Sox2 is required for tumor development and cancer cell proliferation in osteosarcoma.

The stem cell transcription factor Sox2 is highly expressed in many cancers where it is thought to mark cancer stem cells (CSCs). In osteosarcomas, the most common bone malignancy, high Sox2 expression marks and maintains a fraction of tumor-initiating cells that show all the properties of CSC. Knockdown of Sox2 expression abolishes tumorigenicity and suppresses the CSC phenotype. Here we show that, in a mouse model of osteosarcoma, osteoblast-specific Sox2 conditional knockout (CKO) causes a drastic reduction in the frequency and onset of tumors. The rare tumors detected in the Sox2 CKO animals were all Sox2 positive, indicating that they arose from cells that had escaped Sox2 deletion. Furthermore, Sox2 inactivation in cultured osteosarcoma cells by CRISPR/CAS technology leads to a loss of viability and proliferation of the entire cell population. Inactivation of the YAP gene, a major Hippo pathway effector which is a direct Sox2 target, causes similar results and YAP overexpression rescues cells from the lethality caused by Sox2 inactivation. These effects were osteosarcoma-specific, suggesting a mechanism of cell "addiction" to Sox2-initiated pathways. The requirement of Sox2 for osteosarcoma formation as well as for the survival of the tumor cells suggests that disruption of Sox2-initiated pathways could be an effective strategy for the treatment of osteosarcoma.

Myeloid Zinc Finger 1 and GA Binding Protein Co-Operate with Sox2 in Regulating the Expression of Yes-Associated Protein 1 in Cancer Cells.

The transcription factor (TF) yes-associated protein 1 (YAP1) is a major effector of the tumor suppressive Hippo signaling pathway and is also necessary to maintain pluripotency in embryonic stem cells. Elevated levels of YAP1 expression antagonize the tumor suppressive effects of the Hippo pathway that normally represses YAP1 function. High YAP1 expression is observed in several types of human cancers and is particularly prominent in cancer stem cells (CSCs). The stem cell TF Sox2, which marks and maintains CSCs in osteosarcomas (OSs), promotes YAP1 expression by binding to an intronic enhancer element and YAP1 expression is also crucial for the maintainance of OS stem cells. To further understand the regulation of YAP1 expression in OSs, we subjected the YAP1 intronic enhancer to scanning mutagenesis to identify all DNA cis-elements critical for enhancer function. Through this approach, we identified two novel TFs, GA binding protein (GABP) and myeloid zinc finger 1 (MZF1), which are essential for basal YAP1 transcription. These factors are highly expressed in OSs and bind to distinct sites in the YAP1 enhancer. Depletion of either factor leads to drastically reduced YAP1 expression and thus a reversal of stem cell properties. We also found that YAP1 can regulate the expression of Sox2 by binding to two distinct DNA binding sites upstream and downstream of the Sox2 gene. Thus, Sox2 and YAP1 reinforce each others expression to maintain stemness and tumorigenicity in OSs, but the activity of MZF1 and GABP is essential for YAP1 transcription. Stem Cells 2017;35:2340-2350.

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response.

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3ß (GSK3ß) as a novel key mediator of FGF inhibitory response in chondrocytes.

PPARγ agonists promote differentiation of cancer stem cells by restraining YAP transcriptional activity.

Osteosarcoma (OS) is a highly aggressive pediatric bone cancer in which most tumor cells remain immature and fail to differentiate into bone-forming osteoblasts. However, OS cells readily respond to adipogenic stimuli suggesting they retain mesenchymal stem cell-like properties. Here we demonstrate that nuclear receptor PPARγ agonists such as the anti-diabetic, thiazolidinedione (TZD) drugs induce growth arrest and cause adipogenic differentiation in human, mouse and canine OS cells as well as in tumors in mice. Gene expression analysis reveals that TZDs induce lipid metabolism pathways while suppressing targets of the Hippo-YAP pathway, Wnt signaling and cancer-related proliferation pathways. Significantly, TZD action appears to be restricted to the high Sox2 expressing cancer stem cell population and is dependent on PPARγ expression. TZDs also affect growth and cell fate by causing the cytoplasmic sequestration of the transcription factors SOX2 and YAP that are required for tumorigenicity. Finally, we identify a TZD-regulated gene signature based on Wnt/Hippo target genes and PPARγ that predicts patient outcomes. Together, this work highlights a novel connection between PPARγ agonist in inducing adipogenesis and mimicking the tumor suppressive hippo pathway. It also illustrates the potential of drug repurposing for TZD-based differentiation therapy for osteosarcoma.

Sox2 antagonizes the Hippo pathway to maintain stemness in cancer cells.

The repressive Hippo pathway has a profound tumour suppressive role in cancer by restraining the growth-promoting function of the transcriptional coactivator, YAP. We previously showed that the stem cell transcription factor Sox2 maintains cancer stem cells (CSCs) in osteosarcomas. We now report that in these tumours, Sox2 antagonizes the Hippo pathway by direct repression of two Hippo activators, Nf2 (Merlin) and WWC1 (Kibra), leading to exaggerated YAP function. Repression of Nf2, WWC1 and high YAP expression marks the CSC fraction of the tumor population, while the more differentiated fraction has high Nf2, high WWC1 and reduced YAP expression. YAP depletion sharply reduces CSCs and tumorigenicity of osteosarcomas. Thus, Sox2 interferes with the tumour-suppressive Hippo pathway to maintain CSCs in osteosarcomas. This Sox2-Hippo axis is conserved in other Sox2-dependent cancers such as glioblastomas. Disruption of YAP transcriptional activity could be a therapeutic strategy for Sox2-dependent tumours.

SOX2 regulates YAP1 to maintain stemness and determine cell fate in the osteo-adipo lineage.

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 PPARγ, 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 ß-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/ß-catenin signals and regulates PPARγ to determine osteogenic or adipocytic fates.

The B55α regulatory subunit of protein phosphatase 2A mediates fibroblast growth factor-induced p107 dephosphorylation and growth arrest in chondrocytes.

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.

Perspectives on cancer stem cells in osteosarcoma.

Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. These mesenchymal tumors are derived from progenitor cells in the osteoblast lineage that have accumulated mutations to escape cell cycle checkpoints leading to excessive proliferation and defects in their ability to differentiate appropriately into mature bone-forming osteoblasts. Like other malignant tumors, osteosarcoma is often heterogeneous, consisting of phenotypically distinct cells with features of different stages of differentiation. The cancer stem cell hypothesis posits that tumors are maintained by stem cells and it is the incomplete eradication of a refractory population of tumor-initiating stem cells that accounts for drug resistance and tumor relapse. In this review we present our current knowledge about the biology of osteosarcoma stem cells from mouse and human tumors, highlighting new insights and unresolved issues in the identification of this elusive population. We focus on factors and pathways that are implicated in maintaining such cells, and differences from paradigms of epithelial cancers. Targeting of the cancer stem cells in osteosarcoma is a promising avenue to explore to develop new therapies for this devastating childhood cancer.

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins.

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.

Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome.

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 ß-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.

Distinct functions of Sox2 control self-renewal and differentiation in the osteoblast lineage.

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 (U. Basu-Roy et al., Cell Death Differ. 17:1345-1353, 2010; A. Mansukhani, D. Ambrosetti, G. Holmes, L. Cornivelli, and C. Basilico, J. Cell Biol. 168:1065-1076, 2005). Sox2 also interferes with Wnt signaling by binding ß-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 (TA) region is required for binding ß-catenin and interfering with Wnt-induced transcription. The results of gene expression analysis upon Sox2 deletion strongly support the notion that Sox2 maintains stemness. We show also that Sox2 suppresses differentiation by attenuating Wnt signaling by posttranscriptional and transcriptional mechanisms and that adenomatous polyposis coli (APC) and GSK3ß, which are negative regulators of the Wnt pathway, are direct Sox2 targets in osteoblasts. Several genes, such as the FoxP1 and BMI-1 genes, that are associated with stemness 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.

Regulation of non-classical FGF1 release and FGF-dependent cell transformation by CBF1-mediated notch signaling.

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.

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2arrest in RCS chondrocytes.

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 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 G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 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 G2 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 G2 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 G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.

Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology.

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.

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF.

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.

Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways.

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.

Downregulation of Akt activity contributes to the growth arrest induced by FGF in chondrocytes.

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.

A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control.

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.

Mechanisms underlying differential responses to FGF signaling.

Fibroblast growth factors (FGFs) are key regulators of several developmental processes in which cell fate and differentiation to various tissue lineages are determined. The importance of the proper spatial and temporal regulation of FGF signals is evident from human and mouse genetic studies which show that mutations leading to the dysregulation of FGF signals cause a variety of developmental disorders including dominant skeletal diseases and cancer. The FGF ligands signal via a family of receptor tyrosine kinases and, depending on the cell type or stage of maturation, produce diverse biological responses that include proliferation, growth arrest, differentiation or apoptosis. A central issue in FGF biology is to understand how these diverse cellular responses are determined and how similar signaling inputs can generate distinct patterns of gene expression that govern the specificity of the cellular response. In this review we draw upon studies from the past fifteen years and attempt to construct a molecular picture of the different levels of regulation by which such specific cellular responses could be achieved by FGF signals. We discuss whether specificity could lie in the nature of the ligand, the particular receptor, the signal transduction pathways utilized, or the transcriptional regulation of specific genes. Finally, we also discuss how the interplay of FGF signals with other signaling systems could contribute to the cellular response. In particular we focus on the interaction with the Wnt pathway since FGF/Wnt cross-talk is emerging as an important nexus in regulating a variety of biological processes.

Fibroblast growth factors share binding sites in heparan sulphate.

HS (heparan sulphate) proteoglycans bind secreted signalling proteins, including FGFs (fibroblast growth factors) through their HS side chains. Such chains contain a wealth of differentially sulphated saccharide epitopes. Whereas specific HS structures are commonly believed to modulate FGF-binding and activity, selective binding of defined HS epitopes to FGFs has generally not been demonstrated. In the present paper, we have identified a series of sulphated HS octasaccharide epitopes, derived from authentic HS or from biosynthetic libraries that bind with graded affinities to FGF4, FGF7 and FGF8b. These HS species, along with previously identified oligosaccharides that interact with FGF1 and FGF2, constitute the first comprehensive survey of FGF-binding HS epitopes based on carbohydrate sequence analysis. Unexpectedly, our results demonstrate that selective modulation of FGF activity cannot be explained in terms of binding of individual FGFs to specific HS target epitopes. Instead, different FGFs bind to identical HS epitopes with similar relative affinities and low selectivity, such that the strength of these interactions increases with increasing saccharide charge density. We conclude that FGFs show extensive sharing of binding sites in HS. This conclusion challenges the current notion of specificity in HS-FGF interactions, and instead suggests that a set of common HS motifs mediates cellular targeting of different FGFs.