Biomedical subjects

Andre J van Wijnen

Publications and source records attributed to Andre J van Wijnen.

At least 19 recordsLinked to original sources

PubMed2007

HiNF-P is a bifunctional regulator of cell cycle controlled histone H4 gene transcription.

Cell cycle progression beyond the G1/S phase transition requires the activation of a transcription complex containing histone nuclear factor P (HiNF-P) and nuclear protein mapped to ataxia telangiectasia (p220(NPAT)) in response to cyclin dependent kinase 2 (CDK2)/cyclin E signaling. We show here that the potent co-activating properties of HiNF-P/p220(NPAT) on the histone H4 gene promoter, which are evident in the majority of human cell types, are sporadically neutralized in distinct somatic cell lines. In cells where HiNF-P and p220(NPAT) do not activate the H4 gene promoter, HiNF-P instead represses transcription. Our data suggest that the cell type specific expression of the cyclin-dependent kinase inhibitory (CKI) protein p57(KIP2) inhibits the HiNF-P dependent activation of the histone H4 promoter. We propose that, analogous to E2F proteins and other cell cycle regulatory proteins, HiNF-P is a bifunctional transcriptional regulator that can activate or repress cell cycle controlled genes depending on the cellular context.

Animals↗

PubMed2007

Mitotic control of RUNX2 phosphorylation by both CDK1/cyclin B kinase and PP1/PP2A phosphatase in osteoblastic cells.

Skeletal development and osteoblast maturation require the phenotype promoting activity of the transcription factor RUNX2, which controls both cell growth and differentiation in osteoblasts. We have recently shown that in actively proliferating cells RUNX2 regulates the expression of specific target genes as cells enter and exit mitosis. In this study, we addressed whether post-translational modifications of RUNX2 control its activity during mitotic exit. Western blot analysis of proteins from osteoblastic Saos-2 cells released from mitotic inhibition into early G(1) show a phosphatase-sensitive shift in the mobility of RUNX2 in SDS gels. The slowly migrating hyper-phosphorylated form of RUNX2 is immunoreactive with a CDK related phospho-antibody (MPM2) only in mitotic cells and is converted into a faster migrating hypo-phosphorylated RUNX2 when cells complete mitosis. This conversion is inhibited by okadaic acid, an inhibitor of protein phosphatases 1 and 2 (PP1 and PP2A), but not by deltamethrin which blocks PP2B phosphatase. Mitotic phosphorylation of RUNX2 is sensitive to the CDK inhibitors roscovitine and olomoucine. Furthermore, RUNX2 can directly interact with CDK1 and is phosphorylated in vitro by the CDK1/cyclin B kinase complex. Hence, RUNX2 is hyper-phosphorylated by CDK1/cyclin B during mitosis, and dynamically converted into a hypo-phosphorylated form by PP1/PP2A-dependent dephosphorylation after mitosis to support the post-mitotic regulation of RUNX2 target genes.

Blotting, Western↗

PubMed2007

Reconstitution of Runx2/Cbfa1-null cells identifies a requirement for BMP2 signaling through a Runx2 functional domain during osteoblast differentiation.

The Runx2/Cbfa1 transcription factor is a scaffolding protein that promotes osteoblast differentiation; however, the specific Runx2-functional domains required for induction of the osteogenic lineage remain to be identified. We approached this question using a TERT-immortalized cell line derived from calvaria of Runx2-null mice by reconstituting the osteogenic activity with wild-type and deletion mutants of Runx2. The presence or absence of osteogenic media (beta-glycerol phosphate and ascorbic acid) and/or with BMP2 did not stimulate osteoblastic gene expression in the Runx2-null cells. However, cells infected with wild-type Runx2 adenovirus showed a robust temporal increase in the expression of osteoblast marker genes and were competent to respond to BMP2. Early markers (i.e., collagen type-1, alkaline phosphatase) were induced (four- to eightfold) at Days 4 and 8 of culture. Genes representing mature osteoblasts (e.g., Runx2, osteopontin, bone sialoprotein, osteocalcin) were temporally expressed and induced from 18- to 36-fold at Days 8 and 12. Interestingly, TGFbeta and Vitamin D-mediated transcription of osteoblast genes (except for osteopontin) required the presence of Runx2. Runx2 lacking the C-terminal 96 amino acids (Runx2 Delta432) showed a pattern of gene expression similar to wild-type protein, demonstrating the Groucho interaction and part of the activation domain are dispensable for Runx2 osteogenic activity. Upon further deletion of the Runx2 C-terminus containing the nuclear matrix targeting signal and Smad-interacting domain (Delta391), we find none of the osteoblast markers are expressed. Therefore, the Runx2 391-432 domain is essential for execution of the BMP2 osteogenic signal.

Amino Acid Motifs↗

PubMed2007

The 1alpha,25-dihydroxy Vitamin D3 receptor preferentially recruits the coactivator SRC-1 during up-regulation of the osteocalcin gene.

Binding of 1alpha,25-dihydroxy Vitamin D3 to the C-terminal domain (LBD) of its receptor (VDR), induces a conformational change that enables interaction of VDR with transcriptional coactivators such as the members of the p160/SRC family or the DRIP (Vitamin D interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. Recent reports indicate that nuclear receptors, including VDR, interact with p160/SRC members and the DRIP/Mediator complex in a sequential, cyclical, and mutually exclusive manner when bound to a target promoter, exhibiting also a high exchange rate. Here, we present an overview of how these coactivators are recruited to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy Vitamin D3. We find that in intact osteoblastic cells VDR and SRC-1 rapidly bind to the OC promoter in response to the ligand. This recruitment correlates with transcriptional enhancement of the OC gene and with increased histone acetylation at the OC promoter. In contrast, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter after several hours of incubation with 1alpha,25-dihydroxy Vitamin D3. Together, our results indicate that VDR preferentially recruits SRC-1 to enhance basal bone-specific OC gene transcription. We propose a model where specific protein-DNA and protein-protein interactions that occur within the context of the OC gene promoter in osteoblastic cells stabilize the preferential association of the VDR-SRC-1 complex.

Animals↗

PubMed2007

Heparan sulfate regulates the anabolic activity of MC3T3-E1 preosteoblast cells by induction of Runx2.

The transcription factor Runx2 can be controlled by a number of upstream regulators involved in intracellular signalling, including the activation ERK1/2 signaling by fibroblast growth factor-2 (FGF-2). FGFs interact with their cell surface receptors (FGFRs) through an obligate cross-binding interaction with heparan sulfate proteoglycan (HSPG) co-receptors; exogenous HS sugar chains have been shown to potently modulate changes in cell phenotype depending on the stage of tissue differentiation when the HS is harvested, suggesting that HS chain structure and function varies depending on the stage of cell maturity. This study examined the potential of bone-derived heparan sulfate (HS), harvested from differentiating osteoblasts, for the enhancement of preosteoblast growth and differentiation. HS was harvested from conditioned media, cell surface and matrix compartments of postconfluent (differentiating) MC3T3-E1 osteoblasts and dosed back onto preconfluent MC3T3-E1 cells. We show that HS can increase the expression Runx2, ALP, and OPN in preosteoblast cells, suggesting the potential for exogenous HS to shift cells from proliferative to differentiative phenotypes. In line with their structural differences, only HS released into the media was found to co-stimulate the mitogenic effect of FGF-2, whilst exogenous application of all the HSs together with FGF-2 served to increase the expression of OPN. Only the application of cell surface-derived HS triggered a synergistic increase in FGFR1 expression together with FGF-2, although all three HS preparations could trigger transient increases in PI3K, ERK1/2, and stat3 phosphorylation levels. These findings demonstrate that the compartmentally distinct HS species expressed by differentiating MC3T3-E1 cells act in complex ways to coordinate the extracellular conditions that lead to osteoblast differentiation, with the cell surface species coordinating the FGF response.

Alkaline Phosphatase↗

PubMed2007

Establishment of histone gene regulation and cell cycle checkpoint control in human embryonic stem cells.

Rapid self-renewal of human embryonic stem (ES) cells (NIH designation WA01 and WA09) is accommodated by an abbreviated cell cycle due to a reduction in the G1 phase. Thus, molecular mechanisms operative in ES cells may expedite the cellular commitment to progress into S phase to initiate replication of DNA and biosynthesis of histone proteins to form new chromatin. Here we show that the selective cell cycle regulated expression of individual histone H4 gene copies, which is typical for somatic cell types, is already firmly established in human ES cells. This early establishment of H4 gene regulation, which is E2F independent, is consistent with co-expression of the cognate transcriptional regulators HiNF-P and p220(NPAT). Human ES cells differ from somatic cells in the expression of members of the E2F family and RB-related pocket proteins (p105(RB1), p107(RBL1), and p130(RBL2/RB2)) that control expression of genes encoding enzymes for nucleotide metabolism and DNA synthesis. Human ES cells rapidly and robustly (>200-fold) induce the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) upon gamma-irradiation. This DNA damage response promptly reduces histone gene expression as well as mRNA levels for HiNF-P and p220(NPAT) and causes accumulation of unprocessed histone H4 precursor RNAs. Furthermore, while E2F4, E2F5 and p130(RBL2/RB2) are the major E2F and pocket protein mRNAs in actively proliferating ES cells, expression levels of E2F5, E2F6, and p105(RB1) are most strongly elevated during cell cycle arrest in cells responding to DNA damage. Our data suggest that the brief G1 phase of ES cells is supported by a potent p21(WAF1/CIP1) related DNA damage response that functions through several mechanisms to rapidly inhibit cell cycle progression. This response may alter the E2F/pocket protein combinations that control E2F dependent genes and block H4 gene expression by inhibiting histone-specific transcription factors and processing of histone gene transcripts, as well as by destabilizing histone mRNAs.

Cell Cycle Proteins↗

PubMed2006

The histone gene transcription factor HiNF-P stabilizes its cell cycle regulatory co-activator p220NPAT.

Orderly progression through the cell cycle requires the transcriptional activation of histone genes to support packaging of newly replicated DNA. Induction of human histone gene expression is mediated by a co-activation complex containing transcription factor HiNF-P and its cofactor p220NPAT. Here, using cells synchronized in S-phase and in mitosis, as well as serum-stimulated cells, we have investigated how HiNF-P is regulated during the cell cycle and examined its stability relative to p220NPAT. We find that while HiNF-P is maintained at steady-state levels throughout the cell cycle, both HiNF-P and p220NPAT are actively degraded by the proteasome pathway. Importantly, elevation of HiNF-P levels enhances the stability of its co-activator p220NPAT. The HiNF-P-dependent stabilization of p220NPAT may reinforce signaling through the cyclin E/CDK2/p220NPAT pathway and contribute to coordinate control of histone gene expression.

Cell Cycle↗

PubMed2006

BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network.

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation.

Animals↗

PubMed2006

Chromatin remodeling and transcriptional activity of the bone-specific osteocalcin gene require CCAAT/enhancer-binding protein beta-dependent recruitment of SWI/SNF activity.

Tissue-specific activation of the osteocalcin (OC) gene is associated with changes in chromatin structure at the promoter region. Two nuclease-hypersensitive sites span the key regulatory elements that control basal tissue-specific and vitamin D3-enhanced OC gene transcription. To gain understanding of the molecular mechanisms involved in chromatin remodeling of the OC gene, we have examined the requirement for SWI/SNF activity. We inducibly expressed an ATPase-defective BRG1 catalytic subunit that forms inactive SWI/SNF complexes that bind to the OC promoter. This interaction results in inhibition of both basal and vitamin D3-enhanced OC gene transcription and a marked decrease in nuclease hypersensitivity. We find that SWI/SNF is recruited to the OC promoter via the transcription factor CCAAT/enhancer-binding protein beta, which together with Runx2 forms a stable complex to facilitate RNA polymerase II binding and activation of OC gene transcription. Together, our results indicate that the SWI/SNF complex is a key regulator of the chromatin-remodeling events that promote tissue-specific transcription in osteoblasts.

Animals↗

PubMed2006

Lymphocyte enhancer-binding factor 1 (Lef1) inhibits terminal differentiation of osteoblasts.

Lef1 is a transcriptional regulator of the Wnt/beta-catenin signaling cascade. Wnts directly augment bone formation and osteoblast differentiation from mesenchymal stem cells by receptor-mediated pathways involving Lrp5 and Frizzled. We previously reported that Lef1 represses Runx2-dependent activation of the late osteoblast differentiation gene, osteocalcin. Lef1 is expressed in preosteoblasts but is undetectable in fully differentiated osteoblasts. To determine if downregulation of Lef1 is necessary for osteoblast maturation, we constitutively overexpressed Lef1 in MC3T3-E1 preosteoblasts. Lef1-overexpressing cells produced alkaline phosphatase (ALP) and osteocalcin later, and at lower levels than control cells. Moreover, the extracellular matrices of Lef1-overexpressing cell cultures never mineralized. To further examine the role of Lef1 in osteoblasts, we suppressed Lef1 expression in MC3T3-E1 cells by RNA interference. Transient expression of a Lef1 shRNA efficiently reduced murine Lef1 levels and transcriptional activity. Stable suppression of Lef1 in MC3T3 preosteoblasts did not affect proliferation or Runx2 levels; however, ALP production and matrix mineralization were accelerated by 3-4 days. Gene chip analyses identified 14 genes that are differentially regulated in Lef1-suppressed cells. These data outline a role for Lef1 in delaying osteoblast maturation and suggest that Lef1 controls the expression of multiple genes in osteoblasts.

Animals↗

PubMed2006

Brg1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, is required for myeloid differentiation to granulocytes.

Many mammalian SWI/SNF complexes use Brahma-related gene 1 (Brg1) as a catalytic subunit to remodel nucleosomes for transcription regulation. In several mesenchymal cells and tissues, expression of a defective Brg1 protein negates the normal activity of the SWI/SNF complex and delays or blocks differentiation. To investigate the role of SWI/SNF complexes during myelopoiesis, we stably expressed a dominant negative (dn) Brg1 mutant in the myeloid lineage. Forced expression of dnBrg1 in IL-3-dependent murine 32Dcl3 myeloid progenitor cells results in a profound delay in the granulocyte-colony stimulating factor (G-CSF) induced granulocytic maturation. These cells also exhibit a significant decrease in the expression of both CD11b and Gr-1 surface receptors, which are normally upregulated during granulopoiesis, and show sustained expression of myeloperoxidase, which is synthesized primarily during the promyelocytic (blast) stage of myeloid development. Thus, dnBrg1 expression causes a developmental block at the promyelocytic/metamyelocytic stage of myeloid differentiation. Our findings indicate that the normal chromatin remodeling function of Brg1 is necessary for the G-CSF dependent differentiation of myeloid cells towards the granulocytic lineage. This dependency on Brg1 may reflect a stringent requirement for chromatin remodeling at a critical stage of hematopoietic cell maturation.

Adenosine Triphosphatases↗

PubMed2006

Microtubule-dependent nuclear-cytoplasmic shuttling of Runx2.

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol-dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM-1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration-dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co-immunoprecipitates alpha tubulin suggesting that microtubule binding involves the alpha/beta tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear-cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol-induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies.

Active Transport, Cell Nucleus↗

PubMed2006

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 (RUNX2Deltae1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Deltae1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Deltae1 is similar to wild-type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Deltae1, 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 RUNX2Deltae1 in gel shift assay. Finally, RUNX2Deltae1 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.

Adolescent↗

PubMed2006

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells.

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.

Alkaline Phosphatase↗

PubMed2006

Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase.

Competency for self-renewal of human embryonic stem (ES) cells is linked to pluripotency. However, there is a critical paucity of fundamental parameters of human ES cell division. In this study we show that human ES cells (H1 and H9; NIH-designated WA01 and WA09) rapidly proliferate due to a very short overall cell cycle (15-16 h) compared to somatic cells (e.g., normal diploid IMR90 fibroblasts and NT-2 teratocarcinoma cells). The human ES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened. Bromodeoxyuridine (BrdU) incorporation and FACS analysis demonstrated that 65% of asynchronously growing human ES cells are in S phase. Immunofluorescence microscopy studies detecting BrdU labeled mitotic chromosomes, Ki67 domains, and p220(NPAT) containing Cajal bodies revealed that the durations of the S ( approximately 8 h), G2 ( approximately 4 h), and M phases ( approximately 1 h) are similar in ES and somatic cells. We determined that human ES cells remain viable after synchronization with either nocodazole or the anti-tumor drug Paclitaxel (taxol) and have an abbreviated G1 phase of only 2.5-3 h that is significantly shorter than in somatic cells. Molecular analyses using quantitative RT-PCR demonstrate that human ES cells and somatic cells express similar cell cycle markers. However, among cyclins and cyclin-dependent kinases (CDKs), we observed high mRNA levels for the G1-related CDK4 and cyclin D2 genes. We conclude that human ES cells exhibit unique G1 cell cycle kinetics and use CDK4/cyclin D2 related mechanisms to attain competency for DNA replication.

Animals↗

PubMed2006

Alterations in intranuclear localization of Runx2 affect biological activity.

The transcription factor Runx2 controls osteoblast proliferation and differentiation. Runx2 organizes and assembles gene-regulatory complexes in nuclear microenvironments where target genes are activated or suppressed in a context-dependent manner. Intranuclear localization of Runx2 is mediated by the nuclear matrix-targeting signal (NMTS), an autonomous motif with a loop (L1)-turn-loop (L2) structure that forms predicted protein-protein interaction surfaces. Here we examined the functional consequences of introducing mutations in the L1 and L2 loops of the NMTS. These mutant proteins enter the nucleus, interact with the hetero-dimeric partner Cbfbeta, and bind to DNA in vitro and in vivo. In addition, these mutants retain interaction with the carboxy-terminus interacting co-regulatory proteins that include TLE, YAP, and Smads. However, two critical mutations in the L2 domain of the NMTS decrease association of Runx2 with the nuclear matrix. These subnuclear targeting defective (STD) mutants of Runx2 compromise target gene activation or repression. The biological significance of these findings is reflected by decreased osteogenic differentiation of mesenchymal progenitors, concomitant with major changes in gene expression profiles, upon expression of the STD Runx2 mutant. Our results demonstrate that fidelity of temporal and spatial localization of Runx2 within the nucleus is functionally linked with biological activity.

Amino Acids↗

PubMed2006

Networks and hubs for the transcriptional control of osteoblastogenesis.

We present an overview of the concepts of tissue-specific transcriptional control mechanisms essential for development of the bone cell phenotype. BMP2 induced transcription factors constitute a network of activities and molecular switches for bone development and osteoblast differentiation. Among these regulators are Runx2 (Cbfa1/AML3), the principal osteogenic master gene for bone formation, as well as homeodomain proteins and osterix. Runx2 has multiple regulatory activities, including activation or repression of gene expression, and integration of biological signals from developmental cues, such as BMP/TGFbeta, Wnt and Src signaling pathways. Runx2 provides a new paradigm for transcriptional control by functioning as a principal scaffolding protein in nuclear microenvironments to control gene expression in response to physiologic signals (growth factors, cytokines and hormones). The protein serves as a hub for the coordination of activities essential for the expansion and differentiation of osteogenic lineage cells through the formation of co-regulatory protein complexes organized in subnuclear domains. Mechanisms by which Runx2 supports commitment to osteogenesis and determines cell fate involve its retention on mitotic chromosomes. Disruption of a unique protein module, the subnuclear targeting signal of Runx2, has profound effects on osteoblast differentiation and metastasis of cancer cells in the bone microenvironment. Runx2 target genes include regulators of cell growth control, components of the bone extracellular matrix, angiogenesis, and signaling proteins for development of the osteoblast phenotype and bone turnover. The specificity of Runx2 regulatory activities provides a basis for novel therapeutic strategies to correct bone disorders.

Animals↗

PubMed2005

The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells.

Osterix is a bone-related transcription factor that functions genetically downstream of Runx2, which controls both growth and differentiation in osteoblasts. Here we assessed the biological function of Osterix in mesenchymal cells that are not normally committed to the osteogenic lineage. Stably transfected NIH3T3 fibroblasts that express exogenous Osterix were examined for their ability to convert into osteoblastic cells by analyzing gene expression profiles of bone phenotype related markers, as well as by measuring bone nodule formation and cell proliferation. Forced expression of Osterix stimulates osteopontin gene expression but not the expression or activity of other bone-related markers, including collagen type I, alkaline phosphatase, osteocalcin, or osteonectin. Moreover, cells stably expressing Osterix do not induce bone nodule formation. Strikingly, both polyclonal and monoclonal cells expressing Osterix exhibit enhanced proliferation. Collectively, these results indicate that Osterix is insufficient to establish osteogenic lineage commitment, perhaps due to the ability of Osterix to promote cell growth. We propose that regulatory pathways operating upstream of or in parallel with Osterix are required for osteogenic conversion of uncommitted mesenchymal cells.

Animals↗