Wilms tumor suppressor 1 (WT1) and early growth response 1 (EGR1) are regulators of STIM1 expression

Abstract

Store-operated calcium entry (SOCE) is a key evolutionarily conserved process whereby decreases in endoplasmic reticulum Ca(2+) content lead to the influx of Ca(2+) across the plasma membrane. How this process is regulated in specific tumor cell types is poorly understood. In an effort to address this concern, we obtained and tested primary Wilms tumor cells, finding no detectable SOCE in this cell type. Analysis of the expression levels of STIM1 and ORAI1 (the molecular mediators of SOC) revealed poor STIM1 expression. Analysis of the STIM1 promoter using the TESS search system (University of Pennsylvania) revealed four putative response elements to the zinc-finger proteins WT1 (Wilms tumor suppressor 1) and EGR1 (early growth response 1). Either overexpression of WT1 or knockdown of EGR1 resulted in loss of STIM1 expression and a resultant decrease in SOCE. Furthermore, examination of Egr1 knock-out animals revealed loss of STIM1 expression in multiple tissues. Finally, using chromatin immunoprecipitation, we reveal direct binding of both WT1 and EGR1 to putative response elements located within 500 bp of the transcriptional start site of STIM1. Considering that WT1 and EGR1 are well described oncogenes and tumor suppressors, these observations may reveal new mechanisms responsible for distinct Ca(2+) signals in cancer cells.

FIGURE 1.

Loss of SOC in Wilms tumor cells. A, SOCE was measured in Fura-2-loaded WT10, WT11, and HEK293 cells after ER Ca²⁺ depletion via the addition of the SERCA inhibitor thapsigargin (Tg; 2 μm) in nominally Ca²⁺-free medium. Extracellular Ca²⁺ concentration was increased from 0 to 1 mm either before or after store depletion where indicated to differentiate between store-independent (before thapsigargin) and store-dependent (after thapsigargin) Ca²⁺ entry. Shaded areas indicate S.E. B, protein extracted from WT10, WT11, and HEK293 cells was analyzed by Western blotting for STIM1 expression. actin was used as a loading control. C, RNA extracted from WT10, WT11, and HEK293 cells was analyzed by quantitative PCR for expression of TATA-binding protein (TBP), STIM1, WT1, and EGR1. mRNA expression levels are shown as %TBP. *, significant differences from expression levels in HEK293 cells as determined by analysis of variance with Tukey's post-hoc test (p < 0.05). All experiments were completed a minimum of three times.

FIGURE 2.

Control of STIM1 expression by WT1 and EGR1. A, representative traces of Ca²⁺ responses in Fura-2-loaded HEK293 cells after overexpression of WT1A or knockdown of EGR1. Shaded areas indicate S.E. B, quantitation of the change in cytosolic Ca²⁺ concentration after store depletion from three experiments performed as depicted in A. C, STIM1 expression levels in HEK293 cells transfected with either WT1A- or EGR1-targeted siRNA as determined by quantitative PCR. D, Western blot analysis of STIM1, ORAI1, and actin expression in HEK293 cells transfected with WT1A or EGR1 siRNA. KD, knockdown. E, Western blot analysis of STIM1, ORAI1, and actin expression in G401 cells transfected with WT1A or EGR1 siRNA. F, Western blot analysis of STIM1, EGR1, and actin expression in pheochromocytoma 12 or human kidney 2 cells after incubation in either full or 10% growth medium for 48 h. *, significant differences from the control (p < 0.05) as determined by one-way analysis of variance with Tukey's post-hoc test.

FIGURE 3.

Analysis of STIM1 expression in Egr1 knock-out mouse tissues. A, DNA was extracted from C57Bl/6 mouse tails, amplified by PCR, and analyzed by PAGE. B and C, protein was extracted from Egr1^+/+, Egr1^+/−, and Egr1^−/− tissues exhibiting EGR1-dependent STIM1 expression (B; kidney, spleen, liver, and brain) and those exhibiting EGR1-independent STIM1 expression (C; pancreas, thymus, and skeletal muscle). STIM1 expression was determined by Western blot analysis, whereas actin expression was determined as a loading control. WT, wild type; Het, heterozygous; KO, knock-out; Sk., skeletal.

FIGURE 4.

WT1 and EGR1 bind to the genomic region adjacent to the STIM1 transcriptional start site. A, analysis of the DNA sequence of 11p15.5 in the region surrounding the STIM1 transcriptional start site revealed four putative WT1/EGR1 REs (underlined). B, binding of WT1 and EGR1 to the four putative response elements was determined by ChIP of genomic DNA extracted from either HEK293 or G401 cells. Protein-DNA complexes were immunoprecipitated with anti-EGR1 or anti-WT1 antibodies or anti-rabbit IgG. DNA was amplified by PCR using the primers depicted in boldface in A. No template was used as a negative control for each PCR. Each experiment was completed a minimum of three times.

FIGURE 5.

Model for control of STIM1 expression by WT1 and EGR1. Putative EGR1 response elements are depicted relative to the start site for transcription of STIM1 mRNA (marked by the blue arrow). Inhibition of EGR1 binding at RE1 is marked with an arrow.