alpha-Tocopheryl succinate inhibits angiogenesis by disrupting paracrine FGF2 signalling

Malignant mesothelioma (MM) cells enhanced proliferation of endothelial cells (ECs) as well as their angiogenesis in vitro by secretion of fibroblast growth factor-2 (FGF2). This effect was suppressed by pre-treating MM cells with alpha-tocopheryl succinate (alpha-TOS), which inhibited FGF2 secretion by inducing mitochondria-dependent generation of reactive oxygen species. The role of FGF2 was confirmed by its down-regulation by treating MM cells with siRNA, abolishing EC proliferation and wound healing enhancement afforded by MM cells. We conclude that alpha-TOS disrupts angiogenesis mediated by MM cells by inhibiting FGF2 paracrine signalling.     

Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture

One characteristic of skeletal muscle differentiation is the conversion of proliferating cells to a population that is irreversibly postmitotic. This developmental change can be induced in vitro by depriving the cultures of specific mitogens such as fibroblast growth factor (FGF). Analysis of cell surface FGF receptor (FGFR) in several adult mouse muscle cell lines and epidermal growth factor receptor (EGFR) in mouse MM14 cells reveals a correlation between receptor loss and the acquisition of a postmitotic phenotype. Quiescent MM14 cells, mitogen-depleted, differentiation-defective MM14 cells, and differentiated BC3H1 muscle cells (a line that fails to become postmitotic upon differentiation) retained their cell surface FGFR. These results indicate that FGFR loss is not associated with either reversible cessation of muscle cell proliferation or biochemical differentiation and thus, further support a correlation between receptor loss and acquisition of a postmitotic phenotype. Comparison of the kinetics for growth factor receptor loss and for commitment of MM14 cells to a postmitotic phenotype reveals that FGFR rises transiently from approximately 700 receptors/cell to a maximum of approximately 2,000 receptors/cell 12 h after FGF removal, when at the same time, greater than 95% of the cells are postmitotic. FGFR levels then decline to undetectable levels by 24 h after FGF removal. During the interval in which FGFR increases and then disappears there is no change in its affinity for FGF. The transient increase in growth factor receptors appears to be due to a decrease in ligand-mediated internalization because EGFR, which undergoes an immediate decline when cultures are deprived of FGF (Lim, R. W., and S. D. Hauschka. 1984. J. Cell Biol. 98:739-747), exhibits a similar transient rise when cultures are grown in media containing both EGF and FGF before switching the cells to media without these added factors. These results indicate that the loss of certain growth factor receptors is a specific phenotype acquired during skeletal muscle differentiation, but they do not resolve whether regulation of FGFR number is causal for initiation of the postmitotic phenotype. A general model is presented in the discussion.     

Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

Alpha-tocopheryl succinate (alpha-TOS), a redox-silent analogue of vitamin E, induces apoptosis in multiple cell lines in a selective manner, by activating the intrinsic pathway. Since it is a highly hydrophobic compound, it may require a carrier protein for its trafficking to intracellular targets like mitochondria. We studied the role of the ubiquitous tocopherol-associated protein-1 (TAP1 or sec14-like 2) in apoptosis induction by alpha-TOS in malignant mesothelioma (MM) cells. Over-expression of TAP1 in MM cells sensitised them to apoptosis by low doses of alpha-TOS which were sub-apoptotic for the parental cells. Apoptosis induced in TAP1-over-expressing cells was mitochondria- and caspase-dependent, as suggested by dissipation of mitochondrial trans-membrane potential and inhibition by zVAD-fmk, respectively. Binding assays showed affinity of alpha-TOS for TAP1. Finally, TAP1 over-expressing cells accumulated alpha-TOS at higher levels compared to their normal counterparts. We suggest that TAP1 may act as an intracellular shuttle for alpha-TOS, promoting apoptosis initiated by this vitamin E analogue, as shown here for MM cells.     

A Dominant-Negative FGF1 Mutant (the R50E Mutant) Suppresses Tumorigenesis and Angiogenesis

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent (“FGF1 decoy”).     

Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of cyclin E-Cdk2

Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G(1) phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb.     

Role of thioredoxin-1 in apoptosis induction by alpha-tocopheryl succinate and TNF-related apoptosis-inducing ligand in mesothelioma cells

Malignant mesothelioma (MM) is a fatal type of cancer. We studied the role of the redox-active protein thioredoxin-1 (Trx-1) in apoptosis induced in MM cells and their non-malignant counterparts (Met-5A) by alpha-tocopheryl succinate (alpha-TOS) and TNF-related apoptosis-inducing ligand (TRAIL). MM cells were susceptible to alpha-TOS and less to TRAIL, while Met-5A cells were susceptible to TRAIL and resistant to alpha-TOS. MM cells expressed very low level of the Trx-1 protein, which was high in Met-5A cells, while the level of Trx-1 mRNA was similar in all cell lines. Downregulation of Trx-1 further sensitised Met-5A cells to TRAIL but not to alpha-TOS. Our data suggest that the role of Trx-1 in apoptosis modulation is unrelated to its anti-oxidant properties.     

A role for extracellular and transmembrane domains of Sef in Sef-mediated inhibition of FGF signaling

Sef (similar expression to fgf genes) is a member of the fibroblast growth factor (FGF) synexpression group that negatively regulates FGF receptor (FGFR) signaling in zebrafish during early embryonic development and in mammalian cell culture systems. The mechanism by which Sef exerts its inhibitory effect remains controversial. It has been reported that Sef functions either through binding to and inhibiting FGFR1 activation or by acting downstream of FGF receptors at the level of MEK/ERK kinases. In both cases, the intracellular domain of Sef was found to play a role in the inhibitory function of Sef. Here we demonstrated that both extracellular and transmembrane domains of Sef contributed to Sef-mediated negative regulation of FGF signaling. In fact, over-expression studies in NIH3T3 cells showed that a truncated mutant of Sef, which lacks the intracellular domain (SefECTM), exerted the inhibitory activity on FGF signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent activation of the Raf/MEK/ERK signaling cascade. We also showed that SefECTM associated with FGFR1, and inhibited FGF-induced ERK activation in HEK293T cells. Furthermore, we demonstrated that the over-expression of SefECTM was able to inhibit the function of a constitutively activated form of FGFR1, FGFR1-C289R, but not FGFR1-K562E. Finally, we found that SefECTM reduced cell viability when over-expressed in human umbilical vein endothelial cells (HUVEC). These data provide additional insight into the structure-activity relationship in the mechanism of inhibitory action of Sef on FGFR1-mediated signaling.     

Fibroblast growth factor receptor-1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: a role for protein kinase A

The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF-2) or interfere with complex formation between FGF-2 and its high-affinity FGF receptor-1 (FGFR1), yet both native SPARC and a peptide derived from the C-terminal high-affinity Ca(2+)-binding region of protein significantly inhibit ligand-induced autophosphorylation of FGFR1 (>80%), activation of mitogen-activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF-2 (>80%). We also report that in the presence of FGF-2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca(2+)-binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)-deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1-mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell-surface, low-affinity FGF-2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand-binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf-MAPK pathway. SPARC thus modulates the mitogenic effect of FGF-2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade.     

Fgfr1 and Fgfr2 have distinct differentiation- and proliferation-related roles in the developing mouse skull vault

Fibroblast growth factor receptors (FGFRs) play major roles in skeletogenesis, and activating mutations of the human FGFR1, FGFR2 and FGFR3 genes cause premature fusion of the skull bones (craniosynostosis). We have investigated the patterns of expression of Fgfr1, Fgfr2 and Fgfr3 in the fetal mouse head, with specific reference to their relationship to cell proliferation and differentiation in the frontal and parietal bones and in the coronal suture. Fgfr2 is expressed only in proliferating osteoprogenitor cells; the onset of differentiation is preceded by down-regulation of Fgfr2 and up-regulation of Fgfr1. Following up-regulation of the differentiation marker osteopontin, Fgfr1, osteonectin and alkaline phosphatase are down-regulated, suggesting that they are involved in the osteogenic differentiation process but not in maintaining the differentiated state. Fgfr3 is expressed in the cranial cartilage, including a plate of cartilage underlying the coronal suture, as well as in osteogenic cells, suggesting a dual role in skull development. Subcutaneous insertion of FGF2-soaked beads onto the coronal suture on E15 resulted in up-regulation of osteopontin and Fgfr1 in the sutural mesenchyme, down-regulation of Fgfr2, and inhibition of cell proliferation. This pattern was observed at 6 and 24 hours after bead insertion, corresponding to the timing and duration of FGF2 diffusion from the beads. We suggest (a) that a gradient of FGF ligand, from high levels in the differentiated region to low levels in the environment of the osteogenic stem cells, modulates differential expression of Fgfr1 and Fgfr2, and (b) that signalling through FGFR2 regulates stem cell proliferation whereas signalling through FGFR1 regulates osteogenic differentiation.     

Caveolin-1 orchestrates fibroblast growth factor 2 signaling control of angiogenesis in placental artery endothelial cell caveolae

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-β-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis.     

Cooperative dimerization of fibroblast growth factor 1 (FGF1) upon a single heparin saccharide may drive the formation of 2:2:1 FGF1.FGFR2c.heparin ternary complexes

The related glycosaminoglycans heparin and heparan sulfate are essential for the activity of the fibroblast growth factor (FGF) family as they form an integral part of the signaling complex at the cell surface. Using size-exclusion chromatography we have studied the capacities of a variety of heparin oligosaccharides to bind FGF1 and FGFR2c both separately and together in ternary complexes. In the absence of heparin, FGF1 had no detectable affinity for FGFR2c. However, 2:2:1 complexes formed spontaneously in solution between FGF1, FGFR2c, and heparin octasaccharide (dp8). The dp8 sample was the shortest chain length that bound FGFR2c, that dimerized FGF1, and that promoted a strong mitogenic response to FGF1 through FGFR2c. Heparin hexasaccharide and various selectively desulfated heparin dp12s failed to bind FGFR2c and could only interact with FGF1 monomerically. These saccharides formed 1:1:1 complexes with FGF1 and FGFR2c, which had no tendency to self-associate, suggesting that binding of two FGF1 molecules to the same saccharide chain is a prerequisite for subsequent FGFR2c dimerization. We found that FGF1 dimerization upon heparin was favored over monomeric interactions even when a large excess of saccharide was present. A cooperative mechanism of FGF1 dimerization could explain how 2:2:1 signaling complexes form at the cell surface, an environment rich in heparan sulfate.     

Direct binding of integrin alphavbeta3 to FGF1 plays a role in FGF1 signaling

Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin alphavbeta3 (K(D) about 1 microm). Antagonists to alphavbeta3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. alphavbeta3 was the predominant, if not the only, integrin that bound to FGF1, because FGF1 bound only weakly to several beta1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of beta3 plays a role in FGF1 binding. We found that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1 and FRS2alpha and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful tool to identify the role of integrins in FGF signaling.     

Membrane heparan sulfate proteoglycan-supported FGF2-FGFR1 signaling: evidence in support of the "cooperative end structures" model

Fibroblast growth factor 2 (FGF2)-initiated FGF receptor (FGFR)-signaling requires the assistance of heparin/heparan sulfate. Here, we evaluated the effects of different heparan sulfate proteoglycan (HSPG)-expressing cell lines and HSPGs derived from these cells on FGF2-induced FGFR1-phosphorylation in heparan sulfate-negative BaF3 cells. HSPGs supplied in membrane-associated form, by presenting cells, were all effective promotors of FGF2-initiated FGFR1 phosphorylation, independently of their nature (syndecan/glypican) or cellular origin (human lung fibroblasts, transfected Namalwa cells, or transfected K562 cells). A treatment with heparitinase initially stimulated, but finally completely inhibited, the activity of these presenting cells. In comparison, equivalent amounts of soluble HSPGs, obtained by trypsinization of these cells or by immunopurification from cell extracts, did not promote FGF2-induced FGFR1-phosphorylation, yet removal of the less anionic species or a further treatment with heparitinase converted these soluble fractions into potent activators of FGF2/FGFR1 signaling. Extrapolating from current structural models, we suggest that FGFR dimerization and autophosphorylation is supported by cooperative "heparin-like end structures," and that cell surface association and concentration compensate for the relative scarcity of such end structures in native HSPGs. In this model, "proteolytic" shedding of heparan sulfate would act as a diluting, down-regulatory mechanism, while "heparanolytic" shedding might act as an up-regulatory mechanism, by increasing the concentration of these end structures.     

Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis

Two members of the fibroblast growth factor (FGF) family, basic FGF (bFGF) and FGF-18, have been implicated in the regulation of articular and intervertebral disc (IVD) cartilage homeostasis. Studies on bFGF from a variety of species have yielded contradictory results with regards to its precise role in cartilage matrix synthesis and degradation. In contrast, FGF-18 is a well-known anabolic growth factor involved in chondrogenesis and articular cartilage repair. In this review, we examined the biological actions of bFGF and FGF-18 in articular and IVD cartilage, the specific cell surface receptors bound by each factor, and the unique signaling cascades and molecular pathways utilized to exert their biological effects. Evidence suggests that bFGF selectively activates FGF receptor 1 (FGFR1) to exert degradative effects in both human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme activity, inhibition of matrix production, and increased cell proliferation resulting in clustering of cells seen in arthritic states. FGF-18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating FGFR3, increasing matrix formation and cell differentiation while inhibiting cell proliferation, leading to dispersed cells surrounded by abundant matrix. The results from in vitro and in vivo studies suggest the potential usefulness of bFGF and FGFR1 antagonists, as well as FGF-18 and FGFR3 agonists, as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future.     

Gene expression patterns of the fibroblast growth factors and their receptors during myogenesis of rat satellite cells.

Satellite cells are the myogenic precursors in postnatal muscle and are situated beneath the myofiber basement membrane. We previously showed that fibroblast growth factor 2 (FGF2, basic FGF) stimulates a greater number of satellite cells to enter the cell cycle but does not modify the overall schedule of a short proliferative phase and a rapid transition to the differentiated state as the satellite cells undergo myogenesis in isolated myofibers. In this study we investigated whether other members of the FGF family can maintain the proliferative state of the satellite cells in rat myofiber cultures. We show that FGF1, FGF4, and FGF6 (as well as hepatocyte growth factor, HGF) enhance satellite cell proliferation to a similar degree as that seen with FGF2, whereas FGF5 and FGF7 are ineffective. None of the growth factors prolongs the proliferative phase or delays the transition of the satellite cells to the differentiating, myogenin(+) state. However, FGF6 retards the rapid exit of the cells from the myogenin(+) state that routinely occurs in myofiber cultures. To determine which of the above growth factors might be involved in regulating satellite cells in vivo, we examined their mRNA expression patterns in cultured rat myofibers using RT-PCR. The expression of all growth factors, excluding FGF4, was confirmed. Only FGF6 was expressed at a higher level in the isolated myofibers and not in the connective tissue cells surrounding the myofibers or in satellite cells dissociated away from the muscle. By Western blot analysis, we also demonstrated the presence of FGF6 protein in the skeletal musle tissue. Our studies therefore suggest that the myofibers serve as the main source for the muscle FGF6 in vivo. We also used RT-PCR to analyze the expression patterns of the four tyrosine kinase FGF receptors (FGFR1-FGFR4) and of the HGF receptor (c-met) in the myofiber cultures. Depending on the time in culture, expression of all receptors was detected, with FGFR2 and FGFR3 expressed only at a low level. Only FGFR4 was expressed at a higher level in the myofibers but not the connective tissue cell cultures. FGFR4 was also expressed at a higher level in satellite cells compared to the nonmyogenic cells when the two cell populations were released from the muscle tissue and fractionated by Percoll density centrifugation. The unique localization patterns of FGF6 and FGFR4 may reflect specific roles for these members of the FGF signaling complex during myogenesis in adult skeletal muscle.