Accuracy of in vivo microCT imaging in assessing the microstructural properties of the mouse tibia subchondral bone.

Osteoarthritis (OA) is one of the most common musculoskeletal diseases. OA is characterized by degeneration of the articular cartilage as well as the underlying subchondral bone. Post-traumatic osteoarthritis (PTOA) is a subset of OA caused by mechanical trauma. Mouse models, such as destabilization of the medial meniscus (DMM), are useful to study PTOA. Ex vivo micro-Computed Tomography (microCT) imaging is the predominant technique used to scan the mouse knee in OA studies. Nevertheless, in vivo microCT enables the longitudinal assessment of bone microstructure, reducing measurement variability and number of animals required. The effect of image resolution in measuring subchondral bone parameters was previously evaluated only for a limited number of parameters. The aim of this study was to evaluate the ability of in vivo microCT imaging in measuring the microstructural properties of the mouse tibia trabecular and cortical subchondral bone, with respect to ex vivo high resolution imaging, in a DMM model of PTOA. Sixteen male C57BL/6J mice received DMM surgery or sham operation at 14 weeks of age (N=8 per group). The right knee of each mouse was microCT scanned in vivo (10.4µm voxel size) and ex vivo (4.35µm voxel size) at the age of 26 weeks. Each image was aligned to a reference image using rigid registration. The subchondral cortical bone plate thickness was measured at the lateral and medial condyles. Standard morphometric parameters were measured in the subchondral trabecular bone. In vivo microCT imaging led to significant underestimation of bone volume fraction (-14%), bone surface density (-3%) and trabecular number (-16%), whereas trabecular thickness (+3%) and separation (+5%) were significantly overestimated. Nevertheless, most trabecular parameters measured in vivo were well correlated with ex vivo measurements (R2 = 0.69-0.81). Degree of anisotropy, structure model index and connectivity density were measured in vivo with lower accuracy. Excellent accuracy was found for cortical thickness measurements. In conclusion, this study identified what bone morphological parameters can be reliably measured by in vivo microCT imaging of the subchondral bone in the mouse tibia. It highlights that this approach can be used to study longitudinal effects of diseases and treatments on the subchondral cortical bone and on most subchondral trabecular bone parameters, but systematic over- or under-estimations should be considered when interpreting the results.

Non-invasive prediction of the mouse tibia mechanical properties from microCT images: comparison between different finite element models.

New treatments for bone diseases require testing in animal models before clinical translation, and the mouse tibia is among the most common models. In vivo micro-Computed Tomography (microCT)-based micro-Finite Element (microFE) models can be used for predicting the bone strength non-invasively, after proper validation against experimental data. Different modelling techniques can be used to estimate the bone properties, and the accuracy associated with each is unclear. The aim of this study was to evaluate the ability of different microCT-based microFE models to predict the mechanical properties of the mouse tibia under compressive load. Twenty tibiae were microCT scanned at 10.4 µm voxel size and subsequently compressed at 0.03 mm/s until failure. Stiffness and failure load were measured from the load-displacement curves. Different microFE models were generated from each microCT image, with hexahedral or tetrahedral mesh, and homogeneous or heterogeneous material properties. Prediction accuracy was comparable among models. The best correlations between experimental and predicted mechanical properties, as well as lower errors, were obtained for hexahedral models with homogeneous material properties. Experimental stiffness and predicted stiffness were reasonably well correlated (R2 = 0.53-0.65, average error of 13-17%). A lower correlation was found for failure load (R2 = 0.21-0.48, average error of 9-15%). Experimental and predicted mechanical properties normalized by the total bone mass were strongly correlated (R2 = 0.75-0.80 for stiffness, R2 = 0.55-0.81 for failure load). In conclusion, hexahedral models with homogeneous material properties based on in vivo microCT images were shown to best predict the mechanical properties of the mouse tibia.

Optimization of the failure criterion in micro-Finite Element models of the mouse tibia for the non-invasive prediction of its failure load in preclinical applications.

New treatments against osteoporosis require testing in animal models and the mouse tibia is among the most common studied anatomical sites. In vivo micro-Computed Tomography (microCT) based micro-Finite Element (microFE) models can be used for predicting the bone strength non-invasively, after proper validation against experiments. The aim of this study was to evaluate the ability of different microCT-based bone parameters and microFE models to predict tibial structural mechanical properties in compression. Twenty tibiae were scanned at 10.4 µm voxel size and subsequently tested in uniaxial compression at 0.03 mm/s until failure. Stiffness and failure load were measured from the load-displacement curves. Standard morphometric parameters were measured from the microCT images. The spatial distribution of bone mineral content (BMC) was evaluated by dividing the tibia into 40 regions. MicroFE models were generated by converting each microCT image into a voxel-based mesh with homogeneous isotropic material properties. Failure load was estimated by using different failure criteria, and the optimized parameters were selected by minimising the errors with respect to experimental measurements. Experimental and predicted stiffness were moderately correlated (R2 = 0.65, error = 14% ± 8%). Normalized failure load was best predicted by microFE models (R2 = 0.81, error = 9% ± 6%). Failure load was not correlated to the morphometric parameters and weakly correlated with some geometrical parameters (R2 < 0.37). In conclusion, microFE models can improve the current estimation of the mouse tibia structural properties and in this study an optimal failure criterion has been defined. Since it is a non-invasive method, this approach can be applied longitudinally for evaluating temporal changes in the bone strength.

Validation of finite element models of the mouse tibia using digital volume correlation.

The mouse tibia is a common site to investigate bone adaptation. Micro-Finite Element (microFE) models based on micro-Computed Tomography (microCT) images can estimate bone mechanical properties non-invasively but their outputs need to be validated with experiments. Digital Volume Correlation (DVC) can provide experimental measurements of displacements over the whole bone volume. In this study we applied DVC to validate the local predictions of microFE models of the mouse tibia in compression. Six mouse tibiae were stepwise compressed within a microCT system. MicroCT images were acquired in four configurations with applied compression of 0.5 N (preload), 6.5 N, 13.0 N and 19.5 N. Failure load was measured after the last scan. A global DVC algorithm was applied to the microCT images in order to obtain the displacement field over the bone volume. Homogeneous, isotropic linear hexahedral microFE models were generated from the images collected in the preload configuration with boundary conditions interpolated from the DVC displacements at the extremities of the tibia. Experimental displacements from DVC and numerical predictions were compared at corresponding locations in the middle of the bone. Stiffness and strength were also estimated from each model and compared with the experimental measurements. The magnitude of the displacement vectors predicted by microFE models was highly correlated with experimental measurements (R2 >0.82). Higher but still reasonable errors were found for the Cartesian components. The models tended to overestimate local displacements in the longitudinal direction (R2 = 0.69-0.90, slope of the regression line=0.50-0.97). Errors in the prediction of structural mechanical properties were 14% ±â€¯11% for stiffness and 9% ±â€¯9% for strength. In conclusion, the DVC approach has been applied to the validation of microFE models of the mouse tibia. The predictions of the models for both structural and local properties have been found reasonable for most preclinical applications.

SiO2 nanoparticles modulate the electrical activity of neuroendocrine cells without exerting genomic effects.

Engineered silica nanoparticles (NPs) have attracted increasing interest in several applications, and particularly in the field of nanomedicine, thanks to the high biocompatibility of this material. For their optimal and controlled use, the understanding of the mechanisms elicited by their interaction with the biological target is a prerequisite, especially when dealing with cells particularly vulnerable to environmental stimuli like neurons. Here we have combined different electrophysiological approaches (both at the single cell and at the population level) with a genomic screening in order to analyze, in GT1-7 neuroendocrine cells, the impact of SiO2 NPs (50 ± 3 nm in diameter) on electrical activity and gene expression, providing a detailed analysis of the impact of a nanoparticle on neuronal excitability. We find that 20 µg mL-1 NPs induce depolarization of the membrane potential, with a modulation of the firing of action potentials. Recordings of electrical activity with multielectrode arrays provide further evidence that the NPs evoke a temporary increase in firing frequency, without affecting the functional behavior on a time scale of hours. Finally, NPs incubation up to 24 hours does not induce any change in gene expression.

Effect of integration time on the morphometric, densitometric and mechanical properties of the mouse tibia.

Micro-Computed Tomography (microCT) images are used to measure morphometric and densitometric properties of bone, and to develop finite element (FE) models to estimate mechanical properties. However, there are concerns about the invasiveness of microCT imaging due to the X-rays ionising radiation induced by the repeated scans on the same animal. Therefore, the best compromise between radiation dose and image quality should be chosen for each preclinical application. In this study, we investigated the effect of integration time (time the bone is exposed to radiation at each rotation step during microCT imaging) on measurements performed on the mouse tibia. Four tibiae were scanned at 10.4 µm voxel size using four different procedures, characterized by decreasing integration time (from 200 ms to 50 ms) and therefore decreasing nominal radiation dose (from 513 mGy to 128 mGy). From each image, trabecular and cortical morphometric parameters, spatial distribution of bone mineral content (BMC) in the whole tibia and FE-based estimations of stiffness and strength were obtained. A high-resolution scan (4.3 µm voxel size) was used to quantify measurement errors. Integration time had the largest effect on trabecular morphometric parameters (7-28%). Lower effects were observed on cortical parameters (1-3%), BMC (1-10%) distribution, and FE-based estimations of mechanical properties (1-3%). In conclusion, the effect of integration time on image-based measurements has been quantified. This data should be considered when defining the in vivo microCT scanning protocols in order to find the best compromise between nominal radiation exposure and accuracy in the estimation of bone parameters.

Mutations in NOTCH1 PEST domain orchestrate CCL19-driven homing of chronic lymphocytic leukemia cells by modulating the tumor suppressor gene DUSP22.

Even if NOTCH1 is commonly mutated in chronic lymphocytic leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling. CLL patients with a NOTCH1-mutated clone showed low levels of DUSP22 and active chemotaxis to CCL19. Lastly, in xenograft models, NOTCH1-mutated cells displayed a unique homing behavior, localizing preferentially to the spleen and brain. These findings connect NOTCH1, DUSP22, and CCL19-driven chemotaxis within a single functional network, suggesting that modulation of the homing process may provide a relevant contribution to the unfavorable prognosis associated with NOTCH1 mutations in CLL.

TET1 is a tumour suppressor that inhibits colon cancer growth by derepressing inhibitors of the WNT pathway.

Ten eleven translocation (TET) enzymes catalyse the oxidative reactions of 5-methylcytosine (5mC) to promote the demethylation process. The reaction intermediate 5-hydroxymethylcytosine (5hmC) has been shown to be abundant in embryonic stem cells and tissues but strongly depleted in human cancers. Genetic mutations of TET2 gene were associated with leukaemia, whereas TET1 downregulation has been shown to promote malignancy in breast cancer. Here we report that TET1 is downregulated in colon tumours from the initial stage. TET1 silencing in primary epithelial colon cells increase their cellular proliferation while its re-expression in colon cancer cells inhibits their proliferation and the growth of tumour xenografts even at later stages. We found that TET1 binds to the promoter of the DKK gene inhibitors of the WNT signalling to maintain them hypomethylated. Downregulation of TET1 during colon cancer initiation leads to repression, by DNA methylation, the promoters of the inhibitors of the WNT pathway resulting in a constitutive activation of the WNT pathway. Thus the DNA hydroxymethylation mediated by TET1 controlling the WNT signalling is a key player of tumour growth. These results provide new insights for understanding how tumours escape cellular controls.

Identification and characterization of a novel nuclear factor of activated T-cells-1 isoform expressed in mouse brain.

The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3'-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in T-cells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context.

Sp1 and Sp3 physically interact and co-operate with GABP for the activation of the utrophin promoter.

The utrophin gene codes for a large cytoskeletal protein closely related to dystrophin which, in the absence of dystrophin, can functionally substitute it. Utrophin is transcribed by two independently regulated promoters about 50 kb apart. The upstream promoter is TATA-less and contains a functional GABP binding site which, in muscle, restricts the promoter activity to post-synaptic nuclei. Transient transfections analysis of mutant promoters in rhabdomyosarcoma cells showed that the upstream promoter contains three functional GC elements that are recognised by Sp1 and Sp3 factors in vitro. Co-transfections of the promoter with Sp1, Sp3 and GABP factors in Drosophila SL2 Schneider cells, which lack of endogenous Sp factors, demonstrated that both Sp1 and Sp3 are positive regulators of the utrophin promoter and that they activate transcription synergistically with GABP. Consistent with this result, we observed physical interaction of both Sp factors with the GABPalpha subunit in vitro. Functional domain interaction analysis of Sp1 and Sp3 revealed that both factors interact with GABPalpha through their DNA binding zinc finger domain. The modulation and correct interaction between Sp1, Sp3 and GABP in muscle cells may be critical for the regulation of the utrophin promoter, and provide new targets for therapies of Duchenne muscular dystrophy.

In fibroblasts Vegf-D expression is induced by cell-cell contact mediated by cadherin-11.

Vascular endothelial growth factors (VEGFs) are a highly conserved family of growth factors all angiogenic in vivo with mitogenic and chemotactic activity on endothelial cells. VEGFs are expressed in fibroblasts either in hypoxia or in response to growth factors. Here we report that, differently from the other members of the family, Vegf-D is induced by cell-cell contact. By in situ hybridization we demonstrated that noninteracting fibroblasts express low levels of Vegf-D mRNA, whereas contacting cells express high levels of Vegf-D transcripts. By immunostaining we observed that the surface protein cadherin-11 is localized at the opposite sites of interacting cell surfaces. Ca(2+) deprivation from the culture medium determined the loss of cadherin-11 from the cell surfaces and down-regulation of Vegf-D mRNA. Moreover, a cadherin-11 antisense RNA construct inhibited Vegf-D expression in confluent BALB/c fibroblasts, whereas in NIH 3T3 cells, which express low levels of cadherin-11, Vegf-D induction could be obtained by overexpression of cadherin-11. This suggests that cell interaction mediated by cadherin-11 induces the expression of the angiogenic factor Vegf-D in fibroblasts.

Preparation of yeast RNA.

This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample-to-sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.

Utrophin transcription is activated by an intronic enhancer.

The utrophin gene codes for a large cytoskeletal protein closely related to dystrophin. Its transcription is driven by a TATA-less promoter. Here we analyzed 40 kilobases of the 5' end region of the utrophin gene searching for new utrophin regulatory elements in muscle cells. By transient transfection of utrophin genomic fragments in front of a reporter gene, we identified a new enhancer that maps downstream of the transcription start site within the second intron and co-localizes with a DNase I-hypersensitive site. By deletion analysis it was mapped to a sequence of 128 base pairs that retains the whole activity. Linker scanning mutagenesis showed that most of the enhancer sequence is essential for its transcriptional activity. Binding analysis with nuclear cell extracts demonstrated that the enhancer regulatory elements, identified by mutagenesis, are protected from DNase I digestion. Because utrophin can functionally substitute dystrophin, the identification and characterization of new regulatory elements provide new targets for possible therapies of Duchenne muscular dystrophy aiming at the up-regulation of the utrophin expression in muscle cells.

c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro.

c-fos-induced growth factor/vascular endothelial growth factor D (Figf/Vegf-D) is a secreted factor of the VEGF family that binds to the vessel and lymphatic receptors VEGFR-2 and VEGFR-3. Here we report that Figf/Vegf-D is a potent angiogenic factor in rabbit cornea in vivo in a dose-dependent manner. In vitro Figf/Vegf-D induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in primary human umbilical cord vein endothelial cells (HUVECs) and in an immortal cell line derived from Kaposi's sarcoma lesion (KS-IMM). The treatment of HUVECs with Figf/Vegf-D induces dose-dependent cell growth. Figf/VEGF-D also induces HUVEC elongation and branching to form an extensive network of capillary-like cords in three-dimensional matrix. In KS-IMM cells Figf/Vegf-D treatment results in dose-dependent mitogenic and motogenic activities. Taken together with the previous observations that Figf/Vegf-D expression is under the control of the nuclear oncogene c-fos, our data uncover a link between a nuclear oncogene and angiogenesis, suggesting that Figf/Vegf-D may play a critical role in tumor cell growth and invasion.

The dystrophin promoter is negatively regulated by YY1 in undifferentiated muscle cells.

The dystrophin gene transcription is up-regulated during muscle cell differentiation. Its expression in muscle cells is induced by the binding of the positive regulators serum response factor and dystrophin promoter bending factor (DPBF) on a regulatory CArG element present on the promoter. Here we show that the dystrophin CArG box is also recognized by the zinc finger nuclear factor YY1. Transient transfection experiments show that YY1 negatively regulates dystrophin transcription in C2C12 muscle cells. On the dystrophin CArG element YY1 competes with the structural factor DPBF. We further show that YY1 and DPBF binding to the CArG element induce opposite DNA bends suggesting that their binding induces alternative promoter structures. Along with C2C12 myotube formation YY1 is reduced and we observed that YY1, but not DPBF, is a substrate of m-calpain, a protease that is up-regulated in muscle cell differentiation. Thus, high levels of YY1 in non-differentiated muscle cells down-regulate the dystrophin promoter, at least in part, by interfering with the spatial organization of the promoter.

Protein kinase CK2alpha' is induced by serum as a delayed early gene and cooperates with Ha-ras in fibroblast transformation.

Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha') and two noncatalytic (beta) subunits forming a heterotetrameric holoenzyme involved in cell growth and differentiation. Here we report the identification, cloning, and oncogenic activity of the murine CK2alpha' subunit. Serum treatment of quiescent mouse fibroblasts induces CK2alpha' mRNA expression, which peaks at 4 h. The kinetics of CK2alpha' expression correlate with increased kinase activity toward a specific CK2 holoenzyme peptide substrate. The ectopic expression of CK2alpha' (or CK2alpha) cooperates with Ha-ras in foci formation of rat primary embryo fibroblasts. Moreover, we observed that BALB/c 3T3 fibroblasts transformed with Ha-ras and CK2alpha' show a faster growth rate than cells transformed with Ha-ras alone. In these cells the higher growth rate correlates with an increase in calmodulin phosphorylation, a protein substrate specifically affected by isolated CK2 catalytic subunits but not by CK2 holoenzyme, suggesting that unbalanced expression of a CK2 catalytic subunit synergizes with Ha-ras in cell transformation.

Embryonic expression pattern of the murine figf gene, a growth factor belonging to platelet-derived growth factor/vascular endothelial growth factor family.

Morphogenesis, growth and differentiation of tissues and organs require cell interactions mediated by signal molecules, their receptors and transcriptional control systems. c-fos-induced growth factor (figf) is a new secreted member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family with mitogenic activity on fibroblasts. Here we studied figf expression during murine embryonic development. figf expression was detected with a dynamic pattern in several body structures and organs such as limb buds, acoustic ganglion, teeth, heart, anterior pituitary as well as lung and kidney mesenchyme, liver, derma, and periosteum of the vertebral column.

Human FIGF: cloning, gene structure, and mapping to chromosome Xp22.1 between the PIGA and the GRPR genes.

We report the identification, structural characterization, and mapping of the human FIGF gene. FIGF is the human homologue of mouse figf (c-fos-induced growth factor), a new member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. It codes for a secreted factor with mitogenic and morphogenic activity on fibroblast cells. The predicted amino acid sequence of FIGF is 84% identical to that of the mouse protein, and it is highly conserved (up to 40%) in the dimerization domain with respect to the VEGF members of the family. The 2.5-kb mRNA of FIGF was detected in adult lung and heart tissues. The gene spans about 50 kb and is organized into seven exons and six introns. The FIGF promoter contains an optimal AP-1-binding site and lacks a canonical TATA box. Fluorescence in situ hybridization mapped FIGF to chromosomal region Xp22.1. The subsequent identification of YAC positive clones from this region allowed us to refine the map and localize FIGF centromeric to the phosphatidylinositol glycan complementation class A (PIGA) gene and telomeric to the gastrin-releasing peptide receptor (GRPR) gene. FIGF and PIGA genes lie next to each other in a head-to-tail orientation, with the FIGF polyadenylation signal about 12 kb from the PIGA transcriptional start site.

Serum response factor and protein-mediated DNA bending contribute to transcription of the dystrophin muscle-specific promoter.

The minimal muscle-specific dystrophin promoter contains the consensus sequence CC(A/T)6GG, or the CArG element, which can be found in serum-inducible or muscle-specific promoters. The serum response factor (SRF), which mediates the transcriptional activation of the c-fos gene in response to serum stimulation, can bind to different CArG box elements, suggesting that it could be involved in muscle-constitutive transcription. Here we show that SRF binds to the dystrophin promoter and regulates its muscle-specific transcription. In transient transfections, an altered-binding-specificity SRF mutant restores the muscle-constitutive transcription of a dystrophin promoter with a mutation in its CArG box element. The muscle-constitutive transcription of the dystrophin promoter also requires the sequence GAAACC immediately downstream of the CArG box. This sequence is recognized by a novel DNA bending factor which was named dystrophin promoter-bending factor (DPBF). Mutations of the CArG flanking sequence abolish both DPBF binding and the promoter activity in muscle cells. Its replacement with a p62/ternary complex factor binding site changes the promoter specificity from muscle constitutive to serum responsive. These results show that, on the dystrophin promoter, the transcriptional activation induced by SRF requires the DNA bending induced by DPBF. The bending, next to the CArG box, could promote interactions between SRF and other proteins in the transcriptional complex.

Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family.

Using a mRNA differential screening of fibroblasts differing for the expression of c-fos we isolated a c-fos-induced growth factor (FIGF). The deduced protein sequence predicts that the cDNA codes for a new member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. Northern blot analysis shows that FIGF expression is strongly reduced in c-fos-deficient cells. Transfection of exogenous c-fos driven by a constitutive promoter restores the FIGF expression in these cells. In contrast, both PDGF and VEGF expression is unaffected by c-fos. FIGF is a secreted dimeric protein able to stimulate mitogenic activity in fibroblasts. FIGF overexpression induces morphological alterations in fibroblasts. The cells acquire a spindle-shaped morphology, become more refractive, disorganized, and detach from the plate. These results imply that FIGF is a downstream growth and morphogenic effector of c-fos. These results also suggest that the expression of FIGF in response to c-fos activation induces specific differentiation patterns and its aberrant activation contributes to the malignant phenotype of tumors.