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Osteoporosis is one of the most prevalent diseases and the number of patients suffering from this disease is soaring due to the increase in the aged population in the world. The severity of bone loss in osteoporosis is based on the levels of impairment in the balance between bone formation and bone resorption, two arms of the bone metabolism, and bone remodeling. However, determination of bone formation levels is under many layers of control that are as yet fully defined. Bone morphogenetic protein (BMP) plays a key role in regulation of bone formation while its downstream targets are still incompletely understood. Lgr4 gene encodes an orphan receptor and has been identified as a genetic determinant for bone mass in osteoporotic patients. Here, we examine the effects of BMP on the expression of Lgr4 in osteoblastic cells. Lgr4 gene is expressed in an osteoblastic cell line, MC3T3E1 in a time dependent manner during the culture. BMP treatment enhances Lgr4 mRNA expression at least in part via transcriptional event. When Lgr4 mRNA is knocked down, the levels of BMP-induced increase in alkaline phosphatase (Alp) activity and Alp mRNA are suppressed. BMP enhancement of Lgr4 gene expression is suppressed by FGF and reversed by dexamethasone. BMP also enhances Lgr4 expression in primary cultures of calvarial osteoblasts. These data indicate that Lgr4 gene is regulated by BMP and is required for BMP effects on osteoblastic differentiation. J. Cell. Physiol. 231: 887-895, 2016. © 2015 Wiley Periodicals, Inc.

Gene transduction of exogenous factors at local sites in vivo is a promising approach to promote regeneration of tissue defects owing to its simplicity and capacity for expression of a variety of genes. Gene transduction by viral vectors is highly efficient; however, there are safety concerns associated with viruses. As a method for nonviral gene transduction, plasmid DNA delivery is safer and simpler, but requires an efficient carrier substance. Here, we aimed to develop a simple, efficient method for bone regeneration by gene transduction and to identify optimal conditions for plasmid DNA delivery at bone defect sites. We focused on carrier substances and compared the efficiencies of two collagen derivatives, atelocollagen, and gelatin hydrogel, as substrates for plasmid DNA delivery in vivo. To assess the efficiencies of these substrates, we examined exogenous expression of green fluorescence protein (GFP) by fluorescence microscopy, polymerase chain reaction, and immunohistochemistry. GFP expression at the bone defect site was higher when gelatin hydrogel was used as a substrate to deliver plasmids than when atelocollagen was used. Moreover, the gelatin hydrogel was almost completely absorbed at the defect site, whereas some atelocollagen remained. When a plasmid harboring bone morphogenic protein 2 was delivered with the substrate to bony defect sites, more new bone formation was observed in the gelatin group than in the atelocollagen group. These results suggested that the gelatin hydrogel was more efficient than atelocollagen as a substrate for local gene delivery and may be a superior material for induction of bone regeneration.

Sclerostin (SOST), a member of the cystine-knot superfamily, is essential for proper skeletogenesis because a loss-of-function mutation in the SOST gene results in sclerosteosis featured with massive bone growth in humans. To understand the function of SOST in developmental skeletal tissue formation, we examined SOST gene expression in embryonic osteogenesis in vitro and in vivo. During osteoblastic differentiation in primary calvarial cells, the levels of SOST expression were increased along with those of alkaline phosphatase activity and nodule formation. In situ hybridization study revealed that SOST mRNA expression was observed in the digits in embryonic 13-d limb buds, and SOST expression was observed in osteogenic front in embryonic 16.5-d postcoitus embryonic calvariae, and this expression persisted in the peripheral area of cranial bone in the later developmental stage (embryonic 18.5-d post coitum). These temporal and spacial expression patterns in vivo and in vitro were in parallel to those of osterix (Osx), which is a critical transcriptional factor for bone formation. Similar coexpression of SOST and Osx mRNA was observed when the primary osteoblastic calvarial cells were cultured in the presence of bone morphogenetic protein (BMP)2 in vitro. Moreover, endogenous expression of SOST and Osx mRNA was inhibited by infection of noggin-expression adenovirus into the primary osteoblastic calvarial cells, suggesting that endogenous BMPs are required for these cells to express SOST and Osx mRNA. Thus, expression and regulation of SOST under the control of BMP were closely associated with those of Osx in vivo and in vitro.

Osterix is a recently identified zinc-finger-containing transcription factor, which is required for skeletogenesis as no bone formation was observed in osterix-deficient mice. Osterix was first cloned as a gene whose expression was enhanced by BMP in C2C12 cells. As BMP induces ectopic bone formation in vivo via a pathway reminiscent to endochondral bone formation, BMP may also regulate osterix gene expression in chondrocytes. However, no information was available regarding the BMP actions on osterix gene expression in chondrocytes. We therefore examined the effects of BMP-2 on osterix gene expression in chondrocytes in culture. RT-PCR analysis indicated that osterix mRNA was expressed in the primary cultures of chondrocytes derived from mouse rib cartilage. The treatment with BMP-2 enhanced the levels of osterix transcripts within 24 h and the enhancement was still observed at 48 h based on RT-PCR analysis. This BMP effect was specific to this cytokine, as TGF-β did not alter osterix gene expression. BMP effects on the osterix mRNA levels were also confirmed by Northern blot analysis. The enhancing effect of BMP on osterix gene expression was observed in a dose-dependent manner starting at 200 ng/ml. The BMP enhancement of the osterix gene expression in chondrocytes was blocked in the presence of a protein synthesis inhibitor, cycloheximide, while it was still observed in the presence of 5,6-dichloro-1-β D-ribofuranosylbenzimidazol (DRB) suggesting the involvement of post-transcriptional events, which require new protein synthesis. These results indicated that osterix gene is expressed in the primary cultures of chondrocytes and its expression is under the control of BMP-2. J. Cell. Biochem. 88: 1077–1083, 2003. © 2003 Wiley-Liss, Inc.

Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages, Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix-loop-helix (bHLH) type transcription factor. is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG-box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways, To address this point, we investigated scleraxis and Sox9 rnRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons, In the early stage of mesenchymal tissue development at 10.5 d.p.c., scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c.. scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage, scleraxis expression was closely associated with, but distinct from, formation of skeletal primordia, At 13.5 d.p.c., scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then. at 15.5 d.p.c., scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of scleraxis expression during embryonic development of tendon precursor cells in close association with that of So,0 expression in chondrogenic cells in skeletal tissues. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

SOX9 is a transcription factor that plays a key role in chondrogenesis. Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated. TC6 is a clonal chondrocytic cell line derived from articular cartilage. The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by approximately 3-fold the transcriptional activity of the AgCAT-8 construct containing 8-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron. In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines. Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells.

Vitamin D promotes differentiation of cells either by simply enhancing phenotypic gene expression and/or by suppressing expression of inhibitors of differentiation. Previously, we reported that expression of a gene encoding Id1, a negative type helix-loop-helix transcription factor, was transcriptionally suppressed by 1,25-dihydroxyvitamin D3(1,25(OH)2D3) (1). To identify the sequence required for the negative regulation by 1,25(OH)2D3, a 1.5-kilobase 5′-flanking region of murine Id1 gene was examined by transiently transfecting luciferase reporter constructs into ROS17/2.8 osteoblastic cells. The transcriptional activity of this construct was repressed by 10−8 m 1,25(OH)2D3. Deletion analysis revealed that a 57-base pair (bp) upstream response sequence (URS) (−1146/−1090) was required for the suppression by 1,25(OH)2D3. This sequence conferred negative responsiveness to 1,25(OH)2D3 to a heterologous SV40 promoter. The 57-bp URS contained not only Egr-1 consensus sequence (2) but also four direct repeats of a heptamer sequence (C/A)CAGCCC. Electrophoresis mobility shift assay revealed that the 57-bp URS formed specific nuclear protein-DNA complexes, which were neither competed by previously known positive and negative vitamin D response elements nor supershifted by anti-vitamin D receptor antibody, suggesting the absence of vitamin D receptor in these complexes. These results indicate the involvement of the novel 57-bp sequence in the vitamin D suppression of Id1 gene transcription.

Cell differentiation is determined by a certain set of transcription factors such as MyoD in myogenesis. However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis is a helix-loop-helix-type transcription factor whose transcripts are expressed in sclerotome and in a certain set of skeletal cells; however, nothing is known about its function with regard to the regulation of cell function. To examine possible roles of scleraxis, we overexpressed scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of scleraxis. Scleraxis overexpression enhanced expression of the aggrecan gene, which is not normally expressed at high levels in these osteoblastic cells. Overexpression of scleraxis also increased mRNA levels of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkaline phosphatase. Transient transfection experiments indicated that scleraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fragment of the aggrecan gene including both the promoter and its first intron. Deletion analysis identified a 1-kb region that is responsive to scleraxis within the aggrecan gene. This region contains two adjacent E-box sequences. A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extracts as well as to in vitro translated scleraxis. This binding was competed with unlabeled AgE, but not with a mutated E-box DNA sequence (mAgE), indicating the specificity of the binding activity. The AgE binding activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing scleraxis and was supershifted by the antiserum raised against scleraxis. Furthermore, AgE, but not mAgE, conferred responsiveness to scleraxis overexpression to a heterologous promoter. Finally, replacement mutation of the AgE sequence within the 2.5-kb AgCAT-1 construct significantly reduced its responsiveness to scleraxis. These results indicate that overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression via binding to the E-box-containing AgE sequence in ROS17/2.8 cells.

Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of scleraxis mRNA in rat osteoblastic cells and found that the scleraxis gene was expressed as a 1·2 kb mRNA species in osteoblastic osteosarcoma ROS 17/2·8 cells. The scleraxis mRNA expression was enhanced by type-β transforming growth factor (TGFβ) treatment. The TGFβ effect was observed in a dosedependent manner starting at 0·2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGFβ effect was blocked by cycloheximide, while no effect on scleraxis mRNA stability was observed. TGFβ treatment enhanced scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2·8 cells. Furthermore, TGFβ enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGFβ treatment also enhanced scleraxis gene expression in osteoblastenriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helixloop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGFβ. Journal of Endocrinology (1996) 151, 491–499

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.

Tissue-nonspecific-type alkaline phosphatase (TNSALP) is found in the bone, liver, kidney, and other tissues, and its gene consists of 12 exons with the coding sequence beginning in the second exon. Recently, a noncoding first exon was identified in the liver message (liver type) which differed from that of the previously known osteoblast-derived cDNA sequence (bone type). Although these two mRNAs produce an identical protein, they have different promoter regions. It is known that ALPs in dental pulp and periodontal ligament are classified into TNSALP by their enzymatic and immunological properties, but little is known about the expression of ALP mRNAs and the transcriptional mechanisms. In order to examine the expression of their mRNA type, specific oligonucleotide primers corresponding to two types of mRNAs of human TNSALP were designed and amplified by reverse transcription-polymerase chain reaction (RT-PCR). It was found that bone-type mRNA was expressed in the human dental tissues such as dental pulp, periodontal ligament, and dental sac, whereas liver-type mRNA was not expressed. Thus, it was concluded that the human dental tissues express the bone-type isozymes and are regulated by the same transcriptional mechanism as in the bone.

Regulation of alpha 2(I) collagen and osteonectin/SPARC gene expressions in mouse osteoblastic MC3T3-E1 cells by TGF-beta 1 was studied. TGF-beta 1 regulated those differentiation markers of osteoblast phenotypes, although the effects were inconsistent depending on serum concentrations. Exposure of MC3T3-E1 cells to TGF-beta 1 in low serum culture caused stimulation of alpha 2(I) collagen and osteonectin expressions but no stimulation in high serum culture. These inconsistent responses may be due to the presence of some component(s) in serum. bFGF also suppressed stimulation by TGF-beta 1 on alpha 2(I) collagen and osteonectin expressions. Serum factors as well as locally derived growth factors may interact with TGF-beta 1 and modulate the actions of TGF-beta 1 in osteoblastic cells. The present result explains the complexity of TGF-beta 1 actions on bone cells in vitro and also suggests that the actions on bone formation are modified by other factors in vivo.

Osteoporosis due to unloading-induced bone loss is a critical issue in the modern aging society. Although the mechanisms underlying this phenomenon are largely unknown, osteopontin (OPN) is one of the critical mediators required for unloading-induced bone loss [M. Ishijima, S.R. Rittling, T. Yamashita, K. Tsuji, H. Kurosawa, A. Nifuji, D.T. Denhardt, and M. Noda, Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin, J Exp Med, 193 (2001) 399-404]. To clarify the molecular bases for OPN actions, we carried out microarray analyses on the genes expressed in the femoral bone marrow cells in wild type and OPN−/− mice. The removal of the mechanical load induced bone loss in wild type, but not in OPN−/− mice, as previously reported. Expression analysis of 9586 cDNAs on a microarray system revealed that OPN deficiency blocked tail-suspension-induced expression of ten genes (group A). This observation was confirmed based on semi-quantitative RT-PCR analyses. On the other hand, expression of four genes (group B) was not altered by tail suspension in wild type but was enhanced in OPN-deficient mice. NF-κB p105 subunit gene (Nfkb1) was found in group A and Bax in group B. p53 gene expression was upregulated by tail suspension in wild type mice, but it was no longer observed in OPN−/− mice. These data indicate that OPN acts to mediate mechanical stress signaling upstream to the genes encoding apoptosis-related molecules, and its action is associated with alteration of the genes.

SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared from rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox- consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes chondrocyte differentiation through enhancement of SOX9.

Osteoblasts are the cells which form bone under the regulation not only by hormones and cytokines but also by ECM molecules via their attachment. To obtain insights into the role of intracellular signaling molecules operating to mediate the attachment-related regulation of osteoblastic functions, we investigated in osteoblast-like MC3T3E1 cells the effects of the overexpression of CIZ, a novel signaling protein which interacts with p130Cas. In MC3T3E1 cells, CIZ mRNA is expressed constitutively. Endogenous CIZ was localized in the MC3T3E1 cells with relatively high levels of accumulation at the attachment sites when the cells were cultured on fibronectin, collagen, or BSA. CIZ overexpression increased the number of adhesion plaques and reduced proliferation of the cells compared to that of control cells transfected with an empty vector. Furthermore, CIZ overexpression enhanced type I collagen mRNA expression, the most abundant constituent of bone matrix and a major product of osteoblasts. Analysis of the promoter region of type I collagen gene identified the presence of a consensus CIZ-binding sequence, which indeed conferred responsiveness to CIZ overexpression to a heterologous promoter. These data indicate that CIZ acts as a novel regulatory molecule in controlling osteoblastic function.

Core-binding factor A1 (Cbfa1), also called Pebp2 alpha A/AML3, is a transcription factor that belongs to the runt-domain gene family. Cbfa1-deficient mice are completely incapable of both endochondral and intramembranous bone formation, indicating that Cbfa1 is indispensable for osteogenesis. Maturation of chondrocytes in these mice is also disorganized, suggesting that Cbfa1 may also play a role in chondrogenesis. The aim of this study was to examine the expression and regulation of Pebp2 alpha A/AML3/Cbfa1 expression in the chondrocyte-like cell line, TC6. Northern blot analysis indicated that Cbfa1 mRNA was constitutively expressed as a 6.3 kb message in TC6 cells and the level of Cbfa1 expression was enhanced by treatment with bone morphogenetic protein-2 (BMP2) in a time- and dose-dependent manner. This effect was blocked by an RNA polymerase inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, but not by a protein synthesis inhibitor, cycloheximide. Western blot analysis of the cell lysates using polyclonal antibody raised against Cbfa1 indicated that BMP2 treatment increased the Cbfa1 protein level in TC6 cells. In TC6 cells, BMP2 treatment enhanced expression of alkaline phosphatase and type I collagen mRNAs but suppressed that of type II collagen mRNA. In addition to TC6 cells, Cbfa1 mRNA was also expressed in primary cultures of chondrocytes and BMP2 treatment enhanced Cbfa1 mRNA expression in these cells similarly to its effect on TC6 cells. These data indicate that the Pebp2 alpha A/AML3/Cbfa1 gene is expressed in a chondrocyte-like cell line, TC6, and its expression is enhanced by treatment with BMP.