not closely related with mandibular asymmetry. The results showed that the cranial measurement variables were not the dominant factors that determined the degree of facial asymmetry. It seems that the mandibular skeletal factors itself, functional or intrinsic asymmetric growth potential, compensate or aggravate the influence of cranial asymmetry during the growth period.

Runx2 (previously known as Cbfal/Pebp2alphaA/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; pPebp2alphaA, 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. Pebp2alphaA and Osf2 showed tissue-specific expression patterns. The sites of Pebp2alphaA 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 Pebp2alphaA, Osf2 expression did not occur in sutural mesenchyme. Pebp2alphaA 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 Pebp2alphaA isoform participates in a wide variety of cellular activities ranging from early stages of osteoblast differentiation to the final differentiation of osteoblasts.

This study was conducted to verify whether the response to high-dose intravenous immune globulin (IVIG) was related to the effect of splenectomy in chronic idiopathic thrombocytopenic purpura (ITP) patients. A total of 79 patients over 16 years of age were enrolled in this study. The response to the treatment was classified on the basis of the platelet count as no response (NR, <50 x 10(9)/l), incomplete response (IR, (50-150) x 10(9)/l), and complete response (CR, >150 x 10(9)/l). The response was evaluated after the infusion of high-dose IVIG, within 2 weeks after splenectomy (immediate response), and during a follow-up period of more than 6 months after splenectomy (sustained response), respectively. 58 patients (73.4%) showed responses (CR or IR) to high-dose IVIG. After splenectomy, immediate responses were observed in 73 patients (92%). The response to high-dose IVIG had no relationship with the immediate response to splenectomy (P = 0.333). A follow-up evaluation was possible with 58 patients; 6 patients with NR in immediate responses did not show any response during the follow-up period, and 17 patients relapsed within 6 months after immediate responses, so 35 patients (60.3%) had sustained responses. Responders to IVIG had significantly higher sustained response rates to splenectomy than non-responders (62% vs. 38%, P = 0.001). These results indicate that the response to high-dose IVIG could be a valuable factor predicting the sustained response to splenectomy in chronic ITP patients.

When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

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/PEBP2alphaA/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-beta1 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 Cbfal 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.

Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx-1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx-2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain-binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.

Fetal rat calvarial-derived osteoblasts in vitro (ROB) reinitiate a developmental program from growth to differentiation concomitant with production of a bone tissue-like organized extracellular matrix. To identify novel genes which may mediate this sequence, we isolated total RNA from three stages of the cellular differentiation process (proliferation, extracellular matrix maturation, and mineralization), for screening gene expression by the differential mRNA display technique. Of 15 differentially displayed bands that were analyzed by Northern blot analysis, one prominent 310 nucleotide band was confirmed to be proliferation-stage specific. Northern blot analysis showed a 600-650 nt transcript which was highly expressed in proliferating cells and decreased to trace levels after confluency and throughout the differentiation process. We have designated this transcript PROM-1 (for proliferating cell marker). A full length PROM-1 cDNA of 607 bp was obtained by 5' RACE. A short open reading frame encoded a putative 37 amino acid peptide with no significant similarity to known sequences. Expression of PROM-1 in the ROS 17/2.8 osteosarcoma cell line was several fold greater than in normal diploid cells and was not downregulated when ROS 17/2.8 cells reached confluency. The relationship of PROM-1 expression to cell growth was also observed in diploid fetal rat lung fibroblasts. Hydroxyurea treatment of proliferating osteoblasts blocked PROM-1 expression; however, its expression was not cell cycle regulated. Upregulation of PROM-1 in response to TGF-beta paralleled the stimulatory effects on growth as quantitated by histone gene expression. In conclusion, PROM-1 represents a small cytoplasmic polyA containing RNA whose expression is restricted to the exponential growth period of normal diploid cells; the gene appears to be deregulated in tumor derived cell lines.

Okadaic acid, a potent specific inhibitor of serine/threonine protein phosphatases type 1 and 2A, affects several gene expressions in various cells. To determine whether okadaic acid affects the expression of fibronectin in MC3T3-E1 cells, we measured mRNA level and synthesis of fibronectin by Northern blot hybridization and immunoprecipitation methods, respectively. Okadaic acid (10-50 ng/ml) increased both mRNA level and synthesis of fibronectin in a dose-dependent manner. The increase of fibronectin mRNA by okadaic acid was strongly attenuated by the inhibition of new protein synthesis. The results indicate that okadaic acid, inhibitor of protein phosphatases, increases fibronectin synthesis in MC3T3-E1 cells.

We examined the role of the protein kinase C (PKC) signaling pathway in the stimulation of fibronectin synthesis in both normal and transformed human lung fibroblasts. Phorbol myristate acetate (PMA), a potent PKC activator, stimulated fibronectin synthesis in both normal and transformed fibroblasts in a time and dose dependent fashion. Down-regulation of PKC by prior exposure of cells to a high concentration of PMA blocked the increase in fibronectin synthesis and mRNA levels induced by PMA. Bisindolylmaleimide, a specific inhibitor of PKC, also abolished the PMA-induced fibronectin synthesis. 4 alpha-phorbol didecanoate, an inactive phorbol ester, failed to affect fibronectin synthesis. These data suggest that PMA stimulates fibronectin synthesis and gene expression through the PKC signaling pathway in both normal and transformed human lung fibroblasts.

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-beta 1 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-beta 1, 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.

Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.