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Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells.

Impagnatiello MA, Weitzer S, Gannon G, Compagni A, Cotten M, Christofori G - J. Cell Biol. (2001)

Bottom Line: Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered.They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane.The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

ABSTRACT
Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

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mSpry-1 and -2 expression is regulated by FGF2 in endothelial cells. Uninfected 1G11 cells were cultured under exponential growth conditions (exp) or serum starved for 48 h and then stimulated with 10 ng/ml FGF2 for the times indicated. Northern blot analysis was performed on total RNA isolated from each time point, using full-length cDNAs for mSpry-1 and mSpry–2 as probes. Equal loading of RNAs was confirmed by staining of the gels with ethidium bromide. The lengths of Spry mRNAs (Kb) and 18S ribosomal RNA are indicated.
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Figure 4: mSpry-1 and -2 expression is regulated by FGF2 in endothelial cells. Uninfected 1G11 cells were cultured under exponential growth conditions (exp) or serum starved for 48 h and then stimulated with 10 ng/ml FGF2 for the times indicated. Northern blot analysis was performed on total RNA isolated from each time point, using full-length cDNAs for mSpry-1 and mSpry–2 as probes. Equal loading of RNAs was confirmed by staining of the gels with ethidium bromide. The lengths of Spry mRNAs (Kb) and 18S ribosomal RNA are indicated.

Mentions: In Drosophila, DSpry not only interferes with growth factor–mediated signal transduction, but the expression of DSpry is also induced by the factors it subsequently inhibits. For example, DSpry expression is induced by FGF during trachea development (Hacohen et al. 1998) and EGF during oogenesis and eye development (Casci et al. 1999; Reich et al. 1999); in other regions, DSpry expression is induced with active EGF receptor signaling (Kramer et al. 1999). During chicken and mouse embryogenesis, Spry expression was found specifically in centers of FGF signaling or was upregulated in areas where recombinant FGF was applied to cultured embryos (Minowada et al. 1999; Chambers and Mason 2000). To assess whether Sprys are regulated in a similar way in endothelial cells upon stimulation with angiogenic factors, HUVECs (data not shown) and 1G11 cells (Fig. 4) were serum starved and then stimulated with FGF2 for different times. Levels of mRNAs for endogenous Spry-1 and -2 were determined by Northern blot hybridization (Fig. 4). Although Spry-1 mRNA was found at high levels in starved cells, it was transiently downregulated 2 h after treatment with FGF2 and regained high levels of expression 6 h after growth factor stimulation. Inhibition of protein synthesis by cycloheximide during growth factor stimulation prevented the transient degradation of Spry-1 mRNA (data not shown). In contrast, expression of Spry-2 (Fig. 4) and -4 (data not shown) was low in starved cells; it was induced 2 h after treatment with FGF2 and subsequently downregulated at later time points. The results indicate that the expression of Sprys is directly modulated by growth factor stimulation and that the various members of the Spry family may be affected in different ways.


Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells.

Impagnatiello MA, Weitzer S, Gannon G, Compagni A, Cotten M, Christofori G - J. Cell Biol. (2001)

mSpry-1 and -2 expression is regulated by FGF2 in endothelial cells. Uninfected 1G11 cells were cultured under exponential growth conditions (exp) or serum starved for 48 h and then stimulated with 10 ng/ml FGF2 for the times indicated. Northern blot analysis was performed on total RNA isolated from each time point, using full-length cDNAs for mSpry-1 and mSpry–2 as probes. Equal loading of RNAs was confirmed by staining of the gels with ethidium bromide. The lengths of Spry mRNAs (Kb) and 18S ribosomal RNA are indicated.
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Related In: Results  -  Collection

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Figure 4: mSpry-1 and -2 expression is regulated by FGF2 in endothelial cells. Uninfected 1G11 cells were cultured under exponential growth conditions (exp) or serum starved for 48 h and then stimulated with 10 ng/ml FGF2 for the times indicated. Northern blot analysis was performed on total RNA isolated from each time point, using full-length cDNAs for mSpry-1 and mSpry–2 as probes. Equal loading of RNAs was confirmed by staining of the gels with ethidium bromide. The lengths of Spry mRNAs (Kb) and 18S ribosomal RNA are indicated.
Mentions: In Drosophila, DSpry not only interferes with growth factor–mediated signal transduction, but the expression of DSpry is also induced by the factors it subsequently inhibits. For example, DSpry expression is induced by FGF during trachea development (Hacohen et al. 1998) and EGF during oogenesis and eye development (Casci et al. 1999; Reich et al. 1999); in other regions, DSpry expression is induced with active EGF receptor signaling (Kramer et al. 1999). During chicken and mouse embryogenesis, Spry expression was found specifically in centers of FGF signaling or was upregulated in areas where recombinant FGF was applied to cultured embryos (Minowada et al. 1999; Chambers and Mason 2000). To assess whether Sprys are regulated in a similar way in endothelial cells upon stimulation with angiogenic factors, HUVECs (data not shown) and 1G11 cells (Fig. 4) were serum starved and then stimulated with FGF2 for different times. Levels of mRNAs for endogenous Spry-1 and -2 were determined by Northern blot hybridization (Fig. 4). Although Spry-1 mRNA was found at high levels in starved cells, it was transiently downregulated 2 h after treatment with FGF2 and regained high levels of expression 6 h after growth factor stimulation. Inhibition of protein synthesis by cycloheximide during growth factor stimulation prevented the transient degradation of Spry-1 mRNA (data not shown). In contrast, expression of Spry-2 (Fig. 4) and -4 (data not shown) was low in starved cells; it was induced 2 h after treatment with FGF2 and subsequently downregulated at later time points. The results indicate that the expression of Sprys is directly modulated by growth factor stimulation and that the various members of the Spry family may be affected in different ways.

Bottom Line: Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered.They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane.The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

ABSTRACT
Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

Show MeSH
Related in: MedlinePlus