Chunxi Ge

Transcriptional regulation of osteoblasts.

Abstract:  The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. Among these are RUNX2, Osterix (OSX), ATF4, and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations well before the branching of chondrogenic and osteogenic lineages. Given that RUNX2 is expressed so early and participates in several stages of bone formation, it is not surprising that it is subject to a variety of controls. These include regulation by nuclear accessory factors and posttranslational modification, especially phosphorylation. Specific examples of RUNX2 regulation include interactions with DLX proteins and ATF4 and phosphorylation by the ERK/MAP kinase pathway. RUNX2 is regulated via phosphorylation of critical serine residues in the P/S/T domain. MAPK activation of RUNX2 was also found to occur in vivo. Transgenic expression of constitutively active MEK1 in osteoblasts accelerated skeletal development while a dominant‐negative MEK1 retarded development in a RUNX2‐dependent manner. These studies allow us to begin understanding the complex mechanisms necessary to fine‐tune bone formation in response to extracellular stimuli including ECM interactions, mechanical loads, and hormonal stimulation.

Hematopoietic Stem Cells Regulate Mesenchymal Stromal Cell Induction into Osteoblasts Thereby Participating in The Formation of the Stem Cell Niche

Crosstalk between hematopoietic stem cells (HSCs) and the cells comprising the niche is critical for maintaining stem cell activities. Yet little evidence supports the concept that HSCs regulate development of the niche. Here, the ability of HSCs to directly regulate endosteal development was examined. Marrow was isolated 48 hours after “stressing” mice with a single acute bleed or from control nonstressed animals. “Stressed” and “nonstressed” HSCs were cocultured with bone marrow stromal cells to map mesenchymal fate. The data suggest that HSCs are able to guide mesenchymal differentiation toward the osteoblastic lineage under basal conditions. HSCs isolated from animals subjected to an acute stress were significantly better at inducing osteoblastic differentiation in vitro and in vivo than those from control animals. Importantly, HSC‐derived bone morphogenic protein 2 (BMP‐2) and BMP‐6 were responsible for these activities. Furthermore, significant differences in the ability of HSCs to generate a BMP response following stress were noted in aged and in osteoporotic animals. Together these data suggest a coupling between HSC functions and bone turnover as in aging and in osteoporosis. For the first time, these results demonstrate that HSCs do not rest passively in their niche. Instead, they directly participate in bone formation and niche activities.

Identification and Functional Characterization of ERK/MAPK Phosphorylation Sites in the Runx2 Transcription Factor

The Runx2 transcription factor is required for commitment of mesenchymal cells to bone lineages and is a major regulator of osteoblast-specific gene expression. Runx2 is subject to a number of post-transcriptional controls including selective proteolysis and phosphorylation. We previously reported that Runx2 is phosphorylated and activated by the ERK/MAPK pathway (Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K., Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 4453–4459). In this study, we used a combination of in vitro and in vivo phosphorylation analysis, mass spectroscopy, and functional assays to identify two sites at Ser301 and Ser319 within the proline/serine/threonine domain of Runx2 that are required for this regulation. These sites are phosphorylated by activated ERK1 in vitro and in cell culture. In addition to confirming ERK-dependent phosphorylation at Ser319, mass spectroscopy identified two other ERK-phosphorylated sites at Ser43 and Ser510. Furthermore, introduction of S301A,S319A mutations rendered Runx2 resistant to MAPK-dependent activation and reduced its ability to stimulate osteoblast-specific gene expression and differentiation after transfection into Runx2-null calvarial cells and mesenchymal cells. In contrast, S301E,S319E Runx2 mutants had enhanced transcriptional activity that was minimally dependent on MAPK signaling, consistent with the addition of a negative charge mimicking serine phosphorylation. These results emphasize the important role played by Runx2 phosphorylation in the control of osteoblast gene expression and provide a mechanism to explain how physiological signals acting on bone through the ERK/MAPK pathway can stimulate osteoblast-specific gene expression.

Transcriptional Regulation of Osteoblasts

The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. These include RUNX2, Osterix (OSX), ATF4 and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations and precedes the branching of chondrogenic and osteogenic lineages. Given its central role in bone development, it is not surprising that RUNX2 is subject to a variety of controls. These include posttranslational modification, especially phosphorylation, and interactions with accessory nuclear factors. Specific examples of RUNX2 regulation to be reviewed include phosphorylation by the ERK/MAP kinase pathway and interactions with DLX5. RUNX2 is regulated via phosphorylation of critical serine residues in the proline/serine/threonine domain. In vivo, the transgenic expression of constitutively active MAP kinase in osteoblasts accelerated skeletal development, while a dominant-negative MAPK retarded development in a RUNX2-dependent manner. DLX5-RUNX2 complexes can be detected in osteoblasts and this interaction plays a critical role in maintaining osteoblast-specific expression of the bone sialoprotein gene. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation as mesenchymal progenitors progress down the osteoblast lineage.

INTERACTIONS BETWEEN EXTRACELLULAR SIGNAL-REGULATED KINASE 1/2 AND P38 MAP KINASE PATHWAYS IN THE CONTROL OF RUNX2 PHOSPHORYLATION AND TRANSCRIPTIONAL ACTIVITY

RUNX2, a key transcription factor for osteoblast differentiation, is regulated by ERK1/2 and p38 MAP kinase‐mediated phosphorylation. However, the specific contribution of each kinase to RUNX2‐dependent transcription is not known. Here we investigate ERK and p38 regulation of RUNX2 using a unique P‐RUNX2‐specific antibody. Both MAP kinases stimulated RUNX2 Ser319 phosphorylation and transcriptional activity. However, a clear preference for ERK1 versus p38α/β was found when the ability of these MAPKs to phosphorylate and activate RUNX2 was compared. Similarly, ERK1 preferentially bound to a consensus MAPK binding site on RUNX2 that was essential for the activity of either kinase. To assess the relative contribution of ERK1/2 and p38 to osteoblast gene expression, MC3T3‐E1 preosteoblast cells were grown in control or ascorbic acid (AA)‐containing medium ± BMP2/7. AA‐induced gene expression, which requires collagen matrix synthesis, was associated with parallel increases in P‐ERK and RUNX2‐S319‐P in the absence of any changes in P‐p38. This response was blocked by ERK, but not p38, inhibition. Significantly, in the presence of AA, BMP2/7 synergistically stimulated RUNX2 S319 phosphorylation and transcriptional activity without affecting total RUNX2 and this response was totally dependent on ERK/MAPK activity. In contrast, although p38 inhibition partially blocked BMP‐dependent transcription, it did not affect RUNX2 S319 phosphorylation, suggesting the involvement of other phosphorylation sites and/or transcription factors in this response. Based on this work, we conclude that extracellular matrix and BMP regulation of RUNX2 phosphorylation and transcriptional activity in osteoblasts is predominantly mediated by ERK rather than p38 MAPKs.

Foxo1 Mediates Insulin-like Growth Factor 1 (IGF1)/Insulin Regulation of Osteocalcin Expression by Antagonizing Runx2 in Osteoblasts

In this study, we determined the molecular mechanisms whereby forkhead transcription factor Foxo1, a key downstream signaling molecule of insulin-like growth factor 1 (IGF1)/insulin actions, regulates Runx2 activity and expression of the mouse osteocalcin gene 2 (Bglap2) in osteoblasts in vitro. We showed that Foxo1 inhibited Runx2-dependent transcriptional activity and osteocalcin mRNA expression and Bglap2 promoter activity in MC-4 preosteoblasts. Co-immunoprecipitation assay showed that Foxo1 physically interacted with Runx2 via its C-terminal region in osteoblasts or when co-expressed in COS-7 cells. Electrophoretic mobility shift assay demonstrated that Foxo1 suppressed Runx2 binding to its cognate site within the Bglap2 promoter. IGF1 and insulin prevented Foxo1 from inhibiting Runx2 activity by promoting Foxo1 phosphorylation and nuclear exclusion. In contrast, a neutralizing anti-IGF1 antibody decreased Runx2 activity and osteocalcin expression in osteoblasts. Chromatin immunoprecipitation assay revealed that IGF1 increased Runx2 interaction with a chromatin fragment of the proximal Bglap2 promoter in a PI3K/AKT-dependent manner. Conversely, knockdown of Foxo1 increased Runx2 interaction with the promoter. This study establishes that Foxo1 is a novel negative regulator of osteoblast-specific transcription factor Runx2 and modulates IGF1/insulin-dependent regulation of osteocalcin expression in osteoblasts.

Patterning alginate hydrogels using light-directed release of caged calcium in a microfluidic device.

This paper describes a simple reversible hydrogel patterning method for 3D cell culture. Alginate gel is formed in select regions of a microfluidic device through light-triggered release of caged calcium. In the pre-gelled alginate solution, calcium is chelated by DM-nitrophen (DM-n) to prevent cross-linking of alginate. After sufficient UV exposure the caged calcium is released from DM-n causing alginate to cross-link. The effect of using different concentrations of calcium and chelating agents as well as the duration of UV exposure is described. Since the cross-linking is based on calcium concentration, the cross-linked alginate can easily be dissolved by EDTA. We also demonstrate application of this capability to patterned microscale 3D co-culture using endothelial cells and osteoblastic cells in a microchannel.

Healing Cranial Defects with AdRunx2-transduced Marrow Stromal Cells

Marrow stromal cells (MSCs) include stem cells capable of forming all mesenchymal tissues, including bone. However, before MSCs can be successfully used in regeneration procedures, methods must be developed to stimulate their differentiation selectively to osteoblasts. Runx2, a bone-specific transcription factor, is known to stimulate osteoblast differentiation. In the present study, we tested the hypothesis that Runx2 gene therapy can be used to heal a critical-sized defect in mouse calvaria. Runx2-engineered MSCs displayed enhanced osteogenic potential and osteoblast-specific gene expression in vitro and in vivo. Runx2-expressing cells also dramatically enhanced the healing of critical-sized calvarial defects and increased both bone volume fraction and bone mineral density. These studies provide a novel route for enhancing osteogenesis that may have future therapeutic applications for craniofacial bone regeneration.

Physical and functional interactions between Runx2 and HIF‐1α induce vascular endothelial growth factor gene expression

Angiogenesis and bone formation are intimately related processes. Hypoxia during early bone development stabilizes hypoxia‐inducible factor‐1α (HIF‐1α) and increases angiogenic signals including vascular endothelial growth factor (VEGF). Furthermore, stabilization of HIF‐1α by genetic or chemical means stimulates bone formation. On the other hand, deficiency of Runx2, a key osteogenic transcription factor, prevents vascular invasion of bone and VEGF expression. This study explores the possibility that HIF‐1α and Runx2 interact to activate angiogenic signals. Runx2 over‐expression in mesenchymal cells increased VEGF mRNA and protein under both normoxic and hypoxic conditions. In normoxia, Runx2 also dramatically increased HIF‐1α protein. In all cases, the Runx2 response was inhibited by siRNA‐mediated suppression of HIF‐1α and completely blocked by the HIF‐1α inhibitor, echinomycin. Similarly, treatment of preosteoblast cells with Runx2 siRNA reduced VEGF mRNA in normoxia or hypoxia. However, Runx2 is not essential for the HIF‐1α response since VEGF is induced by hypoxia even in Runx2‐null cells. Endogenous Runx2 and HIF‐1α were colocalized to the nuclei of MC3T3‐E1 preosteoblast cells. Moreover, HIF‐1α and Runx2 physically interact using sites within the Runx2 RUNT domain. Chromatin immunoprecipitation also provided evidence for colocalization of Runx2 and HIF‐1α on the VEGF promoter. In addition, Runx2 stimulated HIF‐1α‐dependent activation of an HRE‐luciferase reporter gene without requiring a separate Runx2‐binding enhancer. These studies indicate that Runx2 functions together with HIF‐1α to stimulate angiogenic gene expression in bone cells and may in part explain the known requirement for Runx2 in bone vascularization. J. Cell. Biochem. 112: 3582–3593, 2011.

Biomechanical stimulation of osteoblast gene expression requires phosphorylation of the RUNX2 transcription factor.

Bone can adapt its structure in response to mechanical stimuli. At the cellular level, this involves changes in chromatin organization, gene expression, and differentiation, but the underlying mechanisms are poorly understood. Here we report on the involvement of RUNX2, a bone‐related transcription factor, in this process. Fluid flow shear stress loading of preosteoblasts stimulated translocation of extracellular signal‐regulated kinase (ERK)/mitogen‐activated protein kinase (MAPK) to the nucleus where it phosphorylated RUNX2 on the chromatin of target genes, and increased histone acetylation and gene expression. MAPK signaling and two RUNX2 phosphoacceptor sites, S301 and S319, were critical for this response. Similarly, in vivo loading of mouse ulnae dramatically increased ERK and RUNX2 phosphorylation as well as expression of osteoblast‐related genes. These findings establish ERK/MAPK‐mediated phosphorylation of RUNX2 as a critical step in the response of preosteoblasts to dynamic loading and define a novel mechanism to explain how mechanical signals induce gene expression in bone.