WT1 induction of mitogen-activated protein kinase phosphatase 3 represents a novel mechanism of growth suppression

Abstract

In its role as a tumor suppressor, WT1 transactivates several genes that are regulators of cell growth and differentiation pathways. For instance, WT1 induces the expression of the cell cycle regulator p21, the growth-regulating glycoprotein amphiregulin, the proapoptotic gene Bak, and the Ras/mitogen-activated protein kinase (MAPK) inhibitor Sprouty1. Here, we show that WT1 transactivates another important negative regulator of the Ras/MAPK pathway, MAPK phosphatase 3 (MKP3). In a WT1-inducible cell line that exhibits decreased cell growth and increased apoptosis on expression of WT1, microarray analysis showed that MKP3 is the most highly induced gene. This was confirmed by real-time PCR where MKP3 and other members of the fibroblast growth factor 8 syn expression group, which includes Sprouty 1 and the Ets family of transcription factors, were induced rapidly following WT1 expression. WT1 induction was associated with a block in the phosphorylation of extracellular signal-regulated kinase in response to epidermal growth factor stimulation, an effect mediated by MKP3. In the presence of a dominant-negative MKP3, WT1 could no longer block phosphorylation of extracellular signal-regulated kinase. Lastly, when MKP3 expression is down-regulated by short hairpin RNA, WT1 is less able to block Ras-mediated transformation of 3T3 cells.

Figure 1

Induction of MKP3 and FGF8 syn expression group genes by WT1. Real time PCR analysis of indicated genes in Saos-WT1 inducible cell line following withdrawal of tetracycline. Data were calculated as fold induction relative to pre-WT1-induction condition after normalizing with GAPDH. Western blots corresponding to each induction experiment shows expression of induced WT1 over time.

Figure 2

Regulation of MKP3 promoter activity by WT1 in kidney cell lines. (A) Schematic diagram of the MKP3 promoter fragment highlighting the location of putative WT1 binding sites and PCR primers. (B) Luciferase assays of 293T cells transfected with the MKP3 promoter and increasing concentrations of a vector expressing wild-type or mutant WT1. Data is representative of 3 experiments; each performed in triplicate and normalized to protein concentrations. (C) Chromatin immunoprecipitation from the Wilms tumor cell line, CCG99-11, using a WT1 antibody, IgG or no antibody. PCR primers detect MKP3 promoter regions. Actin primers were used as control.

Figure 3

WT1 regulates the level of phosphorylated ERK1/2 through activation of MKP3. (A) WT1-inducible CCG99-11 clones were incubated with or without doxycycline (Dox) in the presence or absence of EGF. HEK293 clones were transfected with either (B) Flag-tagged MKP3 C294S mutant or (C) MKP3 shRNA vectors prior to induction with Dox. Western blots were performed to detect total and phosphorylated ERK1/2, WT1 and MKP3. Relative expression of endogenous MKP3 mRNA, as determined by qPCR, is indicated.

Figure 4

Reduction of MKP3 expression by shRNA. HEK293 and 3T3 cell lines were transfected with human and mouse MKP3-specific short hairpin interfering RNA (shRNA) vectors, respectively, or with control vector and selected for 4 days. Expression level of MKP3 was determined by qPCR analysis.

Figure 5

Suppression of Ras-induced foci formation by WT1 is mediated by MKP3. (A) NIH3T3 cells were transfected with AU5-Ras in the presence or absence of WT1 or Flag-tagged MKP3 and allowed to grow for 10 days. Foci were counted and plotted as mean number of foci/10cm plate. The relative expression of each protein, as determined by Western blot, is shown. (B) Knock down of endogenous MKP3 expression by shRNA partially blocked the WT1-mediated suppression of foci formation. Experiments were carried out independently three times in triplicate.