Endothelial IL17RD promotes Western diet-induced aortic myeloid cell infiltration.

The Interleukin-17 (IL17) family is a group of cytokines implicated in the etiology of several inflammatory diseases. Interleukin-17 receptor D (IL17RD), also known as Sef (similar expression to fibroblast growth factor) belonging to the family of IL17 receptors, has been shown to modulate IL17A-associated inflammatory phenotypes. The objective of this study was to test the hypothesis that IL17RD promotes endothelial cell activation and consequent leukocyte adhesion. We utilized primary human aortic endothelial cells and demonstrated that RNAi targeting of IL17RD suppressed transcript levels by 83 % compared to non-targeted controls. Further, RNAi knockdown of IL17RD decreased the adhesion of THP-1 monocytic cells onto a monolayer of aortic endothelial cells in response to IL17A. Additionally, we determined that IL17A did not significantly enhance the activation of canonical MAPK and NFκB pathways in endothelial cells, and further did not significantly affect the expression of VCAM-1 and ICAM-1 in aortic endothelial cells, which is contrary to previous findings. We also determined the functional relevance of our findings in vivo by comparing the expression of endothelial VCAM-1 and ICAM-1 and leukocyte infiltration in the aorta in Western diet-fed Il17rd null versus wild-type mice. Our results showed that although Il17rd null mice do not have significant alteration in aortic expression of VCAM-1 and ICAM-1 in endothelial cells, they exhibit decreased accumulation of proinflammatory monocytes and neutrophils, suggesting that endothelial IL17RD induced in vivo myeloid cell accumulation is not dependent on upregulation of VCAM-1 and ICAM-1 expression. We further performed proteomics analysis to identify potential molecular mediators of the IL17A/IL17RD signaling axis. Collectively, our results underscore a critical role for Il17rd in the regulation of aortic myeloid cell infiltration in the context of Western diet feeding.

Interleukin-17 receptor D (Sef) is a multi-functional regulator of cell signaling.

Interleukin-17 receptor D (IL17RD or IL-17RD) also known as Sef (similar expression to fibroblast growth factor), is a single pass transmembrane protein that is reported to regulate several signaling pathways . IL17RD was initially described as a feedback inhibitor of fibroblast growth factor (FGF) signaling during zebrafish and frog development. It was subsequently determined to regulate other receptor tyrosine kinase signaling cascades as well as several proinflammatory signaling pathways including Interleukin-17A (IL17A), Toll-like receptors (TLR) and Interleukin-1α (IL1α) in several vertebrate species including humans. This review will provide an overview of IL17RD regulation of signaling pathways and functions with emphasis on regulation of development and pathobiological conditions. We will also discuss gaps in our knowledge about IL17RD function to provide insight into opportunities for future investigation. Video Abstract.

Macrophage colony-stimulating factor pretreatment of bone marrow progenitor cells regulates osteoclast differentiation based upon the stage of myeloid development.

Osteoclasts (OCs) are large, multinucleated bone resorbing cells originating from the bone marrow myeloid lineage, and share a common progenitor with macrophages and dendritic cells. Bone marrow cells (BMCs) are a common source for in vitro osteoclastogenesis assays but are a highly heterogeneous mixture of cells. Protocols for in vitro osteoclastogenesis vary considerably thus hindering interpretation and comparison of results between studies. Macrophage colony-stimulating factor (M-CSF) pretreatment is commonly used to expand OC progenitors (OCPs) in BMC cultures before in vitro differentiation. However, the failure of osteoclastogenesis of M-CSF primed bone marrow myeloid blasts has been reported. In this study, we used a simple method of differential adherence to plastic to enrich OCP from mouse BMCs. We found that M-CSF pretreatment of plastic-adherent BMCs (adBMCs) increased the number of CD11b-F4/80+ macrophages and decreased the number of CD11b+ monocytes resulting in decreased OC formation. M-CSF pretreatment of purified c-Kit+ progenitors weakly inhibited OC formation, whereas M-CSF pretreatment of purified c-Kit-CD11b+ progenitors promoted the formation of large OC. M-CSF pretreatment increased the proliferation of both purified c-Kit+ and c-Kit-CD11b+ cells and increased the percentage of CD11b-F4/80+ cells from c-Kit+ progenitors. In addition, M-CSF pretreatment increased the percentage of CD11b+ F4/80- cells from purified c-Kit-CD11b+ cells. M-CSF pretreatment increased the percentage of CD14 + CD16 + intermediate monocytes and subsequent OC formation from human 2adBMCs, and increased OC formation of purified CD14 + cells. Together, these results indicate that in vitro OCP expansion in the presence of M-CSF and bone marrow stromal cells is dependent upon the developmental stage of myeloid cells, in which M-CSF favors macrophage differentiation of multipotent progenitors, promotes monocyte maturation and supports differentiation of late-stage OCP cells.

Loss of Spry1 attenuates vascular smooth muscle proliferation by impairing mitogen-mediated changes in cell cycle regulatory circuits.

Signals from growth factors or mechanical stimuli converge to promote vascular smooth muscle cell (VSMC) migration and proliferation, key events in the pathogenesis of intimal hyperplasia upon vascular injury. Spry1, a regulator of receptor tyrosine kinases (RTK), plays a role in maintaining the contractile phenotype of VSMC. The aim of the current study was to determine the role of Spry1 in VSMC proliferation in vitro and injury induced neointimal hyperplasia in vivo. VSMC proliferation and neointima formation were evaluated in cultured human aortic SMC (hAoSMC) and ligation-induced injury of mouse carotid arteries from Spry1 gene targeted mice, and their corresponding wild type littermates. Human Spry1 or non-targeting control lentiviral shRNAs were used to knock down Spry1 in hAoSMC. Time course cell cycle analysis showed a reduced fraction of S-phase cells at 12 and 24 h after growth medium stimulation in Spry1 shRNA transduced hAoSMC. Consistent with reduced S-phase entry, the induction of cyclinD1 and the levels of pRbS807/S811, pH3Ser10, and pCdc2 were also reduced, while the cell cycle inhibitor p27Kip1 was maintained in Spry1 knockdown hAoSMC. In vivo, loss of Spry1 attenuated carotid artery ligation-induced neointima formation in mice, and this effect was accompanied by a decrease in cell proliferation similar to the in vitro results. Our findings demonstrate that loss of Spry1 attenuates mitogen-induced VSMC proliferation, and thus injury-induced neointimal hyperplasia likely via insufficient activation of Akt signaling causing decreased cyclinD1 and increased p27Kip1 and a subsequent decrease in Rb and cdc2 phosphorylation.

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway.

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvß3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvß3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvß3 signaling, this collagen epitope promoted αvß3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvß3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvß3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvß3 rather than the receptor itself.

Inhibition of Ovarian Tumor Growth by Targeting the HU177 Cryptic Collagen Epitope.

Evidence suggests that stromal cells play critical roles in tumor growth. Uncovering new mechanisms that control stromal cell behavior and their accumulation within tumors may lead to development of more effective treatments. We provide evidence that the HU177 cryptic collagen epitope is selectively generated within human ovarian carcinomas and this collagen epitope plays a role in SKOV-3 ovarian tumor growth in vivo. The ability of the HU177 epitope to regulate SKOV-3 tumor growth depends in part on its ability to modulate stromal cell behavior because targeting this epitope inhibited angiogenesis and, surprisingly, the accumulation of α-smooth muscle actin-expressing stromal cells. Integrin α10ß1 can serve as a receptor for the HU177 epitope in α-smooth muscle actin-expressing stromal cells and subsequently regulates Erk-dependent migration. These findings are consistent with a mechanism by which the generation of the HU177 collagen epitope provides a previously unrecognized α10ß1 ligand that selectively governs angiogenesis and the accumulation of stromal cells, which in turn secrete protumorigenic factors that contribute to ovarian tumor growth. Our findings provide a new mechanistic understanding into the roles by which the HU177 epitope regulates ovarian tumor growth and provide new insight into the clinical results from a phase 1 human clinical study of the monoclonal antibody D93/TRC093 in patients with advanced malignant tumors.

Erratum to: Suppression of Spry4 enhances cancer stem cell properties of human MDA-MB-231 breast carcinoma cells.

[This corrects the article DOI: 10.1186/s12935-016-0292-7.].

Sef Regulates Epithelial-Mesenchymal Transition in Breast Cancer Cells.

Sef (similar expression to fgf), also know as IL17RD, is a transmembrane protein shown to inhibit fibroblast growth factor signaling in developmental and cancer contexts; however, its role as a tumor suppressor remains to be fully elucidated. Here, we show that Sef regulates epithelial-mesenchymal transition (EMT) in breast cancer cell lines. Sef expression was highest in the normal breast epithelial cell line MCF10A, intermediate expression in MCF-7 cells and lowest in MDA-MB-231 cells. Knockdown of Sef increased the expression of genes associated with EMT, and promoted cell migration, invasion, and a fibroblastic morphology of MCF-7 cells. Overexpression of Sef inhibited the expression of EMT marker genes and inhibited cell migration and invasion in MCF-7 cells. Induction of EMT in MCF10A cells by TGF-ß and TNF-α resulted in downregulation of Sef expression concomitant with upregulation of EMT gene expression and loss of epithelial morphology. Overexpression of Sef in MCF10A cells partially blocked cytokine-induced EMT. Sef was shown to block ß-catenin mediated luciferase reporter activity and to cause a decrease in the nuclear localization of active ß-catenin. Furthermore, Sef was shown to co-immunoprecipitate with ß-catenin. In a mouse orthotopic xenograft model, Sef overexpression in MDA-MB-231 cells slowed tumor growth and reduced expression of EMT marker genes. Together, these data indicate that Sef plays a role in the negative regulation of EMT in a ß-catenin dependent manner and that reduced expression of Sef in breast tumor cells may be permissive for EMT and the acquisition of a more metastatic phenotype. J. Cell. Biochem. 117: 2346-2356, 2016. © 2016 Wiley Periodicals, Inc.

Lipid Profiling of In Vitro Cell Models of Adipogenic Differentiation: Relationships With Mouse Adipose Tissues.

Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MS(ALL) . Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-derived BAT-C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl- and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3-L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose-derived cell differentiation models, respectively. MS/MS(ALL) data suggest new lipid biomarkers for tissue-specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo. J. Cell. Biochem. 117: 2182-2193, 2016. © 2016 Wiley Periodicals, Inc.

Suppression of Spry4 enhances cancer stem cell properties of human MDA-MB-231 breast carcinoma cells.

BACKGROUND: Cancer stem cells contribute to tumor initiation, heterogeneity, and recurrence, and are critical targets in cancer therapy. Sprouty4 (Spry4) is a potent inhibitor of signal transduction pathways elicited by receptor tyrosine kinases, and has roles in regulating cell proliferation, migration and differentiation. Spry4 has been implicated as a tumor suppressor and in modulating embryonic stem cells. OBJECTIVES: The purpose of this research was to test the novel idea that Spry4 regulates cancer stem cell properties in breast cancer. METHODS: Loss-of function of Spry4 in human MDA-MB-231 cell was used to test our hypothesis. Spry4 knockdown or control cell lines were generated using lentiviral delivery of human Spry4 or non-targeting control shRNAs, and then selected with 2 µg/ml puromycin. Cell growth and migratory abilities were determined using growth curve and cell cycle flow cytometry analyses and scratch assays, respectively. Xenograft tumor model was used to determine the tumorigenic activity and metastasis in vivo. Cancer stem cell related markers were evaluated using immunoblotting assays and fluorescence-activated cell sorting. Cancer stem cell phenotype was evaluated using in vitro mammosphere formation and drug sensitivity tests, and in vivo limiting dilution tumor formation assay. RESULTS: Two out of three tested human Spry4 shRNAs significantly suppressed the expression of endogenous Spry4 in MDA-MB-231 cells. Suppressing Spry4 expression increased MDA-MB-231 cell proliferation and migration. Suppressing Spry4 increased ß3-integrin expression, and CD133(+)CD44(+) subpopulation. Suppressing Spry4 increased mammosphere formation, while decreasing the sensitivity of MDA-MB-231 cells to Paclitaxel treatment. Finally, suppressing Spry4 increased the potency of MDA-MB-231 cell tumor initiation, a feature attributed to cancer stem cells. CONCLUSIONS: Our findings provide novel evidence that endogenous Spry4 may have tumor suppressive activity in breast cancer by suppressing cancer stem cell properties in addition to negative effects on tumor cell proliferation and migration.

Inhibition of tumor-associated αvß3 integrin regulates the angiogenic switch by enhancing expression of IGFBP-4 leading to reduced melanoma growth and angiogenesis in vivo.

A more complete understanding of the mechanisms that regulate the angiogenic switch, which contributes to the conversion of small dormant tumors to actively growing malignancies, is important for the development of more effective anti-angiogenic strategies for cancer therapy. While significant progress has been made in understanding the complex mechanisms by which integrin αvß3 expressed in endothelial cells governs angiogenesis, less is known concerning the ability of αvß3 expressed within the tumor cell compartment to modulate the angiogenic output of a tumor. Here we provide evidence that αvß3 expressed in melanoma cells may contribute to the suppression of IGFBP-4, an important negative regulator of IGF-1 signaling. Given the multiple context-dependent roles for αvß3 in angiogenesis and tumor progression, our novel findings provide additional molecular insight into how αvß3 may govern the angiogenic switch by a mechanism associated with a p38 MAPK and matrix metalloproteinases-dependent regulation of the endogenous angiogenesis inhibitor IGFBP-4.

Fibroblast growth factor signaling in the vasculature.

Despite their discovery as angiogenic factors and mitogens for endothelial cells more than 30 years ago, much remains to be determined about the role of fibroblast growth factors (FGFs) and their receptors in vascular development, homeostasis, and disease. In vitro studies show that members of the FGF family stimulate growth, migration, and sprouting of endothelial cells, and growth, migration, and phenotypic plasticity of vascular smooth muscle cells. Recent studies have revealed important roles for FGFs and their receptors in the regulation of endothelial cell sprouting and vascular homeostasis in vivo. Furthermore, recent work has revealed roles for FGFs in atherosclerosis, vascular calcification, and vascular dysfunction. The large number of FGFs and their receptors expressed in endothelial and vascular smooth muscle cells complicates these studies. In this review, we summarize recent studies in which new and unanticipated roles for FGFs and their receptors in the vasculature have been revealed.

Transitory FGF treatment results in the long-lasting suppression of the proliferative response to repeated FGF stimulation.

FGF applied as a single growth factor to quiescent mouse fibroblasts induces a round of DNA replication, however continuous stimulation results in arrest in the G1 phase of the next cell cycle. We hypothesized that FGF stimulation induces the establishment of cell memory, which prevents the proliferative response to repeated or continuous FGF application. When a 2-5 days quiescence period was introduced between primary and repeated FGF treatments, fibroblasts failed to efficiently replicate in response to secondary FGF application. The establishment of "FGF memory" during the first FGF stimulation did not require DNA synthesis, but was dependent on the activity of FGF receptors, MEK, p38 MAPK and NFκB signaling, and protein synthesis. While secondary stimulation resulted in strongly decreased replication rate, we did not observe any attenuation of morphological changes, Erk1/2 phosphorylation and cyclin D1 induction. However, secondary FGF stimulation failed to induce the expression of cyclin A, which is critical for the progression from G1 to S phase. Treatment of cells with a broad range histone deacetylase inhibitor during the primary FGF stimulation rescued the proliferative response to the secondary FGF treatment suggesting that the establishment of "FGF memory" may be based on epigenetic changes. We suggest that "FGF memory" can prevent the hyperplastic response to cell damage and inflammation, which are associated with an enhanced FGF production and secretion. "FGF memory" may present a natural obstacle to the efficient application of recombinant FGFs for the treatment of ulcers, ischemias, and wounds.

FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development.

Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.

Insulin-like growth factor binding protein-4 differentially inhibits growth factor-induced angiogenesis.

An in-depth understanding of the molecular and cellular complexity of angiogenesis continues to advance as new stimulators and inhibitors of blood vessel formation are uncovered. Gaining a more complete understanding of the response of blood vessels to both stimulatory and inhibitory molecules will likely contribute to more effective strategies to control pathological angiogenesis. Here, we provide evidence that endothelial cell interactions with structurally altered collagen type IV may suppress the expression of insulin-like growth factor binding protein-4 (IGFBP-4), a well documented inhibitor of the IGF-1/IGF-1R signaling axis. We report for the first time that IGFBP-4 differentially inhibits angiogenesis induced by distinct growth factor signaling pathways as IGFBP-4 inhibited FGF-2- and IGF-1-stimulated angiogenesis but failed to inhibit VEGF-induced angiogenesis. The resistance of VEGF-stimulated angiogenesis to IGFBP-4 inhibition appears to depend on sustained activation of p38 MAPK as blocking its activity restored the anti-angiogenic effects of IGFBP-4 on VEGF-induced blood vessel growth in vivo. These novel findings provide new insight into how blood vessels respond to endogenous inhibitors during angiogenesis stimulated by distinct growth factor signaling pathways.

Sprouty4 regulates endothelial cell migration via modulating integrin ß3 stability through c-Src.

Angiogenesis is mediated by signaling through receptor tyrosine kinases (RTKs), Src family kinases and adhesion receptors such as integrins, yet the mechanism how these signaling pathways regulate one another remains incompletely understood. The RTK modulator, Sprouty4 (Spry4) inhibits endothelial cell functions and angiogenesis, but the mechanisms remain to be fully elucidated. In this study, we demonstrate that Spry4 regulates angiogenesis in part by regulating endothelial cell migration. Overexpression of Spry4 in human endothelial cells inhibited migration and adhesion on vitronectin (VTN), whereas knockdown of Spry4 enhanced these behaviors. These activities were shown to be c-Src-dependent and Ras-independent. Spry4 disrupted the crosstalk between vascular endothelial growth factor-2 and integrin αVß3, the receptor for VTN. Spry4 overexpression resulted in decreased integrin ß3 protein levels in a post-transcriptional manner in part by modulating its tyrosine phosphorylation by c-Src. Conversely, knockdown of Spry4 resulted in increased integrin ß3 protein levels and tyrosine phosphorylation. Moreover, in vivo analysis revealed that Spry4 regulated integrin ß3 levels in murine embryos and yolk sacs. Our findings identify an unanticipated role for Spry4 in regulating c-Src activity and integrin ß3 protein levels, which contributes to the regulation of migration and adhesion of endothelial cells. Thus, targeting Spry4 may be exploited as a target in anti-angiogenesis therapies.

Sprouty1 has a protective role in atherogenesis and modifies the migratory and inflammatory phenotype of vascular smooth muscle cells.

BACKGROUND AND AIMS: Sprouty1 (Spry1) regulates the differentiation of vascular smooth muscle cells (VSMC), and our aim was to determine its role in atherogenesis. A significant proportion of cells within atherosclerotic lesions are derived from migration and pathological adaptation of medial VSMC. METHODS: We used global Spry1 null mouse, and Myh11-CreERT2, ROSA26-STOPfl/fl-tdTomato-Spry1fl/fl mice to allow for lineage tracing and conditional Spry1 deletion in VSMC. Atherosclerosis was induced by injection of a mutant form of mPCSK9D377Y-AAV followed by Western diet. Human aortic VSMC (hVSMC) with shRNA targeting of Spry1 were also analyzed. RESULTS: Global loss of Spry1 increased inflammatory markers ICAM1 and Cox2 in VSMC. Conditional deletion of Spry1 in VSMC had no effect on early lesion development, despite increased Sca1high cells. After 26 weeks of Western diet, mice with VSMC deletion of Spry1 had increased plaque burden, with reduced collagen content and smooth muscle alpha actin (SMA) in the fibrous cap. Lineage tracing via tdTomato marking Cre-recombined cells indicated that VSMC with loss of Spry1 had decreased migration into the lesion, noted by decreased proportions of tdTomato+ and tdTomato+/SMA + cells. Loss-of-function of Spry1 in hVSMC increased mesenchymal and activation markers, including KLF4, PDGFRb, ICAM1, and Cox2. Loss of Spry1 enhanced the effects of PDGFBB and TNFa on hVSMC. CONCLUSIONS: Loss of Spry1 in VSMC aggravated plaque formation at later stages, and increased markers of instability. Our results indicate that Spry1 suppresses the mesenchymal and inflammatory phenotype of VSMC, and its expression in VSMC is protective against chronic atherosclerotic disease.

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.

Mechanisms of TGF-ß-induced differentiation in human vascular smooth muscle cells.

BACKGROUND: Transforming growth factor-ß (TGF-ß) plays an important role in vascular homeostasis through effects on vascular smooth muscle cells (SMC). Fine-tuning of TGF-ß signaling occurs at the level of ALK receptors or Smads, and is regulated with cell type specificity. METHODS: Our goal was to understand TGF-ß signaling in regulating SMC differentiation marker expression in human SMC. Activation of Smads was characterized, and loss- and gain-of-function reagents used to define ALK pathways. In addition, Smad-independent mechanisms were determined. RESULTS: TGF-ß type I receptors, ALK1 and ALK5, are expressed in human SMC, and TGF-ß1 phosphorylates Smad1/5/8 and Smad2/3 in a time- and dosage-dependent pattern. ALK5 activity, not bone morphogenetic protein type I receptors, is required for Smad phosphorylation. Endoglin, a TGF-ß type III receptor, is a TGF-ß1 target in SMC, yet endoglin does not modify TGF-ß1 responsiveness. ALK5, not ALK1, is required for TGF-ß1-induction of SMC differentiation markers, and ALK5 signals through an ALK5/Smad3- and MAP kinase-dependent pathway. CONCLUSION: The definition of the specific signaling downstream of TGF-ß regulating SMC differentiation markers will contribute to a better understanding of vascular disorders involving changes in SMC phenotype.

Identification of Sef, a novel modulator of FGF signalling.

Fibroblast growth factors (FGFs) are members of a family of some 30 secreted proteins important in the regulation of cellular proliferation, migration, differentiation and survival. Here we report the identification of a novel modulator of FGF signal transduction, sef, isolated from a zebrafish embryo library through an in situ hybridization screen. The sef gene encodes a transmembrane protein, and belongs to the synexpression group that includes some of the fgf genes. Sef expression is positively regulated by FGF, and ectopic expression of sef in zebrafish or Xenopus laevis embryos specifically inhibits FGF signalling. In co-immunoprecipitation assays, the intracellular domain of Sef interacts with FGF receptors, FGFR1 and FGFR2. Injection of antisense sef morpholino oligos mimicked the phenotypes observed by ectopic fgf8 expression, suggesting that Sef is required to limit FGF signalling during development.