Down-regulation of Sprouty2 via p38 MAPK plays a key role in the induction of cellular apoptosis by tumor necrosis factor-α

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Abstract

Mammalian Sprouty2 (Spry2) is a key regulator of the receptor tyrosine kinase/ERK signaling pathway and involved in many biological processes, including cell growth, migration, and tumor suppression. Here, we demonstrated that the intracellular protein level of Spry2 was significantly down-regulated by tumor necrosis factor-α (TNF-α) in both murine Swiss 3T3 fibroblasts and MLE15 lung epithelial cells. Although TNF-α activates multiple signaling cascades, only the inhibitor of p38 MAPK pathway blocked TNF-α-induced Spry2 down-regulation. Moreover, since both the mRNA level and protein half-life of Spry2 were unaltered by TNF-α treatment, this indicated the possible involvement of a translational mechanism in mediating the inhibitory effect of TNF-α. Importantly, rescue of the TNF-α-induced down-regulation of Spry2 by gene overexpression led to reverse of the apoptotic effect of TNF-α in Swiss 3T3 cells. To our knowledge, this study is the first that reported the association of Spry2 with TNF-α signaling pathway.

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Materials and methods

Materials. Unless otherwise indicated, all reagents were purchased from Sigma–Aldrich (St. Louis, MO). EGF and anti-Spry2 antibody were purchased from Upstate Biotechnology (Lake Placid, NY); TNF-α was obtained from R&D Systems (Minneapolis, Minnesota); SB-431542 was purchased from Tocris Biosciences (Ellisville, MO); PD98059, SP600125, SB203580, and BAY 11-7028 were from Calbiochem (San Diego, CA).

Cell culture. Swiss 3T3 cells were obtained from the American Type Culture Collection (Manassas,

TNF-α down-regulates the protein level of Spry2

To study whether the expression of Spry2 can be regulated by TNF-α, cultured Swiss 3T3 cells were treated with different concentrations of TNF-α for 24 h. Samples were then harvested, and total protein was prepared for immunoblot analysis. As shown in Fig. 1A, Spry2 protein levels declined in a dose-dependent manner in response to treatment with 0.02–10 ng/ml TNF-α. The lower limit for detection of the TNF-α effect was 0.2 ng/ml, and the maximum suppression of Spry2 was attained at a TNF-α

Discussion

TGF-β is the first endogenous cytokine showing capability to down-regulate Spry2 [13], and the functional significance of Spry2 down-regulation has been associated with both cancer formation and enhancement of cellular proliferation. Herein, we further showed that Spry2 down-regulation can be induced by another cytokine, TNF-α and plays a critical role in regulating apoptosis.

Cytokines such as TNF-α are able to regulate gene expression via multiple signal transduction pathways. Therefore, we

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