Faculty Bibliography

The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2-null cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2-null cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage

Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form

Fibroblast growth factor (FGF) and Wnt signals are both critical for proper bone development. We previously reported that the expression of activating FGF receptor mutations in osteoblasts downregulated the expression of many genes reported as targets of Wnt signaling, suggesting an antagonistic effect between Wnt signaling, which promotes osteoblast differentiation and function, and FGF signaling, which inhibits these processes. To analyze the effect of FGF on Wnt signaling in osteoblasts, we created reporter cell lines where a Wnt-responsive promoter drives luciferase expression and showed that Wnt3a-induced luciferase expression was specifically inhibited by FGF treatment. FGF specifically prevented the formation of a Wnt-induced transcriptional complex of TCF1 and -4 with beta-catenin on DNA. FGF did not significantly affect the activation of beta-catenin, although it reduced both the expression of TCF/LEF factors and their induction by Wnt. Microarray analysis using osteoblasts treated with Wnt3a and FGF alone or in combination showed that about 70% of the genes induced by Wnt3a were downregulated by combined FGF treatment. These included novel and previously identified Wnt target genes and genes involved in osteoblast differentiation. Furthermore, FGF alone could downregulate the expression of four Fzd Wnt receptor genes. Our results show that FGF antagonizes Wnt signaling by inhibiting Wnt-induced transcription and suggest that multiple mechanisms, including downregulation of TCFs and Wnt receptors, contribute to this effect

The identification of transcriptional regulatory modules within mammalian genomes is a prerequisite to understanding the mechanisms controlling regulated gene expression. While high-throughput microarray- and sequencing-based approaches have been used to map the genomic locations of sites of nuclease hypersensitivity or target DNA sequences bound by specific protein factors, the identification of regulatory elements using functional assays, which would provide important complementary data, has been relatively rare. Here we present a method that permits the functional identification of active transcriptional regulatory modules using a simple procedure for the isolation and analysis of DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular genome that contains these elements. The more than 100 new active regulatory DNAs identified in this manner from F9 cells correspond to both promoter-proximal and distal elements, and display several features predicted for endogenous transcriptional regulators, including localization within DNase-accessible chromatin and CpG islands, and proximity to expressed genes. Furthermore, comparison with published ChIP-seq data of ES-cell chromatin shows that the functional elements we identified correspond with genomic regions enriched for H3K4me3, a histone modification associated with active transcriptional regulatory elements, and that the correspondence of H3K4me3 with our promoter-distal elements is largely ES-cell specific. The majority of the distal elements exhibit enhancer activity. Importantly, these functional DNA fragments are an average 149 bp in length, greatly facilitating future applications to identify transcription factor binding sites mediating their activity. Thus, this approach provides a tool for the high-resolution identification of the functional components of active promoters and enhancers

Skeletal development requires the correct balance of osteoblast proliferation, survival, and differentiation which is modulated by a network of signaling pathways and transcription factors. We have examined the role of the AKT (PKB), and ERK1/2 signaling pathways in the osteoblast response to FGFs, which inhibit differentiation, and to IGF-1 and Wnt signaling, which promote it. Using osteoblastic cell lines as well as primary calvarial osteoblasts, we show that ERK1/2 and AKT have distinct effects in FGF-induced osteoblast proliferation and differentiation. ERK1/2 is a primary mediator of FGF-induced proliferation, but also contributes to osteoblast differentiation, while AKT is important for osteoblast survival. Signaling by IGF-1, that promotes osteoblast differentiation, strongly activates AKT and weakly ERK1/2, while the opposite results are obtained with FGF, which inhibits differentiation. By introducing a constitutively active form of AKT, we found that increased AKT activity drives osteoblasts to differentiation. Increasing the AKT signal in osteoblasts that harbor FGFR2 activating mutations, found in Crouzon (342Y) and Apert (S22W) syndromes, is also able to drive differentiation in these cells, that normally fail to differentiate. Wnt signals, that promotes differentiation, also induce AKT phosphorylation, and cells expressing active AKT have increased levels of stabilized beta-catenin, a central molecule in Wnt signaling. Our results indicate that the relative strengths of ERK and AKT signaling pathways determine whether osteoblasts are driven into proliferation or differentiation, and that the effects of AKT may be due, in part, to synergy with the Wnt pathway as well as with the Runx2 transcription factor. J. Cell. Physiol. (c) 2007 Wiley-Liss, Inc

FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation

Unregulated FGF signaling produced by activating FGFR3 mutations causes several forms of dwarfism-associated chondrodysplasias in humans and mice. FGF signaling inhibits chondrocyte proliferation by activating multiple signal transduction pathways that all contribute to chondrocyte growth arrest and induction of some aspects of differentiation. Previous studies had identified the Stat1 pathway, dephosphorylation of the Rb family proteins p107 and p130, induction of p21 expression and sustained activation of MAP kinases as playing a role in the FGF response of chondrocytes. We have examined the role of Akt (PKB) in the response of chondrocytes to FGF signaling. Differently from what is observed in many other cell types, FGF does not activate Akt in chondrocytes, and Akt phosphorylation is actually downregulated after FGF treatment. By expressing a constitutively activated, myristylated form of Akt (myr-Akt) in the RCS chondrosarcoma cell line, we show that Akt activation partially counteracts the inhibitory effect of FGF signaling. The response of myr-Akt expressing cells to FGF is identical to parental RCS in the first few hours after treatment, but then diverges as myr-Akt cells show decreased p130 phosphorylation, increased cyclin E/cdk2 activity and continue to proliferate at a slow rate. Constitutive Akt activation does not affect p21 expression but appears to influence directly cdk/cyclin activity. On the other hand, the induction of differentiation-related genes is unchanged in myr-Akt cells. These results identify Akt downregulation as an important aspect of the response of chondrocytes to FGF that, however, only affects chondrocyte proliferation and not the ability of FGF to induce differentiation genes

Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a 'promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl-/- or IL-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha