The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.

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Sef was recently identified as a negative regulator of fibroblast growth factor (FGF) signaling in a genetic screen of zebrafish and subsequently in mouse and humans. By inhibiting FGFR1 tyrosine phosphorylation and/or Ras downstream events, Sef inhibits FGF-mediated ERK activation and cell proliferation as well as PC12 cell differentiation. Here we show that Sef and a deletion mutant of Sef lacking the extracellular domain (SefIC) physically interact with TAK1 (transforming growth factor-␤-associated kinase) and activate JNK through a TAK1-MKK4-JNK pathway. Sef and SefIC overexpression also resulted in apoptotic cell death, while dominant negative forms of MKK4 and TAK1 blocked Sef-mediated JNK activation and attendant 293T cell apoptosis. These investigations reveal a novel activating function of Sef that is distinct from its inhibitory effect on FGF receptor signaling and ERK activation.Cell growth and differentiation are mediated in part by the actions of receptor tyrosine kinases (RTKs) 1 that signal via Ras-MAPK pathways (1, 2). Dysregulation of RTK signaling is associated with human diseases including cancer, skeletal dwarfism, and craniosynostosis, thus RTK signaling must be tightly regulated (3)(4)(5). One mode involves negative feedback loops that limit the duration and/or intensity of RTK signals.Within the Ras-MAPK pathway, several feedback inhibitors have recently been identified and include members of the Sprouty and SPRED (Sprouty-related EVH1-domain containing) families of polypeptides (6 -8). Sef (similar expression to fgf genes), also called IL-17R-D, is a newly identified feedback inhibitor of fibroblast growth factor receptor signaling and encodes a type I transmembrane domain protein that is highly conserved in zebrafish, mouse, and humans (9). Studies in each system indicate that Sef acts as a feedback inhibitor of FGFmediated Ras-MAPK signaling and ERK activation (10 -13). Studies in mouse also indicate Sef inhibition of Akt (14). Sef has been proposed to inhibit ERK activation in NIH3T3 cells by physically interacting with the FGFR1 and decreasing its tyrosine phosphorylation (14). Other studies indicate that Sef may act downstream of Ras (12,15). In addition, a splice variant of Sef has been identified that gives rise to a cytoplasmic form of Sef that likewise associates with FGFR1 and inhibits ERK activation (15).In this study, we show that ectopic expression of Sef activates c-Jun amino-terminal kinase (JNK) and apoptosis. We also present evidence that Sef activates JNK through a TAK1-MKK4-JNK pathway and that TAK1 associates with Sef in co-immunoprecipitated complexes. These studies demonstrate for the first time the multifunctional potential of this FGFR and ERK modulating protein as a mediator of JNK and apoptosis. EXPERIMENTAL PROCEDURESReagents-Monoclonal antibodies to FLAG and ␤-actin were from Sigma. The V5 monoclonal antibody was from Invitrogen. Antibodies to JNK, phospho-JNK, c-Jun, phospho-c-Jun, MKK4, phospho-MKK4, caspase-3, and poly(ADP-ribose) polymerase (PARP) were fr...

Fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) are major regulators of skeletal growth and development. Signal transduction via FGFRs is complex and mediates proliferation, differentiation, or migration depending upon the cellular context. Members of the Spry gene family antagonize the FGFR signal transduction pathway and inhibit lung morphogenesis, angiogenesis, and chondrogenesis. We examined the expression of Spry2 in the osteoblastic MC3T3-E1 cell line. MC3T3-E1 cells express Spry2 in response to FGF1 stimulation. Treatment of MC3T3-E1 cells with FGF1 results in the expression of Spry2 in a manner consistent with an early response gene. Pharmacological inhibitors of mitogen-activated protein kinase activation inhibit FGF1-induced expression of Spry2 mRNA. Transient overexpression of Spry2 in MC3T3-E1 resulted in decreased FGF1-mediated extracellular signal-regulated kinase phosphorylation and FGF1-stimulated osteopontin promoter activity. Furthermore, we show that Spry2 interacts with Raf-1 in a glutathione-S-transferase pulldown assay and that this interaction may involve multiple sites. Finally, Spry2 expression precedes the onset of the expression of osteoblast differentiation markers in an in vitro assay of primary osteoblast differentiation. Taken together, these results indicate that Spry2 expression is an early response to stimulation by FGF1 in MC3T3-E1 cells and acts as a feedback inhibitor of FGF1-induced osteoblast responses, possibly through interaction with Raf1.

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