Faculty Bibliography

The osteoblastic and adipocytic lineages arise from mesenchymal stem cells (MSCs), but few regulators of self-renewal and early cell-fate decisions are known. Here, we show that the Hippo pathway effector YAP1 is a direct target of SOX2 and can compensate for the self-renewal defect caused by SOX2 inactivation in osteoprogenitors and MSCs. Osteogenesis is blocked by high SOX2 or YAP1, accelerated by depletion of either one, and the inhibition of osteogenesis by SOX2 requires YAP1. SOX2 favors adipogenesis and induces PPARgamma, but adipogenesis can only occur with moderate levels of YAP1. YAP1 induction by SOX2 is restrained in adipogenesis, and both YAP1 overexpression and depletion inhibit the process. YAP1 binds beta-catenin and directly induces the Wnt antagonist Dkk1 to dampen pro-osteogenic Wnt signals. We demonstrate a Hippo-independent regulation of YAP1 by SOX2 that cooperatively antagonizes Wnt/beta-catenin signals and regulates PPARgamma to determine osteogenic or adipocytic fates.

Precise regulation of the levels and timing of gene expression is fundamental to all biological processes and is largely determined by the activity of cis-regulatory modules, containing the binding sites for transcription factors, within promoters and enhancers. The global identification of these transcriptional regulatory elements within mammalian genomes, and understanding when and where they are active, is an important effort that will require the development and implementation of several different technologies. Here we detail a means for the identification of transcriptional regulatory elements using functional assays. The success of this approach relies on focusing the functional assay on DNA derived from nucleosome-free regions (NFRs), i.e., the 2% of the genome within a given cell in which regulatory elements reside. Accordingly, we present a simple method for isolating NFR DNA, and a functional assay that can be used for the identification of promoter and enhancers components within this population.

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1-3, and inactivating mutations of TWIST1. In a mouse model of TWIST1 haploinsufficiency, cell mixing between the neural crest-derived frontal bone and mesoderm-derived parietal bone accompanies coronal suture fusion during embryonic development. However, the relevance of lineage mixing in craniosynostosis induced by activating FGFR mutations is unknown. Here, we demonstrate a novel mechanism of suture fusion in the Apert Fgfr2(S252W) mouse model. Using Cre/lox recombination we simultaneously induce expression of Fgfr2(S252W) and beta-galactosidase in either the neural crest or mesoderm of the skull. We show that mutation of the mesoderm alone is necessary and sufficient to cause craniosynostosis, while mutation of the neural crest is neither. The lineage border is not disrupted by aberrant cell migration during fusion. Instead, the suture mesenchyme itself remains intact and is induced to undergo osteogenesis. We eliminate postulated roles for dura mater or skull base changes in craniosynostosis. The viability of conditionally mutant mice also allows post-natal assessment of other aspects of Apert syndrome.

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.

Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor-initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma (mOS) cell lines as well as in the tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by short-hairpin RNAs in independent mOS-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these cells maintain a requirement for Sox2 for tumorigenicity. Our data indicate that Sox2 is required for osteosarcoma cell self renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.Oncogene advance online publication, 19 September 2011; doi:10.1038/onc.2011.405

The characterization of mesenchymal progenitors is central to understanding development, postnatal pathology and evolutionary adaptability. The precise identity of the mesenchymal precursors that generate the coronal suture, an important structural boundary in mammalian skull development, remains unclear. We show in mouse that coronal suture progenitors originate from hedgehog-responsive cephalic paraxial mesoderm (Mes) cells, which migrate rapidly to a supraorbital domain and establish a unidirectional lineage boundary with neural crest (NeuC) mesenchyme. Lineage tracing reveals clonal and stereotypical expansion of supraorbital mesenchymal cells to form the coronal suture between E11.0 and E13.5. We identify engrailed 1 (En1) as a necessary regulator of cell movement and NeuC/Mes lineage boundary positioning during coronal suture formation. In addition, we provide genetic evidence that En1 functions upstream of fibroblast growth factor receptor 2 (Fgfr2) in regulating early calvarial osteogenic differentiation, and postulate that it plays an additional role in precluding premature osteogenic conversion of the sutural mesenchyme.

The transcription factor Sox2 is a key player in the maintenance of pluripotency and stemness. We have previously shown that Sox2 maintains self-renewal in the osteoblast lineage while inhibiting differentiation. Sox2 also interferes with Wnt signaling by binding beta-catenin, a central mediator of the Wnt pathway. Here we show that these multiple functions of Sox2 are encoded in distinct domains. The self-renewal function of Sox2 is dependent on its transcriptional activity, and requires both its DNA-binding and C-terminal activation regions, while only the third C-terminal-transactivation region is required for binding beta-catenin and interfering with Wnt-induced transcription. Gene expression analysis upon Sox2 deletion strongly supports the notion that Sox2 maintains stemness. We also show that Sox2 suppresses differentiation by attenuating Wnt signaling by posttranscriptional and transcriptional mechanisms and that negative regulators of the Wnt pathway, APC and GSK3beta are direct Sox2 targets in osteoblasts. Several genes associated with stemness such as FoxP1 and BMI-1 are downregulated upon Sox2 inactivation. Constitutive expression of the polycomb complex member, BMI-1, can bypass the Sox2 requirement for self-renewal, but does not affect differentiation. Our results establish a connection between Sox2 and BMI-1 in maintaining self-renewal and identify BMI-1 as a key mediator of Sox2 function

FGF1, a widely expressed proangiogenic factor involved in tissue repair and carcinogenesis, is released from cells through a non-classical pathway independent of endoplasmic reticulum and Golgi. Although several proteins participating in FGF1 export were identified, genetic mechanisms regulating this process remained obscure. We found that FGF1 export and expression are regulated through Notch signaling mediated by transcription factor CBF1 and its partner MAML. The expression of a dominant negative (dn) form of CBF1 in 3T3 cells induces transcription of FGF1 and sphingosine kinase 1 (SphK1), which is a component of FGF1 export pathway. dnCBF1 expression stimulates the stress-independent release of transduced FGF1 from NIH 3T3 cells and endogenous FGF1 from A375 melanoma cells. NIH 3T3 cells transfected with dnCBF1 form colonies in soft agar and produce rapidly growing highly angiogenic tumors in nude mice. The transformed phenotype of dnCBF1 transfected cells is efficiently blocked by dn forms of FGF receptor 1 and S100A13, which is a component of FGF1 export pathway. FGF1 export and acceleration of cell growth induced by dnCBF1 depend on SphK1. Similar to dnCBF1, dnMAML transfection induces FGF1 expression and release, and accelerates cell proliferation. The latter effect is strongly decreased in FGF1 null cells. We suggest that the regulation of FGF1 expression and release by CBF1-mediated Notch signaling can play an important role in tumor formation.

We have previously shown that in osteoblasts Sox2 expression can be induced by Fgfs, and can inhibit Wnt signaling and differentiation. Furthermore, in mice in which Sox2 is conditionally deleted in the osteoblastic lineage, bones are osteopenic, and Sox2 inactivation in cultured osteoblasts leads to a loss of proliferative ability with a senescent phenotype. To help understand the role of Sox2 in osteoblast development we have specifically expressed Sox2 in bone from a Col1alpha1 promoter, which extended Sox2 expression into more mature osteoblasts. In long bones, trabecular cartilage remodeling was delayed and the transition from endochondral to cortical bone was disrupted, resulting in porous and undermineralized cortical bone. Collagen deposition was disorganized, and patterns of osteoclast activity were altered. Calvarial bones were thinner and parietal bones failed to develop the diploic space. Microarray analysis showed significant up- or downregulation of a variety of genes coding for non-collagenous extracellular matrix proteins, with a number of genes typical of mature osteoblasts being downregulated. Our results position Sox2 as a negative regulator of osteoblast maturation in vivo.

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G(1) phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G(2) phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G(1) arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G(2) arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G(2) checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G(2) arrest. Additionally our results indicate that the transient G(2) arrest is induced by FGF in RCS cell through mechanisms that are independent of the G(1) arrest, and that the G(2) block is not strictly required for the sustained G(1) arrest but may provide a pausing mechanism that allows the FGF response to be fully established