Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein

Abstract

Receptor Tyrosine Kinase (RTK) signaling plays a major role in tumorigenesis and normal development. Sprouty2 (Spry2) attenuates RTK signaling and inhibits processes such as angiogenesis, cell proliferation, migration and survival, which are all upregulated in tumors. Indeed in cancers of the liver, lung, prostate and breast, Spry2 protein levels are markedly decreased correlating with poor patient prognosis and shorter survival. Thus, it is important to understand how expression of Spry2 is regulated. While prior studies have focused on the post-translation regulation of Spry2, very few studies have focused on the transcriptional regulation of SPRY2 gene. Here, we demonstrate that in the human hepatoma cell line, Hep3B, the transcription of SPRY2 is inhibited by the transcription regulating hypoxia inducible factors (HIFs). HIFs are composed of an oxygen regulated alpha subunit (HIF1α or HIF2α) and a beta subunit (HIF1β). Intriguingly, silencing of HIF1α and HIF2α elevates SPRY2 mRNA and protein levels suggesting HIFs reduce the transcription of the SPRY2 promoter. In silico analysis identified ten hypoxia response elements (HREs) in the proximal promoter and first intron of SPRY2. Using chromatin immunoprecipitation (ChIP), we show that HIF1α/2α bind near the putative HREs in the proximal promoter and intron of SPRY2. Our studies demonstrated that not only is the SPRY2 promoter methylated, but silencing HIF1α/2α reduced the methylation. ChIP assays also showed DNA methyltransferase1 (DNMT1) binding to the proximal promoter and first intron of SPRY2 and silencing HIF1α/2α decreased this association. Additionally, silencing of DNMT1 mimicked the HIF1α/2α silencing-mediated increase in SPRY2 mRNA and protein. While simultaneous silencing of HIF1α/2α and DNMT1 increased SPRY2 mRNA a little more, the increase was not additive suggesting a common mechanism by which DNMT1 and HIF1α/2α regulate SPRY2 transcription. Together these data suggest that the transcription of SPRY2 is inhibited by HIFs, in part, via DNMT1- mediated methylation.

Fig 1. HIF1α and HIF2α regulate mRNA and protein levels of Spry2.

(A) Cells transfected with siRNA against HIF1α, HIF2α or both isoforms were incubated under hypoxic conditions (3% O₂) for 24 hours. RNA was isolated and mRNA levels of HIF1α, HIF2α (right panels) and SPRY2 (left panel) were monitored by qRT-PCR with specific primers/probe and normalized with 18S rRNA. Cells transfected with mutant siRNA were used as control. Graphs are mean + SEM of 5 independent experiments. (B) Cells were treated same as in (A) except hypoxic incubation was for 32 hours. The protein levels of HIF1α, HIF2α and Spry2 were analyzed by Western blotting. Actin was used as loading control. Graph is mean + SEM from six independent experiments. (C) HEK293T cells transfected with vector alone or HIF1β along with vector, HIF1α, HIF2α, or both HIF1α and HIF2α were incubated under normoxic conditions for 40 hours after transfection. The mRNA amounts of SPRY2 (left panel) or PGK1 (right panel) were monitored by qRT- PCR and normalized with 18S rRNA. Graphs are mean + SEM from four independent experiments. Each group was compared with cells transfected with pcDNA3-HIF1β only. Statistical significance was assessed using unpaired Student t-tests (A & B) or one-way ANOVA with Dunnett’s multiple comparison test (C) **: p<0.01, ***: p<0.001, ****: p<0.0001.

Fig 2. HIF1α and HIF2α do not regulate the stability of SPRY2 mRNA, but they bind to the proximal promoter and intron of SPRY2.

(A) Hep3B cells transfected with control or HIF1α/HIF2α siRNAs were incubated in hypoxia for 24 hours and then treated with actinomycin D (3 μg/mL). Total RNA was extracted at the indicated times and the mRNA levels of SPRY2 were monitored using qRT-PCR. (B) Schematic of SPRY2 from -3850 to 3395 encompassing the promoter, transcription start site (+1), exon 1 (Ex1), intron, and exon 2 (Ex2). Each grey rectangle labeled with a letter represents a putative HRE and the location of each HRE is labeled underneath. Each numbered line above shows the location of a primer pair designed to amplify a region of DNA with specific putative HREs in a ChIP. (C) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. Proteins, cross-linked to DNA, were immunoprecipitated with control rabbit IgG or HIF1β antibody. The DNA was sheared and the amounts of co-immunoprecipitated DNA were examined by qRT-PCR with the indicated primer sets. Graphs are the mean + SEM from five independent experiments. (D) Hep3B cells transfected with control, HIF1α, HIF2α, or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. ChIP assays were performed as stated in (C) except primers were used that encompass the HREs located in the promoter of the HIF1α-responsive gene PFK-1 or the HIF2α-responsive gene EPO. Graph shows the mean + SEM from three independent experiments. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test (C & D) *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Fig 3. HIF1α and HIF2α repress SPRY2 mRNA levels by enhancing the methylation of the SPRY2 promoter.

(A) Upper panel: Hep3B cells treated with vehicle (V) or decitabine (DAC) were incubated in hypoxia for 24 hours. DNA was extracted, bisulfite-converted, and the methylation status was assessed with methylation specific (M) and unmethylated specific (U) PCR primers. The amount of β-actin DNA was monitored to control for DNA amount loaded into each PCR. The amounts of methylated and unmethylated SPRY2 promoter DNA were quantified by densitometry and normalized to β-actin. Graph shows the mean + SEM for three independent experiments. Lower panel: Schematic of hSPRY2 promoter and gene showing the positions of PCR primers for both methylation specific and unmethylated specific PCRs. The arrow shows the transcription start site. Grey rectangles depict putative HREs. (B) Hep3B cells were treated with vehicle or decitabine (DAC, 5 μM), transfected with control or HIF1α and HIF2α siRNAs and incubated in hypoxia for 24 hours. RNA was isolated and the mRNA amounts of SPRY2 (left panel), HIF1α and HIF2α (right panels) were monitored by qRT-PCR and normalized with 18S rRNA. Graphs show the mean + SEM from three independent experiments repeated in duplicate or triplicate. (C) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 24 hours. The methylation status of the SPRY2 promoter was analyzed as in (A). Graph shows the mean + SEM from five independent experiments. Statistical significance was assessed using unpaired student t-tests (A, B & C) *: p<0.05, **: p<0.01, ***: p<0.001, n.s: not significant.

Fig 4. DNMT1 contributes toward the suppression of SPRY2 mRNA expression by HIF1α and HIF2α.

(A) Hep3B cells were treated with vehicle or laccaic acid A (LCA, 50 μg/mL), transfected with control or HIF1α and HIF2α siRNAs and incubated in hypoxia for 24 hours. RNA was isolated and mRNA amounts of SPRY2 (left panel), HIF1α, and HIF2α (right panels) were quantified by qRT-PCR and normalized with 18S rRNA. Graphs show the mean + SEM from three independent experiments in duplicate. (B) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. Proteins, cross-linked to DNA, were immunoprecipitated with control mouse IgG or a DNMT1 antibody. The DNA was sheared and the amounts of co-immunoprecipitated DNA were examined by qRT-PCR with the indicated primer sets. Location of binding of primers is indicated in Fig 2B. Graphs show the mean + SEM from three independent experiments performed in singles or duplicates. Statistical significance was assessed using unpaired student t-tests (A) or one-way ANOVA with Dunnett’s multiple comparison test (B). *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Fig 5. Silencing DNMT1 attenuates the increase in SPRY2 mRNA and protein levels when HIF1α and HIF2α expression is silenced.

(A) Hep3B and (B) HuH7 cells transfected with control, HIF1α and HIF2α, DNMT1, or DNMT1 and HIF1α and HIF2α siRNAs were incubated in hypoxia for 24 hours. RNA was isolated and mRNA amounts of SPRY2, HIF1α, HIF2α, and DNMT1 were quantified by qRT-PCR and normalized with (A) 18S rRNA or (B) 18S rRNA and RPLP0. Graphs show the mean + SEM from three independent experiments in duplicate or triplicate. (C) Hep3B or (D) HuH7 cells were treated as in (A&B). The protein levels of DNMT1, HIF1α, HIF2α and Spry2 were analyzed by Western blotting. Actin was used as a loading control. Graph shows the mean + SEM from (C) three or (D) four independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparison test (A, B) or Sidak’s multiple comparison test (D) or unpaired student t-tests (B) *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, n.s: not significant. Key for bar graphs is at top.