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PubMed1991

Transcriptional activation through ETS domain binding sites in the cytochrome c oxidase subunit IV gene.

A mutational analysis of the rat cytochrome c oxidase subunit IV (RCO4) promoter region revealed the presence of a major control element consisting of a tandemly repeated pair of binding sites for a nuclear factor from HeLa cells. This factor was designated NRF-2 (nuclear respiratory factor 2) because a functional recognition site was also found in the human ATP synthase beta-subunit gene. Deletion or site-directed point mutations of the NRF-2 binding sites in the RCO4 promoter resulted in substantial loss of transcriptional activity, and synthetic oligomers of the NRF-2 binding sites from both genes stimulated a heterologous promoter when cloned in cis. NRF-2 binding and transcriptional activation required a purine-rich core sequence, GGAA. This motif is characteristic of the recognition site for a family of activators referred to as ETS domain proteins because of the similarity within their DNA-binding domains to the ets-1 proto-oncogene product. NRF-2 recognized an authentic Ets-1 site within the Moloney murine sarcoma virus long terminal repeat, and this site was able to compete for NRF-2 binding to the RCO4 promoter sequence. In addition, a single polypeptide of 55 kDa was detected following cross-linking of a partially purified NRF-2 fraction to RCO4, the human ATP synthase beta subunit, or Moloney murine sarcoma virus binding sites. However, in contrast to Ets-1, which appears to be exclusive to lymphoid tissues, NRF-2 has the broad tissue distribution expected of a regulator of respiratory chain expression.

Animals↗
PubMed2004

Structure of the archaeal translation initiation factor aIF2 beta from Methanobacterium thermoautotrophicum: implications for translation initiation.

aIF2 beta is the archaeal homolog of eIF2 beta, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met-tRNA(i)(Met) to the 40S ribosomal subunit. We have determined the solution structure of the intact beta-subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2 beta is composed of an unfolded N terminus, a mixed alpha/beta core domain and a C-terminal zinc finger. NMR data shows the two folded domains display restricted mobility with respect to each other. Analysis of the aIF2 gamma structure docked to tRNA allowed the identification of a putative binding site for the beta-subunit in the ternary translation complex. Based on structural similarity and biochemical data, a role for the different secondary structure elements is suggested.

Amino Acid Sequence↗
PubMed2005

EBF2 regulates osteoblast-dependent differentiation of osteoclasts.

Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts.

Animals↗
PubMed2003

Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation.

Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, we demonstrate a novel function of the Stat1 transcription factor in the regulation of bone remodeling. In the bone of the Stat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-beta. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. Our study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling.

Animals↗
PubMed2007

Oxysterol-induced osteoblastic differentiation of pluripotent mesenchymal cells is mediated through a PKC- and PKA-dependent pathway.

Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in circulation, and in human and animal tissues. The discovery of osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblastic cells and therefore enhance bone formation is crucial for better management of bone fractures and osteoporosis. We previously reported that specific oxysterols have potent osteoinductive properties and induce the osteoblastic differentiation of pluripotent mesenchymal cells. In the present report we demonstrate that the induction of osteoblastic differentiation by oxysterols is mediated through a protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanism(s). Furthermore, oxysterol-induced-osteoblastic differentiation is marked by the prolonged DNA-binding activity of Runx2 in M2-10B4 bone marrow stromal cells (MSCs) and C3H10T1/2 embryonic fibroblastic cells. This increased activity of Runx2 is almost completely inhibited by PKC inhibitors Bisindolylmaleimide and Rottlerin, and only minimally inhibited by PKA inihibitor H-89. PKC- and PKA-dependent mechanisms appear to also regulate other markers of osteoblastic differentiation including alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in response to oxysterols. Finally, osteogenic oxysterols induce osteoblastic differentiation with BMP7 and BMP14 in a synergistic manner as demonstrated by the enhanced Runx2 DNA-binding activity, ALP activity, and osteocalcin mRNA expression. Since Runx2 is an indispensable factor that regulates the differentiation of osteoblastic cells and bone formation in vitro and in vivo, its increased activity in oxysterol-treated cells further validates the potential role of oxysterols in lineage-specific differentiation of pluripotent mesenchymal cells and their potential therapeutic use as bone anabolic factors.

Alkaline Phosphatase↗
PubMed2004

Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts.

Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

3T3 Cells↗
PubMed2005

Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells.

AIM: To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. METHODS: AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7) with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAV-BMP7 at multiplicities of infection of 1 x 10(5) vector genomes per cell and subsequent culture, C2C12 cells were assessed qualitatively for BMP7 production, alkaline phosphatase activity, osteocalcin production and Cbfal and MyoD expression. RESULTS: C2C12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated. Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. CONCLUSION: The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.

Alkaline Phosphatase↗
PubMed2004

Molecular mechanisms of leukemogenesis by AML1/EVI-1.

The AML1/EVI-1 chimeric gene is generated by the t(3;21)(q26;q22) translocation and plays a pivotal role in progression of hematopoietic stem cell malignancies such as chronic myelocytic leukemia and myelodysplastic syndrome. In AML1/EVI-1, an N-terminal half of AML1 including a runt homology domain is fused to the entire zinc-finger EVI-1 protein. AML1 is essential for hematopoietic cell development in fetal liver and its lineage-specific differentiation in adult. In contrast, EVI-1 is barely expressed in normal hematopoietic cells, but it is overexpressed in chronic myelocytic leukemia in blastic crisis and myelodysplastic syndrome-derived leukemia. There are at least four mechanisms identified in AML1/EVI-1 fusion protein that possibly lead into malignant transformation of hematopoietic stem cells. Firstly, AML1/EVI-1 exerts dominant-negative effects over AML1-induced transcriptional activation. Although target genes repressed by AML1/EVI-1 are still not known, binding competition to a specific DNA sequence and histone deacetylase recruitment through a co-repressor CtBP in EVI-1 part are conceivable underlying mechanisms for the dominant-negative effects. Secondly, AML1/EVI-1 interferes with TGF beta signaling and antagonizes the growth-inhibitory effects of TGF beta. The first zinc-finger domain of EVI-1 associates with Smad3, a TGF beta signal transducer, and represses its transcriptional activity by recruiting histone deacetylase through CtBP that interacts with EVI-1. Thirdly, AML1/EVI-1 blocks JNK activity and prevents stress-induced apoptosis. AML1/EVI-1 associates with JNK through the first zinc-finger domain of EVI-1 and disturbs the association between JNK and its substrates. Lastly, AML1/EVI-1 enhances AP-1 activity by activating the c-Fos promoter depending on the second zinc-finger domain of EVI-1, and promotes cell proliferation. All these functions cooperatively contribute to the malignant transformation of the hematopoietic stem cells by AML1/EVI-1.

Cell Division↗
PubMed2004

Effect of carnosine on runt-related transcription factor-2/core binding factor alpha-1 and Sox9 expressions of human periodontal ligament cells.

BACKGROUND: Previous studies have reported that beta-alanyl-l-histidine (carnosine) enhanced the process of wound healing by stimulating effusion at the initial stage of inflammation, and also enhanced runt-related transcription factor-2/core binding factor alpha-1 (RUNX2/Cbfa1), Sox9, bone morphogenetic protein-2 (BMP-2) and BMP-7 expressions of human periodontal ligament cells. OBJECTIVES: In order to clarify the relationship between RUNX2/Cbfa1 or Sox9 expressions and BMP-2 or BMP-7 expressions enhanced by carnosine, we determined the effect of carnosine on the expression of BMP receptors and activation of their downstream signaling molecules in human periodontal ligament cells. MATERIAL AND METHODS: Human periodontal ligament cells were cultured with alpha-minimum essential medium containing 10% fetal bovine serum with or without 10(-4) or 10(-5) m carnosine for up to 10 days. The gene expression of BMP receptors, RUNX2/Cbfa1 and Sox9 was measured using semiquantitative reverse transcription-polymerase chain reaction. Phosphorylation of Smad1 was determined using a western blot analysis. RESULTS: Alkaline phosphatase activity increased in cultures with carnosine. Among the BMP receptors, expression of ActR-I and BMPR-II increased in cultures with carnosine, whereas expression of BMPR-IA, BMPR-IB, ActR-IIA and ActR-IIB was not affected. Culture with carnosine increased phosphorylation of Smad1, a signal-transducing molecule for BMP-2 and BMP-7. Noggin reduced carnosine-induced up-regulation of RUNX2/Cbfa1 and Sox9 mRNA, suggesting that BMPs were responsible for up-regulating RUNX2/Cbfa1 gene expression in human periodontal ligament cells. CONCLUSION: These results suggest that carnosine enhance RUNX2/Cbfa1 and Sox9 expression of human periodontal ligament cells via the autocrine action of BMP-2 or BMP-7 produced by the cells.

Alkaline Phosphatase↗
PubMed2006

Influence of an LRP5 cytoplasmic SNP on Wnt signaling and osteoblastic differentiation.

The low density lipoprotein receptor-related protein 5 (LRP5) is a key determinant of bone mass, via the Wnt signaling pathway control of osteoblast function. This study examined human LRP5 signaling and the effects of an intracellular domain single nucleotide polymorphism (SNP: p.V1525A) on osteoblast differentiation and mineralization. Constitutively active LRP5 was constructed by deletion of the extracellular domain of LRP5 (LRP5DeltaN). Expression of LRP5DeltaN-V, which carries the allele p.1525V, induced higher beta-catenin/TCF-LEF activity compared to LRP5DeltaN-A, which carries the allele p.1525A. In a yeast two-hybrid assay, LRP5DeltaN-V also demonstrated a stronger interaction with AXIN than LRP5DeltaN-A. Expression of either of the alleles did not change cell proliferation. However, cells expressing LRP5DeltaN-V showed increased alkaline phosphatase activity and bone nodule formation compared to cells transfected with empty vector or LRP5DeltaN-A after osteogenic supplement (OS: beta-glycerophosphate and l-ascorbic acid) treatment. Cells expressing LRP5DeltaN-V revealed significantly increased bone sialoprotein (BSP) expression after 7 days of OS treatment and maintained elevated expression until day 21. Osteocalcin (OCN) mRNA levels were increased after 14-21 days of OS treatment in LRP5DeltaN-V expressing cells. LRP5DeltaN-V expressing cells demonstrated positive interaction with BMP-2 signaling of transcription at the SBE-luc promoter. LRP5 signaling is affected by the cytoplasmic SNP, p.V1525A. mRNA levels of Runx2 and Osterix were not affected by this SNP.

Alkaline Phosphatase↗
PubMed2000

The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5.

The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand-specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine-dependent and factor-independent activation of the receptor.

Amino Acid Motifs↗
PubMed2002

Analysis of regulator of G-protein signaling-2 (RGS-2) expression and function in osteoblastic cells.

Regulator of G-protein signaling-2 (RGS-2) belongs to a novel family of GTPase-activating proteins that rapidly turn-off G-protein coupled receptor signaling. RGS proteins contain a characteristic RGS domain by which they interact with the alpha-subunit of G-proteins and drive them into their inactive GDP-bound forms. Previously, we have reported that RGS-2 mRNA is rapidly and transiently increased by PTH in rat bone and in osteoblast cultures in vitro. In this study, we further explored the molecular basis for the regulation of RGS-2 by cloning and functionally characterizing the RGS-2 gene promoter. We cloned 2.3- and 2.8-kb fragments of the 5'-flanking regions of the rat and mouse RGS-2 genes, respectively, and generated a stable clone of UMR106 osteoblastic cells containing the rat RGS-2 promoter driving the beta-gal reporter gene (p2.3RGS-2-beta-gal). Treatment of the stable clone with PTH resulted in a maximal 2.2- to 3.6-fold increase in promoter activity at 8 h, reminiscent of the early response observed with endogenous RGS-2 mRNA regulation. Further, PTH (1-38), (1-31), PTHrP (1-34), and forskolin, which elevate cAMP levels, stimulated the promoter, while PTH (3-34) and (7-34), which do not readily stimulate cAMP accumulation, and PMA that directly activates protein kinase C, had no effect on promoter activity. Taken together, these results implicate the involvement of the Galpha(s)-adenylate cyclase-protein kinase A pathway in stimulating RGS-2 expression. Maintenance of a hyperphosphorylated state via the inhibition of type 2A protein phosphatases by okadaic acid, resulted in a strong dose-dependent increase in transcriptional activity of the RGS-2 promoter as well as that of the endogenous RGS-2 gene. Furthermore, overexpression of the osteoblast-specific transcription factor Runx2 also led to a stimulation of RGS-2 promoter activity. Functional analysis using RGS-2 overexpression suggests the potential negative regulatory effects of RGS-2 on PTH- and forskolin-induced cAMP production in osteoblastic cells. In summary, our data suggest that PTH treatment results in a direct transcriptional stimulation of RGS-2 that in turn may play a role in modulating the duration/intensity of PTH receptor signaling.

Animals↗
PubMed2005

RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAF1/Cip1) expression in cooperation with transforming growth factor {beta}-activated SMAD.

RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor beta1 (TGF-beta1). It is known that TGF-beta1 induces cell growth arrest by activating CDKN1A (p21(WAF1)(/Cip1)), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-beta-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-beta-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.

Animals↗
PubMed2002

The interaction between sigmaS, the stationary phase sigma factor, and the core enzyme of Escherichia coli RNA polymerase.

BACKGROUND: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure alpha2betabeta') associated with one of the sigma subunits, required for promoter recognition. Different sigma factors compete for core binding. Among the seven sigma factors present in E. coli, sigma70 controls gene transcription during the exponential phase, whereas sigmaS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled sigmaS and sigma70, we compared the affinities of both sigma factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. RESULTS: Using native polyacrylamide gel electrophoresis, we demonstrate that sigmaS binds to the core enzyme with fivefold reduced affinity compared to sigma70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of sigmaS, while increasing the accessibility in region 4.1 of sigmaS. We have also analysed the positioning of sigmaS on the holoenzyme by the proximity-dependent protein cleavage method using sigmaS derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the beta and beta' subunits, but not alpha. Regions 2.5, 3.1 and 3.2 of sigmaS are close to both beta and beta' subunits, in agreement with iron chelate protein footprinting data. CONCLUSIONS: A comparison between these results using sigmaS and previous data from sigma70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2.

Bacterial Proteins↗
PubMed2007

The suppressive effect of myeloid Elf-1-like factor (MEF) in osteogenic differentiation.

Myeloid Elf-1 like factor (MEF) is a member of the Ets transcription factor family. Ets family proteins control the expression of genes that are critical for biological processes such as proliferation, differentiation, and cell death. Some of Ets factors are also known to regulate bone development. In this study, we investigated the role of MEF in osteoblast differentiation. MEF expression was highest early in the differentiation of MC3T3-E1 osteoblasts and was reduced by treatment with BMP-2. The expression of MEF suppressed the alkaline phosphatase activity and expression induced by BMP-2 stimulation and mediated by Runx2. The expression of MEF also reduces osteocalcin mRNA levels, and mineralization in MC3T3-E1 cells. We found that the MEF-mediated suppression of osteogenic differentiation was critically related to Runx2 regulation. The MEF and Runx2 proteins physically interact to form a complex, and this interaction interferes with Runx2 binding to the cis-acting element OSE2 derived from the osteocalcin promoter. Co-transfection of MEF inhibited the 6xOSE2-luciferase reporter activity induced by Runx2. In addition, MEF stimulated the transcription of a negative mediator Msx2, and a transcriptional repressor, Mab21L1, and suppressed the transcription of a positive mediator, Dlx5 in osteoblast differentiation. MEF overexpression stimulated C2C12 cell proliferation. Together, our findings suggest that MEF promotes cell proliferation and functions as a negative regulator of osteogenic differentiation by directly interacting with Runx2 and suppressing its transcriptional activity.

Alkaline Phosphatase↗
PubMed2001

Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells.

Physical shock wave (SW) has shown effectiveness on promotion of bone growth. We have recently demonstrated that SW could promote bone marrow stromal cell differentiation toward osteoprogenitor associated with induction of TGF-beta1. We have further demonstrated that SW-induced membrane hyperpolarization and Ras activation acted an early signal for the osteogenesis in human bone marrow stromal cells. An optimal dose of SW treatment at 0.16 mJ/mm(2) for 500 impulses induced a rapid membrane hyperpolarization in 5 min, activation of Ras in 30 min, and cell proliferation in 2 days. The SW-promoted cell growth was related to osteogenesis as demonstrated by increase of bone alkaline phosphatase activity in 6 days and osteocalcin mRNA expression in 12 days. In support that SW-induced Ras activation mediated osteogenesis of human bone marrow stromal cells, we further demonstrated that transfection of bone marrow stromal cells with a dominant negative Ras mutant (Asn-17 ras(H)) abrogated the SW enhancement of osteogenic transcription factor (CBFA1) activation, osteocalcin mRNA expression, and bone nodule formations. These results suggest that physical SW promotes bone marrow stromal cell differentiation toward osteogenic lineage via membrane hyperpolarization, followed by Ras activation and specific osteogenic transcription factor CBFA1 expression. A link between physical SW and biomembrane perturbation-mediated Ras activation may highlight how noninvasive physical agents could be used to promote fracture healing and to rescue patients with osteoporosis and osteopenic disorders in the future.

Bone Marrow Cells↗
PubMed1998

Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages.

Cumulative evidence indicates that osteoblasts and adipocytes share a common mesenchymal precursor and that bone morphogenetic proteins (BMPs) can induce both osteoblast and adipocyte differentiation of this precursor. In the present study, we investigated the roles of BMP receptors in differentiation along these separate lineages using a well-characterized clonal cell line, 2T3, derived from the mouse calvariae. BMP-2 induced 2T3 cells to differentiate into mature osteoblasts or adipocytes depending upon culture conditions. To test the specific roles of the type IA and IB BMP receptor components, truncated and constitutively active type IA and IB BMP receptor cDNAs were stably expressed in these cells. Overexpression of truncated type IB BMP receptor (trBMPR-IB) in 2T3 cells completely blocked BMP-2-induced osteoblast differentiation and mineralized bone matrix formation. Expression of trBMPR-IB also blocked mRNA expression of the osteoblast specific transcription factor, Osf2/ Cbfa1, and the osteoblast differentiation-related genes, alkaline phosphatase (ALP) and osteocalcin (OC). BMP-2-induced ALP activity could be rescued by transfection of wild-type (wt) BMPR-IB into 2T3 clones containing trBMPR-IB. Expression of a constitutively active BMPR-IB (caBMPR-IB) induced formation of mineralized bone matrix by 2T3 cells without addition of BMP-2. In contrast, overexpression of trBMPR-IA blocked adipocyte differentiation and expression of caBMPR-IA induced adipocyte formation in 2T3 cells. Expression of the adipocyte differentiation-related genes, adipsin and PPARgamma, correlated with the distinct phenotypic changes found after overexpression of the appropriate mutant receptors. These results demonstrate that type IB and IA BMP receptors transmit different signals to bone-derived mesenchymal progenitors and play critical roles in both the specification and differentiation of osteoblasts and adipocytes.

Adipocytes↗
PubMed2002

Causal relationship between the loss of RUNX3 expression and gastric cancer.

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.

Animals↗