Regulation of sprouty expression by PLCγ and calcium-dependent signals

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Abstract

Sprouty, an essential antagonist of fibroblast growth factor receptor signaling, is induced following fibroblast growth factor receptor activation. The signaling pathways that induce sprouty have been incompletely characterized. However, studies show that MAP kinase signaling stimulates sprouty induction in various cell lines. Here we report that activation of sprouty expression by basic fibroblast growth factor required phospholipase Cγ (PLCγ) and calcium-dependent signaling. We showed that the induction of sprouty was inhibited by chelation of intracellular or extracellular calcium and that a fibroblast growth factor receptor deficient for PLCγ signaling only weakly induced sprouty expression. Additionally, inhibition of PLCγ with a pharmacological antagonist repressed the induction of sprouty by basic fibroblast growth factor. These findings indicate that calcium-dependent signaling regulates sprouty expression and that PLCγ is vital for this process. This pathway of sprouty induction may be critical at sites such as limb bud mesenchyme where MAP kinases are inactive.

Section snippets

Materials and methods

Cell culture conditions. ATDC5 cells were maintained in a log growth phase in DMEM/Ham’s F12 hybrid medium containing 5% (v/v) FBS (Hyclone), 10 μg/ml human transferrin (Calbiochem), and 3 × 10−8 M sodium selenite (Sigma) at 37 °C in a humidified 5% CO2/95% air atmosphere. In the sprouty induction study, we plated ATDC5 cells in six-multiwell plastic plates (Falcon) at an initial density of 30 × 104 cells/well and cultured these cells without changing the medium for 48 h to obtain quiescent state. For

bFGF induces sprouty1 and sprouty2 in ATDC5 cells

The mouse chondrogenic cell line, ATDC5, was stimulated with bFGF and Spry1 and Spry2 gene expression was assessed by RT-PCR (Fig. 1). Expression of both genes was induced after 1 h of bFGF addition. Spry1 expression achieved maximal expression 4 h after stimulation with bFGF and elevated levels of sprouty expression were maintained for at least 12 h. Similarly, Spry2 was induced 1 h after addition of bFGF and high expression continued at least 12 h after bFGF addition. No amplification product was

Discussion

Others [15] and we have shown that erk MAP kinases contribute to FGF-induced sprouty gene expression. However, a requirement for alternative pathways to induce sprouty expression is suggested by the finding that erk MAP kinases are repressed in certain tissues where sproutys are expressed. For example, in limb bud mesenchyme erk MAP kinases are largely inactive [16], [17], yet sproutys are strongly expressed. Therefore, in this report we investigated if calcium-dependent pathways regulate

Acknowledgments

We thank Victoria Frohlich and James Lechleiter for assistance with confocal imaging and analysis. This work was supported by NIH Grants AR47070, AR050024 and the Paul Beeson Physician Faculty Scholars in Aging Research Program.

References (26)

  • C.W. Taylor

    Controlling calcium entry

    Cell

    (2002)
  • G. Christofori

    The implications of angiogenesis on tumor invasiveness

    Angiogenesis

    (1998)
  • D.C. Sullivan et al.

    New molecular pathways in angiogenesis

    Br. J. Cancer

    (2003)
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